Method for evaluating risk of toxicity exposure of microplastics to fishes by taking wild grass carp as sample

Abstract

The invention discloses a risk evaluation method for toxicity exposure of microplastics to fishes by taking wild grass carp as a sample, relates to the field of risk evaluation for toxicity exposure of microplastics, and in particular relates to a risk evaluation method for toxicity exposure of microplastics to fishes by taking wild grass carp as a sample. The invention aims to solve the problem that the existing evaluation method is single and cannot comprehensively and accurately evaluate the toxicity exposure risk of microplastics to fishes. The method comprises: 1, setting an experiment group; 2, putting wild grass carp into the experimental group, and recording body length and weight; 3, breeding with feed, respectively adding corresponding micro-plastic particles, and not adding the micro-plastic particles into a control group; 4, calculating by combining the weight change, body length change, feeding rate, death rate and intestinal microbial diversity index of the wild grass carp; evaluating the risk level of the microplastics to the fish toxicity exposure through the comprehensive index R, wherein the higher the R value is, the higher the toxicity risk is. The method is used for evaluating the risk of toxicity exposure of the microplastics taking the wild grass carp as a sample to the fish.

Description

Technical Field

[0001] The present invention relates to the field of microplastic toxicity exposure risk assessment, and specifically to a risk assessment method for fish toxicity exposure to microplastics using wild grass carp as a sample. Background Art

[0002] In recent years, the widespread use of plastic products has led to increasing environmental pollution. As a highly malleable polymer, plastic has become ubiquitous in every aspect of our lives due to its corrosion resistance, cost-effectiveness, and ease of molding. However, the frequent use of plastic products, low recycling rates, and the lack of effective waste management strategies have led to the continuous accumulation of plastic waste in the natural environment, severely impacting both terrestrial and marine environments.

[0003] Numerous studies have shown that the ubiquitous microplastics in water have varying degrees of toxicological effects on aquatic species (such as mussels, fish, and shrimp). Aquatic species are extremely susceptible to harm from unintentional ingestion of microplastics, which can penetrate into their various tissues and organs, produce toxic effects at the cellular and molecular levels, and even cause death from suffocation. Eating fish containing microplastics may threaten human health because they may carry harmful chemicals such as plasticizers and bisphenol A. These substances accumulate in organisms and can cause health risks such as endocrine disruption, reproductive and developmental problems. In addition, the surface of microplastics may also adsorb heavy metals and organic pollutants, further enhancing their potential toxicity to organisms. Therefore, the impact and harm of microplastics on aquatic animals have received widespread attention worldwide. It is of great practical significance to explore the damage of microplastics to fish and conduct ecological risk assessments.

[0004] Currently, toxicity assessment indices are based solely on the exposure dose of microplastics in fish, combined with a toxicity coefficient. This method is relatively simplistic, lacks persuasiveness when sample sizes are insufficient, and cannot represent the overall level of a particular fish species in the study area. The gut microbiome is composed of various microorganisms that inhabit the animal's intestines. The composition of this microbial community is host-dependent, fluctuating throughout the host's life cycle and susceptible to internal and external factors. Therefore, a comprehensive assessment of microplastic toxicity using multiple indices is essential. Summary of the Invention

[0005] The present invention aims to solve the problem that the existing evaluation method is single and cannot comprehensively and accurately assess the risk of microplastic toxic exposure to fish. A risk assessment method for microplastic toxic exposure to fish is provided using wild grass carp as a sample.

[0006] The present invention provides a risk assessment method for the toxicity of microplastics to fish using wild grass carp as a sample, comprising the following steps:

[0007] Step 1: Wild grass carp was selected as the model animal, and five experimental groups were set up. One group did not add microplastic particles as a blank control experimental group, and the other groups were 300-mesh PA particle experimental group, 1000-mesh PA particle experimental group, 300-mesh PET particle experimental group, and 1000-mesh PET particle experimental group.

[0008] Step 2: 15 wild grass carps were added to each experimental group, and the length and weight of each wild grass carp were recorded;

[0009] Step 3: Feed each experimental group, add corresponding microplastic particles to each experimental group, and do not add microplastic particles to the control group;

[0010] Step 4: The feed culture process lasts for one month, and the growth and feeding status of wild grass carp are recorded throughout the process. Once dead, their body length and weight are measured. At the same time, each dead wild grass carp is dissected and the intestinal contents are collected for model animal microbial diversity analysis;

[0011] Step 5: Calculate the weight change, length change, feeding rate, mortality rate and intestinal microbial diversity index of wild grass carp using the following formula:

[0012]

[0013] Where CDR represents mortality rate, N d represents the number of deaths, and N represents the total number; substitute formula (1) into formula (2);

[0014]

[0015] R represents the comprehensive index of microplastics in fish species, W0 represents body weight before feeding, W represents body weight after feeding, L0 represents body length before feeding, L represents body length after feeding, α represents intestinal microbial diversity index, G represents feeding rate; s represents the mesh size of microplastic particles, and cFi represents the toxicity coefficient of microplastics;

[0016] 6. The risk level of toxic exposure of microplastics to fish is evaluated by the comprehensive index R of microplastics in fish species. The higher the R value, the higher the toxicity risk.

[0017] Beneficial effects:

[0018] The present invention is not limited to calculating the toxicity index of a compound based on concentration dosage and toxicity coefficients calculated from existing studies. This invention records data from multiple aspects for calculation. Microplastics in fish affect their growth and development and may even cause death, and also affect the activity of intestinal microorganisms. Comprehensive consideration of these aspects can make the calculation results more convincing and more valuable for reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the mortality control curve in the embodiment;

[0020] Figure 2 is the feeding rate control curve in the embodiment;

[0021] Figure 3 is the body length change comparison curve in the embodiment;

[0022] Figure 4 is the weight change control curve in the embodiment;

[0023] Figure 5 It is the Simpson comparison bar graph in the embodiment. DETAILED DESCRIPTION

[0024] Specific embodiment 1: The risk assessment method for microplastic toxicity exposure to fish using wild grass carp as a sample described in this embodiment includes the following steps:

[0025] Step 1: Wild grass carp was selected as the model animal, and five experimental groups were set up. One group did not add microplastic particles as a blank control experimental group, and the other groups were 300-mesh PA particle experimental group, 1000-mesh PA particle experimental group, 300-mesh PET particle experimental group, and 1000-mesh PET particle experimental group.

[0026] Step 2: 15 wild grass carps were added to each experimental group, and the length and weight of each wild grass carp were recorded;

[0027] Step 3: Feed each experimental group, add corresponding microplastic particles to each experimental group, and do not add microplastic particles to the control group;

[0028] Step 4: The feed breeding process lasted for one month, and the conditions of the wild grass carp in each experiment were recorded throughout the process to obtain the weight change, body length change, feeding rate, mortality rate and intestinal microbial diversity index of the wild grass carp;

[0029] Step 5: Calculate the weight change, length change, feeding rate, mortality rate and intestinal microbial diversity index of wild grass carp using the following formula:

[0030]

[0031] Where CDR represents mortality rate, N d represents the number of deaths, and N represents the total number; substitute formula (1) into formula (2);

[0032]

[0033] R represents the comprehensive index of microplastics in fish species, W0 represents body weight before feeding, W represents body weight after feeding, L0 represents body length before feeding, L represents body length after feeding, α represents intestinal microbial diversity index, G represents feeding rate; s represents the mesh size of microplastic particles, and cFi represents the toxicity coefficient of microplastics;

[0034] 6. The risk level of toxic exposure of microplastics to fish is evaluated by the comprehensive index R of microplastics in fish species. The higher the R value, the higher the toxicity risk.

[0035] In this embodiment, a blank control group is set up to ensure experimental accuracy and to observe whether the experiment is operating normally. During the experiment, the control group is ensured to be unaffected by the experimental treatment to ensure the accuracy of the experimental results. After the experiment is completed, the results of the control group can be used as a reference to evaluate the effectiveness of the experimental treatment and help interpret and verify the research results.

[0036] In this embodiment, two common plastics and two different sizes are set for comparison, and such a setting can be used for horizontal and vertical comparison.

[0037] In order to ensure the accuracy of the data including the activity of intestinal microorganisms, this embodiment measures the length and weight of the specimen and performs an autopsy immediately after death, and then performs microbial testing.

[0038] During the entire experimental process of this embodiment, the use of plastic tools and contact with plastic materials are avoided, so as to achieve the purpose of pollution control and improve the accuracy of the experiment.

[0039] In the calculation formula (2) of this algorithm, the value of the parameter microplastic toxicity coefficient cFi is based on the international academic journal "Science of the Total Environment" [Delilah Lithner ＊ , Larsson, Dave]. [Environmental and health hazard ranking and assessment of plastic polymers based on chemical composition].

[2011] . [3309-3324] The document is a well-known source in the art for determining such parameters. Based on its content, a person skilled in the art can clearly obtain the value of the parameter from the hazard score in Table 2 of the document.

[0040] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the experimental environment was set to a constant room temperature of 20°C, and the fish tank water temperature was maintained stable by air conditioning. The experiment was conducted in October, the coldest month of the year. The fish tank water was changed every two weeks, and the fish were fed with Tuna brand micro-granular small fish feed. The fish tanks were equipped with an oxygen pump. Feeding was conducted once a day at 9:00 AM. The previous day's leftover bait and feces were collected with a straw and refrigerated in a vacuum device pending the next experiment. The water temperature was tested before and after the water was changed. Other aspects were the same as specific embodiment 1.

[0041] Specific embodiment three: This embodiment differs from specific embodiment one in that: 100 wild grass carp, as described in step two, are temporarily housed in a laboratory under ventilated conditions for one week. 75 individuals in good physical condition are screened and selected as model animals. Each wild grass carp is measured three times and the average value is calculated to obtain the average body length of the 75 wild grass carp. The wild grass carp are evenly distributed in five groups of fish tanks. Other procedures are the same as specific embodiment one.

[0042] Specific embodiment 4: This embodiment differs from specific embodiment 3 in that the average body length of the wild grass carp is 6.26 cm; the water temperature is maintained at a constant indoor temperature of 20 degrees Celsius, and the water in the fish tank is kept stable by air conditioning. The fish tank water needs to be changed every two weeks. Each fish tank of the same specifications is equipped with an oxygen pump, and the water temperature is tested before and after water changes. Other aspects are the same as specific embodiment 3.

[0043] Specific embodiment 5: This embodiment differs from specific embodiment 1 in that the feces collected in the experiment are filtered through a 2500-mesh filter before analysis. Other aspects are the same as specific embodiment 1.

[0044] Specific embodiment 6: This embodiment differs from specific embodiment 1 in that, in step 3, the wild grass carp were not fed any feed for the first ten days of the feed culture. Feeding began on the eleventh day, and all five experimental groups were fed a feed without microplastics for five days. Afterward, each experimental group was fed the appropriate microplastic particles. Furthermore, the number of microplastic particles was examined microscopically before the wild grass carp were introduced. All other aspects are the same as specific embodiment 1.

[0045] Specific embodiment 7: This embodiment differs from specific embodiment 1 in that a metal sieve is used for sieving during the experiment. Other aspects are the same as specific embodiment 1.

[0046] Specific Embodiment 8: This embodiment differs from Specific Embodiment 1 in that: in step 4, the weight change, length change, feeding rate, mortality rate, and intestinal microbial diversity index of wild grass carp are obtained by the following method: high-throughput sequencing technology is used to amplify the 16S rRNA gene V3-V4 region of the intestinal microbiota of wild grass carp exposed to PA and PET environments, and the growth and development of the wild grass carp and the intestinal microbiota are evaluated based on feeding, growth rate, and intestinal microbial changes. The growth and development of the wild grass carp and the intestinal microbiota are recorded one month after exposure. A Venn diagram is used to analyze the number of OTUs in the gastrointestinal bacterial communities of wild grass carp before the experiment and the bacterial communities of different control groups after the experiment, followed by alpha diversity analysis. The taxonomic levels in the OTU table are statistically analyzed, and the relative abundance of each taxonomic level is calculated. Taxonomic groups with a relative abundance greater than 1% are screened, and the top 15 taxonomic groups with the highest relative abundance are selected. A relative abundance distribution diagram is then drawn for inter-group comparison to obtain the weight change, length change, feeding rate, mortality rate, and intestinal microbial diversity index of the experimental samples. Other aspects are the same as Specific Embodiment 1.

[0047] Specific embodiment 9: This embodiment differs from specific embodiment 1 in that: the α specifically refers to the Simpson diversity index in the intestinal microbial diversity index, and its calculation formula is:

[0048]

[0049] P i is the relative abundance of species i, which is analyzed and plotted using the Meige Gene platform. Other aspects are the same as those in the first embodiment.

[0050] Specific embodiment 10: This embodiment differs from specific embodiment 1 in that the risk level classification described in step 6 is: R < 1000 is defined as Level I, which is a low risk level; R 1000-5000 is defined as Level II, which is a medium risk level; R 5000-10000 is defined as Level III, which is a high risk level; R > 10000 is defined as Level IV, which is a significant risk level. Other aspects are the same as specific embodiment 1.

[0051] The beneficial effects of the present invention are verified by the following examples:

[0052] Experimental process:

[0053] 1. The experimental environment was maintained at a constant indoor temperature of 20°C, with air conditioning used to maintain a stable aquarium water temperature. During the cold season of October, the aquarium water required biweekly changes. The aquarium feed used was Tuna brand micro-granular small fish feed, specifically formulated for juvenile fish. Each aquarium of the same size should be equipped with an oxygen pump and house a maximum of 15 grass carp. Feed the aquarium once daily around 9:00 AM, then use a straw to collect leftover bait, feces, and other impurities from the previous day. The water temperature was tested before and after water changes. The aquarium was then refrigerated in a vacuum chamber pending the next step in the experiment.

[0054] Second, keep the fish in a ventilated laboratory for one week and select individuals with good physical condition. Measure the same sample three times and take the average value to reduce errors and improve experimental accuracy. Then, select individuals with strong vitality and good health and place them evenly in five groups of fish tanks.

[0055] 3. The feces in the experiment were filtered through a 2500-mesh filter. The grass carp did not eat any feed for the first ten days of breeding, and started eating on the eleventh day. All five groups were fed feed without added microplastics for five days, after which microplastic particles of corresponding particle sizes were added. Before adding the grass carp, the number of microplastic particles was checked under a microscope. There is no guarantee that the actual exposure concentration of PA and PET particles of the same particle size in the culture water is the same. A metal sieve, not a polyester sieve, was used for sieving. Throughout the experiment, the use of plastic tools and contact with plastic materials was avoided to achieve the purpose of pollution control and improve the accuracy of the experiment.

[0056] One month later, the five experimental grass carp groups were weighed, and microplastic ingestion rates and mortality rates were recorded. High-throughput sequencing was used to amplify the V3-V4 region of the 16S rRNA gene in the gut microbiota of the grass carp exposed to PA and PET. Parameters such as food intake, growth rate, and changes in the gut microbiota were assessed to understand the growth and development of the grass carp and their gut microbiota after one month of exposure. Venn diagrams were used to analyze the number of OTUs in the gastrointestinal bacterial communities of the grass carp before the experiment and in the different control groups after the experiment. Alpha diversity analysis was also performed. Alpha diversity refers to the diversity within a specific region or ecosystem and is often measured as species richness. It focuses on the number of species within a locally homogeneous ecological environment and is therefore also called within-habitat diversity, referring to the species diversity within a sample, independent of other samples. To a certain extent, alpha diversity remains the most direct and intuitive method for comparing communities. The taxonomic levels (phylum, class, order, family, genus, species, etc.) in the OTU table were counted, and the relative abundance of each taxonomic level was calculated. The taxonomic groups with a relative abundance higher than 1% were screened out, and the top 15 taxonomic groups with the highest relative abundance were selected. Then, a relative abundance distribution diagram was drawn for inter-group comparison.

[0057] 5. The calculation formula is based on the weight change, body length change, feeding rate, mortality rate and intestinal microbial diversity index of wild grass carp:

[0058]

[0059] Where CDR represents mortality rate, N d represents the number of deaths, and N represents the total number; substitute formula (1) into formula (2);

[0060]

[0061] R represents the comprehensive index of microplastics in fish species, W0 represents body weight before feeding, W represents body weight after feeding, L0 represents body length before feeding, L represents body length after feeding, α represents intestinal microbial diversity index, G represents feeding rate; s represents the mesh size of microplastic particles, and cFi represents the toxicity coefficient of microplastics;

[0062] 6. The risk level of toxic exposure of microplastics to fish is evaluated by the comprehensive index R of microplastics in fish species. The higher the R value, the higher the toxicity risk.

[0063] The present invention is not limited to calculating the toxicity of compounds based on concentration, dosage and toxicity coefficients calculated from existing studies. This invention records data from multiple aspects for calculation. Microplastics in fish bodies affect their growth and development and even cause mortality, and also affect the activity of intestinal microorganisms. Comprehensive consideration of these aspects can make the calculation results more convincing and more valuable for reference.

[0064] Figure 1 is the mortality control curve in the embodiment; Figure 2 is the feeding rate control curve in the embodiment; Figure 3 is the body length change comparison curve in the embodiment; Figure 4 is the weight change control curve in the embodiment; Figure 5 It is the Simpson comparison bar graph in the embodiment.

[0065] Verification process:

[0066] The experiment was set up with S1 group, PA group, PET group and control CK group before the experiment.

[0067] The Simpson index results of this experimental data are:

[0068]

[0069] Experimental calculation:

[0070] Calculation formula: Taking the 30th day as an example:

[0071] The first group of 300 mesh PETR = [28.57% × (0.14 + 0.17) × 300 × 4] / (28.57% × 0.1) = 3720

[0072] The R value result is within the range of Ⅱ (1000-5000), so the risk level is medium risk.

Claims

1. A risk assessment method for the toxicity exposure of microplastics to fish using wild grass carp as a sample, characterized in that The risk assessment method for microplastic toxicity exposure to fish using wild grass carp as a sample includes the following steps: Step 1: Select wild grass carp as the model animal, and set up five experimental groups, one of which was a blank control experimental group without the addition of microplastic particles, and the other groups were a 300-mesh PA particle experimental group, a 1000-mesh PA particle experimental group, a 300-mesh PET particle experimental group, and a 1000-mesh PET particle experimental group; Step 2: 15 wild grass carps were added to each experimental group, and the length and weight of each wild grass carp were recorded; Step 3: Feed each experimental group, add corresponding microplastic particles to each experimental group, and do not add microplastic particles to the control group; Step 4: The feed breeding process lasted for one month, and the conditions of the wild grass carp in each experiment were recorded throughout the process to obtain the weight change, body length change, feeding rate, mortality rate and intestinal microbial diversity index of the wild grass carp; Step 5: Calculate the weight change, length change, feeding rate, mortality rate and intestinal microbial diversity index of wild grass carp using the following formula: Where CDR represents mortality rate, N d represents the number of deaths, and N represents the total number; Substitute formula (1) into formula (2); R represents the comprehensive index of microplastics in fish species, W0 represents body weight before feeding, W represents body weight after feeding, L0 represents body length before feeding, L represents body length after feeding, α represents intestinal microbial diversity index, G represents feeding rate; s represents the mesh size of microplastic particles, and cFi represents the toxicity coefficient of microplastics; 6. The risk level of toxic exposure of microplastics to fish is evaluated by the comprehensive index R of microplastics in fish species. The higher the R value, the higher the toxicity risk.

2. According to claim 1, a risk assessment method for the toxic exposure of microplastics to fish using wild grass carp as a sample is characterized in that The experimental environment was set to a constant room temperature of 20°C, and the water temperature of the fish tank was kept stable by air conditioning; the experiment was conducted in October, the low temperature season, and the fish tank water was replaced every two weeks. The feed was Tuna brand micro-granular small fish feed; the fish tanks were equipped with oxygen pumps; feeding was carried out once a day at 9 am, and then the leftover bait and feces of the previous day were collected with a straw and refrigerated in a vacuum device awaiting the next experiment. The water temperature needed to be tested before and after the water was changed.

3. According to claim 1, a risk assessment method for the toxic exposure of microplastics to fish using wild grass carp as a sample is characterized in that In step 2, a total of 100 wild grass carps are temporarily raised under laboratory conditions for one week with ventilation, and 75 individuals with good physical conditions are selected as model animals. Each wild grass carp is measured three times to take the average value, and the average value of the body length of the 75 wild grass carps is calculated; the wild grass carps are evenly placed in five groups of fish tanks.

4. According to claim 3, a risk assessment method for the toxic exposure of microplastics to fish using wild grass carp as a sample is characterized in that The average body length of the wild grass carp is 6.26 cm; the water temperature is maintained at a constant indoor temperature of 20 degrees Celsius, and the water temperature in the fish tank is kept stable by air conditioning. The fish tank water needs to be replaced every two weeks. Each fish tank of the same specifications is equipped with an oxygen pump, and the water temperature needs to be tested before and after the water is changed.

5. According to claim 1, a method for risk assessment of microplastic toxicity exposure to fish using wild grass carp as a sample is characterized in that The feces collected in the experiment were filtered through a 2500-mesh filter before analysis.

6. According to claim 1, a method for risk assessment of microplastic toxicity exposure to fish using wild grass carp as a sample is characterized in that In step three, the wild grass carp were not fed any feed for the first ten days of feed farming, and started eating on the eleventh day. The five experimental groups were all fed feed without microplastics for 5 days, after which each experimental group was added with corresponding microplastic particles; and before the wild grass carp were released, the number of microplastic particles was checked under a microscope.

7. According to claim 1, a method for risk assessment of microplastic toxicity exposure to fish using wild grass carp as a sample is characterized in that Experimental process: Metal sieve was used for sieving.

8. According to claim 1, a method for risk assessment of microplastic toxicity exposure to fish using wild grass carp as a sample is characterized in that In step 4, the weight change, body length change, feeding rate, mortality rate and intestinal microbial diversity index of wild grass carp were obtained in the following way: high-throughput sequencing technology was used to amplify the V3-V4 region of the 16S rRNA gene in the intestinal flora of wild grass carp exposed to PA and PET environments, respectively, and the feeding, growth rate and intestinal flora change parameters were evaluated, and the growth and development of wild grass carp and intestinal flora after one month of exposure were recorded; the OTU quantity analysis was performed on the gastrointestinal bacterial community of wild grass carp before the experiment and the bacterial community of different control groups after the experiment using a Venn diagram, and then an Alpha diversity analysis was performed; the classification levels in the OTU table were counted, the relative abundance of each classification level was calculated, and the classification groups with a relative abundance higher than 1% were screened out, the top 15 classification groups with the highest relative abundance were selected, and then the relative abundance distribution map was drawn for inter-group comparison to obtain the weight change, body length change, feeding rate, mortality rate and intestinal microbial diversity index of the experimental samples.

9. According to claim 1, a method for risk assessment of microplastic toxicity exposure to fish using wild grass carp as a sample, characterized in that The α specifically refers to the Simpson diversity index in the intestinal microbial diversity index, and its calculation formula is: P i is the relative abundance of the ith species, which is analyzed and plotted with the assistance of the Meige gene platform.

10. The method for risk assessment of microplastic toxicity exposure to fish using wild grass carp as a sample according to claim 1, characterized in that The risk level classification described in step six is: R<1000, defined as Level I, the risk level is low risk; R is 1000-5000, defined as Level II, the risk level is medium risk; R is 5000-10000, defined as Level III, the risk level is high risk; R>10000, defined as Level IV, the risk level is major risk.

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