A method for determining a marine ecological risk assessment threshold of microplastic particles

Abstract

The application discloses a method for determining a seawater ecological risk evaluation threshold of microplastic particles, and comprises the following steps: (1) preparing pure microplastics, (2) biological testing, and (3) threshold derivation. Through the method, the toxic effect of pure microplastics can be determined instead of the combined toxic effect of microplastics and leaching additives, so that the toxicity of microplastic particles to marine organisms can be more accurately evaluated. In addition, the tested organisms used in the method are all marine organisms, so that the risk of microplastic particles to seawater ecology can be more accurately reflected. The method can also derive the toxicity threshold of microplastic particles to marine organisms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of water ecological risk assessment, and particularly relates to a method for determining a seawater ecological risk assessment threshold of microplastic particles. BACKGROUND

[0002] Microplastics widely exist in marine environments, and the biological and ecological environmental problems caused by microplastics 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 activities and metabolic pathways, and the like. However, the current toxicological studies on the effects of enzyme activities mainly focus on polystyrene (PS) microplastics, and various additives such as antioxidants, flame retardants and stabilizers contained in microplastics will leach out during the experiment, and the toxicity of these additives to marine organisms may be greater than that of microplastic particles, thereby causing a "false positive" of the toxicity effects of microplastics and causing certain interference to the scientific evaluation of the true toxicity effects. In addition, although a large number of studies have explored the toxicity effects of microplastics on enzyme activities of marine organisms, there is still a lack of clear microplastic threshold standard, making it difficult to determine the specific reference point when evaluating the seawater ecological risk of microplastics, and it is necessary to rely on complex models and assumptions to infer the effects of microplastics, which increases the uncertainty and complexity of the evaluation. SUMMARY

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

[0004] The purpose of the present application is achieved by the following scheme:

[0005] A method for determining a seawater ecological risk assessment threshold of microplastic particles, comprising the following steps:

[0006] (1) Preparing pure microplastics: placing the microplastics to be cleaned into a semi-permeable membrane container, sealing the container, and then placing the semi-permeable membrane container containing the microplastics into ultrapure water for stirring and cleaning, collecting the microplastics in the semi-permeable membrane container after cleaning, drying, and obtaining pure microplastics;

[0007] (2) Biological test: selecting marine organisms as test organisms, and then preparing different concentrations of pure microplastic suspensions, respectively adding them into the test organism exposure system, and collecting the test organisms after reaction; for cell samples, washing and centrifuging the collected cell samples, then lysing with a lysis solution and taking the supernatant; for tissue samples, after freezing and killing the collected biological samples, adding them into phosphate buffered saline (PBS) or saline, then crushing the biological tissues, centrifuging, and taking the supernatant; and then measuring the protein concentration, superoxide dismutase (SOD) and catalase (CAT) enzyme activities of the supernatant;

[0008] (3) Threshold derivation: obtaining the acute toxicity value through the result of step 2; calculating the 5% species hazard concentration (HC5) according to the acute toxicity value, and deriving the predicted no effect concentration (PNEC) through the 5% species hazard concentration.

[0009] Further, the microplastics in step (1) include at least one of polypropylene (PP) microplastics, polyethylene (PE) microplastics, and polyethylene terephthalate (PET) microplastics.

[0010] Further, the stirring and cleaning in the ultrapure water in step (1) is stirring and cleaning for 6-10 days, and the ultrapure water is replaced every 20-30 hours during the stirring and cleaning; further, the stirring and cleaning in the ultrapure water in step (1) is stirring and cleaning for 7 days, and the ultrapure water is replaced every 24 hours during the stirring and cleaning.

[0011] Further, the marine organisms in step (2) include at least one of Vibrio fischeri, Chaetoceros muelleri, and Perinereis nuntia.

[0012] Further, the acute toxicity value in step (3) is the half effect concentration (EC 50 ) or the lowest effective concentration (LOEC).

[0013] Further, the 5% species hazard concentration in step (3) is calculated by the average value of the acute toxicity value, minus the standard deviation of the acute toxicity value multiplied by the result of the extrapolation constant; further, it is calculated by the following calculation formula:

[0014] logHC5= average (log (L⁄EC 50 values))-k*SD (log (L⁄EC 50 values)) (1);

[0015] Wherein average is the average value of the toxicity value after logarithmic conversion, SD is the standard deviation of the toxicity value after logarithmic conversion, and k is the extrapolation constant;

[0016] Further, it is calculated by the following calculation formula:

[0017] logHC5= average (log LOEC)-k*SD (log LOEC) (2);

[0018] Wherein average is the average value of the toxicity value after logarithmic conversion, SD is the standard deviation of the toxicity value after logarithmic conversion, and k is the extrapolation constant.

[0019] Further, when the sample size of the species is three, the value of k is 1.938.

[0020] Further, the predicted no effect concentration in step (3) is calculated by the following formula:

[0021] PNEC=HC5 / extra safety factor (3) ;

[0022] Wherein, the extra safety factor is indicated by the REACH guidance document that the extra safety factor of 1 to 5 should be adopted to consider the problems of less species number, deviation and uncertainty; further, in the case of three species sample amount, the extra safety factor is 5.

[0023] The present application has the following advantages and effects relative to the prior art:

[0024] 1. The method of the present application measures the toxicity effect of pure microplastics, rather than the combined toxicity effect of microplastics and leaching additives, which can more accurately evaluate the toxicity of microplastic particles to marine organisms.

[0025] 2. The test organisms used in the method of the present application are all marine organisms, which can more accurately reflect the risk of microplastic particles to seawater ecology.

[0026] 3. The method of the present application can derive the toxicity threshold of microplastic particles to marine organisms, providing a theoretical basis for formulating scientific pollution control strategies. BRIEF DESCRIPTION OF DRAWINGS

[0027] Fig. 1 is the protein concentration result graph of Vibrio fischeri, Chaetoceros muelleri and Perinereis aibuhitensis after microplastic exposure; wherein, (a) is the protein concentration result graph of Vibrio fischeri after PP microplastic exposure, (b) is the protein concentration result graph of Chaetoceros muelleri after PP microplastic exposure, (c) is the protein concentration result graph of Perinereis aibuhitensis after PP microplastic exposure, (d) is the protein concentration result graph of Vibrio fischeri after PE microplastic exposure, (e) is the protein concentration result graph of Chaetoceros muelleri after PE microplastic exposure, (f) is the protein concentration result graph of Perinereis aibuhitensis after PP microplastic exposure, (g) is the protein concentration result graph of Vibrio fischeri after PET microplastic exposure, (h) is the protein concentration result graph of Chaetoceros muelleri after PET microplastic exposure, and (i) is the protein concentration result graph of Perinereis aibuhitensis after PET microplastic exposure.

[0028] Fig. 2 is the result graph of the influence of PE, PET and PP microplastic exposure on the SOD and CAT enzyme activity of Vibrio fischeri.

[0029] Fig. 3 is the result graph of the influence of PE, PET and PP microplastic exposure on the SOD and CAT enzyme activity of Chaetoceros muelleri.

[0030] Fig. 4 Figure for the effect of PE, PET, PP microplastic exposure on SOD and CAT enzyme activity of Perinereis exserta. DETAILED DESCRIPTION

[0031] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0032] Example 1

[0033] A method for determining the ecological risk assessment threshold of microplastic particles in water, comprising the following steps:

[0034] 1. Preparation of pure microplastics

[0035] In this embodiment, three different microplastics (PP, PE, and PET) are selected, and the specific operation steps are as follows:

[0036] (1) Cut a piece of dialysis bag (dialysis bag is a bag container made of semi-permeable membrane, dialysis bag model: MD34-5M, Wide flat: 34 mm) with scissors, and cut the two sides open. The length is about 3-4 cm, and the width is about 1-2 cm. Put it into boiling water at 100°C for 5-10 min, and take it out after the temperature drops to room temperature. Dry and reserve;

[0037] (2) Take a 1 L beaker and a magnetic stirring rotor, and clean them with pure water and ultrapure water in turn. Then add 2 / 3 of ultrapure water into the beaker and put the magnetic stirring rotor into it for standby;

[0038] (3) Take a semi-permeable membrane clamp to hold a piece of cleaned semi-permeable membrane, and keep the other end open. Weigh 3-4 g of microplastics to be cleaned and put them into the prepared semi-permeable membrane, and clamp the other end of the semi-permeable membrane to make both ends closed. Then put it into the beaker;

[0039] (4) Place the beaker on the magnetic stirrer, adjust the position so that the rotor is adsorbed at the bottom of the beaker, and turn on the magnetic stirrer to make the semi-permeable membrane completely immersed in water, and keep the speed;

[0040] (5) The ultrapure water in the beaker needs to be replaced every 24 h, and 7 d is a leaching and washing period. After 7 d, take out the semi-permeable membrane with tweezers, open the semi-permeable membrane clamp on one side, pour out the microplastics inside, and rinse the residual on the bag with ultrapure water. Pour out the microplastics and the rinsing liquid to form a microplastic suspension. Collect the microplastic suspension and perform filtration and drying to obtain pure microplastic samples.

[0041] 2. Biological test

[0042] Three marine organisms were used as test organisms: Vibrio fischeri, Chaetoceros muelleri, and Nematoda (Vibrio fischeri was purchased from the China Industrial Microbial Culture Collection Center (CICC 10483), Nematoda was purchased from Donghai Island Jindi Fishing Bait Farm in Mazhang District, Zhanjiang City, and Chaetoceros muelleri was purchased from Pinduoduo Fengyun Algae Industry).

[0043] For Vibrio fischeri, microplastic suspensions were prepared using an aqueous solution containing 0.05% sodium dodecyl sulfate (SDS) and 2% NaCl, with microplastic concentrations of 10, 100, and 10⁻⁶. 3 10 4 10 5 10 6 10 7 μg / L. In the experimental group, 100 μL of microplastic suspension and 100 μL of bacterial culture were added to 10 mL of 2216e liquid medium (purchased from Shanghai Ruichu Biotechnology Co., Ltd.). In the control group, 100 μL of aqueous solution containing 0.05% sodium dodecyl sulfonate (SDS) and 2% NaCl and 100 μL of bacterial culture were added to 10 mL of 2216e liquid medium. After Vibrio fischeri reacted for 24 h, the exposed bacterial culture was obtained after microplastic exposure. 1 mL of the exposed bacterial culture was taken, washed twice with 2% NaCl aqueous solution to remove the liquid medium, centrifuged, and then 100 μL of lysis buffer was added for lysis treatment. The supernatant was then used for subsequent assays.

[0044] For Chaetoceros muelleri, seven microplastic suspensions of the same concentration were prepared using phosphate-buffered saline (PBS). In the experimental group, 200 μL of microplastic suspension and 2 mL of algal solution were added to 20 mL of f / 2 liquid medium (Guillard F / 2 Medium, purchased from Coolaber), while in the control group, 200 μL of PBS and 2 mL of algal solution were added to 20 mL of f / 2 liquid medium. After reacting for 96 h, the microplastic-exposed algal solution was obtained. 12 mL of the exposed algal solution was washed twice with 2% NaCl aqueous solution to remove the liquid medium, centrifuged, and then lysed with 200 μL of lysis buffer. The supernatant was then used for subsequent assays.

[0045] For *Nematostella didentata*, a microplastic suspension was prepared using an aqueous solution containing 0.05% sodium dodecyl sulfate (SDS) and 2% NaCl, at concentrations of 100 and 10... 3 10 4 10 5 10 6μg / L. The experimental group respectively took 500 μL of microplastic suspension and 5 sandworms into 500 mL of 2% NaCl solution (2 L beaker), and the control group took 500 μL of water solution containing 0.05% sodium dodecyl sulfate (SDS) and 2% NaCl by mass percentage and 5 sandworms into 500 mL of 2% NaCl solution (2 L beaker), and after 96 h of exposure, 3 sandworms were taken out, frozen and killed, and then the middle part of the sandworm body about 0.1 g was cut off, put into a 1.5 mL centrifuge tube, and then 1 mL of 2% saline was added for preservation. The sandworm tissue in the centrifuge tube was broken by a fast tissue cell crusher, and then centrifuged, and the supernatant was used for subsequent determination of the experiment.

[0046] Then the protein concentration, SOD enzyme activity and CAT enzyme activity of the three supernatants were determined by using the Biyun Tian BCA protein concentration determination kit, total SOD activity detection kit (WST-8 method) and hydrogen peroxidase detection kit respectively.

[0047] 3. Threshold derivation

[0048] The acute toxicity value of the test organism is further used to estimate the species sensitivity distribution (SSD), and the HC5 (concentration causing harmful effects on 5% of the species) is calculated on the basis of the SSD value. The specific calculation method of HC5 is to take the average value of toxicity, minus the standard deviation of the acute toxicity value multiplied by the result of the extrapolation constant:

[0049] log HC5 = average (log (L⁄EC 50 values))-k*SD (log (L⁄EC 50 values)) (1)

[0050] Wherein average is the average value of the toxicity value after logarithmic conversion, SD is the standard deviation of the toxicity value after logarithmic conversion, and k is the extrapolation constant, which is 1.938 when the sample size of the species is three.

[0051] This embodiment uses the lowest effective concentration (LOEC) instead of L / EC 50 to calculate, and the formula is as follows:

[0052] log HC5 = average (log LOEC)-k*SD (log LOEC) (2);

[0053] The REACH guidance document indicates that an additional safety factor of 1 to 5 should be applied to account for issues such as small number of species, bias and uncertainty. In this study, since only three species were used to estimate SSD, the maximum value of 5 was applied when calculating HC5, and further derived PNEC estimates:

[0054] PNEC = HC5 / 5 (4)

[0055] The effects of three kinds of microplastics on the protein concentration and enzyme activity of Vibrio fischeri, Chaetoceros muelleri and Perinereis aibuhitensis were analyzed by one-way ANOVA using Graph pad, and the results are shown in Figs. 1-4 , wherein * indicates that there is a significant difference between the experimental group and the control group ( P <0.05), and the LOEC, HC5 and PNEC results are shown in Tables 1-4.

[0056] Table 1 LOEC values of microplastics on the enzyme activity of three organisms

[0057] .

[0058] Table 2 Ecological risk assessment value of PE microplastics in seawater

[0059] .

[0060] Table 3 Ecological risk assessment value of PET microplastics in seawater

[0061] .

[0062] Table 4 Ecological risk assessment value of PP microplastics in seawater

[0063] .

[0064] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method of determining a water ecological risk assessment threshold value for microplastic particles, characterized in that, The method comprises the following steps: (1) preparing pure microplastics: placing the microplastics to be cleaned into a semi-permeable membrane, then stirring and cleaning in ultrapure water, collecting the microplastics in the semi-permeable membrane, and drying to obtain pure microplastics; (2) biological test: selecting marine organisms as test organisms, then preparing different concentrations of pure microplastic suspensions, respectively adding them into the test organism exposure system, and collecting the test organisms after a period of reaction; for cell samples, the collected cell samples are washed, centrifuged, then lysed with a lysis solution, and the supernatant is taken; for tissue samples, after the collected biological samples are frozen and killed, they are added into phosphate buffered saline (PBS) or saline, then the biological tissues are broken, centrifuged, and the supernatant is taken; then the protein concentration, superoxide dismutase and catalase enzyme activity of the supernatant are determined; (3) threshold derivation: obtaining the acute toxicity value from the results of step 2; calculating the 5% species hazard concentration according to the acute toxicity value, and deriving the predicted no effect concentration from the 5% species hazard concentration; the acute toxicity value in step (3) is the half effect concentration or the lowest effective concentration; the 5% species hazard concentration in step (3) is calculated by the following formula: log HC5= average (log (L / EC values)) - k * SD (log (L / EC values)); or 50 log HC5= average (log (L / EC values)) - k * SD (log (L / EC values)); or 50 log HC5= average (log (L / EC values)) - k * SD ( logHC5= average (log LOEC)-k*SD (log LOEC); where HC5 is the 5% species hazard concentration, average is the mean of the log-transformed toxicity values, SD is the standard deviation of the log-transformed toxicity values, k is the extrapolation constant, and L / EC 50 values is the median effect concentration, and LOEC is the lowest observed effect concentration. the predicted no effect concentration in step (3) is calculated by the following formula: PNEC=HC5 / additional safety factor; wherein, HC5 is the 5% species hazard concentration.

2. The method of claim 1, wherein: the microplastics in step (1) comprise at least one of polypropylene microplastics, polyethylene microplastics and polyethylene terephthalate microplastics.

3. The method of claim 1, wherein: in step (1), the stirring and cleaning in ultrapure water is stirring and cleaning for 6-10 days, and the ultrapure water is replaced every 20-30 hours during the stirring and cleaning.

4. The method of claim 1, wherein: the marine organisms in step (2) comprise at least one of Vibrio fischeri, Chaetoceros muelleri and Perinereis nuntia.

5. The method of claim 1, wherein: in the case of three species sample amounts, the k value is 1.

938.

6. The method of claim 5, wherein: in the case of three species sample amounts, the additional safety factor is 5.

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