Evaluation of low activity radioactivity based on oceanic blue gourami 137 Methods of cs toxicity

Abstract

The present application relates to the field of radioactive toxicity detection, and more particularly to a method for evaluating low-activity radioactive 137 Cs toxicity based on marine Oryzias latipes, which comprises the following steps: step 1, setting the breeding conditions of Oryzias latipes, starting the exposure experiment in the adjusted exposure parameters of the radioactive 137 Cs; step 2, observing the reproductive state parameters of Oryzias latipes after the exposure experiment; step 3, dissecting the Oryzias latipes to obtain gonadal tissue; step 4, analyzing and comparing the significant level differences of each index of the control group and the experimental group by using single factor variance statistics of the data information in steps 2 and 3, and determining the toxicity level of the radioactive 137 Cs by the index differences. The present application realizes rapid evaluation of low-activity radioactive 137 Cs toxicity in seawater, and has the characteristics of simple processing process, high operation repeatability and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of radiotoxicity detection, specifically to a method for evaluating low-activity radioactivity based on marine medaka. 137 Methods for detecting Cs toxicity. Background Technology

[0002] Nuclear power is a clean, low-carbon, safe, and efficient form of energy. Due to its safety, reliability, and strong supply capacity, it has become an important component of energy strategy. However, nuclear power plants are built along waterways, and during operation, some radioactive nuclides may be released, impacting marine life. 137 Cs is a radioactive nuclide that is inevitably produced during nuclear fission, with a half-life of up to 30.17 years. It enters the marine environment... 137 Cs accumulates in organisms and continuously releases gamma rays, affecting the health of marine life.

[0003] However, current research on the toxicity of radionuclides to marine organisms is limited, and available data for toxicity assessment of radioactive pollutants are scarce. Xu et al. (2023) used the stable isotope substitution method to study the ionic properties of Cs on mussels (C5) under experimental ecological conditions. Mytilis edulis The toxic effects of ) were found 133 Cs exposure leads to the death of mussels and inhibits their feeding behavior, manifested as a decrease in feeding rate and filtration rate; Trijau et al. (2018) investigated this by utilizing Cs... 137 Exposure to gamma rays produced by Cs in water fleas revealed altered DNA methylation, which was inherited in the F3 generation. However, existing studies mostly used stable nuclides or radiation sources as substitutes, which differs greatly from reality. Furthermore, the doses used were far greater than actual values, and currently, low-activity radioactivity studies are lacking. 137 Research reports on the application of Cs in marine biological risk assessment. Therefore, there is an urgent need to establish a low-radioactivity... 137 Methods for assessing the toxic effects of Cs on marine organisms.

[0004] Ocean medaka ( Oryzias melastigma With its wide temperature and salinity range, strong environmental tolerance, relatively small size, high spawning capacity, and ease of laboratory culture, this species is highly sensitive to pollutants and can rapidly reflect environmental pollution levels, making it an ideal marine bony fish model. (Detection) 137 This study investigates the impact of Cs exposure on marine medaka and proposes simple and feasible indicators for environmental monitoring in the waters surrounding nuclear power plants. 137 Cs provides technical support for environmental monitoring and comprehensive management. Summary of the Invention

[0005] In response to the current lack of low radioactivity in seawater 137The problems with Cs toxicity assessment methods are that toxicity studies often use stable nuclides or radioactive sources as substitutes, resulting in toxicity effect data that differs greatly from actual conditions and often involves excessively high doses. The purpose of this invention is to provide a low-radioactivity method for seawater. 137 The Cs toxicity evaluation method uses marine medaka as the test organism to determine its reproductive toxicity-related indicators. It is simple to operate, has good repeatability, and is sensitive to changes under low radioactivity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a method for evaluating low-activity radioactivity based on marine medaka. 137 The method for detecting Cs toxicity includes the following steps:

[0007] Step 1: Set up the breeding conditions for medaka. After the medaka have been domesticated, set up a control group and an experimental group. 137 The exposure concentration of Cs, after adjusting the exposure parameters... 137 Exposure experiments begin in Cs;

[0008] Step 2: Observe the reproductive status parameters of the medaka after the exposure experiment, including the number of eggs laid in seven days, fertilization rate, hatching rate, and hatching time, and compare the reproductive status parameters of the control group and the experimental group.

[0009] Step 3: Dissect the medaka to obtain gonadal tissue. Classify the gonadal tissue according to the difference between male and female medaka. After processing, take the supernatant to determine the activities of total superoxide dismutase, catalase and malondialdehyde content. Measure the protein in the sample, calculate the enzyme activity per unit protein, and statistically analyze the measurement results of the control group and the experimental group.

[0010] Step 4: Use one-way ANOVA to analyze and compare the differences in various indicators of significance levels between the control group and the experimental group based on the data from Steps 2 and 3. P <0.05 indicates a significant difference; P <0.01 indicates highly significant differences between groups, allowing for the selection of male and female medaka pairs. 137 The sensitivity indicators of Cs are determined by the differences among various indicators. 137 The toxicity level of Cs.

[0011] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 The method for detecting Cs toxicity, wherein the culture conditions include a culture temperature of 28±0.5℃, a light-dark cycle of 14:10h, a salinity of 30±1‰, dissolved oxygen >6.0 mg / L, and alternating feeding of live brine shrimp and feed 3-5 times a day, and acclimatization in the laboratory for 7 days.

[0012] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 The method for measuring Cs toxicity, wherein the control group consisted of medaka growing in normal waters, and the experimental group consisted of...137 Cs exposure concentration was 1×10 3 Bq / L, 1×10 4 Bq / L and 1×10 5 Bq / L, medaka samples were obtained for testing after 21 days.

[0013] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 In the method for Cs toxicity, step 2:

[0014] Seven-day egg production: After the exposure ended, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. Then they were transferred to beakers containing 5 L of clean seawater and raised under the conditions in step 1. Eggs were collected from the bottom of the beakers at 10:00 am every day and counted. The experiment was repeated three times for seven consecutive days.

[0015] Fertilization rate: After the exposure period, five pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to beakers containing 5 L of clean seawater and cultured under the conditions described in step 1 for 24 hours. After that, the eggs at the bottom of the beakers were collected and spread out in a culture dish containing clean seawater. The eggs were gently separated manually with tweezers and observed under a stereomicroscope. The fertilization rate was calculated using the formula: Fertilization rate = Number of fertilized eggs / Total number of eggs × 100%. 50 eggs were measured in each group, and the experiment was repeated three times.

[0016] Hatching rate and hatching time: 100 embryos that were normally fertilized after the exposure test were placed in a culture dish containing clean seawater and raised under the culture conditions in step 1. Bad eggs were removed and the culture water was changed daily. After 10 days, the number of successfully hatched fry was counted and the hatching time was recorded. The hatching rate was calculated using the formula: Hatching rate = Number of fertilized eggs hatched / Total number of fertilized eggs × 100%.

[0017] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 The method for Cs toxicity, step 3 includes:

[0018] Step 3-1: Place the medaka in the control group and experimental group in clean seawater, wash them three times, and then anesthetize them with MS-222 at 200 mg / L for 2 minutes before dissecting them and removing the gonadal tissue.

[0019] Step 3-2: After rinsing the gonadal tissue three times with pre-cooled PBS buffer, suspend it in a centrifuge tube containing 1 mL of PBS buffer, break the tissue using a rapid sample preparation instrument, centrifuge, take 800 μL of supernatant and perform protein quantification using a BCA protein concentration assay kit, read the value at a wavelength of 562 nm, and calculate the protein concentration of the sample.

[0020] Step 3-3: Take 200 μL of gonadal tissue supernatant from the control group and the experimental group respectively, and use the total superoxide dismutase assay kit, catalase assay kit, and malondialdehyde assay kit to detect the oxidative stress level.

[0021] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 The method for Cs toxicity involves setting the instrument parameters of the rapid sample preparation instrument to a speed of 6.0 m / s, a time of 10 s, and performing 3 cycles; a centrifugation speed of 6000 rpm / min, a centrifugation time of 10 min, and a centrifugation temperature of 4℃.

[0022] This invention provides a method for evaluating low-activity radioactivity based on marine medaka. 137 The beneficial effect of the Cs toxicity method is that the method of the present invention achieves low radioactivity in seawater. 137 Rapid evaluation of Cs toxicity is achieved through a simple process with high repeatability and sensitivity. This fills a gap in the evaluation of low-activity radioactive materials. 137 Research on the toxic effects of Cs on marine organisms provides insights into the marine environment. 137 This provides an effective approach for evaluating the toxicity of cytotoxicity. Using marine killifish as the test organism is a universal, sensitive, and representative method. Measuring reproductive toxicity-related indicators is simple, reproducible, and sensitive to changes at low radioactivity levels. Low radioactivity can be accurately detected through multiple biological indicators such as egg production. 137 The content of Cs, for 137 Cs seawater has high detection sensitivity. Attached Figure Description

[0023] Figure 1 This is a volcano diagram of differentially expressed genes in the ovary, as shown in this embodiment of the invention.

[0024] Figure 2 This is a volcano diagram of differentially expressed genes in the testes, as shown in this embodiment of the invention.

[0025] Figure 3 As described in the embodiments of the present invention 137 Cs exposure analysis of differential expression of gonadal genes in marine medaka; ovarian GO enrichment entries.

[0026] Figure 4 As described in the embodiments of the present invention 137 Differential expression enrichment analysis of gonadal genes in marine medaka exposed to Cs; GO enrichment entries in testes.

[0027] Figure 5 As described in the embodiments of the present invention 137 The effect of Cs on the seven-day spawning of marine medaka;

[0028] Figure 6 As described in the embodiments of the present invention 137The effect of Cs on the fertilization rate of marine medaka;

[0029] Figure 7 As described in the embodiments of the present invention 137 The effect of Cs on the hatching rate of marine medaka;

[0030] Figure 8 As described in the embodiments of the present invention 137 The effect of Cs on the hatching time of marine medaka;

[0031] Figure 9 As described in the embodiments of the present invention 137 The effect of Cs on SOD, a redox-related indicator in the ovary;

[0032] Figure 10 As described in the embodiments of the present invention 137 The effect of Cs on ovarian redox-related index CAT;

[0033] Figure 11 As described in the embodiments of the present invention 137 The effect of Cs on ovarian redox-related marker MDA;

[0034] Figure 12 As described in the embodiments of the present invention 137 The effect of Cs on SOD, a redox-related index in the testes;

[0035] Figure 13 As described in the embodiments of the present invention 137 The effect of Cs on testicular redox-related index CAT;

[0036] Figure 14 As described in the embodiments of the present invention 137 The effect of Cs on MDA, a redox-related index in the testes;

[0037] Figure 15 As described in the embodiments of the present invention 137 Heatmap of the correlation between Cs radioactivity and biological indicators. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described below in conjunction with specific embodiments and accompanying drawings.

[0039] Example 1

[0040] A method for evaluating low-activity radioactivity based on marine medaka 137 Methods for treating Cs toxicity include the following steps.

[0041] Step 1: Set up the breeding conditions for medaka. After the medaka have been domesticated, set up a control group and an experimental group. 137The exposure concentration of Cs, after adjusting the exposure parameters... 137 Exposure experiments begin in Cs.

[0042] The breeding conditions included a breeding temperature of 28±0.5℃, a light-dark cycle of 14:10h, a salinity of 30±1‰, dissolved oxygen >6.0mg / L, and alternating feeding of live brine shrimp and feed 3-5 times a day, followed by 7 days of domestication in the laboratory.

[0043] The control group consisted of medaka growing in normal waters, while the experimental group... 137 Cs exposure concentration was 1×10 3 Bq / L, 1×10 4 Bq / L and 1×10 5 Bq / L, medaka samples were obtained for testing after 21 days.

[0044] Step 2: Observe the reproductive status parameters of the medaka after the exposure experiment, including the number of eggs laid in seven days, fertilization rate, hatching rate, and hatching time, and compare the reproductive status parameters of the control group and the experimental group.

[0045] Seven-day egg production: After the exposure ended, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. Then they were transferred to beakers containing 5 L of clean seawater and raised under the conditions described in step 1. Eggs were collected from the bottom of the beakers at 10:00 AM every day and counted. This was repeated for seven consecutive days, and the experiment was repeated three times.

[0046] Fertilization rate: After the exposure period, five pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to beakers containing 5 L of clean seawater and cultured under the conditions described in step 1 for 24 hours. After that, the eggs from the bottom of the beakers were collected and spread out in a petri dish containing clean seawater. The eggs were gently separated manually with tweezers and placed under a stereomicroscope to observe the fertilization status. The fertilization rate was calculated using the formula: Fertilization rate = Number of fertilized eggs / Total number of eggs × 100%. Fifty eggs were measured in each group, and the experiment was repeated three times.

[0047] Hatching rate and hatching time: 100 embryos that were normally fertilized after the exposure test were placed in a culture dish containing clean seawater and raised under the culture conditions in step 1. Bad eggs were removed and the culture water was changed daily. After 10 days, the number of successfully hatched fry was counted and the hatching time was recorded. The hatching rate was calculated using the formula: Hatching rate = Number of fertilized eggs hatched / Total number of fertilized eggs × 100%.

[0048] Step 3: Dissect the medaka to obtain gonadal tissue. Classify the gonadal tissue according to the difference between male and female medaka. After processing, take the supernatant to determine the activities of total superoxide dismutase, catalase and malondialdehyde content. Measure the protein in the sample, calculate the enzyme activity per unit protein, and statistically analyze the measurement results of the control group and the experimental group.

[0049] include:

[0050] Step 3-1: Place the medaka in the control group and experimental group in clean seawater, wash them three times, and then anesthetize them with MS-222 at 200 mg / L for 2 minutes before dissecting them and removing the gonadal tissue.

[0051] Step 3-2: After rinsing the gonadal tissue three times with pre-cooled PBS buffer, suspend it in a centrifuge tube containing 1 mL of PBS buffer. Use a rapid sample preparation instrument to disrupt the tissue. Set the instrument parameters to speed 6.0 m / s and time 10 s, and perform 3 cycles. After completion, centrifuge at 6000 rpm / min for 10 min at a temperature of 4℃.

[0052] Take 800 μL of supernatant and use the BCA protein concentration assay kit to quantify the protein concentration. Read the value at a wavelength of 562 nm and calculate the protein concentration of the sample.

[0053] Step 3-3: Take 200 μL of gonadal tissue supernatant from the control group and the experimental group respectively, and use the total superoxide dismutase assay kit, catalase assay kit, and malondialdehyde assay kit to detect the oxidative stress level.

[0054] Step 4: Use one-way ANOVA to analyze and compare the differences in various indicators between the control group and the experimental group. P < 0.05 indicates a significant difference; P < 0.01 indicates an extremely significant difference between the groups. This allows for the selection of male and female medaka pairs. 137 The sensitivity indicators of Cs are determined by the differences among various indicators. 137 The toxicity level of Cs.

[0055] This technical solution is the first to propose using the reproductive toxicity of the marine model organism, the marine killifish, as a measure of low radioactivity in seawater. 137 Methods for detecting Cs content were developed, and multiple biological indicators were screened for use in actual seawater. 137 Cs content.

[0056] Example 2

[0057] This embodiment is a detailed expression of Embodiment 1, and specifically includes the following steps.

[0058] 1. Experimental methods.

[0059] 1.1: Marine medaka farming and exposure experiment design.

[0060] Adult marine killifish were reared in a culture environment of 28±0.5℃, with a light-dark cycle of 14:10h, salinity of 30±1‰, and dissolved oxygen >6.0 mg / L. Adults were fed 3-5 times daily, alternating between live brine shrimp and commercial feed. They were then domesticated in the laboratory for 7 days before use. 137 Cs exposure treatment, with an exposure concentration set at 1×10⁻⁶. 3 10 4 10 5 Bq / L, after 21 days, marine medaka were obtained for testing.

[0061] Transcriptome analysis of the gonads of marine medaka.

[0062] 1×10 5 Bq / L group 137 Marine medaka treated with Cs for 21 days were rinsed three times with clean seawater, placed in culture dishes, anesthetized with 200 mg / L MS-222 for 2 min, dissected, and the gonads were removed. After washing three times with PBS buffer, the gonads were placed in Trizon for lysis. The lysate was sent to OE Bio for transcriptome sequencing. The analysis protocol is shown below.

[0063] Species name: Oryzias melastigma Source: NCBI; Reference genome version: GCA_002922805.2.

[0064] Total RNA was extracted using Trizon, and RNA purity and quantification were determined using a NanoDrop2000 spectrophotometer (ThermoScientific, USA). The library was sequenced using an Illumina Novaseq 6000 sequencing platform, generating 150bp paired-end reads. The HISAT2 software was used for reference genome alignment, and the DESeq2 software was used for differentially expressed gene analysis.

[0065] Genes with thresholds are defined as differentially expressed genes (DEGs).

[0066] The transcriptome results were obtained by sending the gonads to a sequencing company; the entire analysis focused on reproductive toxicity, with the target organs being the male and female gonads. 137 Cs has a significant impact on active cells, leading us to believe it also significantly affects gamete production, specifically the site of gamete production—the gonads. However, this is merely an inference; transcriptome sequencing of the male and female gonads in section 2.1 confirms this. 137 Cs can lead to differential expression of various gonadal genes. This indicates that reproductive toxicity-related indicators can be used to evaluate... 137 The toxicity of Cs is accurate.

[0067] The transcriptome data used here are all existing technologies, and the purpose is to demonstrate that evaluating reproductive toxicity is appropriate. Transcriptome data is not the focus; the specific procedures are the same as those used in existing technologies and will not be described in detail here.

[0068] 1.2: Seven-day egg production, fertilization rate, hatching rate, and incubation time were measured.

[0069] After the exposure ended, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to beakers containing 5L of clean seawater and raised under the above-mentioned conditions. Eggs were collected from the bottom of the beakers at 10:00 AM every day and counted. This was repeated for 7 consecutive days, and the experiment was repeated three times.

[0070] After exposure, five pairs of male and female medaka from each group were rinsed three times with clean seawater and then transferred to beakers containing 5 L of clean seawater. After 24 hours of culture under the aforementioned conditions, eggs were collected from the bottom of the beakers. The collected eggs were then spread evenly in a petri dish containing clean seawater, and each egg was gently separated manually using tweezers. Fertilization was observed under a stereomicroscope. The fertilization rate was calculated using the formula: Fertilization rate = (Number of fertilized eggs / Total number of eggs) × 100%. Fifty eggs were measured from each group, and the experiment was repeated three times.

[0071] One hundred normally fertilized embryos from each of the above treatment groups were placed in a culture dish containing clean seawater and raised under the above culture conditions. Bad eggs were produced and the culture water was changed daily. After 10 days, the number of successfully hatched fry was counted and the hatching time was recorded. The hatching rate was calculated using the formula: Hatching rate = Number of fertilized eggs hatched / Total number of fertilized eggs × 100%.

[0072] 1.3: Detection of redox-related indicators.

[0073] 1×10 3 10 4 10 5 Bq / L 137Marine killifish exposed to Cs for 21 days were placed in clean seawater and washed three times. After anesthetizing with 200 mg / L MS-222 for 2 minutes, they were dissected, and gonadal tissue was removed. The tissue was washed three times with pre-cooled PBS buffer and then suspended in centrifuge tubes containing 1 mL of PBS buffer. The tissue was then lysed using a rapid sample preparation instrument at a speed of 6.0 m / s for 10 s, with three cycles. After centrifugation (6000 rpm, 10 min, 4℃), 800 μL of the supernatant was collected for protein quantification using a BCA kit at 562 nm to calculate the protein concentration. 200 μL of the supernatant was then used to detect oxidative stress levels using a total superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) assay kit.

[0074] 1.4: Data processing and statistical analysis.

[0075] Data were statistically analyzed using SPSS Statistics 20 software. One-way ANOVA was used to compare the differences in each indicator between the control and treatment groups. All data are expressed as mean ± standard deviation (SD), and the significance level is expressed as p-value. P <0.05 indicates a significant difference; P <0.01 indicates highly significant differences between groups. GraphPad Prism 8 software was used for graphing.

[0076] 2. Experimental results.

[0077] 2.1: 137 Differential gene expression and enrichment in the gonads of marine medaka under Cs exposure.

[0078] Differentially expressed genes with functional annotation information are plotted as a volcano plot. The markers in the plot represent the names of the top 10 differentially expressed genes, such as... Figure 1 and Figure 2 As shown. In the ovaries, compared with the control group, 10 5 In the Bq / L activity treatment group, 1206 genes were significantly upregulated and 3276 genes were significantly downregulated. In the testes, compared with the control group, 10 5 In the Bq / L activity treatment group, 1824 genes were significantly upregulated and 576 genes were significantly downregulated. The results show that 10 5 The Bq / L activity treatment group had a significant effect on the gonads of both male and female fish.

[0079] Gene Ontology Enrichment Analysis is the most commonly used type of enrichment analysis. It is used to examine the enrichment of Gene Ontology (GO) entries in a gene set. It is a very common type of analysis in transcriptome data analysis, which can integrate differentially expressed genes into some biological processes.

[0080] like Figure 3 and Figure 4 As shown, GO enrichment of differentially expressed genes revealed... 137 Differentially expressed genes in the ovaries of marine medaka exposed to Cs were mainly enriched in biological processes such as negative regulation of NF-kappaB transcription factor activity, negative regulation of cytokine production involved in inflammatory responses, negative regulation of innate immune responses, angiogenesis, negative regulation of chemokine-6 ​​production, negative regulation of interleukin-12 production, innate immune response, angiogenesis, and inflammatory responses. They were mainly enriched in cellular components such as membrane components, collagen trimers, cell surfaces, collagen-containing extracellular matrix, extracellular matrix components, plasma membrane, extracellular regions, and extracellular spaces. The main molecular functions of differentially expressed genes include peptidase inhibitors, binding of active cytokines, transmembrane signal receptor activity, DNA-binding transcription factor activity, growth factor activity, Serine-type endopeptidase activity, binding of phosphoside 3-enzyme regulatory subunits, galactose-type endopeptidase inhibitor activity, heparin binding, and extracellular matrix structure. Differentially expressed genes in the testes of marine medaka are mainly enriched in biological processes such as prevention of polyspermia, cholesterol homeostasis, righting reflex, acrosome reaction, triglyceride biosynthesis, G protein-coupled receptor signaling pathway, antigen synthesis, sperm capacitation, lipid catabolism, and digestion. They are mainly enriched in cellular components such as the internal body cavity, chylomicrons, NMDA selective glutamate receptor complex, synaptic complex, basement membrane, hemoglobin complex, cortical granule protein complex, extracellular region, and extracellular space. The main molecular functions of differentially expressed genes include phosphokinase A2 inhibitor activity, efflux transmembrane transporter activity, 2-glycerol-O-glycerol lipotransferase activity, CCR chemokine receptor binding, carbonic acid dehydratase activity, phosphate ion binding, oxygen binding, oxygen carrier activity, serine hydrolase activity, and serine endopeptidase activity.

[0081] This suggests that sea medaka... 137 Cs exposure produces reproductive toxicity, suggesting that reproductive toxicity should be used to evaluate marine medaka. 137 The toxicity of Cs is accurate.

[0082] 2.2: 137 Cs exposure affects the seven-day egg production, fertilization rate, hatching rate, and hatching time of marine medaka.

[0083] like Figure 5 , Figure 6 , Figure 7 , Figure 8 As shown, 137 Cs exposure affected the seven-day spawning rate, fertilization rate, hatching rate, and hatching time of marine medaka, with 10% of the results compared to the control group. 4 ( P <0.05), 10 5 Bq / L ( P <0.01) Reduction in seven-day egg production and hatching rate under radiation activity exposure (* indicates P-value less than 0.05; ** indicates P-value less than 0.01; no marking is made for P-value greater than 0.05), 10 5 Bq / L ( P <0.01) Radioactivity exposure significantly reduced the fertilization rate of marine medaka; with 137 Increased Cs radioactivity leads to prolonged embryo incubation time.

[0084] 2.3: 137 Cs affects redox-related indicators in marine medaka.

[0085] Changes in SOD and CAT activity and MDA content, such as Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 As shown. 10 5 Bq / L ( P <0.01) Radiation exposure significantly increased the activity of SOD and CAT in the ovaries of female fish, but had no significant effect on the content of MDA; 10 5 Bq / L ( P <0.01) Radiation activity exposure significantly reduced the activity of SOD in the testes of male fish, while 10 4 10 5 Exposure to Bq / L radioactivity significantly increased the MDA content in the testes of male fish. P <0.01).

[0086] like Figure 15 As shown, after screening, the SOD enzyme activity in the gonads of marine medaka changed in both sexes; the CAT enzyme activity in females increased; the MDA content in males increased; and the 7-day egg production, fertilization rate, and hatching rate decreased. P <0.01, significant changes in the above indicators indicate that there is a high concentration of [something] in the environment or sample. 137 Cs radioactivity exceeds 1×10 5 Bq / L. Increased MDA levels in male marine medaka (Bq / L) P <0.01); Egg production decreased on day 7 ( P <0.05), hatching rate decreased ( P<0.05) indicates that the environment or sample contains... 137 Cs radioactivity exceeds 1×10 4 Bq / L.

[0087] The above embodiments are merely illustrative of the inventive concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for evaluating low-activity radioactivity based on marine medaka. 137 The method for detecting Cs toxicity is characterized by... Includes the following steps: Step 1: Set up the breeding conditions for medaka. After the medaka have been domesticated, set up a control group and an experimental group. 137 The exposure concentration of Cs, after adjusting the exposure parameters... 137 Exposure experiments begin in Cs; Step 2: Observe the reproductive status parameters of the medaka after the exposure experiment, including the number of eggs laid in seven days, fertilization rate, hatching rate, and hatching time, and compare the reproductive status parameters of the control group and the experimental group. Step 3: Dissect the medaka to obtain gonadal tissue. Classify the gonadal tissue according to the difference between male and female medaka. After processing, take the supernatant to determine the activities of total superoxide dismutase, catalase and malondialdehyde content. Measure the protein in the sample, calculate the enzyme activity per unit protein, and statistically analyze the measurement results of the control group and the experimental group. Step 4: Using one-way ANOVA, analyze and compare the differences in various indicators of significance levels between the control group and the experimental group based on the data from Steps 2 and 3. P < 0.05 indicates a significant difference; P < 0.01 indicates extremely significant differences between groups, allowing for the selection of male and female medaka pairs. 137 The sensitivity indicators of Cs are determined by the differences among various indicators. 137 The toxicity level of Cs.

2. The method for evaluating low-activity radioactivity based on marine medaka as described in claim 1 137 The method for detecting Cs toxicity is characterized by... The breeding conditions included a breeding temperature of 28±0.5℃, a light-dark cycle of 14:10h, a salinity of 30±1‰, dissolved oxygen >6.0mg / L, and alternating feeding of live brine shrimp and feed 3-5 times a day, followed by 7 days of domestication in the laboratory.

3. The method for evaluating low-activity radioactivity based on marine medaka as described in claim 1 137 The method for detecting Cs toxicity is characterized by... The control group consisted of medaka that grew in normal waters, while the experimental group... 137 Cs exposure concentration was 1×10 3 Bq / L, 1×10 4 Bq / L and 1×10 5 Bq / L, medaka samples were obtained for testing after 21 days.

4. The method for evaluating low-activity radioactivity based on marine medaka as described in claim 1 137 The method for detecting Cs toxicity is characterized by... In step 2: Seven-day egg production: After the exposure ended, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. Then they were transferred to beakers containing 5 L of clean seawater and raised under the conditions in step 1. Eggs were collected from the bottom of the beakers at 10:00 am every day and counted. The experiment was repeated three times for seven consecutive days. Fertilization rate: After the exposure, five pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. Then they were transferred to beakers containing 5L of clean seawater and cultured under the conditions in step 1 for 24 hours. After that, the eggs at the bottom of the beaker were taken and spread out in a culture dish containing clean seawater. The eggs were gently separated by hand with tweezers and placed under a stereomicroscope to observe the fertilization status. The fertilization rate was calculated using the formula: Fertilization rate = Number of fertilized eggs / Total number of eggs × 100%. 50 eggs were measured in each group, and the experiment was repeated three times. Hatching rate and hatching time: 100 embryos that were normally fertilized after the exposure test were placed in a culture dish containing clean seawater and raised under the culture conditions in step 1. Bad eggs were removed and the culture water was changed daily. After 10 days, the number of successfully hatched fry was counted and the hatching time was recorded. The hatching rate was calculated using the formula: Hatching rate = Number of fertilized eggs hatched / Total number of fertilized eggs × 100%.

5. The method for evaluating low-activity radioactivity based on marine medaka as described in claim 1 137 The method for detecting Cs toxicity is characterized by... Step 3 includes: Step 3-1: Place the medaka in the control group and experimental group in clean seawater, wash them three times, and then anesthetize them with MS-222 at 200 mg / L for 2 minutes before dissecting them and removing the gonadal tissue. Step 3-2: After rinsing the gonadal tissue three times with pre-cooled PBS buffer, suspend it in a centrifuge tube containing 1 mL of PBS buffer, break the tissue using a rapid sample preparation instrument, centrifuge, take 800 μL of supernatant and perform protein quantification using a BCA protein concentration assay kit, read the value at a wavelength of 562 nm, and calculate the protein concentration of the sample. Step 3-3: Take 200 μL of gonadal tissue supernatant from the control group and the experimental group respectively, and use the total superoxide dismutase assay kit, catalase assay kit, and malondialdehyde assay kit to detect the oxidative stress level.

6. The method for evaluating low-activity radioactivity based on marine medaka as described in claim 5 137 The method for detecting Cs toxicity is characterized by... The instrument parameters of the rapid sample preparation instrument are set as follows: speed 6.0 m / s, time 10s, with 3 cycles; centrifugation speed 6000 rpm / min, centrifugation time 10min, and centrifugation temperature 4℃.

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