Method for evaluating influence of extracellular polymeric substances on microplastic toxicity based on algae growth indexes

Abstract

The invention discloses a method for evaluating the influence of an extracellular polymeric substance (EPS) on the toxicity of microplastics based on an algae growth index, which can effectively evaluate the influence of the EPS on the toxicity of the tire microplastics by taking an algae growth inhibition rate as an evaluation parameter, and can evaluate the toxicity of the tire microplastics by comparing the EPS algae growth conditions under the exposure of the tire microplastics (TWPs). The method realizes quantitative discrimination of influence of EPS on microplastic toxicity, and has the advantages of short evaluation period, simple operation, sensitive response and low cost.

Description

Technical Field

[0001] This invention relates to the field of environmental ecology technology, specifically to a method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators. Background Technology

[0002] Plastics in the environment fragment under weathering, forming microplastics. Once microplastics enter water bodies, they typically negatively impact phytoplankton photosynthesis, affecting their growth and potentially reducing phytoplankton community biodiversity. In studies of the ecological effects of aquatic pollutants, microalgae, due to their short growth cycle and sensitivity to toxic substances, have become key indicator organisms for assessing the environmental risks of pollutants such as microplastics. Algal extracellular polymers (EPS) are complex mixtures produced by microalgal metabolism, mainly composed of proteins and polysaccharides, with surfaces rich in functional groups such as carboxyl, amino, and hydroxyl groups. These functional groups can interact with external organic compounds, forming a protective barrier on the surface of algal cells against external substances. However, the impact of algal EPS on microplastic toxicity is currently poorly understood; therefore, there is an urgent need to design an evaluation method for mitigating microplastic toxicity using algal EPS. Summary of the Invention

[0003] The purpose of this invention is to provide a method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators, which can accurately assess the impact of algal extracellular polymers on microplastic toxicity in the aquatic environment.

[0004] To achieve the above objectives, the solution of the present invention is: A method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators includes the following steps: Step 1: Select *Phaeocystis globosa* as a sample. First, inoculate *Phaeocystis globosa* containing EPS and *Phaeocystis globosa* without EPS into 250mL Erlenmeyer flasks containing 100ml F / 2 medium, respectively, to obtain algal culture systems containing EPS and without EPS. The initial cell density of *Phaeocystis globosa* in each algal culture system is 2×10⁻⁶ cells / mL. 6 cells / mL; Step 2: Then, different masses of tire microplastics were added to the algae culture system containing EPS and cultured in a light incubator to obtain multiple experimental groups A. Another algae culture system containing EPS but without added tire microplastics was used as control group A for comparison. In the algae culture system without EPS, different masses of tire microplastics were added and cultured in a light incubator to obtain multiple experimental groups B. Another algae culture system without EPS and without added tire microplastics was used as control group B for comparison. Step 3: Then, measure the absorbance (OD) of algal cells in the algal solution of each experimental group A, control group A, experimental group B, and control group B every 24 hours. 680 The algal cell density is obtained from the linear equation of algal cell density versus absorbance: The cell density of *Phaeocystis globosa* is 104.972 × OD. 680 -12.166, cell density is measured in units of 10-10. 5 cells / mL; Step 4: Finally, evaluate the toxicity of microplastics based on the growth inhibition rate (IR) of algal cells. The formula is as follows: IR (%) = (1 - T / C) × 100% In the formula, T represents the cell density of *Phaeocystis globosa* calculated for each experimental group, in units of 10T. 5 Cells / mL, where C is the cell density of *Phaeocystis globosa* calculated for the corresponding control group, in units of 10-1. 5 per mL.

[0005] In step 1, the method for preparing the EPS-free spherical Phaeocystis is to first centrifuge 10 mL of spherical Phaeocystis at 4°C for 15 min, then discard the supernatant and add 10 mL of PBS buffer to resuspend, then centrifuge at 4°C for 15 min, and finally discard the supernatant to obtain spherical Phaeocystis without EPS.

[0006] In step 1, the centrifugal force of the first centrifugation is 4000 g, and the centrifugal force of the second centrifugation is 10000 g.

[0007] In step 2, the method for preparing the tire microplastics is to first remove impurities from the surface of the waste tires, then rinse them with deionized water, then grind the waste tires with a stainless steel file, and finally sieve them through 60-mesh and 100-mesh screens to obtain tire microplastics with particle sizes of 250 μm and 150 μm.

[0008] In step 2, the mass of the tire microplastics added is 10 mg, 50 mg, 100 mg and 500 mg respectively.

[0009] In step 2, the cultivation conditions in the light incubator are as follows: light intensity of 3000~4000 lx, light-dark cycle of 12h / 12h, shaking at least 3 times a day to prevent algae deposition, temperature of 25±1℃, and continuous cultivation for 96h.

[0010] By adopting the above technical solution, the present invention provides a method for evaluating the influence of extracellular polymers (EPS) on microplastic toxicity based on algal growth indicators. Using algal growth inhibition rate as the evaluation parameter, the method can effectively assess the influence of EPS on tire microplastic toxicity. This method achieves quantitative discrimination of the influence of EPS on microplastic toxicity by comparing algal growth with and without EPS under tire microplastic (TWP) exposure. It has the advantages of short evaluation cycle, simple operation, sensitive response and low cost. Attached Figure Description

[0011] Figure 1 The results of the growth inhibition rate of algal cells are shown in the figure. (a) shows the growth inhibition rate of *Phaeocystis globosa* with extracellular polymeric substances (EPS) under exposure to tire microplastics (TWPs) with a particle size of 250 μm; (b) shows the growth inhibition rate of *Phaeocystis globosa* without EPS under exposure to TWPs with a particle size of 250 μm; (c) shows the growth inhibition rate of *Phaeocystis globosa* with EPS under exposure to TWPs with a particle size of 150 μm; and (d) shows the growth inhibition rate of *Phaeocystis globosa* without EPS under exposure to TWPs with a particle size of 150 μm. Detailed Implementation

[0012] To further explain the technical solution of the present invention, the present invention will be described in detail below through specific embodiments.

[0013] Example 1. Preparation of *Phaeocystis globosa* without EPS First, centrifuge 10 mL of *Phaeocystis globosa* at 4000 g for 15 min at 4℃. Then, discard the supernatant and resuspend in 10 mL of PBS buffer. Centrifuge again at 10000 g for 15 min at 4℃. Finally, discard the supernatant to obtain *Phaeocystis globosa* with EPS removed.

[0014] 2. Preparation of tire microplastics First, remove impurities from the surface of the waste tires, then rinse them with deionized water, then grind the waste tires with a stainless steel file, and finally sieve them through 60-mesh and 100-mesh screens to obtain tire microplastics with particle sizes of 250 μm and 150 μm, denoted as T250 and T150.

[0015] 3. Composition of F / 2 culture medium F / 2 medium is a conventional and widely used general-purpose seawater enrichment medium, the composition of which is shown in Tables 1 and 2.

[0016] Table 1. Composition of F / 2 culture medium

[0017] Table 2 Composition of F / 2 metal

[0018] 4. Evaluation Methods A method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators includes the following steps: Step 1: Select *Phaeocystis globosa* as a sample. First, inoculate *Phaeocystis globosa* containing EPS and *Phaeocystis globosa* without EPS into 250 mL Erlenmeyer flasks containing 100 ml of F / 2 medium, respectively, to obtain algal culture systems containing EPS and without EPS. The initial cell density in each algal culture system is 2 × 10⁻⁶ cells / mL. 6 pcs / mL (corresponding absorbance is 0.307); Step 2: Then, 10 mg, 50 mg, 100 mg, and 500 mg of T250 and T150 were added to the algal culture system containing EPS, and the systems were incubated in a light incubator to obtain experimental group A with tire microplastic concentration gradients of 10 mg / L, 50 mg / L, 100 mg / L, and 500 mg / L. A total of 8 groups were formed (4 groups each for T250 and T150). Another algal culture system containing EPS but without added tire microplastics was used as control group A for comparison. In the algal culture system without EPS, 10 mg, 50 mg, 100 mg, and 500 mg of T250 and T150 were added, and the systems were incubated in a light incubator to obtain tire microplastic concentration gradients of 10 mg / L, 50 mg / L, 100 mg / L, and 500 mg / L. Experimental group B with mg / L was divided into 8 groups (4 groups each of T250 and T150), and another algae culture system without EPS and without tire microplastics was used as control group B for comparison. The cultivation conditions in the light incubator were as follows: light intensity of 3000~4000 lx, light-dark cycle of 12h / 12h, shaking at least 3 times a day to prevent algae deposition, temperature of 25±1℃, and continuous cultivation for 96h. The cultivation conditions of each experimental group A, control group A, experimental group B and control group B were kept consistent. Step 3: Then, measure the absorbance (OD) of algal cells in the algal solution of each experimental group A, control group A, experimental group B, and control group B every 24 hours. 680 The algal cell density is obtained from the linear equation of algal cell density versus absorbance: The cell density of *Phaeocystis globosa* is 104.972 × OD. 680 -12.166, cell density is measured in units of 10-10. 5 cells / mL; Step 4: Finally, evaluate the toxicity of microplastics based on the growth inhibition rate (IR) of algal cells. The formula is as follows: IR (%) = (1 - T / C) × 100% In the formula, T represents the cell density of *Phaeocystis globosa* calculated for each experimental group, in units of 10T. 5 Cells / mL, where C is the cell density of *Phaeocystis globosa* calculated for the corresponding control group, in units of 10-1. 5 per mL.

[0019] 5. Evaluation Results The method described above for evaluating the effect of extracellular polymers on microplastic toxicity based on algal growth indicators was repeated three times. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 27 with multivariate analysis of variance (ANOVA) and 95% confidence intervals. Visualization was created in Origin (2024). The algal cell growth inhibition rate results are as follows: Figure 1 As shown.

[0020] like Figure 1 As shown in (a), under exposure to tire microplastics (TWPs) with a particle size of 250 μm, the growth inhibition rate of *Phaeocystis globosa* with extracellular polymeric substances (EPS) remained negative at TWP concentration gradients of 10 mg / L and 50 mg / L, indicating that growth was promoted. Conversely, the growth inhibition rate of *Phaeocystis globosa* at TWP concentration gradients of 100 mg / L and 500 mg / L remained positive, indicating that growth was inhibited. Furthermore, the inhibitory effect gradually leveled off over time. Figure 1 As shown in (b), after removing EPS, TWPs microplastics inhibited the growth of *Phaeocystis globosa*, and the inhibitory effect increased with increasing TWPs microplastic concentration. Figure 1 As shown in (c), when exposed to TWPs microplastics with a particle size of 150 μm, *Phaeocystis globosum* with EPS exhibited growth-promoting effects at low concentrations (10 mg / L) and growth-inhibiting effects at high concentrations (50 mg / L, 100 mg / L, and 500 mg / L). Figure 1 As shown in (d), after removing EPS, the growth of *Phaeocystis globosa* was inhibited to varying degrees by TWPs microplastics, and the inhibition increased with increasing concentration.

[0021] The results show that the method of evaluating the effect of extracellular polymers (EPS) on microplastic toxicity based on algal growth indicators of the present invention, using algal growth inhibition rate as evaluation parameter, can effectively assess the effect of EPS on tire microplastic toxicity. This method achieves quantitative discrimination of the effect of EPS on microplastic toxicity by comparing algal growth with and without EPS under tire microplastic (TWP) exposure. It has the advantages of short evaluation cycle, simple operation, sensitive response and low cost.

[0022] The above embodiments and figures are not intended to limit the product form and style of the present invention. Any appropriate changes or modifications made by those skilled in the art should be considered as not departing from the patent scope of the present invention.

Claims

1. A method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators, characterized in that: Includes the following steps: Step 1: Select *Phaeocystis globosa* as a sample. First, inoculate *Phaeocystis globosa* containing EPS and *Phaeocystis globosa* without EPS into 250mL Erlenmeyer flasks containing 100ml F / 2 medium, respectively, to obtain algal culture systems containing EPS and without EPS. The initial cell density of *Phaeocystis globosa* in each algal culture system is 2×10⁻⁶ cells / mL. 6 cells / mL; Step 2: Then, different masses of tire microplastics were added to the algae culture system containing EPS and cultured in a light incubator to obtain multiple experimental groups A. Another algae culture system containing EPS but without added tire microplastics was used as control group A for comparison. In the algae culture system without EPS, different masses of tire microplastics were added and cultured in a light incubator to obtain multiple experimental groups B. Another algae culture system without EPS and without added tire microplastics was used as control group B for comparison. Step 3: Then, measure the absorbance of algal cells in the algal solution of each experimental group A, control group A, experimental group B, and control group B every 24 hours. The algal cell density is obtained by the linear equation of algal cell density-absorbance. The cell density of *Phaeocystis globosa* is 104.972 × OD. 680 -12.166, cell density is measured in units of 10-10. 5 pcs / mL, where OD 680 Absorbance of algal cells; Step 4: Finally, evaluate the toxicity of microplastics based on the growth inhibition rate of algal cells. The formula is as follows: IR (%) = (1 - T / C) × 100% In the formula, IR represents the growth inhibition rate of algal cells, and T represents the cell density of *Phaeocystis globosa* calculated for each experimental group, in units of 10T. 5 Cells / mL, where C is the cell density of *Phaeocystis globosa* calculated for the corresponding control group, in units of 10-1. 5 per mL.

2. The method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators according to claim 1, characterized in that: In step 1, the method for preparing the EPS-free Phaeocystis globosa is to first centrifuge 10 mL of Phaeocystis globosa at 4℃ for 15 min, then discard the supernatant and add 10 mL of PBS buffer to resuspend, then centrifuge at 4℃ for 15 min, and finally discard the supernatant to obtain Phaeocystis globosa without EPS.

3. The method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators according to claim 2, characterized in that: In step 1, the centrifugal force of the first centrifugation is 4000 g, and the centrifugal force of the second centrifugation is 10000 g.

4. The method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators according to claim 1, characterized in that: In step 2, the method for preparing the tire microplastics is to first remove impurities from the surface of the waste tires, then rinse them with deionized water, then grind the waste tires with a stainless steel file, and finally sieve them through 60-mesh and 100-mesh screens to obtain tire microplastics with particle sizes of 250 μm and 150 μm.

5. The method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators according to claim 1, characterized in that: In step 2, the mass of the tire microplastics added is 10 mg, 50 mg, 100 mg and 500 mg respectively.

6. The method for evaluating the influence of extracellular polymers on microplastic toxicity based on algal growth indicators according to claim 1, characterized in that: In step 2, the cultivation conditions in the light incubator are as follows: light intensity of 3000~4000 lx, light-dark cycle of 12h / 12h, shaking at least 3 times a day to prevent algae deposition, temperature of 25±1℃, and continuous cultivation for 96h.

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