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

Abstract

The invention discloses a method for researching the influence of heavy metal cadmium 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 cadmium on the survival and antioxidant enzyme activity of crassostrea hongkongensis through an acute toxicity experiment. The LC50 of the Crassostrea hongkongensis to the cadmium ions is determined to be 5.452 mg / L, and the SC of the Crassostrea hongkongensis to When the determined semi-lethal concentration is used for stress treatment, the activity of the antioxidant enzyme in different tissues of the oysters in different time periods is in a trend of firstly increasing and then decreasing, which indicates that the antioxidant enzyme system plays a key role in resisting heavy metal stress in organisms. As time goes on, the activity of antioxidant enzyme is gradually reduced, which shows that the resistance of Crassostrea hongkongensis to cadmium stress is limited. The invention not only provides a new perspective for understanding the defense mechanism of Crassostrea hongkongensis on cadmium toxicity, but also is beneficial to understanding the heavy metal pollution condition in the marine environment, and provides a scientific basis for further toxicity research and ecological environment protection.

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 cadmium on the acute toxicity and antioxidant enzyme activity of Hong Kong oysters. Background Art

[0002] Hong Kong oysters (Crassostreahongkongensis), commonly known as Qinzhou oysters or white oysters, are classified as Pterioida, Ostreidae, and Crassostrea. Hong Kong oysters are mainly distributed in the South my country Sea and are the main aquaculture species in the coastal areas of Guangdong and Guangxi. They are also distributed in Hainan and Fujian. Hong Kong oysters mainly grow in shallow sea mudflats and mangroves. They are typical representative species of intertidal zones. Their living environment is exposed to air and submerged in seawater every day, so Hong Kong oysters have a strong ability to adapt to the environment. Hong Kong oysters are important economic shellfish in my country and one of the three major aquaculture oyster varieties in my country. In 2020, their production was about 1.7885 million tons, accounting for about 32.97% of the national oyster production. Because of their rich nutrition, delicious taste and tender meat, they are very popular in the market. Hong Kong oysters are very sensitive to seawater pollution and changes in the ecological environment, so they are widely used in the monitoring and evaluation of the marine ecological environment. In addition, Hong Kong oysters are often used as experimental animals to study the impact of environmental pollution and climate change on the physiological functions and ecology of marine organisms.

[0003] In recent years, with the rapid development of industry, a large amount of industrial waste has been discharged into the natural environment. Among them, heavy metals are one of the key pollutants in the environment. Due to their very stable chemical properties, they are difficult to degrade once released into the environment, so they can cause serious damage to water bodies, soil, air and human health. Heavy metals can induce the occurrence of diseases in organisms through various pathways, mainly through the oxidative stress of free radicals, damaging cellular macromolecules such as DNA, proteins and lipids. Cadmium (Cd) is a widely distributed pollutant in aquatic environments. Cadmium poisoning usually occurs when cadmium ions (Cd 2+) is absorbed and accumulated in tissues beyond the tolerable limit. Once absorbed by the human body, cadmium can combine with metallothionein (MT), albumin, cysteine, glutathione and other thiol-rich proteins containing thiol groups to form various cadmium compounds. In aquatic animals, long-term cadmium stress can have a serious impact on the growth and development, intestinal tissue flora, antioxidant capacity and DNA methylation of aquatic animals. The total amount of cadmium discharged into seawater from industrial waste in my country reaches more than 680 tons each year. Due to their own living habits and environmental relations, shellfish are susceptible to heavy metal pollution and poisoning. Hong Kong oysters are affected by environmental pollutants during their growth, and a lot of heavy metals such as cadmium may be enriched in their bodies. These harmful substances will threaten the health and food safety of Hong Kong oysters. Studies have shown that after ingesting oysters infected with cadmium, 78.3%-87.8% of the cadmium in the oyster digestate can be absorbed by the human body. Given the potential toxic effects of cadmium on oysters, in-depth toxicity research and strict testing are particularly urgent, which is not only related to food safety, but also directly affects public health and the sustainability of the ecological environment. Summary of the invention

[0004] In view of this, the present invention provides a method for studying the effects of heavy metal cadmium on the acute toxicity and antioxidant enzyme activity of Hong Kong oysters, which specifically adopts the following technical scheme:

[0005] A method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters, characterized in that it comprises the following steps:

[0006] Step 1: Select Hong Kong oysters of uniform size and without any disease or injury, clean them and keep them for a week for later use;

[0007] Step 2: Acute toxicity test

[0008] Set up temporary culture water containing cadmium chloride hexahydrate of different concentrations, and then place Hong Kong oysters in temporary culture water of different concentrations for culture. Change the water every 24 hours, remove dead Hong Kong oysters in time and record the number of dead shellfish. Continue culture for 96 hours, and calculate the median lethal concentration and safe concentration of Hong Kong oysters after 96 hours of heavy metal cadmium stress.

[0009] Step 3: Stress experiment

[0010] According to the results of the acute toxicity test in step 2, the 96h 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 digestive caeca were sampled under sterile conditions, and then the antioxidant enzyme activity was determined.

[0011] Preferably, in step 1, two-year-old Hong Kong oysters are selected, with a shell length of 6.5±0.5 cm.

[0012] The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1 is characterized in that during the temporary culture period in step 1, the salinity is 19±1, the pH is 8±0.2, the water temperature is 26±2°C, Chlorella powder is fed at 8:00 and 20:00 every day, the feeding amount each time is 3% of the oyster body mass, and the dead shellfish are removed and the water is changed every 24 hours.

[0013] Preferably, in step 2, five concentrations of cadmium chloride hexahydrate-containing temporary water are set at 1 mg / L, 2.78 mg / L, 6.75 mg / L, 17.55 mg / L and 30 mg / L, with 3 parallels in each group.

[0014] Preferably, in step 2, temporary water with the same temporary water quality conditions as in step 1 is used as a control group.

[0015] Preferably, in step 2, SPSS 26.0 is used to calculate the median lethal concentration and safety concentration of Hong Kong oysters after 96 hours of heavy metal cadmium stress, and the calculation formula is as follows:

[0016] SC=f·96h LC50

[0017] Among them, SC is the safe concentration and the f value is 0.01.

[0018] Preferably, in step three, at least three horizontal groups are set up, each group has at least 20 fish, and temporary water with the same temporary water quality conditions as in step one is used as a control group.

[0019] Preferably, the antioxidant enzyme in step three is catalase, superoxide dismutase, glutathione peroxidase and glutathione-S transferase.

[0020] Furthermore, the catalase assay adopts the ammonium molybdate termination method, the superoxide dismutase assay adopts the pigment reduction method, the glutathione peroxidase assay adopts the colorimetric method, and the glutathione-S transferase assay adopts the ultraviolet spectrophotometric method.

[0021] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for studying the influence of heavy metal cadmium on the acute toxicity and antioxidant enzyme activity of Hong Kong oysters, which has the following beneficial effects:

[0022] The present invention takes Hong Kong oyster as the object and explores the acute toxicity of cadmium and its effect on antioxidant enzyme activity. The study found that the 96h LC50 of Hong Kong oyster for cadmium ions is 5.452mg / L, and the safe concentration is 0.05452mg / L. Under cadmium stress, the antioxidant enzyme activity of different tissues of Hong Kong oysters showed a trend of first increasing and then decreasing. It was significantly induced and reached a peak within 24-48h, and then the activity decreased and was significantly inhibited at 96h. The research results provide a new perspective for understanding the defense mechanism of oysters against cadmium toxicity. Antioxidant enzymes play a key role in resisting heavy metal stress. The experimental data confirmed the impact of cadmium on the life activities of oysters and enriched the theory of aquatic organisms' response to heavy metal stress. The study provides a scientific basis for ecological and environmental protection. As an important organism for marine ecological monitoring, the response of Hong Kong oysters to cadmium helps to understand the heavy metal pollution in the marine environment. In addition, since oysters are important economic shellfish, the enrichment of cadmium in the body may pose a threat to human health. This study helps to strengthen the monitoring and control of heavy metal pollution in aquatic products and ensure food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 This is the concentration-mortality curve of the official experiment of Hong Kong oysters;

[0025] Figure 2 This is the change of SOD enzyme activity in Hong Kong oysters under cadmium stress;

[0026] Figure 3 This is the change of CAT enzyme activity in Hong Kong oysters under cadmium stress;

[0027] Figure 4 This is the change of GPX enzyme activity in Hong Kong oysters under cadmium stress;

[0028] Figure 5 This is a graph showing the changes in GST enzyme activity in Hong Kong oysters under cadmium stress. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] Step 1: Select two-year-old Hong Kong oysters (shell length of 6.5±0.5cm) purchased from Zhanjiang City, Guangdong Province. Select Hong Kong oysters of uniform size, disease-free and uninjured, clean their shell sludge, and then temporarily culture them in the laboratory for one week. During the temporary culture period, the salinity is 19±1, pH 8±0.2, and the water temperature is 26±2℃. Feed chlorella powder at 8:00 and 20:00 every day, and the feeding amount each time is 3% of the oyster body weight. Remove dead shells and change water every 24 hours;

[0032] Step 2: Use cadmium chloride hexahydrate (CdCl2·6H2O) to analyze the pure solution used in the experiment. First, through preliminary experiments, determine that the lowest cadmium concentration at which Hong Kong oysters survive 100% within 48 hours under heavy metal cadmium stress and the highest cadmium concentration at which 100% die are 1-30 mg / L. According to this concentration range, set up 5 experimental groups with concentration gradients, namely 1 mg / L, 2.78 mg / L, 6.75 mg / L, 17.55 mg / L and 30 mg / L, with 3 parallels in each group. The control group has the same water quality conditions as during the temporary culture period. The water is changed every 24 hours at the same concentration, and the dead shellfish are removed in time, and the number of dead shellfish is recorded. The experiment lasts for 96 hours; finally, use SPSS26.0 to calculate the half-lethal concentration and safe concentration of Hong Kong oysters after 96 hours of heavy metal cadmium stress. The calculation formula is as follows:

[0033] SC=f·96h LC50

[0034] Among them, SC is the safe concentration, f value is 0.01, and the results are shown in the attached Figure 1 As shown, according to the attached Figure 1 The data showed that the LC50 of Hong Kong oysters under acute toxic stress of heavy metal cadmium for 96 hours was 5.452 mg / L, and the SC was 0.05452 mg / L. SPSS26.0 was used for a two-way chi-square test, and the chi-square value was 13.478, the degree of freedom was 4, and the P value was 0.018. There were significant differences between the experimental group and the control group at different cadmium ion concentrations, and their mortality rate showed an obvious filling trend. With the increase of cadmium ion concentration, the harm to the experimental group gradually intensified.

[0035] Step three: According to the results of the acute toxicity experiment in step two, the 96h LC50 was selected as the stress concentration to stress Hong Kong oysters. Three parallel groups were set up, with 20 shellfish in each group. The water quality conditions of the control group were the same as those during the temporary culture period. Six shellfish were randomly selected after 0h, 24h, 48h, 72h, and 96h of stress, and their mantles, gills, gonads, and digestive caeca were sampled. The entire process was carried out under sterile conditions, and the tissues were quickly frozen in liquid nitrogen and finally stored in a -80℃ refrigerator for later determination of enzyme activity;

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

[0037] First, take out the tissue from the cryopreservation tube and weigh it, then grind it into a homogenate, then add physiological saline to make a mixture (the weight ratio is 1:9), put it into a 15ml centrifuge tube and centrifuge it at 2500r / min for 10 minutes. After the centrifugation, insert the centrifuge tube vertically into ice. Take the supernatant and then measure the biochemical indicators. All biochemical indicators are measured using kits. The measurement method is as follows:

[0038] Table 1 Biochemical index determination method

[0039] category Abbreviation Determination method unit Catalase CAT Ammonium molybdate termination method U / mgprot Superoxide dismutase SOD Pigment reduction method U / mgprot Glutathione peroxidase GPX Colorimetry U / mgprot Glutathione-S transferase GST UV spectrophotometry U / mgprot

[0040] The experimental data were tested for significant differences and two-way chi-square tests using SPSS 26.0, and the regression equation was calculated. The statistical significance level was set at 0.05. Finally, Excel 2021 software was used to draw the graph. The results are shown in the attached figure. Figure 2-5 shown.

[0041] Figure 2 This is a graph showing changes in SOD enzyme activity in Hong Kong oysters under cadmium stress. In the figure, * indicates a significant difference between the experimental group and the control group (P < 0.05), and ** indicates an extremely significant difference from the control group (P < 0.01). Figure 2 The results showed that compared with the control group, at the beginning of the experiment, the difference in SOD activity was not significant (P>0.05). Since the beginning of the stress, the activity of SOD began to rise compared with the control group. At 24h, the SOD activity in the gills reached its peak and was significantly higher than that in the control group (P<0.05), an increase of 10.59% compared with the control group. Thereafter, the SOD activity began to decline, and was significantly lower than that in the control group from 72h (P<0.05), and reached the lowest level at 96h, a decrease of 40.91% compared with the control group. In the mantle and digestive caeca, the activity of SOD reached its peak at 48h, an increase of 14.12% and 22.68% respectively compared with the control group, and then the activity of SOD began to decline, and was significantly lower than that in the control group at 96h (P<0.05), a decrease of 40.70% and 13.39% respectively. In the gonads, the activity of SOD also increased after the start of the experiment and reached a peak at 48 hours. However, its activity did not differ significantly from that of the control group during the entire experiment (P>0.05).

[0042] Figure 3 This is a graph showing the changes in CAT enzyme activity in Hong Kong oysters under cadmium stress. In the figure, * indicates a significant difference between the experimental group and the control group (P < 0.05), and ** indicates an extremely significant difference from the control group (P < 0.01). Figure 3The results showed that in the initial stage of the experiment, there was no significant difference in CAT activity in various tissues between the experimental group and the control group (P>0.05). For the mantle and gonads, CAT activity showed an upward trend from 0h to 24h, reaching a peak at 24h, which was 28.48% and 23.10% higher than the control group, respectively. After that, CAT activity gradually decreased, and was lower than the control group level after 72h. At 96h, the activity dropped to the lowest level, and was significantly lower than the control group (P<0.01), and was 213.16% and 48.90% lower than the control group, respectively. For the gills and digestive caeca, CAT activity showed an upward trend from 0h to 48h, and reached the highest level at 48h, which was significantly higher than the control group (P<0.01), and was 47.89% and 76.99% higher than the control group, respectively. After that, CAT activity began to decline, and was lower than the control group at 96h, and was 13.51% and 21.34% lower than the control group, respectively.

[0043] Figure 4 This is a diagram showing the changes in GPX enzyme activity in Hong Kong oysters under cadmium stress. In the figure, * indicates a significant difference between the experimental group and the control group (P < 0.05), and ** indicates an extremely significant difference from the control group (P < 0.01). Figure 4 The results showed that at 0h, the GPX activity of the experimental group did not show significant difference compared with the control group (P>0.05). With the extension of cadmium stress time, GPX activity gradually increased, reaching the highest level at 24h, and was significantly higher than that of the control group (P<0.05). Compared with the control group, the GPX activity in the mantle, gills, gonads, and digestive caeca increased by 41.24%, 29.45%, 39.48%, and 43.39%, respectively. Subsequently, the GPX activity began to decrease, but the GPX activity was still significantly higher than that of the control group in the interval of 24h-48h (P<0.05). In the mantle, gills, and gonads, the GPX activity was significantly lower than that of the control group at 96h (P<0.05), decreasing by 37.46%, 32.67%, and 44.82%, respectively. However, the activity of GPX in the digestive caeca was always higher than that in the control group, but its activity was almost the same as that in the control group, with no significant difference (P>0.05).

[0044] Figure 5 Figure 2 shows the changes in GST enzyme activity of Hong Kong oysters under cadmium stress. In the figure, * indicates that the experimental group was significantly different from the control group (P < 0.05), and ** indicates that the difference from the control group was extremely significant (P < 0.01). Figure 5The results showed that at the beginning of the experiment, the GST activity of the experimental group did not show significant differences compared with the control group (P>0.05). With the progress of cadmium stress, GST activity began to rise. In the mantle and digestive caeca, this upward trend reached a peak at 24h, and was significantly higher than the level of the control group (P<0.05), increasing by 39.85% and 24.42%, respectively. After that, GST activity began to decline, but within 24h to 48h, its activity was still significantly higher than that of the control group (P<0.05). Finally, at 96h, GST activity dropped to the lowest point and was significantly lower than that of the control group (P<0.05), decreasing by 49.76% and 17.49%, respectively. In the gills and gonads, GST activity reached a peak at 48h, and was also significantly higher than that of the control group (P<0.05), increasing by 56.84% and 46.08%, respectively. After that, GST activity began to decline. In gills, GST activity dropped to the lowest level at 96h and was significantly lower than that of the control group (P<0.05), with a decrease of 34.04%. However, in gonads, GST activity was significantly higher than that of the control group throughout the observation period (P<0.05).

[0045] The above research results show that under 5.452 mg / L cadmium stress, as the stress time increases, the activities of SOD and CAT in various tissues of Hong Kong oysters also increase successively, and begin to decline after 24h-48h of stress, and even fall below the control group. This may be because in the early stage of cadmium stress, the body senses external pressure and activates the antioxidant defense mechanism, which increases the activity of SOD and CAT to cope with the oxidative stress caused by cadmium. This is a self-protective stress response. However, as the stress time increases, maintaining a high level of activity of the two enzymes requires a lot of energy and material resources. When the consumption of these resources exceeds the body's supply capacity, the activity of SOD and CAT cannot be maintained continuously and begins to decline. In addition, long-term cadmium stress will induce the continuous production of ·O in the body. 2- , and those that are not cleared in time 2- Continuously accumulate in the body, excessive ·O 2- It may lead to the accumulation of damage to cell structure and function, including the related mechanisms involved in the synthesis and regulation of SOD and CAT, which may reduce the synthesis of these two enzymes or inhibit their function, thereby reducing their activity.

[0046] The activities of GPX and GST in various tissues of Hong Kong oysters showed a trend of first increasing and then decreasing under the condition of cadmium concentration of 5.452 mg / L. In the digestive caeca, GPX activity was always higher than that in the control group. In addition, in the gonads, GST activity was always significantly higher than that in the control group under cadmium stress. This is because when Hong Kong oysters are exposed to cadmium, these enzymes are involved in the process of scavenging free radicals and repairing oxidative damage, so their activities change. In the digestive caeca, GPX activity is always higher than that in the control group, which may be because the digestive caeca is the main place for Hong Kong oysters to process food and exogenous substances. Cadmium will accumulate in the digestive caeca of oysters, so it is more sensitive to oxidative stress and needs to maintain a higher GPX activity to protect cells from oxidative damage. In the gonads, GST activity is always significantly higher than that in the control group under cadmium stress, which may be because the gonads are the key part of oyster reproduction and are very sensitive to environmental changes. GST enzymes play an important role in protecting reproductive cells from oxidative damage and maintaining normal reproductive function. Cadmium exposure may activate the expression of GST to enhance antioxidant capacity and protect reproductive cells from oxidative stress. In summary, the changes in GPX and GST activities in oysters under cadmium stress are an adaptive response of their antioxidant system to environmental stress, aimed at protecting cells from oxidative damage and maintaining normal physiological functions.

[0047] 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 cadmium 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 uniform size and without disease or injury, clean them and keep them for a week for later use; Step 2: Acute toxicity test Set up temporary culture water containing cadmium chloride hexahydrate of different concentrations, and then place Hong Kong oysters in temporary culture water of different concentrations for culture. Change the water every 24 hours, remove dead Hong Kong oysters in time and record the number of dead shellfish. Continue culture for 96 hours, and calculate the median lethal concentration and safe concentration of Hong Kong oysters after 96 hours of heavy metal cadmium stress. Step 3: Stress experiment According to the results of the acute toxicity test in step 2, the 96h 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 digestive caeca were sampled under sterile conditions, and then the antioxidant enzyme activity was determined.

2. The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: In step 1, two-year-old Hong Kong oysters with a shell length of 6.5±0.5 cm were selected.

3. The method for studying the effects of heavy metal cadmium 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 19±1, the pH was 8±0.2, and the water temperature was 26±2°C. Chlorella powder was fed at 8:00 and 20:00 every day, and the feeding amount each time was 3% of the oyster body weight. Dead shellfish were removed and the water was changed every 24 hours.

4. The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: In step 2, five concentrations of cadmium chloride hexahydrate containing 1 mg / L, 2.78 mg / L, 6.75 mg / L, 17.55 mg / L and 30 mg / L were set up in temporary water, with 3 parallels in each group.

5. The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: In step 2, temporary water with the same water quality conditions as in step 1 is used as the control group.

6. The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: Step 2: SPSS26.0 was used to calculate the median lethal concentration and safety concentration of Hong Kong oysters after 96 hours of heavy metal cadmium stress. The calculation formula is as follows: SC=f·96h LC50 Among them, SC is the safe concentration and the f value is 0.

01.

7. The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 1, characterized in that: Step 3: set up at least 3 level groups, with at least 20 fish in each group, and use temporary water with the same water quality conditions as step 1 as the control group.

8. The method for studying the effects of heavy metal cadmium 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 catalase, superoxide dismutase, glutathione peroxidase and glutathione-S transferase.

9. The method for studying the effects of heavy metal cadmium on acute toxicity and antioxidant enzyme activity of Hong Kong oysters according to claim 8, characterized in that: The catalase assay adopts the ammonium molybdate quenching method, the superoxide dismutase assay adopts the pigment reduction method, the glutathione peroxidase assay adopts the colorimetric method, and the glutathione-S transferase assay adopts the ultraviolet spectrophotometric method.

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