Method for evaluating toxicity of low-activity radioactive 137Cs based on oryzias latipe

Abstract

The invention relates to the field of radioactive substance toxicity detection, in particular to a method for evaluating low-activity radioactive 137Cs toxicity based on oryzias latipes, which comprises the following steps: step 1, setting culture conditions of the oryzias latipes, and starting an exposure experiment in 137Cs with adjusted exposure parameters; 2, observing reproductive state parameters of the oryzias latipes after the exposure experiment; 3, dissecting the oryzias latipe and taking gonad tissues; and 4, counting the data information in the step 2 and the step 3 by adopting single-factor variance, analyzing and comparing the difference of each index of the significance level of the control group and the experimental group, and determining the toxicity level of 137Cs according to the difference of each index. According to the method, the toxicity of the low-radiation-activity 137Cs in the seawater is rapidly evaluated, the treatment process is simple, and the method has the characteristics of high operation repeatability and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of radioactive material toxicity detection, and specifically to a method for evaluating low-activity radioactivity based on marine medaka 137 Cs toxicity method. Background Art

[0002] Nuclear power is a clean, low-carbon, safe and efficient form of energy. It has become an important part of energy strategy due to its safety, reliability and strong supply capacity. Nuclear power plants are built along the coast of waters. During operation, some radioactive nuclides will flow out of the nuclear power plants, which will have an impact on marine life. 137 Cs is a radioactive nuclide that is inevitably produced during nuclear fission. Its half-life is as long as 30.17 years. 137 Cs will accumulate in organisms and continuously release gamma rays, affecting the health of marine life.

[0003] However, there is little research on the toxicity of radionuclides to marine organisms, and there is little data available for the toxicity evaluation of radioactive pollutants. Xu et al. (2023) used the stable isotope substitution method to study the effects of the ionic properties of Cs on the blue mussel ( Mytilis edulis ) toxic effects, and found 133 Cs exposure can cause the death of blue mussels and inhibit their feeding behavior, which is manifested by a decrease in feeding rate and water filtration rate; Trijau et al. (2018) used 137 Exposure of Daphnia to gamma rays produced by Cs revealed changes in DNA methylation and inheritance to the F3 generation. However, the only studies that have been conducted have mostly used stable nuclides or radiation sources as substitutes, which is very different from the actual situation. At the same time, the dose used is far beyond the actual situation. Currently, there is no low-radioactivity 137 Related research reports on the risk assessment of Cs to marine organisms. Therefore, it is urgent to establish a low-radioactivity 137 Methods for assessing the toxic effects of Cs on marine organisms.

[0004] Ocean Medaka Oryzias melastigma ) It has a wide range of temperatures and salt content, strong environmental tolerance, small size, large egg-laying capacity, is easy to culture in the laboratory, is sensitive to pollutants, and can quickly respond to environmental pollution conditions. It is an ideal marine bony fish model. 137 The impact of Cs exposure on the marine medaka, proposed a simple and feasible indicator for environmental monitoring, and provided a reference for the marine environment around nuclear power plants. 137 Provide technical support for Cs environmental monitoring and comprehensive management. Summary of the invention

[0005] In view of the current lack of low radioactivity in seawater 137The problem of Cs toxicity evaluation method is that toxicity research is mostly substituted by stable nuclides or radioactive sources. The toxic effect data obtained are very different from the actual situation, and the dose is too high. The purpose of this invention is to provide a low radioactivity in seawater 137 The Cs toxicity evaluation method uses the 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 purpose, the technical solution adopted by the present invention is: a method for evaluating low-activity radioactivity based on marine medaka 137 The method for Cs toxicity comprises the following steps: Step 1: Set up the breeding conditions for medaka, domesticate the medaka, and set up the control group and experimental group. 137 The exposure concentration of Cs, after adjusting the exposure parameters 137 The exposure experiment started in Cs; Step 2: Observe the reproductive status parameters of medaka after the exposure experiment, including the seven-day egg production, fertilization rate, hatching rate, and hatching time, and compare the reproductive status parameters of the control group and the experimental group; Step 3: dissecting the medaka to obtain gonadal tissue, classifying the gonadal tissue according to the difference between male and female medaka, taking the supernatant after treatment to determine the activity of total superoxide dismutase and catalase and the content of malondialdehyde, determining the protein in the sample, calculating the enzyme activity per unit protein, and statistically analyzing the determination results of the control group and the experimental group; Step 4: Use one-way variance statistics to analyze the data information in steps 2 and 3, and compare the differences in the significance levels of various indicators between the control group and the experimental group. P <0.05 indicated significant differences; P <0.01 indicates that the difference between the groups is extremely significant, and the male and female medaka pairs were selected. 137 The sensitive index of Cs is determined by the difference of each index. 137 Toxicity level of Cs.

[0007] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 The method for detecting Cs toxicity, the culture conditions include culture temperature of 28±0.5℃, light-dark cycle of 14:10h, salinity of 30±1‰, dissolved oxygen>6.0 mg / L, fresh brine shrimps and feeds are fed alternately 3-5 times a day, and the shrimps are acclimated in the laboratory for 7 days.

[0008] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 Cs toxicity method, the control group is medaka growing in normal waters, the experimental group 137 The Cs exposure concentration was 1×10 3 Bq / L, 1×10 4Bq / L and 1×10 5 Bq / L, medaka for testing was obtained after 21 days.

[0009] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 Cs toxicity method, in said step 2: Seven-day egg production: After the exposure, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to a beaker containing 5 L of clean seawater and raised according to the culture conditions in step 1. The eggs at the bottom of the beaker were collected and counted at 10 am every day for 7 consecutive days. The experiment was repeated three times. Fertilization rate: After the exposure, 5 pairs of male and female medaka were taken from each group and rinsed three times with clean seawater, then transferred to a beaker containing 5 L of clean seawater. After culturing for 24 hours according to the culture conditions in step 1, the eggs at the bottom of the beaker were taken and spread flat on a culture dish containing clean seawater. The eggs were separated manually one by one with tweezers and placed under a stereoscope 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: Take 100 normally fertilized embryos after the exposure test and place them in a culture dish filled with clean seawater. Raise them according to the culture conditions in step 1. Remove bad eggs and replace the culture water every day. Count the number of successfully hatched fry after 10 days, and record the hatching time. Calculate the hatching rate using the formula: Hatching rate = number of hatched fertilized eggs / total number of fertilized eggs × 100%.

[0010] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137 Cs toxicity method, said step 3 comprises: Step 3-1: Place the medaka in the control group and the experimental group in clean seawater, wash them three times, anesthetize them with 200 mg / L MS-222 for 2 minutes, and then dissect them to remove the gonadal tissue; Step 3-2: After the gonad tissue was rinsed three times with pre-cooled PBS buffer, it was suspended in a centrifuge tube containing 1 mL of PBS buffer. The tissue was broken using a rapid sample preparation instrument. After completion, the tissue was centrifuged and 800 μL of the supernatant was taken for protein quantification using a BCA protein concentration assay kit. The reading was performed at a wavelength of 562 nm to calculate the protein concentration of the sample. Step 3-3: Take 200 μL of the gonadal tissue supernatant from the control group and the experimental group respectively, and use the total superoxide dismutase detection kit, catalase detection kit, and malondialdehyde detection kit to detect the oxidative stress level.

[0011] The above-mentioned evaluation of low-activity radioactivity based on marine medaka 137The method for detecting Cs toxicity is as follows: the instrument parameters of the rapid sample preparation instrument are set to a speed of 6.0 m / s, a time of 10 s, and 3 cycles; the centrifugal speed is 6000 rpm / min, the centrifugal time is 10 min, and the centrifugal temperature is 4°C.

[0012] The invention discloses a method for evaluating low-activity radioactivity based on marine medaka 137 The beneficial effect of the method for Cs toxicity is that the method of the present invention achieves low radioactivity in seawater. 137 Rapid evaluation of Cs toxicity has the characteristics of simple processing, high repeatability and high sensitivity. 137 The study of the toxic effects of Cs on marine organisms provides a reference for the research on the toxic effects of Cs on marine organisms. 137 It provides an effective way to evaluate Cs toxicity. It uses the marine medaka as the test organism, which is universal, sensitive and representative. It measures the reproductive toxicity related indicators. It is easy to operate, has good repeatability and is sensitive to changes under low radioactivity. It can accurately detect low radioactivity through multiple biological indicators such as egg laying. 137 The content of Cs, for 137 Cs seawater has high sensitivity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a volcano plot of differentially expressed genes in the ovary in the embodiment of the present invention; Figure 2 This is a volcano plot of testis differentially expressed genes in the embodiment of the present invention; Figure 3 In the embodiment of the present invention 137 Differential expression enrichment analysis of genes in the gonad of marine medaka exposed to Cs Ovarian GO enrichment terms; Figure 4 In the embodiment of the present invention 137 Differential expression enrichment analysis of genes in the gonad of marine medaka exposed to Cs, testis GO enrichment terms; Figure 5 In the embodiment of the present invention 137 Effects of Cs on seven-day spawning of medaka; Figure 6 In the embodiment of the present invention 137 Effects of Cs on the fertilization rate of marine medaka; Figure 7 In the embodiment of the present invention 137 Effects of Cs on the hatching rate of marine medaka; Figure 8 In the embodiment of the present invention 137 Effects of Cs on the hatching time of marine medaka; Fig. 9 In the embodiment of the present invention 137Effects of Cs on SOD, an ovarian redox-related index; Fig.10 In the embodiment of the present invention 137 Effects of Cs on ovarian redox-related index CAT; Fig.11 In the embodiment of the present invention 137 Effects of Cs on ovarian redox-related index MDA; Fig.12 In the embodiment of the present invention 137 Effects of Cs on SOD, an index related to testicular redox; Fig.13 In the embodiment of the present invention 137 Effects of Cs on testicular redox-related index CAT; Fig.14 In the embodiment of the present invention 137 Effects of Cs on testicular redox-related index MDA; Fig.15 In the embodiment of the present invention 137 Heat map of the correlation between Cs radioactivity and biological indicators. DETAILED DESCRIPTION

[0014] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is described below in conjunction with specific implementation methods and drawings.

[0015] Example 1 A method for evaluating low-activity radioactivity based on marine medaka 137 The method for detecting Cs toxicity comprises the following steps.

[0016] Step 1: Set up the breeding conditions for medaka, domesticate the medaka, and set up the control group and experimental group. 137 The exposure concentration of Cs, after adjusting the exposure parameters 137 The exposure experiment was started in Cs.

[0017] The culture conditions included a culture temperature of 28 ± 0.5 °C, a light-dark cycle of 14:10 h, a salinity of 30 ± 1‰, dissolved oxygen > 6.0 mg / L, and feeding fresh brine shrimp alternately with feed 3-5 times a day, and acclimation in the laboratory for 7 days.

[0018] The control group consisted of medaka growing in normal waters, and the experimental group 137 The Cs exposure concentration was 1×10 3 Bq / L, 1×10 4 Bq / L and 1×10 5 Bq / L, medaka for testing was obtained after 21 days.

[0019] Step 2: Observe the reproductive status parameters of medaka after the exposure experiment, including seven-day egg production, fertilization rate, hatching rate, and hatching time, and compare the reproductive status parameters of the control group and the experimental group.

[0020] Seven-day egg production: After the exposure, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to a beaker containing 5 L of clean seawater and raised according to the culture conditions in step 1. The eggs at the bottom of the beaker were collected and counted at 10 am every day for 7 consecutive days. The experiment was repeated three times.

[0021] Fertilization rate: After the exposure, 5 pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. Then they were transferred to a beaker containing 5 L of clean seawater. After culturing for 24 h according to the culture conditions in step 1, the eggs at the bottom of the beaker were taken and spread flat on a culture dish filled with clean seawater. The eggs were manually separated one by one with tweezers and placed under a stereoscope 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.

[0022] Hatching rate and hatching time: Take 100 normally fertilized embryos after the exposure test and place them in a culture dish filled with clean seawater. Raise them according to the culture conditions in step 1. Remove bad eggs and replace the culture water every day. Count the number of successfully hatched fry after 10 days, and record the hatching time. Calculate the hatching rate using the formula: Hatching rate = number of hatched fertilized eggs / total number of fertilized eggs × 100%.

[0023] Step 3: dissect the medaka to obtain gonadal tissue, classify the gonadal tissue according to the difference between male and female medaka, take the supernatant after treatment to determine the activity of total superoxide dismutase and catalase and the content of malondialdehyde, determine 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.

[0024] include: Step 3-1: The medaka in the control group and the experimental group were placed in clean seawater, washed three times, anesthetized with 200 mg / L MS-222 for 2 minutes, and then dissected to remove the gonadal tissue.

[0025] Step 3-2: After the gonadal tissue was rinsed three times with pre-cooled PBS buffer, it was suspended in a centrifuge tube containing 1 mL of PBS buffer and the tissue was broken using a rapid sample preparation instrument. The instrument parameters were set to a speed of 6.0 m / s and a time of 10 s for three cycles. After completion, the tissue was centrifuged at a speed of 6000 rpm / min, a time of 10 min, and a temperature of 4°C.

[0026] Take 800 μL of supernatant and use BCA protein concentration determination kit for protein quantification, read at a wavelength of 562 nm, and calculate the protein concentration of the sample.

[0027] Step 3-3: Take 200 μL of the gonadal tissue supernatant from the control group and the experimental group respectively, and use the total superoxide dismutase detection kit, catalase detection kit, and malondialdehyde detection kit to detect the oxidative stress level.

[0028] Step 4: Use one-way ANOVA to analyze and compare the data information in steps 2 and 3, and compare the differences in the significance level of each index 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. 137 The sensitive index of Cs is determined by the difference of each index. 137 Toxicity level of Cs.

[0029] This technical solution proposes for the first time to use the reproductive toxicity of the marine model organism, the marine medaka, as a measure of low-level radioactivity in seawater. 137 Cs content method, and screened out multiple biological indicators for detecting Cs in actual seawater 137 Cs content.

[0030] Example 2 This embodiment is a concrete expression of Embodiment 1, and specifically includes the following steps.

[0031] 1. Experimental method.

[0032] 1.1: Design of the marine medaka breeding and exposure experiment.

[0033] Adult marine medaka were raised in a culture environment at 28±0.5℃, with a light-dark cycle of 14:10h, a salinity of 30±1‰, and dissolved oxygen>6.0 mg / L. Adult fish were fed 3-5 times a day, alternating between fresh brine shrimp and feed. 137 Cs exposure treatment, the exposure concentration was set to 1×10 3 , 10 4 , 10 5 , Bq / L, and the marine medaka for detection was obtained after 21 days.

[0034] Transcriptome analysis of the gonad of marine medaka.

[0035] 1×10 5 Bq / L group 137The medaka treated with Cs for 21 days was rinsed three times with clean seawater, placed in a culture dish, anesthetized with 200 mg / L MS-222 for 2 minutes, and then dissected. The gonads were washed three times with PBS buffer and then lysed in Trizon. The lysate was sent to Ouyi Biotechnology for transcriptome sequencing. The analysis plan is shown below.

[0036] Species Name: Oryzias melastigma ; Source: NCBI; Reference genome version: GCA_002922805.2.

[0037] Total RNA was extracted with Trizon, and the purity and quantification of RNA were identified using a NanoDrop2000 spectrophotometer (ThermoScientific, USA). The library was sequenced using the Illumina Novaseq 6000 sequencing platform to generate 150 bp paired-end reads. The HISAT2 software was used for reference genome alignment, and the DESeq2 software was used for differentially expressed gene analysis.

[0038] The genes with the threshold were defined as differentially expressed genes (DEGs).

[0039] The transcriptome results are obtained by sending the gonads to a sequencing company. The entire content focuses on reproductive toxicity, and the target organs are the male and female gonads. 137 Cs has a great impact on active cells, so we think it has a great impact on gamete production or the place where gametes are produced, that is, the gonads. However, this is an inference. After sequencing the transcriptome of male and female gonads in 2.1, it can be verified that 137 Cs can cause differences in the expression of various genes in the gonads. This indicates that the use of reproductive toxicity-related indicators to evaluate 137 Cs toxicity is accurate.

[0040] The use of transcriptomes here is all prior art, and the purpose is to prove that the evaluation of reproductive toxicity is appropriate. Transcriptome data is not the focus, and the specific process is the same as the means used in the prior art, so it will not be repeated.

[0041] 1.2: Seven-day egg production, fertilization rate, hatching rate and hatching time testing.

[0042] After the exposure, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to a beaker containing 5 L of clean seawater and raised according to the above-mentioned culture conditions. The eggs at the bottom of the beaker were collected and counted at 10 am every day for 7 consecutive days. The experiment was repeated three times.

[0043] After the exposure, 5 pairs of male and female medaka were taken from each group and rinsed three times with clean seawater, then transferred to a beaker containing 5 L of clean seawater. After 24 hours of culture under the above conditions, the eggs at the bottom of the beaker were taken and laid flat in a culture dish containing clean seawater. The eggs were separated manually one by one with tweezers and placed under a stereoscope 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.

[0044] 100 normally fertilized embryos from the above treatment groups were placed in a culture dish filled with clean seawater and raised under the above culture conditions. Bad eggs were laid and the culture water was replaced every day. 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 hatched fertilized eggs / total number of fertilized eggs × 100%.

[0045] 1.3: Detection of redox related indicators.

[0046] 1×10 3 , 10 4 , 10 5 Bq / L 137 The medaka exposed to Cs for 21 days was placed in clean seawater, washed three times, anesthetized with 200 mg / L MS-222 for 2 minutes, and then dissected. The gonadal tissue was removed, rinsed three times with pre-cooled PBS buffer, and suspended in a centrifuge tube containing 1 mL PBS buffer. The tissue was then broken using a rapid sample preparation instrument. The instrument parameters were set to a speed of 6.0 m / s, a time of 10 s, and three cycles. After completion, centrifugation (6000 rpm, 10 min, 4 ° C) was performed, and 800 μL of the supernatant was taken for protein quantification using a BCA kit, and the reading was taken at a wavelength of 562 nm to calculate the protein concentration of the sample. 200 μL of each of the above supernatants was taken to detect the level of oxidative stress using total superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) detection kits.

[0047] 1.4: Data processing and statistical analysis.

[0048] SPSS Statistics 20 software was used for statistical analysis of the data. One-way analysis of variance (ANOVA) was used to compare and analyze the differences in various indicators between the control group and the treatment group. All data are expressed as mean ± standard deviation (SD), and the P value represents the significance level. P <0.05 indicated significant differences; P<0.01 indicated that the difference between the groups was extremely significant. GraphPad Prism 8 software was used for drawing.

[0049] 2. Experimental results.

[0050] 2.1: 137 Differential gene expression and enrichment in the gonad of marine medaka exposed to Cs.

[0051] The differentially expressed genes with functional annotation information are plotted into a volcano plot, and the punctuation in the plot represents the gene names of the top 10 differentially expressed genes, such as Figure 1 and Figure 2 In the ovaries, compared with the control group, 10 5 In the Bq / L activity treatment group, 1206 genes were significantly up-regulated and 3276 genes were significantly down-regulated. 5 In the Bq / L activity treatment group, 1824 genes were significantly up-regulated and 576 genes were significantly down-regulated. 5 The Bq / L activity treatment group had a significant effect on the gonads of male and female fish.

[0052] Gene Ontology Enrichment Analysis is the most commonly used type of enrichment analysis. It is used to test the enrichment of Gene Ontology (GO) entries in a gene set. It is a very commonly used analysis type in transcriptome data analysis and can integrate differentially expressed genes into some biological processes.

[0053] like Figure 3 and Figure 4 As shown, the differentially expressed genes were enriched in GO 137The 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 response, negative regulation of innate immune response, angiogenesis, negative regulation of chemotactic interleukin 6 production, negative regulation of interleukin 12 production, innate immune response, angiogenesis, and inflammatory response. They were mainly enriched in cellular components such as membrane components, collagen trimers, cell surface, collagen-containing extracellular matrix, components of the cell membrane outside the extracellular matrix cell membrane, plasma membrane, extracellular region, and extracellular space. The main molecular functions of the differentially expressed genes were peptidase inhibitors, active cytokine binding, transmembrane signaling receptor activity, DNA binding transcription factor activity, growth factor activity, Serine-type endopeptidase activity, phosphatase 3-enzyme regulatory subunit binding, galactose-type endopeptidase inhibitor activity, heparin binding, and extracellular matrix structure. The differentially expressed genes in the testis of the marine medaka were mainly enriched in biological processes such as prevention of polyspermy, cholesterol homeostasis, righting reflex, acrosome reaction, triglyceride biosynthesis, G protein-coupled receptor signaling pathway, antigen synthesis, sperm capacitation, lipid decomposition and digestion. They were mainly enriched in cellular components such as the endosomal cavity, chylomicrons, NMDA-selective glutamate receptor complex, synaptonemal complex, basement membrane, hemoglobin complex, cortical granule protein complex, extracellular region and extracellular space. The main molecular functions of the differentially expressed genes were phosphatase A2 inhibitor activity, efflux transmembrane transporter activity, 2-glycerolipid o-glycerol transferase activity, CCR chemokine receptor binding, carbonic anhydrase activity, phosphate ion binding, oxygen binding, oxygen carrier activity, serine hydrolase activity and serine endopeptidase activity.

[0054] This suggests that the ocean medaka 137 Cs exposure produces reproductive toxicity, and suggests evaluating the reproductive toxicity of marine medaka 137 The toxicity of Cs is accurate.

[0055] 2.2: 137 Cs exposure affects the seven-day egg production, fertilization rate, hatching rate and hatching time of marine medaka.

[0056] like Figure 5 , Figure 6 , Figure 7 , Figure 8 As shown, 137 Cs exposure affected the seven-day egg production, fertilization rate, hatching rate, and hatching time of medaka. Compared with the control group, Cs exposure had a significant effect on the number of eggs laid, fertilization rate, hatching rate, and hatching time of medaka. 4 ( P <0.05), 10 5 Bq / L( P<0.01) radiation activity exposure reduced the seven-day egg production and hatching rate (* indicates P value less than 0.05; ** indicates P value less than 0.01; no mark is made if P value is greater than 0.05), 10 5 Bq / L( P <0.01) radiation activity exposure significantly reduced the fertilization rate of marine medaka; 137 The increase of Cs radioactivity prolongs the embryo incubation time.

[0057] 2.3: 137 Cs affects redox-related indicators of marine medaka.

[0058] Changes in SOD and CAT activities and MDA content Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 10 5 Bq / L( P <0.01) The radiation activity exposure significantly increased the activities 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 testis of male fish, while 10 4 , 10 5 Bq / L radiation exposure will significantly increase the content of MDA in the testis of male fish ( P <0.01).

[0059] like Fig.15 As shown in the figure, after screening, the SOD enzyme activity of the male and female gonads of the marine medaka changed; the CAT enzyme activity of females increased; the MDA content of males increased; the 7-day egg production decreased, the fertilization rate decreased, and the hatching rate decreased P <0.01, the significant changes in the above indicators indicate that the environment or samples 137 The Cs radioactivity exceeds 1×10 5 Bq / L. The MDA content of male medaka increased ( P <0.01); 7-day egg production decreased ( P <0 .05), reduced hatching rate ( P <0.05) indicates that the environment or sample 137 The Cs radioactivity exceeds 1×10 4 Bq / L.

[0060] The above embodiments are only for illustrating the inventive concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for evaluating low-activity radioactivity based on marine medaka 137 Cs toxicity method, characterized in that, The following steps are involved: Step 1: Set up the breeding conditions for medaka, domesticate the medaka, and set up the control group and experimental group. 137 The exposure concentration of Cs, after adjusting the exposure parameters 137 The exposure experiment started in Cs; Step 2: Observe the reproductive status parameters of medaka after the exposure experiment, including the seven-day egg production, fertilization rate, hatching rate, and hatching time, and compare the reproductive status parameters of the control group and the experimental group; Step 3: dissecting the medaka to obtain gonadal tissue, classifying the gonadal tissue according to the difference between male and female medaka, taking the supernatant after treatment to determine the activity of total superoxide dismutase and catalase and the content of malondialdehyde, determining the protein in the sample, calculating the enzyme activity per unit protein, and statistically analyzing the determination results of the control group and the experimental group; Step 4: Use one-way variance statistics to analyze the data information in steps 2 and 3, and compare the differences in the significance levels of various indicators between the control group and the experimental group. P < 0.05 indicated significant differences; P < 0.01 indicates that the difference between the groups is extremely significant, and the male and female medaka pairs were selected. 137 The sensitive index of Cs is determined by the difference of each index. 137 Toxicity level of Cs.

2. The method for evaluating low-activity radioactivity based on marine medaka according to claim 1 137 Cs toxicity method, characterized in that, The culture conditions include a culture temperature of 28±0.5°C, a light-dark cycle of 14:10h, a salinity of 30±1‰, a dissolved oxygen>6.0mg / L, fresh brine shrimp and feed are fed alternately 3-5 times a day, and the shrimps are acclimated in the laboratory for 7 days.

3. The method for evaluating low-activity radioactivity based on marine medaka according to claim 1 137 Cs toxicity method, characterized in that, The control group consisted of medaka growing in normal waters, and the experimental group 137 The Cs exposure concentration was 1×10 3 Bq / L, 1×10 4 Bq / L and 1×10 5 Bq / L, medaka for testing was obtained after 21 days.

4. The method for evaluating low-activity radioactivity based on marine medaka according to claim 1 137 Cs toxicity method, characterized in that, In step 2: Seven-day egg production: After the exposure, three pairs of male and female medaka were taken from each group and rinsed three times with clean seawater. They were then transferred to a beaker containing 5 L of clean seawater and raised according to the culture conditions in step 1. The eggs at the bottom of the beaker were collected and counted at 10 am every day for 7 consecutive days. The experiment was repeated three times. Fertilization rate: After the exposure, 5 pairs of male and female medaka were taken from each group and rinsed three times with clean seawater, then transferred to a beaker containing 5L clean seawater. After culturing for 24 hours according to the culture conditions in step 1, the eggs at the bottom of the beaker were taken and spread flat on a culture dish containing clean seawater. The eggs were separated manually one by one with tweezers and placed under a stereoscope 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: Take 100 normally fertilized embryos after the exposure test and place them in a culture dish filled with clean seawater. Raise them according to the culture conditions in step 1. Remove bad eggs and replace the culture water every day. Count the number of successfully hatched fry after 10 days, and record the hatching time. Calculate the hatching rate using the formula: Hatching rate = number of hatched fertilized eggs / total number of fertilized eggs × 100%.

5. The method for evaluating low-activity radioactivity based on marine medaka according to claim 1 137 Cs toxicity method, characterized in that, The step 3 comprises: Step 3-1: Place the medaka in the control group and the experimental group in clean seawater, wash them three times, anesthetize them with 200 mg / L MS-222 for 2 minutes, and then dissect them to remove the gonadal tissue; Step 3-2: After the gonad tissue was rinsed three times with pre-cooled PBS buffer, it was suspended in a centrifuge tube containing 1 mL of PBS buffer. The tissue was broken using a rapid sample preparation instrument. After completion, the tissue was centrifuged and 800 μL of the supernatant was taken for protein quantification using a BCA protein concentration assay kit. The reading was performed at a wavelength of 562 nm to calculate the protein concentration of the sample. Step 3-3: Take 200 μL of the gonadal tissue supernatant from the control group and the experimental group respectively, and use the total superoxide dismutase detection kit, catalase detection kit, and malondialdehyde detection kit to detect the oxidative stress level.

6. The method for evaluating low-activity radioactivity based on marine medaka according to claim 5. 137 Cs toxicity method, characterized in that, The instrument parameters of the rapid sample preparation instrument were set at a speed of 6.0 m / s, a time of 10 s, and 3 cycles, a centrifugal speed of 6000 rpm / min, a centrifugal time of 10 min, and a centrifugal temperature of 4°C.

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