A method for effectively reducing the damage of nano ZnO particles to Takifugu obscurus

Abstract

The present invention relates to a method for effectively reducing damage of nano ZnO particles to dark-striped pufferfish, characterized in that the method comprises the following steps: adopting a laboratory controlled variable experiment, setting treatment groups of different concentrations of ZnO NPs and different salt concentrations in pairs; placing dark-striped pufferfish fry in different treatment groups, feeding them with bait regularly every day, cleaning them in time, and replacing new treatment solutions every day; the experiment lasts for 4 days, observing the survival of the fry every day, and taking gill, kidney and intestinal tissues for physiological and biochemical detection on the last day; combining the survival of the fry under each treatment, the half-effective concentration EC50 of each time period, physiological and biochemical indicators and the integrated biomarker response index (IBR), analyzing and evaluating the effect of salinity on alleviating the toxicity of ZnO NPs. The present invention has the advantages of simple method, wide source of raw materials, low price, easy operation, good effect, etc., and is convenient for popularization and application in production and nature protection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for effectively reducing damage caused by nano ZnO particles to Takifugu obscurus. Background Art

[0002] 1. With the acceleration of the industrialization of nanotechnology, many nanomaterials have penetrated into various industries such as medicine, dyes, coatings, food, cosmetics and environmental pollution control. In the process of large-scale preparation, use and treatment, they are bound to be released into the environment. Due to the difficulty of degradation, these nanoparticles will be enriched in the body, causing certain damage. Among them, nano ZnO has become another nano functional material that has attracted much attention after carbon nanotubes due to its excellent characteristics such as surface effect, volume effect, quantum size effect and high absorption rate. It is one of the most mature nano materials in production process and detection method in recent years, and it is also one of the earliest materials for commercial application. In the process of production, use and treatment, nano ZnO products are bound to be released into the water environment. Italian scientist Danovaro calculated that about 25% of sunscreen will be washed away during swimming and bathing. These nano materials will eventually enter rivers and oceans, which will affect the ecological environment of water bodies, especially aquatic organisms. As a high-level trophic level in the aquatic ecosystem, fish have an irreplaceable role in responding to and enriching environmental pollution. At the same time, fish is also one of the main sources of human food, so the degree of fish pollution is closely related to human health. The dark-striped pufferfish, commonly known as pufferfish, is mainly distributed in the coastal waters of China and the middle and lower reaches of the Yangtze River. It is a sea-freshwater migratory fish. In the spring of each year, sexually mature dark-striped pufferfish swim upstream from the sea to the middle reaches of the Yangtze River or lakes connected to the river to spawn and reproduce, while the young fish are fattened in fresh water and return to the sea at the end of the year or the spring of the following year. The main channel for the dark-striped pufferfish to migrate is located in the Yangtze River Delta, one of the most economically developed regions in China. The region has a very developed industry and is also seriously polluted. Nanoparticles are likely to become a potential emerging pollutant. Therefore, the potential toxic effects of nano-ZnO on dark-striped pufferfish cannot be ignored.

[0003] 2. Although the content of ZnO NPs in water bodies is currently very low, it will be enriched through the food chain. When it passes through algae-zooplankton-benthic organisms and finally ingested by fish, its actual exposure concentration may be thousands of times the concentration in natural water bodies. Therefore, its potential threat cannot be underestimated. The present invention can well evaluate the potential threat of nanoparticles to aquatic organisms in the future, and find a method that can effectively alleviate the toxicity of ZnO NPs to dark-striped pufferfish, providing data support for the protection of fish, which has certain economic value. At the same time, it can also be applied to the risk management of new pollutants in the water environment. Summary of the invention

[0004] The purpose of the present invention is to provide a method for effectively reducing the damage of nano ZnO particles to Takifugu obscurus, so as to solve the problem of toxic damage caused by ZnO NPs proposed in the above background technology and improve the survival rate of Takifugu obscurus.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for effectively reducing the damage of nano ZnO particles to dark-striped pufferfish, the method comprising the following steps:

[0006] (1) Using laboratory controlled variable experiments, treatment groups with different concentrations of ZnO NPs and different salt concentrations were set up in pairs;

[0007] (2) Place the dark-striped pufferfish fry in different treatment groups, feed them with bait regularly every day, clean them in time, and replace the treatment solution with new one every day;

[0008] (3) The experiment lasted for 4 days, and the survival status of the fry was observed every day. On the last day, gill, kidney, and intestinal tissues were collected for physiological and biochemical tests;

[0009] (4) Combined with the survival of fry under each treatment, the half effective concentration EC50 in each time period, physiological and biochemical indicators and the integrated biomarker response index (IBR), the effect of salinity on alleviating the toxicity of ZnO NPs was analyzed and evaluated.

[0010] Preferably, the step (1) specifically comprises:

[0011] (1-1) The average particle size of single-crystal ZnO NPs was 50 nm. They were dissolved by ultrasonication and divided into 6 concentration gradients: 0, 10, 20, 50, 100, and 200 mg / L;

[0012] (1-2) Salinity was prepared using sea salt and divided into two salinity groups: 0 and 15 ppt;

[0013] (1-3) Place healthy fry in tanks, each containing 30 L of treatment solution and continuously aerating.

[0014] Preferably, the step (2) specifically comprises: selecting healthy young fish, two to three months old, with a body length of 6.3±1.0 cm and a body weight of 19.2±1.9 g.

[0015] Preferably, the step (3) specifically comprises: recording the number of surviving juvenile Takifugu fugu every day, extracting proteins from gill, kidney and intestinal tissues, and then determining the contents of MDA, SOD, CAT and GSH in the tissues using a kit.

[0016] Preferably, the step (4) specifically includes: performing variance analysis on the survival rate of the fry using SigmaPlot software and making a graph, and calculating the half effective concentration EC50 of ZnO NPs on the fry based on the survival rate; performing variance analysis on the detected enzyme activity index and making a graph; calculating the IBR value based on the enzyme activity parameters, thereby reflecting the damage of different treatments to the fry.

[0017] Preferably, the algorithm of the IBR value is as follows: for each marker, first calculate its mean value (x) under each treatment and the total mean value (m) and standard deviation (s) under all treatments, and perform normalization on x under each treatment according to formula (1): Y = (xm) / s (1) In formula (1), Y is the normalized value of x, and the calculation formula of the score S value of the biomarker under each treatment is: S = Z + |Min| (2) In formula (2), if the activity of the marker is activated due to the influence of environmental factors, let Z = x; otherwise, let Z = -Y, |Min| is all The absolute value of the minimum value of the normalized data of the marker under a certain treatment. The S value of each marker under a certain treatment is represented by the length of the radial line in the star diagram. The IBR value under a certain treatment is obtained by calculating the area of ​​the star diagram (that is, the sum of the triangular areas Ai surrounded by the radial lines of adjacent biomarkers in the diagram): (4)(5) In formulas (3) to (5), n is the number of biomarkers selected, α is the angle between two adjacent radial lines, α=2π / n; Si+1=S1, when n=4, α=π / 2, and the corresponding Ai calculation formula can be simplified to Ai=S1Si+1 / 2.

[0018] Technical advantages of the present invention:

[0019] (1) The present invention can quickly and effectively alleviate the damage of ZnO NPs in water to dark-striped pufferfish and improve the survival rate of dark-striped pufferfish.

[0020] (2) The present invention is easy to operate, has low raw material cost and significant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the survival of dark-striped pufferfish juveniles within 96 hours under different concentrations of ZnO NPs and salinities;

[0022] Figure 2 This is a schematic diagram of the response of related enzyme activities in the gills, kidneys, and intestines of dark-striped pufferfish juveniles to different concentrations of ZnO NPs and salinity;

[0023] Figure 3 This is a schematic diagram of the median effective concentration of ZnO NPs on dark-striped pufferfish juveniles;

[0024] Figure 4 It is a schematic diagram of the results of the integrated biological indicator response index (IBR). DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in 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. Please refer to Figure 1-4 The present invention provides an embodiment: a method for effectively reducing the damage of nano ZnO particles to dark-striped pufferfish, the method comprising the following steps:

[0026] (1) Using laboratory controlled variable experiments, treatment groups with different concentrations of ZnO NPs and different salt concentrations were set up in pairs;

[0027] (2) Place the dark-striped pufferfish fry in different treatment groups, feed them with bait regularly every day, clean them in time, and replace the treatment solution with new one every day;

[0028] (3) The experiment lasted for 4 days, and the survival status of the fry was observed every day. On the last day, gill, kidney, and intestinal tissues were collected for physiological and biochemical tests;

[0029] (4) Combined with the survival of fry under each treatment, the half effective concentration EC50 in each time period, physiological and biochemical indicators and the integrated biomarker response index (IBR), the effect of salinity on alleviating the toxicity of ZnO NPs was analyzed and evaluated.

[0030] Step (1) specifically includes:

[0031] (1-1) The average particle size of single-crystal ZnO NPs was 50 nm. They were dissolved by ultrasonication and divided into 6 concentration gradients: 0, 10, 20, 50, 100, and 200 mg / L;

[0032] (1-2) Salinity was prepared using sea salt and divided into two salinity groups: 0 and 15 ppt;

[0033] (1-3) Place healthy fry in tanks, each containing 30 L of treatment solution and continuously aerating.

[0034] Step (2) specifically includes: selecting healthy young fish, two to three months old, with a body length of 6.3±1.0 cm and a body weight of 19.2±1.9 g.

[0035] Step (3) specifically includes: recording the number of surviving fry of dark-striped pufferfish every day, extracting proteins from gill, kidney and intestinal tissues, and then determining the contents of MDA, SOD, CAT and GSH in the tissues using a kit.

[0036] Step (4) specifically includes: using SigmaPlot software to perform variance analysis on the survival rate of the fry and draw a graph, and at the same time calculating the half effective concentration EC50 of ZnO NPs on the fry based on the survival rate; performing variance analysis on the detected enzyme activity index and drawing a graph; calculating the IBR value based on the enzyme activity parameters, thereby reflecting the damage of different treatments to the fry.

[0037] The algorithm of IBR value is as follows: for each marker, first calculate its mean value (x) under each treatment and the total mean value (m) and standard deviation (s) under all treatments, and then normalize x under each treatment according to formula (1): Y = (xm) / s (1) In formula (1), Y is the value after normalization of x. The calculation formula of the score S value of the biomarker under each treatment is: S = Z + |Min| (2) In formula (2), if the activity of the marker is activated due to the influence of environmental factors, let Z = x; otherwise, let Z = -Y, and |Min| is the value under all treatments. The absolute value of the minimum value of the normalized data of the marker. The S value of each marker under a certain treatment is represented by the length of the radial line in the star diagram. The IBR value under a certain treatment is obtained by calculating the area of ​​the star diagram (that is, the sum of the triangular areas Ai surrounded by the radial lines of adjacent biomarkers in the diagram): (4)(5) In formulas (3) to (5), n is the number of selected biomarkers, α is the angle between two adjacent radial lines, α=2π / n; Si+1=S1, when n=4, α=π / 2, and the corresponding Ai calculation formula can be simplified to Ai=S1Si+1 / 2.

[0038] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for effectively reducing the damage of nano ZnO particles to Takifugu obscurus, characterized in that: The method comprises the following steps: (1) Using laboratory controlled variable experiments, we set up treatment groups with different concentrations of ZnO NPs and different salt concentrations in pairs; (2) Place the dark-striped pufferfish fry in different treatment groups, feed them with bait regularly every day, clean them in time, and replace the treatment solution with new one every day; (3) The experiment lasted for 4 days. The survival status of the fry was observed every day, and on the last day, the gill, kidney, and intestinal tissues were collected for physiological and biochemical tests. (4) The effect of salinity on alleviating the toxicity of ZnO NPs was analyzed and evaluated based on the survival of juvenile fish under each treatment, the half effective concentration (EC50) at each time period, physiological and biochemical indicators, and the integrated biomarker response index (IBR); The step (1) specifically includes: (1-1) The average particle size of single-crystalline ZnO NPs was 50 nm. They were dissolved by ultrasonication and divided into 6 concentration gradients: 0, 10, 20, 50, 100, and 200 mg / L; (1-2) Salinity was prepared using sea salt and divided into two salinity groups: 0 and 15 ppt; (1-3) Place healthy fry in tanks, each containing 30 L of treatment solution and continuously aerating; The step (2) specifically comprises: selecting healthy juvenile fish, two to three months old, with a body length of 6.3±1.0 cm and a body weight of 19.2±1.9 g; The step (3) specifically comprises: recording the number of juvenile dark-striped pufferfish surviving each day, and extracting protein from gills, kidneys, and intestinal tissues, and then using a kit to determine the content of MDA, SOD, CAT, and GSH in the tissues; The step (4) specifically includes: performing variance analysis on the survival rate of the fry using SigmaPlot software and drawing a graph, and calculating the half effective concentration EC50 of ZnO NPs on the fry based on the survival rate; performing variance analysis on the detected enzyme activity index and drawing a graph; calculating the IBR value based on the enzyme activity parameter, thereby reflecting the damage of different treatments to the fry; The algorithm of the IBR value is as follows: for each marker, first calculate its mean value x under each treatment and the total mean value m and standard deviation s under all treatments, and perform normalization on x under each treatment: Y= x−m / s, where Y is the normalized value of x. The calculation formula of the score S value of the biomarker under each treatment is: S=Z+|Min|. If the activity of the marker is activated due to the influence of environmental factors, let Z=x; otherwise, let Z=-Y, |Min| is the absolute value of the minimum value of the normalized data of the marker under all treatments. The S value of each marker under each treatment is represented by the length of the radial line in the star diagram. The IBR value under each treatment is obtained by calculating the area of ​​the star diagram, that is, the sum of the areas of the triangles Ai surrounded by the radial lines of adjacent biomarkers in the diagram: n is the number of selected biomarkers, α is the angle between two adjacent radial lines, α=2π / n, when n=4, α=π / 2, and the corresponding Ai calculation formula is Ai=S1Si+1 / 2, Si+1=S1.

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