Method for researching influence of heavy metal copper on acute toxicity and antioxidant enzyme activity of crassostrea hongkongensis

Abstract

The invention discloses a method for researching the influence of heavy metal copper on the acute toxicity and antioxidant enzyme activity of crassostrea hongkongensis, which comprises the following steps: selecting crassostrea hongkongensis as a research object, and evaluating the influence of heavy metal copper on the survival and antioxidant enzyme activity of crassostrea hongkongensis through an acute toxicity experiment. The LC50 of the crassostrea hongkongensis on the copper ions is determined to be 65.32 mg / L, and the SC of the crassostrea hongkongensis on When the determined semi-lethal concentration is used for stress treatment, the antioxidant indexes of different tissues of the crassostrea hongkongensis are remarkably influenced along with the prolonging of the experiment time. CAT in hepatopancreas of crassostrea hongkongensis, T-SOD and GST in gill tissues and GPX in gonad show specificity in corresponding tissues, and the discovery can be used as an important basis for ecological monitoring of the Cu < 2 + > content. The test result can provide reference for healthy cultivation of crassostrea hongkongensis.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture research, and more particularly to a method for studying the influence of heavy metal copper on the acute toxicity and antioxidant enzyme activity of Hong Kong oysters. Background Art

[0002] Hong Kong oyster (Crassostrea hongkongensis), also known as Hong Kong giant oyster, commonly known as white consumption, belongs to Mollusca, Lamellibranchia, Pterimorpjia, Pterioida, Ostreidae, Crassostrea, and usually grows near the low tide line and estuaries. Hong Kong oysters have a history of more than 2,000 years of farming in my country. They are characterized by delicious meat and rich nutritional value. They are called "milk in the sea". They are high-protein, low-fat, low-cholesterol economic species rich in trace elements such as zinc and selenium. They have been favored by the market and the attention of scientific and technological workers. Studies have shown that the annual output of Hong Kong oysters in China in 2018 was 1.8 million tons, accounting for 34.7% of the annual output of oysters in China, second only to Fujian oysters.

[0003] In recent years, with the large-scale input of heavy metals produced by non-ferrous metal smelting, coal combustion and electronic waste treatment in industrial production processes into the coastal waters through surface runoff and other means, as well as improper aquaculture practices such as the release of feed and the application of fish medicines during aquaculture, the water quality of the coastal waters has been seriously polluted. Studies have shown that in the flow of food chains and food webs, the content of heavy metals gradually decreases with the increase of trophic levels. Most bivalve molluscs are filter feeders, and their enrichment levels of heavy metals are relatively high. Once the threshold is reached, the physiological response of the shellfish will be enhanced, causing tissue damage and decreased blood cell activity, ultimately affecting their survival. According to surveys, the current heavy metal pollution in the coastal waters is mainly Hg and Cu, while the heavy metal pollution in the aquaculture areas is mainly Cu and Zn. Therefore, many scholars have conducted research on the impact of heavy metal Cu on farmed shellfish. Compared with the control group, the single heavy metal ion Cu 2+ The lethal mud cockle (Tegillarca granosa) has obvious specific death symptoms: Cu 2+ The lethal mud clams showed gill erosion. The changes in blood cell activity were measured by flow cytometry with PI staining. The results showed that after 6 hours of stress, 10 -3 M C 2+ The mortality rate of blood cells is significant and is related to Hg 2+ 、Cd 2+ 、Zn 2+ Comparison revealed that Cu 2+The toxicity to the blood cells of Abalone variegatede was the lowest. The static acute toxicity test method was used to study the heavy metal Cu 2+ The combined effects of Cu stress and seawater acidification on Philippine clams showed that Cu stress significantly reduced the oxygen consumption rate and water filtration rate of clams (P<0.05). As the stress time increased, the heart rate of clams decreased significantly (P<0.05).

[0004] Water quality physical and chemical indicators are important factors affecting the healthy growth of Hong Kong oysters. Studies have shown that temperature, salinity, dissolved oxygen, pH, etc. will affect the health of Hong Kong oysters. 2+ There are no reports on the effects of stress on Hong Kong oysters. Given the potential toxic effects of copper on oysters, in-depth toxicity studies and rigorous testing are particularly urgent, as this is not only related to food safety, but also directly affects public health and the sustainability of the ecological environment. Summary of the invention

[0005] In view of this, the present invention provides a method for studying the effects of heavy metal copper on the acute toxicity and antioxidant enzyme activity of Hong Kong oysters, which specifically adopts the following technical scheme:

[0006] A method for studying the effects of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters comprises the following steps:

[0007] Step 1: Select Hong Kong oysters of the same size, clean them and keep them for 4 days for later use;

[0008] Step 2: Acute toxicity test

[0009] The aquaculture seawater containing Cu(NO3)·2.5H2O of different concentrations was set, and then Hong Kong oysters were placed in it for breeding. The water was changed every 24 hours with the same concentration, and the dead Hong Kong oysters were removed in time and the survival rate was recorded. The breeding was continued for 4 days, and the half-lethal concentration and safe concentration of Hong Kong oysters after 4 days of heavy metal copper stress were calculated.

[0010] Step 3: Stress experiment

[0011] According to the results of the acute toxicity test in step 2, the 4d LC 50 In order to stress Hong Kong oysters with different stress concentrations, multiple level groups were set up with the same culture number in each group. The same number of Hong Kong oysters were randomly selected after 0h, 24h, 48h, 72h, and 96h of stress, and their mantle, gills, gonads, and hepatopancreas were sampled under sterile conditions, and then the antioxidant enzyme activity was determined.

[0012] Preferably, in step 1, the shell height of Hong Kong oysters is 140.15±2.56 mm and the shell length is 71.28+±1.89 mm.

[0013] Preferably, during the temporary culture period in step 1, the salinity is 18.75±0.67‰ and the water temperature is 25±0.5°C, and the Zhanjiang Isochrysis algae is fed twice a day, with each feeding amount of 1×10 4 cell / mL, with 100% water changes performed daily and continuous aeration.

[0014] Preferably, the temporary culture water in step one is the same as the aquaculture seawater in step two, and both meet the Class I seawater standard. In step two, five concentrations of aquaculture seawater containing Cu(NO3)·2.5H2O are set at 40.00 mg / L, 56.62 mg / L, 79.98 mg / L, 113.24 mg / L and 160.00 mg / L, with 3 parallels in each group.

[0015] Preferably, in step 2, aquaculture seawater is used as a control group, no feeding is given during the experiment, the other culture conditions are consistent with the temporary culture period, and the survival of Hong Kong oysters is checked every 3 hours.

[0016] Preferably, in step 2, the Probit process in SPSS 27.0 software is used to construct a linear regression equation of probability unit-drug mass concentration logarithm, and the half-lethal concentration (LC50) at 24h, 48h, 72h, and 96h is obtained by the equation. 50 ), and then use the safe concentration calculation formula MPC = 96hLC 50 / 100, calculate the safe concentration (SC).

[0017] Preferably, in step three, aquaculture seawater is used as a control group, and at least three level groups are set.

[0018] Preferably, the antioxidant enzymes in step three are CAT, T-SOD, GSH-PX and GSH-ST.

[0019] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a method for studying the influence of heavy metal copper on the acute toxicity and antioxidant enzyme activity of Hong Kong oysters, which has the following beneficial effects:

[0020] The present invention uses Hong Kong oysters as the object to explore the acute toxicity of copper and its effect on the activity of antioxidant enzymes. The study found that the 96h LC50 of Hong Kong oysters for copper ions is 65.32mg / L, and the safe concentration is 0.65mg / L. Under copper stress, CAT in the hepatopancreas, T-SOD and GST in the gill tissue, and GPX in the gonad of Hong Kong oysters are specific in the antioxidant stress levels in different tissues. This finding can be used as a Cu 2+ The results of this experiment can provide a reference for the healthy cultivation of oysters in Hong Kong. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 This is a graph showing changes in CAT activity in different tissues of Hong Kong oysters under copper stress;

[0023] Figure 2 This is a graph showing the changes in T-SOD activity in different tissues of Hong Kong oysters under copper stress;

[0024] Figure 3 This is a graph showing the changes in GPX activity in different tissues of Hong Kong oysters under copper stress;

[0025] Figure 4 This is a graph showing the changes in GST activity in different tissues of Hong Kong oysters under copper stress. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] The experimental seawater used in the experiment was the seawater from the Donghai Island Aquaculture Experimental Base of the Fisheries College of Guangdong Ocean University, which was treated by sedimentation and sand filtration. The results of water quality analysis showed that heavy metals: Cd was 0.04μg / L, Pb was 0.3μg / L, Cu was 0.89μg / L, and Zn was 7.23μg / L, which met the Class I seawater standard. The experiment used analytically pure heavy metal pollutant Cu(NO3)·2.5H2O to prepare a mother solution with a mass concentration of 30mg / mL. Before the experiment, it was diluted with seawater to the required concentration and prepared for immediate use.

[0029] Step 1: The Hong Kong oysters used in the experiment were purchased from a coastal farm in Zhanjiang in April 2023. 500 healthy individuals were randomly selected with a shell height of (140.15±2.56) mm and a shell length of (71.28+±1.89) mm. After the oysters were transported back, the surface attached organisms were removed and placed in a 500L plastic barrel for temporary culture for 4 days. During the temporary culture period, the salinity was (18.75±0.67)‰ and the water temperature was (25±0.5)℃. Isochrysis zhanjiangensis was fed twice a day, with a feeding amount of 1×104 cell / mL, 100% water change was performed daily with continuous aeration;

[0030] Step 2: Use analytical pure Cu(NO3)·2.5H2O to prepare the solution used in the experiment. According to the preliminary experimental results (Hong Kong oysters died when the copper ion concentration was 40.00 mg / L, and all Hong Kong oysters died when the copper ion concentration was 160.00 mg / L), the drug mass concentration range was determined. Using the equal logarithmic interval method, 5 mass concentration gradients were set: 40.00 mg / L, 56.62 mg / L, 79.98 mg / L, 113.24 mg / L and 160.00 mg / L experimental groups, and a normal seawater control group. The experiment was carried out in a 55cm×45cm×30cm square plastic box. Three parallel groups were set for each concentration gradient, and 30 oysters were placed in each plastic box. No bait was added during the experiment, and the experimental group Cu was replaced every 24 hours. 2+ The concentration of seawater was consistent, and the other culture conditions were consistent with the temporary culture period. During the experiment, the survival of Hong Kong oysters was checked every 3 hours. If any Hong Kong oysters were found to have opened their shells and there was no contraction in the mantle after acupuncture, they were determined to be dead and removed. The experiment lasted for 4 days, and the survival rate was recorded every 24 hours. The Probit process in SPSS27.0 software was used to construct a linear regression equation of probability unit-drug mass concentration logarithm. Through this equation, the half-lethal concentration (LC50) of 24h, 48h, 72h, and 96h was obtained, respectively. 50 ), and then use the formula MPC = 96hLC 50 / 100

[12] , the safe concentration (SC) was calculated. Finally, one-way analysis of variance (ANOVA) and Duncan's multiple comparison analysis were performed. All charts were drawn using Excel 2021;

[0031] The results are shown in Table 1:

[0032]

[0033] As shown in Table 1, within a certain period of time, as Cu 2+ As the stress concentration increased, the mortality rate of Hong Kong oysters gradually increased. 2+ Hong Kong oysters began to die after 72 hours of stress, with an average mortality rate of 4.44%, 160.00 mg / LCu 2+ All died at 96h of stress. The mortality rate of the control group was 72h and 96h, and that of the experimental group was 40.00mg / L Cu. 2+ There was a significant difference in mortality (P<0.05). The mortality of the control group was significantly higher than that of the experimental groups at 56.62 mg / L, 79.98 mg / L, 113.24 mg / L and 160.00 mg / L Cu within 96 hours. 2+There were significant differences in mortality between the two groups (P<0.05). 2+ LC of Hong Kong oysters at 24h, 48h, 72h and 96h stress 50 As the stress time increased, the values ​​gradually decreased, reaching the highest at 24 h and the lowest at 96 h. 50 They are 230.13 mg / L, 116.01 mg / L, 84.33 mg / L, 65.32 mg / L respectively, and the SC is 0.65 mg / L.

[0034] Step 3: According to the acute toxicity test results of step 2, select the 96h LC 50 The stress concentration was used to stress the Hong Kong oysters, that is, the experiment set up a control group and a treatment group, the concentration of the treatment group was set as the half-lethal concentration of 65.32 mg / L at 96 hours as the stress concentration, and normal seawater was used as the control group. The experiment was carried out in a 55cm×45cm×30cm square plastic box, and 3 Hong Kong oysters were randomly taken from each group at 0h, 24h, 48h, 72h, and 96h to determine their immune indicators. The experiment was set up with 3 replicates;

[0035] The specific determination of immune indexes is as follows: the mantle, gills, gonads and hepatopancreas of Hong Kong oysters are dissected out in turn, about 500 mg of tissue is mixed with 0.86% saline at a mass: volume ratio of 1:9, a homogenizer is used to make a 10% tissue homogenate, centrifuged at 2500r for 10min at 4℃, and the supernatant is stored at 4℃;

[0036] The enzyme activity determination is specifically as follows:

[0037] The kits produced by Nanjing Jiancheng Biochemical Reagent Company were used to measure CAT, T-SOD, GSH-PX, and GSH-ST. All operations were carried out according to the instructions of the kits provided by Nanjing Jiancheng Company. The principle of the determination is as follows:

[0038] CAT: Ammonium molybdate can terminate the reaction of CAT decomposing H2O2, and react with the remaining H2O2 to produce a light yellow complex. The amount of H2O2 decomposed by 1μmol per milligram of tissue protein per second is defined as a CAT activity unit. The change is measured at 405nm and the CAT activity is calculated.

[0039] SOD:WST-1 can react with superoxide anion (O 2- ) reaction to form a water-soluble formazan dye. SOD inhibits the reaction process. The amount of enzyme corresponding to 50% SOD inhibition rate in this reaction system is defined as one SOD activity unit. The product of WST-1 is colorimetrically measured at 450nm and the activity of T-SOD is calculated.

[0040] GSH-PX: Reduced glutathione and dithiodinitrobenzoic acid can react to generate a stable 5-thiodinitrobenzoic acid anion, and the product is yellow. The absorbance is measured at 412nm to calculate the amount of reduced glutathione.

[0041] GSH-ST: GST catalyzes the reaction between GSH and 1-chloro-2,4-dinitrobenzene. Within a certain reaction time, the GST enzyme activity is calculated by detecting the concentration of the reactants in the solution.

[0042] The results are attached Figure 1-4 shown.

[0043] Figure 1 The changes of CAT activity in different tissues under copper stress. Figure 1 Show, in Cu 2+ Within 96 hours of stress, the extension of the experimental time had a significant effect on the CAT of Hong Kong oysters. The CAT of its gills and gonads showed a trend of first rising and then falling, while the CAT of its mantle and hepatopancreas showed an upward trend. There were significant differences between the mantle, gills, gonads, and liver treatment groups and the control group at 24h, 48h, 72h, and 96h of stress (P<0.05). 2+ There were significant differences in the effects of stress on CAT in different tissues (P<0.05), and the sensitivity was hepatopancreas>gills>gonads>mantle, and the maximum values ​​within 96 were 0.545U / mg prot, 0.557U / mg prot, 0.636U / mg prot, and 0.615U / mg prot, respectively.

[0044] Figure 2 This is a graph showing the changes in T-SOD activity in different tissues under copper stress. Figure 2 Show, in Cu 2+ Within 96 hours of stress, the extension of the experimental time had a significant effect on the T-SOD of the Hong Kong oyster. The T-SOD of its mantle showed a trend of first decreasing and then increasing, the T-SOD of its gills showed a trend of first increasing and then decreasing, and the T-SOD of its gonads and hepatopancreas showed a trend of first decreasing, then increasing, and then decreasing. There were significant differences between the mantle, gills, gonads, and liver treatment groups and the control group at 24 hours, 48 ​​hours, 72 hours, and 96 hours of stress (P<0.05). Heavy metal Cu 2+ There were significant differences in the effects of stress on T-SOD in different tissues (P<0.05), and the sensitivity showed the following antioxidant levels: gills> gonads> hepatopancreas> mantle. The maximum values ​​within 96 were 0.740U / mg prot, 0.806U / mg prot, 0.770U / mg prot, and 0.785U / mg prot, respectively.

[0045] Figure 3 This is a graph showing changes in GPX activity in different tissues under copper stress. Figure 3 Show, in Cu 2+ Within 96 hours of stress, the extension of the experimental time had a significant effect on the GPX of the Hong Kong oyster. The GPX of its gonads, mantle, and hepatopancreas showed a trend of first rising and then falling, and the GPX of its gill tissue showed a wave-like change. There were significant differences between the mantle, gill, gonad, and liver treatment groups and the control group at 24h, 48h, 72h, and 96h of stress (P<0.05). 2+ There were significant differences in the effects of stress on GPX in different tissues (P<0.05), and the sensitivity was gonad>mantle>gill>hepatopancreas. The maximum values ​​within 96 were 0.420U / mg prot, 0.275U / mg prot, 0.217U / mg prot, and 0.349U / mg prot, respectively.

[0046] Figure 4 Changes in GST activity in different tissues under copper stress. Figure 4 Show, in Cu 2+ Within 96 hours of stress, the extension of the experimental time had a significant effect on the GST of Hong Kong oysters. The GST of its mantle and hepatopancreas showed a trend of first increasing and then decreasing, the GST of its gills showed a trend of first decreasing, then increasing and then decreasing, and the GST of its gonads showed a trend of first decreasing and then increasing. There were significant differences between the mantle, gill, gonad, and liver treatment groups and the control group at 24 hours, 48 ​​hours, 72 hours, and 96 hours of stress (P<0.05). Heavy metal Cu 2+ There were significant differences in the effects of stress on GST in different tissues (P<0.05), and the sensitivity was gills>mantle>hepatopancreas>gonads. The maximum values ​​within 96 were 0.271U / mg prot, 0.294U / mg prot, 0.211U / mg prot, and 0.406U / mg prot, respectively.

[0047] The above research results show that in c(Cu 2+ )=65.32mg / L, as the experimental time prolonged, the CAT of the gills and gonads of the Hong Kong oysters showed a trend of first rising and then falling, while the CAT of the mantle and hepatopancreas showed an upward trend. This result is related to the concentration, type and stress time of the experimental toxin. The hepatopancreas is the main organ for detoxification and metabolism of the Hong Kong oyster and is the main organ for the heavy metal Cu 2+ The results showed that CAT of Hong Kong oysters could accumulate Cu 2+ The sensitivity of Cu shows the rule of hepatopancreas>gills>gonads>mantle. 2+When entering the hepatopancreatic tissue, it catalyzes CAT to produce a large number of reactive oxygen free radicals. The level has remained at a high level and has not decreased. This is because when tissue cells are slightly damaged, new CAT is continuously produced due to the stress of pollutants to maintain the balance of the redox system. This is the body's own normal stress response.

[0048] In Cu 2+ Under acute stress with a concentration of 65.32 mg / L, as the experimental time prolonged, the T-SOD of the mantle of the Hong Kong oyster showed a trend of first decreasing and then increasing, the T-SOD of the gills showed a trend of first increasing and then decreasing, and the T-SOD of its gonads and hepatopancreas showed a trend of first decreasing, then increasing, and then decreasing. This result is related to the concentration of the experimental poison and different tissues. The gill tissue is the respiratory organ of mollusks. Damage to the gills will have an important impact on its respiration, causing ion imbalance in the body and ultimately leading to the death of the organism. The results show that the T-SOD of the Hong Kong oyster is sensitive to Cu 2+ The sensitivity of the tissues is as follows: gills> gonads> hepatopancreas> mantle. 2+ Within 72 hours of stress, high expression of Cu / Zn-SOD gene can effectively eliminate excessive oxygen free radicals, reduce and prevent cell damage, and SOD shows an upward trend. After 72 hours, gill tissue is damaged by oxidation, which inhibits the activity of SOD and SOD shows a downward trend.

[0049] Cu 2+ Under acute stress of 65.32 mg / L, as the experimental time prolonged, the GPX of the mantle and hepatopancreas of Hong Kong oysters showed a trend of first rising and then falling, the GPX of the gonads showed a trend of first rising, then falling, and then rising, and the GPX of the gills showed a wave-like change, and the sensitivity of different tissues showed the rule of gonads>mantle>gills>hepatopancreas. When the body is stressed by heavy metals, the gonads will accumulate a large amount of ROS, so the GPX activity in the gonads increases until the gonad tissue is oxidatively damaged, the reproductive function is impaired, and the GPX activity begins to decline.

[0050] Hong Kong oyster GST to Cu 2+ The sensitivity of the mantle GST shows a pattern of gills > mantle > hepatopancreas > gonads. The reason is that due to the increase of reactive oxygen, tissue damage occurs, resulting in a stronger sensitivity of mantle GST.

[0051] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will comply with the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A method for studying the effects of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters, characterized in that: The following steps are involved: Step 1: Select Hong Kong oysters of the same size, clean them and keep them for 4 days for later use; Step 2: Acute toxicity test The aquaculture seawater containing Cu(NO3)·2.5H2O of different concentrations was set, and then Hong Kong oysters were placed in it for breeding. The water was changed every 24 hours with the same concentration, and the dead Hong Kong oysters were removed in time and the survival rate was recorded. The breeding was continued for 4 days, and the half-lethal concentration and safe concentration of Hong Kong oysters after 4 days of heavy metal copper stress were calculated. Step 3: Stress experiment According to the results of the acute toxicity test in step 2, the 4d LC 50 In order to stress Hong Kong oysters with different stress concentrations, multiple level groups were set up with the same culture number in each group. The same number of Hong Kong oysters were randomly selected after 0h, 24h, 48h, 72h, and 96h of stress, and their mantle, gills, gonads, and hepatopancreas were sampled under sterile conditions, and then the antioxidant enzyme activity was determined.

2. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: In step 1, the shell height of Hong Kong oysters is 140.15±2.56mm and the shell length is 71.28+±1.89mm.

3. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: During the temporary culture period in step 1, the salinity was 18.75±0.67‰ and the water temperature was 25±0.5℃. The Isochrysis Zhanjiangensis was fed twice a day, with a feeding amount of 1×10 4 cell / mL, with 100% water changes performed daily and continuous aeration.

4. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: The temporary culture water in step 1 is the same as the aquaculture seawater in step 2, both of which meet the Class I seawater standards. In step 2, five concentrations of aquaculture seawater containing Cu(NO3)·2.5H2O, namely 40.00 mg / L, 56.62 mg / L, 79.98 mg / L, 113.24 mg / L and 160.00 mg / L, are set, with 3 parallels in each group.

5. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: In step 2, aquaculture seawater was used as the control group. No feeding was performed during the experiment. The other culture conditions were the same as those during the temporary culture period. The survival of Hong Kong oysters was checked every 3 hours.

6. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: Step 2: Use the Probit process in SPSS27.0 software to construct a linear regression equation of probability unit-drug mass concentration logarithm. Through this equation, the half-lethal concentration LC at 24h, 48h, 72h, and 96h is obtained respectively. 50 , and then use the safe concentration calculation formula MPC = 96hLC 50 / 100, calculate the safe concentration SC.

7. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: Step 3: Using aquaculture seawater as the control group, set up at least 3 level groups.

8. The method for studying the effect of heavy metal copper on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: The antioxidant enzymes in step three are CAT, T-SOD, GSH-PX and GSH-ST.

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