Method for determining micro-plastic particle seawater ecological risk evaluation threshold value

Abstract

The invention discloses a method for determining a micro-plastic particle seawater ecological risk evaluation threshold value. The method comprises the following steps: (1) preparing pure micro-plastic; (2) carrying out biological testing; and (3) deducing the threshold value. According to the method, the toxic effect of pure micro-plastic can be measured, instead of the composite toxic effect of the micro-plastic and the leaching additive, and the toxicity of the micro-plastic particles to marine organisms can be evaluated more accurately; in addition, the tested organisms adopted by the method are marine organisms, so that the risk of the micro-plastic particles to seawater ecology can be reflected more accurately; the method can also derive the toxicity threshold value of the micro-plastic particles to marine organisms.

Description

Technical Field

[0001] The present invention belongs to the field of water ecological risk assessment, and in particular relates to a method for determining a threshold value for seawater ecological risk assessment of microplastic particles. Background Art

[0002] Microplastics are widely present in the marine environment, and the biological and ecological environmental problems they cause have attracted much attention. Microplastics can have adverse effects on the physiological functions and metabolism of marine organisms, such as damaging the integrity of cell membranes, interfering with enzyme activity and metabolic pathways. However, the microplastics used in current toxicological studies on the effects of enzyme activity mainly focus on polystyrene (PS) microplastics, and the various additives contained in microplastics, such as antioxidants, flame retardants and stabilizers, will leach out during the experiment, and these additives may be more toxic to marine organisms than the microplastic particles themselves, resulting in "false positives" in the toxic effects of microplastics, which interferes with the scientific evaluation of their true toxic effects. In addition, although a large number of studies have explored the toxic effects of microplastics on the enzyme activity of marine organisms, there is still a lack of clear threshold standards for microplastics, making it difficult to determine specific reference points when assessing the ecological risks of microplastics in seawater. Complex models and assumptions need to be relied on to infer the impact of microplastics, which increases the uncertainty and complexity of the assessment. Summary of the invention

[0003] In order to overcome the shortcomings and deficiencies of the above-mentioned existing methods, the purpose of the present invention is to provide a method for determining the threshold value of the ecological risk assessment of microplastic particles in seawater.

[0004] The purpose of the present invention is achieved through the following solutions: A method for determining the threshold value of microplastic particle seawater ecological risk assessment comprises the following steps: (1) Preparation of pure microplastics: placing the microplastics to be cleaned into a semipermeable membrane container, sealing the container, and then placing the semipermeable membrane container containing the microplastics into ultrapure water for stirring and cleaning. After cleaning, the microplastics in the semipermeable membrane container are collected and dried to obtain pure microplastics; (2) Biological testing: Marine organisms were selected as test organisms, and pure microplastic suspensions of different concentrations were prepared and added to the test organism exposure system. The test organisms were collected after the reaction. For cell samples, the collected cell samples were washed and centrifuged, and then lysed with lysis buffer to obtain the supernatant. For tissue samples, the collected biological samples were frozen and killed, added to phosphate buffered saline (PBS) or saline, and then the biological tissues were broken and centrifuged to obtain the supernatant. The protein concentration, superoxide dismutase (SOD) and catalase (CAT) enzyme activities of the supernatant were then measured. (3) Threshold derivation: The acute toxicity value is obtained through the results of step 2; the 5% species hazard concentration (HC5) is calculated based on the acute toxicity value, and the predicted no-effect concentration (PNEC) is derived from the 5% species hazard concentration.

[0005] Furthermore, the microplastics described in step (1) include at least one of polypropylene (PP) microplastics, polyethylene (PE) microplastics and polyethylene terephthalate (PET) microplastics.

[0006] Furthermore, the stirring and cleaning in ultrapure water described in step (1) is: stirring and cleaning for 6 to 10 days, and the ultrapure water is replaced every 20 to 30 hours during the stirring and cleaning process; further, the stirring and cleaning in ultrapure water described in step 1 is: stirring and cleaning for 7 days, and the ultrapure water is replaced every 24 hours during the stirring and cleaning process.

[0007] Furthermore, the marine organisms described in step (2) include at least one of Vibrio fischeri, Chaetoceros muehii and Perineuridae bicolor.

[0008] Furthermore, the acute toxicity value in step (3) is the half effective concentration (EC 50 ) or lowest effective concentration (LOEC).

[0009] Further, the 5% species hazard concentration in step (3) is calculated by subtracting the standard deviation of the acute toxicity value from the average acute toxicity value multiplied by the extrapolation constant; further, it is calculated by the following calculation formula: logHC5 = average (log (L⁄EC 50 values))-k*SD (log (L⁄EC 50 values))(1); Where average is the mean of the log-transformed toxicity values, SD is the standard deviation of the log-transformed toxicity values, and k is the extrapolation constant; Furthermore, it is calculated by the following formula: logHC5= average (log LOEC)-k*SD (log LOEC) (2); where average is the mean of the log-transformed toxicity values, SD is the standard deviation of the log-transformed toxicity values, and k is the extrapolation constant.

[0010] Furthermore, when the sample size of species is three, the k value is 1.938.

[0011] Furthermore, the predicted no-effect concentration described in step (3) is calculated by the following calculation formula: PNEC = HC5 / additional safety factor (3); Among them, the additional safety factor indicated by the REACH guidance document should be an additional safety factor of 1 to 5 to take into account problems such as the small number of species, bias and uncertainty; further, when the sample size of species is three, the additional safety factor is 5.

[0012] Compared with the prior art, the present invention has the following advantages and effects: 1. The method of the present invention measures the toxic effects of pure microplastics, rather than the combined toxic effects of microplastics and leached additives, and can more accurately assess the toxicity of microplastic particles themselves to marine organisms.

[0013] 2. The test organisms used in the method of the present invention are all marine organisms, which can more accurately reflect the risks of microplastic particles to seawater ecology.

[0014] 3. The method of the present invention can deduce the toxicity threshold of microplastic particles to marine organisms, providing a theoretical basis for formulating scientific pollution control strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The graphs show the protein concentrations of Vibrio fischeri, Chaetoceros muehli, and Perinereux bipinnatus after exposure to microplastics; (a) shows the protein concentration of Vibrio fischeri after exposure to PP microplastics, (b) shows the protein concentration of Chaetoceros muehli after exposure to PP microplastics, (c) shows the protein concentration of Perinereux bipinnatus after exposure to PP microplastics, (d) shows the protein concentration of Vibrio fischeri after exposure to PE microplastics, (e) shows the protein concentration of Chaetoceros muehli after exposure to PE microplastics, (f) shows the protein concentration of Perinereux bipinnatus after exposure to PP microplastics, (g) shows the protein concentration of Vibrio fischeri after exposure to PET microplastics, (h) shows the protein concentration of Chaetoceros muehli after exposure to PET microplastics, and (i) shows the protein concentration of Perinereux bipinnatus after exposure to PET microplastics. Figure 2 This is the result of the effects of exposure to PE, PET, and PP microplastics on the SOD and CAT enzyme activities of Vibrio fischeri.

[0016] Figure 3 This is a graph showing the effects of exposure to PE, PET, and PP microplastics on the SOD and CAT enzyme activities of Chaetoceros muelleri.

[0017] Figure 4 This is a graph showing the effects of exposure to PE, PET, and PP microplastics on the SOD and CAT enzyme activities of Perinectus bicolor. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0019] Example 1 A method for determining a water ecological risk assessment threshold of microplastic particles comprises the following steps: 1. Preparation of pure microplastics This example uses three different microplastics (PP, PE, PET), and the specific operation steps are as follows: (1) Use scissors to cut a section of dialysis bag (dialysis bag is a bagged container made of semipermeable membrane, dialysis bag model: MD34-5M, Wide flat: 34 mm), open on both sides after cutting. The length is about 3 to 4 cm and the width is about 1 to 2 cm. Put it into boiling water at 100℃ and boil for 5 to 10 minutes. After the temperature drops to room temperature, take it out and dry it in the shade for later use; (2) Take a 1 L beaker and a magnetic stirring rotor, wash them with pure water and ultrapure water in turn, add 2 / 3 of ultrapure water to the beaker and put in the magnetic stirring rotor for later use; (3) Take a semipermeable membrane clamp and clamp one end of the cleaned semipermeable membrane, leaving the other end open. Weigh 3-4 g of the microplastics to be cleaned and put them into the prepared semipermeable membrane, and clamp the other end of the semipermeable membrane so that both ends are sealed, and then put it into the beaker; (4) Place the beaker on the magnetic stirrer and adjust the position so that the rotor is adsorbed on the bottom of the beaker. Turn on the magnetic stirrer so that the semipermeable membrane is completely immersed in water and maintain the rotation speed. (5) The ultrapure water in the beaker needs to be replaced every 24 hours. One leaching and washing cycle is 7 days. After 7 days, use tweezers to remove the semipermeable membrane, open the semipermeable membrane clip on one side, pour out the microplastics inside, and the residue on the bag can be rinsed with ultrapure water. The poured out microplastics and the residual rinsing liquid form a microplastic suspension. The collected microplastic suspension is filtered and dried to obtain a pure microplastic sample.

[0020] 2. Biological testing Three marine organisms, namely Vibrio fischeri, Chaetoceros muehii, and Perinectus didentatus (Vibrio fischeri was purchased from China Industrial Culture Collection Center (CICC 10483), lugworm was purchased from Donghai Island Jindi Bait Farm in Mazhang District, Zhanjiang City, and Chaetoceros muehii was purchased from Pinduoduo Fengyun Algae Industry) were used as test organisms.

[0021] For Vibrio fischeri, a microplastic suspension was prepared with an aqueous solution containing 0.05% by mass sodium dodecyl sulfate (SDS) and 2% by mass NaCl. The concentrations of microplastics were 10, 100, and 10 3 , 10 4 , 10 5 , 106 , 10 7 μg / L. In the experimental group, 100 μL of microplastic suspension and 100 μL of bacterial solution were added to 10 mL of 2216e liquid culture medium (purchased from Shanghai Ruichu Biotechnology Co., Ltd.), and in the control group, 100 μL of an aqueous solution containing 0.05% by mass of sodium dodecyl sulfate (SDS) and 2% by mass of NaCl and 100 μL of bacterial solution were added to 10 mL of 2216e liquid culture medium. After 24 hours of reaction with Vibrio fischeri, the exposed bacterial solution after microplastic exposure was obtained. 1 mL of the exposed bacterial solution was taken, washed twice with a 2% by mass NaCl aqueous solution to remove the liquid culture medium, centrifuged, and then 100 μL of lysis solution was added for lysis treatment, and then the supernatant was taken for subsequent measurement experiments.

[0022] For Chaetoceros muelleri, 7 microplastic suspensions with the same concentrations as above were prepared with phosphate buffered saline (PBS). The experimental group took 200 μL of microplastic suspension and 2 mL of algae solution into 20 mL f / 2 liquid culture medium (Guillard F / 2 Medium, purchased from Coolable), and the control group took 200 μL of PBS and 2 mL of algae solution into 20 mL f / 2 liquid culture medium. After 96 hours of reaction, the exposed algae solution after microplastic exposure was obtained. 12 mL of the exposed algae solution was taken, washed twice with a 2% by mass NaCl aqueous solution to remove the liquid culture medium, centrifuged, and then 200 μL of lysis solution was added for lysis treatment, and then the supernatant was taken for subsequent measurement experiments.

[0023] For Perineuridae bicolor, a microplastic suspension was prepared with an aqueous solution containing 0.05% by mass of sodium dodecyl sulfate (SDS) and 2% by mass of NaCl at concentrations of 100, 10 3 , 10 4 , 10 5 , 10 6 μg / L. The experimental group took 500 μL of microplastic suspension and 5 nereids into 500 mL of 2% NaCl solution (2 L beaker), and the control group took 500 μL of an aqueous solution containing 0.05% by mass sodium dodecyl sulfate (SDS) and 2% by mass NaCl and 5 nereids into 500 mL of 2% NaCl solution (2 L beaker). After 96 h of exposure, 3 nereids were taken, first frozen to death, and then about 0.1 g of the middle section of the nereid was cut off and placed in a 1.5 mL centrifuge tube, and then 1 mL of 2% saline was added for storage. The nereid tissue in the centrifuge tube was crushed with a rapid tissue cell disruptor, and then centrifuged, and the supernatant was taken for subsequent measurement experiments.

[0024] Then, the protein concentration, SOD enzyme activity and CAT enzyme activity of the three supernatants were determined using the Bio-Tech BCA protein concentration assay kit, the total SOD activity assay kit (WST-8 method) and the catalase assay kit, respectively.

[0025] 3. Threshold derivation The acute toxicity values ​​of the tested organisms are further used to estimate the species sensitivity distribution (SSD), and the HC5 (concentration that causes harmful effects to 5% of the species) is calculated based on this SSD value. The specific calculation method of HC5 is to take the average value of toxicity, subtract the standard deviation of the acute toxicity value and multiply it by the extrapolation constant: logHC5 = average (log (L⁄EC 50 values))-k*SD (log (L⁄EC 50 values)) (1) where average is the mean of the log-transformed toxicity values, SD is the standard deviation of the log-transformed toxicity values, and k is the extrapolation constant, which is 1.938 when the sample size is three.

[0026] In this example, the lowest effective concentration (LOEC) was used instead of L / EC 50 The calculation formula is as follows: logHC5= average (log LOEC)-k*SD (log LOEC) (2); The REACH guidance document indicates that an additional safety factor of 1 to 5 should be used to account for issues such as small numbers of species, bias and uncertainty. In this study, since only three species were used to estimate the SSD, the recommended maximum value of 5 was used in calculating the HC5 and further deriving the PNEC estimate: PNEC=HC5 / 5 (4) The effects of three types of microplastics on the protein concentration and enzyme activity of Vibrio fischeri, Chaetoceros muehii, and Perineuri bilaterian were analyzed by one-way ANOVA using Graph pad. The results are shown in the figure. Figures 1 to 4 As shown. * indicates that there is a significant difference between the experimental group and the control group ( P <0.05), LOEC, HC5 and PNEC results are shown in Tables 1 to 4 below.

[0027] Table 1 LOEC values ​​of microplastics on the activities of three biological enzymes .

[0028] Table 2 Ecological risk assessment values ​​of PE microplastics in seawater .

[0029] Table 3 Ecological risk assessment values ​​of PET microplastics in seawater .

[0030] Table 4 Seawater ecological risk assessment values ​​of PP microplastics .

[0031] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for determining the threshold value of microplastic particle water ecological risk assessment, characterized in that: The following steps are involved: (1) Preparation of pure microplastics: Place the microplastics to be cleaned into a semipermeable membrane, then place them into ultrapure water for stirring and cleaning, collect the microplastics in the semipermeable membrane, and dry them to obtain pure microplastics; (2) Biological testing: Select marine organisms as test organisms, prepare pure microplastic suspensions of different concentrations, add them to the test organism exposure system, and collect the test organisms after a period of reaction; For cell samples, the collected cell samples were washed and centrifuged, and then lysed with lysis buffer to obtain the supernatant; For tissue samples, the collected biological samples were frozen and killed, added to phosphate buffered saline (PBS) or saline, and then the biological tissues were broken, centrifuged, and the supernatant was collected; then the protein concentration, superoxide dismutase and catalase activities of the supernatant were measured; (3) Threshold derivation: The acute toxicity value is obtained through the results of step 2; the 5% species hazard concentration is calculated based on the acute toxicity value, and the no-effect concentration is predicted by deriving the 5% species hazard concentration.

2. The method according to claim 1, characterized in that: The microplastics described in step (1) include at least one of polypropylene microplastics, polyethylene microplastics and polyethylene terephthalate microplastics.

3. The method according to claim 1, characterized in that: The stirring and cleaning in ultrapure water described in step (1) is: stirring and cleaning for 6 to 10 days, and the ultrapure water is replaced every 20 to 30 hours during the stirring and cleaning process.

4. The method according to claim 1, characterized in that: The marine organisms described in step (2) include at least one of Vibrio fischeri, Chaetoceros muehii and Perineuridae bicolor.

5. The method according to claim 1, characterized in that: The acute toxicity value described in step (3) is the half-maximum effect concentration or the minimum effective concentration.

6. The method according to claim 5, characterized in that: The 5% species hazard concentration described in step (3) is calculated using the following formula: logHC5= average (log (L⁄EC 50 values))-k*SD (log (L⁄EC 50 values)) ; Where HC5 is the 5% species hazard concentration, average is the average of the logarithmic-transformed toxicity values, SD is the standard deviation of the logarithmic-transformed toxicity values, k is the extrapolation constant, and L / EC 50 The values ​​are the half-maximal effect concentrations.

7. The method according to claim 5, characterized in that: The 5% species hazard concentration described in step (3) is calculated using the following formula: logHC5= average (log LOEC)-k*SD (log LOEC); Where HC5 is the 5% species hazard concentration, average is the mean of the logarithmic-transformed toxicity values, SD is the standard deviation of the logarithmic-transformed toxicity values, k is the extrapolation constant, and LOEC is the lowest effective concentration.

8. The method according to claim 6 or 7, characterized in that: When the sample size of species is three, the k value is 1.

938.

9. The method according to claim 1, characterized in that: The predicted no-effect concentration described in step (3) is calculated using the following formula: PNEC=HC5 / additional safety factor; Among them, HC5 is the 5% species hazard concentration.

10. The method according to claim 8, characterized in that: In the case of a sample size of three species, an additional safety factor of 5 was stated.

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