A method for risk assessment of microplastics toxicity exposure to fish by taking wild grass carp as sample

Abstract

A kind of microplastic toxicity exposure risk evaluation method for fish with wild grass carp as sample, the present application relates to the field of microplastic toxicity exposure risk evaluation, specifically relates to a kind of microplastic toxicity exposure risk evaluation method for fish with wild grass carp as sample.The present application is to solve the problem that the existing evaluation method is single, cannot comprehensively and accurately evaluate the toxicity exposure risk of microplastic to fish.Method: I, set experimental group;II, input wild grass carp to experimental group, record body length and weight;III, feed cultivation, respectively add corresponding microplastic particles, the control group does not add microplastic particles;IV, combine the weight change of wild grass carp, body length change, feeding rate, mortality and intestinal microbial diversity index for calculation;The higher the value of comprehensive index R, the higher the toxicity risk of microplastic to fish is evaluated.The present application is used to evaluate the toxicity exposure risk of microplastic to fish with wild grass carp as sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microplastic toxicity exposure risk evaluation, and in particular to a method for evaluating the toxicity exposure risk of microplastics to fish by taking wild grass carp as samples. BACKGROUND

[0002] In recent years, the pollution of the environment by plastic products has been increasing year by year due to their wide application. As a kind of high polymer with strong plasticity, plastic has been widely used in all aspects of our life due to its strong corrosion resistance, economy, practicality and easy molding. However, the frequent use of plastic products, the low recycling efficiency and the lack of effective waste management strategies have led to the accumulation of plastic waste in the natural environment, which has harmed both land and marine environments.

[0003] A large number of studies have shown that microplastics, which are ubiquitous in water, have different degrees of toxicological effects on aquatic species such as mussels, fish and shrimps. Aquatic species are easily harmed by unintentionally ingesting microplastics, which can penetrate various tissues and organs, causing toxic effects at the cellular and molecular levels, and even leading to suffocation death. Humans may be threatened by eating fish containing microplastics, as they may carry harmful chemicals such as plasticizers and bisphenol A, which accumulate in the body and can cause endocrine disruption, reproductive and developmental problems, and other health risks. 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, and it is of great practical significance to explore the damage of microplastics to fish and conduct ecological risk assessment.

[0004] Currently, the toxicity evaluation index is only based on the exposure dose of microplastics in fish combined with the toxicity coefficient to calculate the formula, which is relatively single and lacks persuasiveness when the sample size is insufficient, and cannot represent the overall level of the fish species in the study area. The intestinal flora is composed of various microorganisms inhabiting in the intestinal tract of animals, and the composition of the microbial community is related to the host and changes in the host's life cycle, and is easily affected by internal and external factors. Therefore, it is necessary to comprehensively evaluate the toxicity of microplastics by multiple indexes. SUMMARY

[0005] The present application is to solve the problem that the existing evaluation method is single and cannot comprehensively and accurately evaluate the toxicity exposure risk of microplastics to fish, and provides a method for evaluating the toxicity exposure risk of microplastics to fish by taking wild grass carp as samples.

[0006] The method for evaluating the toxicity exposure risk of microplastics to fish by taking wild grass carp as samples provided by the present application comprises the following steps:

[0007] Step one: select wild grass carp as a model animal, set five experimental groups, one group without adding microplastic particles as a blank control experimental group, the rest of each group is 300 PA particle experimental group, 1000 PA particle experimental group, 300 PET particle experimental group, 1000 PET particle experimental group;

[0008] Step two: respectively into 15 wild grass carp in each experimental group, record the body length and weight of each wild grass carp;

[0009] Step three: feed each experimental group, each experimental group is added to the corresponding microplastic particles, and the control group is not added to the microplastic particles;

[0010] Step four: the feed cultivation process lasts for one month, the growth and feeding conditions of wild grass carp are recorded throughout the process, once dead, the body length and weight are measured, and each dead wild grass carp is dissected and treated, and the fish intestinal content model animal is collected for microbial diversity analysis;

[0011] Step five: combine the body weight change, body length change, feeding rate, mortality and intestinal microbial diversity index of wild grass carp, and the formula is:

[0012]

[0013] Where CDR represents mortality, N d The number of deaths, N represents the total number; formula (1) is substituted into formula (2);

[0014]

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

[0016] Six, the risk grade of microplastic toxicity exposure to fish is evaluated by the comprehensive index R of microplastic in fish species, and the higher the R value, the higher the toxicity risk.

[0017] Beneficial effects:

[0018] The present application is not limited to calculating the toxicity index of the compound by the concentration dose and the toxicity coefficient calculated by the existing research, but the present application calculates the data from multiple aspects, the growth and development of microplastics in fish body are affected, even dead, and the activity of intestinal microorganisms is also affected, which can make the calculation result more convincing and more valuable. DETAILED DESCRIPTION

[0019] Figure 1 Mortality control curve for the example;

[0020] Figure 2 Feeding rate control curve for the example;

[0021] Figure 3 Body length change control curve for the example;

[0022] Figure 4 Body weight change control curve for the example;

[0023] Figure 5 Simpson control column chart for the example. DETAILED DESCRIPTION

[0024] Embodiment one: a risk assessment method for microplastic toxicity exposure to fish by taking wild grass carp as a sample, comprising the following steps:

[0025] Step one: select wild grass carp as a model animal, set five experimental groups, one group without adding microplastic particles as a blank control experimental group, and the rest of each group is 300 mesh PA particle experimental group, 1000 mesh PA particle experimental group, 300 mesh PET particle experimental group, 1000 mesh PET particle experimental group;

[0026] Step two: respectively put 15 wild grass carps into each experimental group, and record the body length and body weight of each wild grass carp;

[0027] Step three: feed each experimental group, and add corresponding microplastic particles to each experimental group, and the control group does not add microplastic particles;

[0028] Step four: the feeding process lasts for one month, and the conditions of each experimental wild grass carp are recorded throughout the process to obtain the body weight change, body length change, feeding rate, mortality and intestinal microbial diversity index of wild grass carp;

[0029] Step five: combine the body weight change, body length change, feeding rate, mortality and intestinal microbial diversity index of wild grass carp to calculate, and the formula is:

[0030]

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

[0032]

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

[0034] Sixth, the risk level of fish toxicity exposure of microplastics is evaluated by the comprehensive index R of microplastics in fish species, and the higher the R value, the higher the toxicity risk.

[0035] In this embodiment, a group is set as a blank control group to ensure the accuracy of the experiment and observe whether the experiment is in a normal state. During the experiment, it is ensured that the control group is not affected 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 for evaluating the effect of the experimental treatment, which helps to interpret and verify the research results.

[0036] In this embodiment, two common plastics and two different sizes are set for comparison, which can be compared horizontally and vertically.

[0037] In order to ensure the accuracy of the data, the activity of intestinal microorganisms is included in this embodiment. The body length and body weight data measurement and dissection operation are performed at the first time after the sample dies, and then the microorganism detection is performed.

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

[0039] In the calculation formula (2) of the algorithm, the value of the parameter toxicity coefficient cFi of microplastics is obtained according to the method determined in 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], which is a commonly known source for determining such parameters in the art, and based on the content thereof, a person skilled in the art can clearly obtain the value of the parameter selected from the hazard score in Table 2 of the literature.

[0040] Specific implementation two: the difference between this embodiment and specific implementation one is that the experimental environment is set to a constant room temperature of 20°C, and the water temperature of the fish tank is kept stable by air conditioning; the experiment is carried out in October in the low temperature season, the fish tank water is replaced every two weeks, and the feed is Pig brand micro-particle small fish feed; the fish tank is provided with an oxygen pump; feeding is carried out once a day at 9 am, then the previous day's leftover feed and feces are collected with a straw and refrigerated in a vacuum device for the next step of the experiment, and the water temperature needs to be tested before and after water replacement. The same as specific implementation one.

[0041] Specific implementation three: the difference between this embodiment and specific implementation one is that 100 wild grass carps in step two are temporarily raised in a laboratory condition for one week, 75 individuals with better body conditions are obtained by screening as model animals, each wild grass carp is measured three times to obtain 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 fish tanks. The same as specific implementation one.

[0042] Specific implementation four: the difference between this embodiment and specific implementation three is that the average body length of the wild grass carps is 6.26 cm; the water temperature is kept at a constant indoor temperature of 20°C, and the water temperature of the fish tank is kept stable by air conditioning, and the fish tank water needs to be replaced every two weeks, each fish tank of the same specification is provided with an oxygen pump, and the water temperature needs to be tested before and after water replacement. The same as specific implementation three.

[0043] Specific implementation five: the difference between this embodiment and specific implementation one is that the feces collected in the experiment is filtered by a 2500-mesh filter before analysis. The same as specific implementation one.

[0044] Specific implementation six: the difference between this embodiment and specific implementation one is that the wild grass carps are not fed for the first ten days of step three, and start to eat on the eleventh day, and the five experimental groups are fed for 5 days without adding microplastics, and then each experimental group adds corresponding microplastic particles; and before the wild grass carps are put in, the number of microplastic particles is checked by a microscope. The same as specific implementation one.

[0045] Specific implementation seven: the difference between this embodiment and specific implementation one is that a metal sieve is used during the experiment. The same as specific implementation one.

[0046] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Method One in that the changes in body weight, body length, feeding rate, mortality rate, and gut microbiota diversity index of wild grass carp obtained in step four are specifically obtained as follows: High-throughput sequencing technology is used to amplify the V3-V4 region of the 16S rRNA gene in the gut microbiota of wild grass carp exposed to PA and PET environments, respectively. Evaluation is conducted using parameters related to feeding, growth rate, and changes in gut microbiota. The growth, development, and gut microbiota status of wild grass carp one month after exposure are recorded. Venn diagrams are used to analyze the number of OTUs in the gastrointestinal bacterial community of wild grass carp before the experiment and in 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 higher than 1% are selected, and the top 15 taxonomic groups with the highest relative abundance are chosen. A relative abundance distribution map is then plotted for inter-group comparison to obtain the changes in body weight, body length, feeding rate, mortality rate, and gut microbiota diversity index of the experimental samples. Everything else is the same as in Specific Implementation Method One.

[0047] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method One in that: α specifically refers to the Simpson diversity index in the gut microbiome diversity index, and its calculation formula is:

[0048]

[0049] P i This represents the relative abundance of the i-th species, plotted using the MegGene platform. Everything else is the same as in Specific Implementation Method 1.

[0050] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method One in that the risk level classification in step six is ​​as follows: R < 1000 is defined as Level I, with a low risk level; R 1000–5000 is defined as Level II, with a medium risk level; R 5000–10000 is defined as Level III, with a high risk level; and R > 10000 is defined as Level IV, with a significant risk level. Everything else is the same as in Specific Implementation Method One.

[0051] The beneficial effects of the present invention are verified through the following embodiments:

[0052] Experimental procedure:

[0053] I. The experimental environment was set at a constant indoor temperature of 20 degrees Celsius, with air conditioning used to maintain a stable aquarium water temperature. The experiment was conducted in October, during the colder season. The aquarium water needed to be changed every two weeks, and the fish food should be specially formulated for juvenile fish, using the "Pig Brand" micro-particle small fish food. Each aquarium of the same size should be equipped with an air pump, and a maximum of 15 grass carp should be placed in each tank. Feeding was performed once daily around 9:00 AM, followed by the collection of uneaten food, feces, and other debris from the previous day using a pipette. The water temperature needed to be tested before and after each water change. The water was then refrigerated in a vacuum chamber until the next stage of the experiment.

[0054] Second, the grass carp were temporarily kept in a well-ventilated laboratory for one week, and individuals with good physical condition were selected. The same sample was measured three times and the average value was taken to reduce errors and improve the accuracy of the experiment. The average body length was then measured. Subsequently, individuals with strong vitality and good health were selected and the grass carp were evenly placed into five groups of aquariums.

[0055] III. The feces used in the experiment were filtered through a 2500-mesh filter. For the first ten days of rearing, the grass carp did not eat any feed. Feeding began on the eleventh day. All five groups were fed a diet free of microplastics for five days, after which microplastic particles of the corresponding particle size were added. Before introducing the grass carp, the number of microplastic particles was checked under a microscope; however, it cannot be guaranteed that the actual exposure concentration of PA and PET particles of the same particle size in the culture water would be the same. Metal sieves, not polyester sieves, were used for sieving. Throughout the experiment, the use of plastic tools and contact with plastic materials were avoided to control contamination and improve experimental accuracy.

[0056] IV. One month later, the body weight of the five groups of grass carp was measured, and the microplastic feeding rate and mortality rate were recorded throughout the process. High-throughput sequencing technology was used to amplify the 16S rRNA gene V3-V4 region in the gut microbiota of grass carp exposed to PA and PET environments, respectively. Feeding, growth rate, and changes in gut microbiota were used to assess the growth, development, and gut microbiota status of the grass carp one month after exposure. Venn diagrams were used to analyze the number of OTUs in the gastrointestinal bacterial community of grass carp before the experiment and in different control groups after the experiment. Alpha diversity analysis was performed. Alpha diversity refers to the diversity within a specific region or ecosystem, often measured by species richness. It mainly focuses on the number of species in a locally homogeneous ecological environment, and is therefore also called within-habitat diversity, i.e., the species diversity within a sample, independent of other samples. To a certain extent, alpha diversity remains the most direct and intuitive way to compare communities. The taxonomic levels (phylum, class, order, family, genus, species, etc.) in the OTU table were statistically analyzed, the relative abundance of each taxonomic level was calculated, taxa with relative abundance higher than 1% were selected, the top 15 taxa with the highest relative abundance were selected, and then a relative abundance distribution map was drawn for intergroup comparison.

[0057] V. The formula for calculating the weight change, body length change, feeding rate, mortality rate, and intestinal microbial diversity index of wild grass carp is as follows:

[0058]

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

[0060]

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

[0062] VI. The risk level of microplastic toxicity exposure 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] This invention is not limited to calculating the toxicity of compounds based on concentration, dosage, and toxicity coefficients calculated from existing studies. Instead, it calculates toxicity by recording data from multiple aspects. Microplastics affect the growth and development of fish and can even cause death, and they can also affect the activity of intestinal microorganisms. Taking these aspects into account can make the calculation results more convincing and more valuable for reference.

[0064] Figure 1 This is a mortality rate control curve in the examples; Figure 2 This is a control curve showing the feeding rate in the examples; Figure 3 This is a comparison curve of body length changes in the examples; Figure 4 This is a control curve showing weight change in the examples; Figure 5 This is a Simpson control histogram from the examples.

[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 exponent results for this experiment are as follows:

[0068]

[0069] Experimental calculations:

[0070] Calculation formula: Taking day 30 as an example for calculation:

[0071] Group 1 300 mesh PETR = [28.57% × (0.14 + 0.17) × 300 × 4] / (28.57% × 0.1) = 3720

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

Claims

1. A method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, characterized in that... The risk assessment method for microplastic toxicity exposure in wild grass carp samples includes the following steps: Step 1: Select wild grass carp as the model animal and set up five experimental groups. One group was a blank control group without microplastic particles. The other groups were 300 mesh PA particles, 1000 mesh PA particles, 300 mesh PET particles, and 1000 mesh PET particles, respectively. Step 2: 15 wild grass carp were introduced into each experimental group, and the body length and weight of each wild grass carp were recorded; Step 3: Feed the experimental groups and add the corresponding microplastic particles to each experimental group, while no microplastic particles are added to the control group; Step 4: The feed culture process lasted for one month, and the condition of each experimental wild grass carp was recorded throughout the process to obtain the changes in body weight, body length, feeding rate, mortality rate and intestinal microbial diversity index of the wild grass carp; Step 5: Calculate the following based on changes in body weight, body length, feeding rate, mortality rate, and gut microbiota diversity index of wild grass carp: The formula is as follows: (1)； Where CDR represents the mortality rate, N d Let N represent the number of deaths and N represent the total number of deaths; substitute formula (1) into formula (2); (2)； R represents the comprehensive index of microplastics in fish species, W0 represents the body weight before feeding, W represents the body weight after feeding, L0 represents the body length before feeding, L represents the body length after feeding, α represents the gut microbial diversity index, G represents the feeding rate, s represents the microplastic particle size, and cFi represents the microplastic toxicity coefficient; specifically, α refers to the Simpson diversity index in the gut microbial diversity index, which is calculated using the following formula: ； P i The relative abundance of the i-th species is shown in the figure, which was generated with the assistance of the Meggen platform. Step 6: Evaluate the risk level of microplastic toxicity exposure to fish by using the comprehensive index R of microplastic performance in fish species. The higher the R value, the higher the toxicity risk.

2. The method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, as described in claim 1, is characterized in that... The experimental environment was set at a constant room temperature of 20℃, and the aquarium water temperature was kept stable by air conditioning. The experiment was conducted in October, the cold season, with the aquarium water changed every two weeks. The fish were fed with Pig Brand micro-particle small fish food. Each aquarium was equipped with an air pump. The fish were fed once a day at 9:00 am, and the uneaten food and feces from the previous day were collected with a straw and refrigerated in a vacuum device for the next step of the experiment. The water temperature was tested before and after each water change.

3. The method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, as described in claim 1, is characterized in that... In step two, 100 wild grass carp were temporarily kept in a well-ventilated laboratory for one week. 75 individuals with better physical condition were selected as model animals. Each wild grass carp was measured three times and the average value was taken. The average body length of the 75 wild grass carp was calculated. The wild grass carp were then placed evenly into five groups of aquariums.

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

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

6. The method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, as described in claim 1, is characterized in that... In step three, wild grass carp were not fed for the first ten days of feed culture. They started feeding on the eleventh day. All five experimental groups were fed feed without added microplastics for five days. After that, each experimental group was given the corresponding microplastic particles. Before the wild grass carp were introduced, the number of microplastic particles was checked under a microscope.

7. The method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, as described in claim 1, is characterized in that... Metal sieves were used for sieving during the experiment.

8. The method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, as described in claim 1, is characterized in that... Step four involved obtaining data on weight changes, body length changes, feeding rates, mortality rates, and gut microbiota diversity indices in wild grass carp, specifically as follows: High-throughput sequencing was used to amplify the V3-V4 region of the 16S rRNA gene in the gut microbiota of wild grass carp exposed to PA and PET environments, respectively. Feeding, growth rate, and changes in gut microbiota parameters were used for evaluation. The growth, development, and gut microbiota status of wild grass carp were recorded one month after exposure. Venn diagrams were used to analyze the number of OTUs in the gastrointestinal bacterial communities of wild grass carp before the experiment and in different control groups after the experiment, followed by Alpha diversity analysis. The taxonomic levels in the OTU table were statistically analyzed, and the relative abundance of each taxonomic level was calculated. Taxonomic groups with a relative abundance higher than 1% were selected, and the top 15 taxonomic groups with the highest relative abundance were chosen. A relative abundance distribution map was then plotted for inter-group comparison to obtain the weight changes, body length changes, feeding rates, mortality rates, and gut microbiota diversity indices of the experimental samples.

9. The method for risk assessment of microplastic toxicity exposure in fish using wild grass carp as a sample, as described in claim 1, is characterized in that... The risk level classification described in step six is ​​as follows: R < 1000 is defined as Level I, with a low risk level; R = 1000~5000 is defined as Level II, with a medium risk level; R = 5000~10000 is defined as Level III, with a high risk level; R > 10000 is defined as Level IV, with a significant risk level.

Images