Method for evaluating environmental risk of perfluorooctane sulfonic acid by using euglena gracilis

Abstract

The invention discloses a method for evaluating the environmental risk of perfluorooctane sulfonic acid by using euglena gracilis, and belongs to the field of environmental pollutant biological monitoring and ecotoxicology evaluation. The method comprises the following steps: detecting the photosynthetic activity and the movement ability of Euglena gracilis exposed to perfluorooctane sulfonic acid with environmental concentration and the organelle distribution condition of the perfluorooctane sulfonic acid in euglena gracilis cells in situ; the photosynthetic activity comprises in-situ monitoring of chlorophyll fluorescence parameters and maximum non-photochemical quenching coefficient of euglena gracilis, and quantitative analysis of chloroplast pigment content; the movement ability comprises horizontal light-driving movement speed and vertical light-driving movement speed measurement of the euglena gracilis and flagellum shedding rate calculation. The method breaks through the limitation of a traditional single autotrophic mode, reveals a regulation and control mechanism of nutritional conditions on toxic effects, provides a scientific basis for water environment safety monitoring and optimization, and has the advantages of being convenient to operate and low in cost.

Description

Technical Field

[0001] The present invention belongs to the field of biological monitoring and ecotoxicological assessment of environmental pollutants, and particularly relates to a method for evaluating the environmental risk of perfluorooctane sulfonic acid using Euglena gracilis. Background Art

[0002] Aquatic environmental safety is a crucial element in safeguarding human health and well-being. Water shortages and severe water pollution have become environmental issues of grave concern to the international community. Emerging pollutants are newly discovered synthetic or natural chemicals or biological agents detected in the environment that pose potential or recently confirmed hazards to humans and ecosystems. Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a representative example. Perfluorooctane sulfonic acid (PFOS) is one of the most common PFAS compounds. PFOS is widely distributed in aquatic media, and its biological toxicity and environmental risks have garnered significant attention.

[0003] In nature, many microorganisms do not rely on a single nutritional model to survive. Most photosynthetic bacteria and microalgae adapt their nutritional patterns in response to changing environmental conditions. Among them, Euglena gracilis is a single-celled eukaryote with both plant and animal characteristics, belonging to the class Euglena of the phylum Protista. Euglena's cell structure also exhibits characteristics of both plants and animals: it lacks a cell wall, but instead consists of a periplasmic membrane, containing chloroplasts (containing chlorophylls a and b) and eyespots (photosensitive structures), and uses flagella to enable phototaxis. Euglena exhibits a mixed nutritional model, capable of both photosynthetic autotrophy through chloroplasts and heterotrophic activity through endocytosis, making it an overlooked model organism for studying the biotoxicity of pollutants. Furthermore, Euglena holds great potential in wastewater treatment, demonstrating highly efficient removal of heavy metals, as well as nitrogen, phosphorus, and chemical oxygen demand (COD) in domestic wastewater. Furthermore, Euglena harbors its own unique microbial community, which has significant environmental significance for the biogeochemical cycles of elements such as carbon, nitrogen, phosphorus, and sulfur.

[0004] Assessing the biotoxicity of environmental pollution to microorganisms and exploring the environmental risks of pollutants are commonly used methods, but the nutritional patterns of the test model organisms are often relatively simple and lack representativeness. Previous toxicity assessments of algae have often only examined a single nutritional pattern (autotrophic behavior), with representative characterization primarily reflected in the maximum photochemical efficiency of PSⅡ and chlorophyll quantification, while ignoring the responses of algae to pollutants under different nutritional patterns (mixotrophic), such as the distribution of pollutants in cellular organelles, algal motility, and flagellar shedding.

[0005] In order to explore a reasonable method to evaluate the environmental risk of PFOS, Euglena gracilis was used as a model organism to reveal the binding mechanism and toxic effects of PFOS on organisms at environmental concentrations. Summary of the Invention

[0006] The present invention aims to address the shortcomings of existing studies that use a single model organism to assess environmental risks, and to provide a more representative, scientifically sound basis and method for accurately assessing the environmental risks of PFOS. Specifically, a method for assessing the environmental risk of perfluorooctane sulfonate using Euglena gracilis is provided.

[0007] The specific technical solutions adopted in the present invention are as follows:

[0008] The present invention provides a method for evaluating the environmental risk of perfluorooctane sulfonate (PFOS) using Euglena gracilis, which in situ detects the photosynthetic activity and motility of Euglena gracilis exposed to environmental concentrations of PFOS, as well as the organelle distribution of PFOS in Euglena gracilis cells.

[0009] The photosynthetic activity includes in-situ monitoring of chlorophyll fluorescence parameters and maximum non-photochemical quenching coefficient of Euglena gracilis, and quantitative analysis of chloroplast pigment content; the movement ability includes the measurement of horizontal light-driven movement speed and vertical light-driven movement speed of Euglena gracilis, and calculation of flagella shedding rate.

[0010] Preferably, the Euglena gracilis is deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences with the deposit number FACHB-848, and the deposit address is the Freshwater Algae Seed Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, No. 7, Shandong Hunan Road, Luojia Mountain, Wuhan.

[0011] Preferably, the environmental concentration of perfluorooctane sulfonic acid is selected to be 0.005 to 500 μg / L.

[0012] Preferably, the horizontal light-driving motion speed is detected using a cuboid microscale experimental device, and the specific method is as follows: placing Euglena gracilis exposed to an environmental concentration in the cuboid microscale experimental device, irradiating the device from one side with an LED light source, and transmitting light only to the side of the device closest to the light source; after 20 minutes of irradiation, algae liquid is sampled every 3 cm along the length of the cuboid microscale experimental device;

[0013] The vertical light-driven motion speed was measured using a cylindrical microscale experimental device. The specific method was as follows: Euglena gracilis, exposed to ambient concentrations, was placed in the cylindrical microscale experimental device and illuminated from the top of the device using an LED light source, with the side walls of the device opaque. After 20 minutes of irradiation, algal liquid was sampled at 3 cm intervals along the height of the cylindrical microscale experimental device.

[0014] The calculation formula of the horizontal light driving motion speed or the vertical light driving motion speed is as follows:

[0015]

[0016] Where: V is the horizontal or vertical light-driven movement speed of Euglena gracilis, μm / s; n is the number of samples; Ci is the cell density of Euglena gracilis in the algae solution, cells / mL; Vi is the volume of the algae solution, mL; t is the irradiation time, s; Si is the movement distance, μm.

[0017] Furthermore, the dimensions of the rectangular micro-scale experimental device are 4×4×9 cm; the inner diameter of the cylindrical micro-scale experimental device is 4.4 cm and the height is 10.0 cm; and the illumination intensity of the LED light source is 3500 lux.

[0018] Preferably, the organelle distribution of PFOS in Euglena gracilis cells is observed using a scanning transmission electron microscope, as shown below:

[0019] S1: Euglena gracilis cells exposed to environmental concentrations of PFOS were fixed in a 2.5% glutaraldehyde solution for 2 days; the fixed Euglena gracilis cells were suspended in 2.5% agar.

[0020] S2: After the agar solidifies, cut the sample into cubes of appropriate size; fix the cubes in a 1% osmium tetroxide solution for 1 hour using a Na2HPO4-KH2PO4 buffer solution with a pH of 7.2;

[0021] S3: Dehydrating the sample fixed in step S2 in an aqueous solution with increasing ethanol concentrations; embedding the dehydrated sample in Durcupan resin, then sectioning the sample using a microtome and mounting it on a copper grid;

[0022] S4: Sections mounted on copper grids were contrast-stained with lead citrate solution and 5% uranyl acetate solution, respectively; the stained samples were observed using a scanning transmission electron microscope to observe the accumulation of PFOS in organelles such as chloroplasts, mitochondria, and eyespots.

[0023] Preferably, the Euglena gracilis is cultured autotrophically using EM autotrophic medium, or mixotrophically using HUT medium;

[0024] The EM autotrophic culture medium includes: 1.6 g / L NH4Cl, 60 mg / L CO(NH2)2, 1.0 g / L KH2PO4, 0.6 g / L MgSO4, 0.02 g / L CaCl2, 3 mg / L Fe2(SO4)3, 0.48 mg / L Na2EDTA, 1 mg / L VitaminB1, 5 μg / L Vitamin B 12 , 1mL / L of the first mother liquor and 1mL / L of the second mother liquor; the first mother liquor includes 0.01molHCl, 1.8g / L of MnCl2 and 1.6g / L of CoSO4; the second mother liquor includes 0.5g / L of ZnSO4, 0.4g / L of Na2MoO4 and 0.4g / L of CuSO4.

[0025] Preferably, the chlorophyll fluorescence parameters and the maximum non-photochemical quenching coefficient are detected in situ using a handheld chlorophyll fluorescence instrument.

[0026] Preferably, the quantitative analysis of chloroplast pigments is specifically as follows: after extracting the Euglena gracilis cells exposed to environmental concentrations of perfluorooctane sulfonic acid with 80% volume fraction acetone, a UV-visible spectrophotometer is used to measure the absorbance at 470 nm, 646 nm, and 663 nm, respectively, to calculate the content of chlorophyll a, chlorophyll b, and carotenoids.

[0027] Preferably, the cell density of the gracilis algae exposed to the environmental concentration of perfluorooctane sulfonic acid is 10 4 -10 5 pieces / mL.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) This study uses Euglena gracilis as a model organism to evaluate the environmental risks of perfluorooctane sulfonate (PFOS). Euglena gracilis can be cultivated both autotrophically and mixotrophically, revealing the dynamic regulatory mechanisms of different nutrient modes on the toxic effects of PFOS. This overcomes the limitations of traditional algal assessments based on a single autotrophic model and significantly improves the authenticity of ecological simulations of complex nutrient scenarios in natural water bodies.

[0030] (2) The present invention is based on chlorophyll fluorescence parameters (F v / F m , QP lss) non-invasive in situ monitoring technology effectively avoided the interference of the sampling process on the physiological state of naked algae, and achieved real-time dynamic tracking of photosynthetic activity damage. Further combining the micro-scale phototaxis device with microscopic flagellar integrity analysis, a new standard for the quantification of movement behavior toxicity was successfully constructed, providing technical support for the early warning of trace pollutants. Ultimately, by integrating the dual dimensions of photosynthetic efficiency and movement ability (phototaxis speed and flagellar shedding rate), the combined stress effects of PFOS on aquatic organisms were analyzed from the perspective of biological functional integrity, significantly enhancing the ecological relevance of the assessment results. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The micro-scale experimental device used in the present invention, wherein (a) is used to measure the vertical driving light movement speed, and (b) is used to measure the horizontal driving light movement speed;

[0032] Figure 2 The photosynthetic activity of Euglena gracilis under different environmental concentrations of PFOS exposure in Example 1, where (a) and (b) are the chlorophyll fluorescence parameters (F v / F m ) and maximum non-photochemical quenching (QP lss ) data, (c) and (d) are the chlorophyll fluorescence parameters (F v / F m ) and maximum non-photochemical quenching (QP lss )data;

[0033] Figure 3 The chloroplast pigment content in the cells of Euglena gracilis exposed to different environmental concentrations of PFOS in Example 1, where (a) is the autotrophic mode and (b) is the mixotrophic mode;

[0034] Figure 4 This is the S-TEM-EDS mapping image of Euglena gracilis under exposure to different environmental concentrations of PFOS in Example 2;

[0035] Figure 5 The vertical light-repelling movement speed (a) and horizontal light-repelling movement speed (b) of Euglena gracilis under exposure to different environmental concentrations of PFOS in Example 3;

[0036] Figure 6 This is the flagella shedding rate of Euglena gracilis under exposure to different environmental concentrations of PFOS in Example 4. DETAILED DESCRIPTION

[0037] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention may be combined accordingly, provided that there is no conflict between them.

[0038] The Euglena gracilis used in the following examples was purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences with the deposit number FACHB-848. The deposit address is the Freshwater Algae Seed Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, No. 7 Shandong Hunan Road, Luojia Mountain, Wuhan.

[0039] The specific components of the EM autotrophic medium used in the following examples are as follows: 1.6 g / L NH4Cl, 60 mg / L CO(NH2)2, 1.0 g / L KH2PO4, 0.6 g / L MgSO4, 0.02 g / L CaCl2, 3 mg / L Fe2(SO4)3, 0.48 mg / L Na2EDTA, 1 mg / L Vitamin B1, 5 μg / L Vitamin B 12 , 1mL / L of first mother liquor and 1mL / L of second mother liquor; the first mother liquor includes 0.01mol HCl, 1.8g / L of MnCl2 and 1.6g / L of CoSO4; the second mother liquor includes 0.5g / L of ZnSO4, 0.4g / L of Na2MoO4 and 0.4g / L of CuSO4.

[0040] The specific components of the HUT medium used in the following examples are as follows: 0.02 g / L KH2PO4, 0.6 g / L peptone, 0.025 g / L MgSO4·7H2O, 0.4 g / L yeast extract, 0.4 g / L sodium acetate, 0.5 μg / L Vitamin B 12 , 0.4mg / L Vitamin B1 and 0.04g / L Potassium citrate.

[0041] Example 1

[0042] This example provides a method for measuring the photosynthetic activity of Euglena gracilis under exposure to different environmental concentrations of PFOS, as follows:

[0043] (1) Chlorophyll fluorescence parameters (F v / F m ) and maximum non-photochemical quenching (QP lss ) In-situ detection

[0044] The autotrophic culture medium containing 0.5, 5 and 50 μg / L PFOS was prepared, and the control group was an autotrophic culture medium without PFOS. Euglena gracilis was inoculated into the autotrophic culture medium containing different concentrations of PFOS. The cell density of Euglena gracilis after inoculation was 10 4 Sampling was done once every 24 hours, and the chlorophyll fluorescence parameters (F v / F m) and maximum non-photochemical quenching (QP lss ), and monitored continuously for 10 days.

[0045] Mixotrophic culture media containing 0.5, 5, and 50 μg / L PFOS were prepared, and the control group was a mixotrophic culture medium without PFOS. Euglena gracilis was inoculated into the mixotrophic culture media containing different concentrations of PFOS. The cell density of Euglena gracilis after inoculation was 10 4 Sampling was performed every 6 hours, and the chlorophyll fluorescence parameters (F v / F m ) and maximum non-photochemical quenching (QP lss ), continuously monitored for 24 hours. Figure 2 shown.

[0046] like Figure 2 (a) and Figure 2 As shown in (b), in this embodiment, when Euglena gracilis is exposed to PFOS at a concentration of 0.5 to 50.0 μg / L, whether in autotrophic or mixotrophic mode, its PFOS v / F m In the autotrophic mode, on the 7th day after PFOS exposure, the v / F m and QP lss However, as the exposure time increased, the promoting effect of PFOS on the photosynthetic efficiency of Euglena gradually weakened. For Euglena in the mixotrophic mode, PFOS had a significant effect on the photosynthetic efficiency of Euglena 18h after exposure to PFOS. v / F m The promoting effect of PFOS is very obvious, and the effect of high concentration of PFOS in improving photosynthetic efficiency is more significant. In addition, under the autotrophic mode, the QP of Euglena lss There was basically no significant change in the 10 days of exposure to PFOS at all concentrations. However, the response of Euglena in the mixotrophic mode to PFOS exposure was faster. After 6 hours of PFOS exposure, the QP of Euglena in the mixotrophic mode was lss This shows that under the mixotrophic mode, although the photosynthetic efficiency of Euglena after PFOS exposure is v / F m It also showed a promoting effect, but because PFOS could enter the naked algae cells faster under the mixotrophic mode, it showed cytotoxicity earlier.

[0047] (2) Chloroplast pigment determination

[0048] Take 10mL of cell suspension cultured in autotrophic and mixotrophic modes for 7 days, filter them separately, and rinse them three times with distilled water. Then place the cells together with glass sand and 80% (v / v) acetone solution in an ice water bath for grinding until the pigment is completely transferred to the acetone solution. The extract slurry is filtered again to remove insoluble matter. After mixing the filtrates obtained in batches, dilute to 10mL with acetone solution. Finally, use a UV-visible spectrophotometer to measure the absorbance at three wavelengths of 470, 646, and 663nm. Chlorophyll a (Chl a ), chlorophyll b (Chl b The calculation formulas for the contents of ) and carotenoids (Cx+c) are as follows:

[0049] Chl a =12.21×Abs 663 -2.81×Abs 646 ;

[0050] Chl b =20.13×Abs 646 -5.03×Abs 663 ;

[0051] Cx+c=(1000×Abs 470 -3.27×Chl a -104×Chl b ) / 229.

[0052] like Figure 3 (a) and Figure 3 As shown in (b), in this embodiment, the amount of chlorophyll synthesised by Euglena in the mixotrophic mode is higher than that in the autotrophic mode, which is consistent with its mixed nutritional characteristics. After exposure to environmental concentrations of PFOS, the total amount of chlorophyll in Euglena cells showed an upward trend, among which chlorophyll a responded most sensitively. In the autotrophic mode, a low concentration of 0.5 μg / L PFOS significantly increased the chlorophyll a content, chlorophyll b did not change significantly, and the other pigments increased synchronously; while exposure to a high concentration of 50.0 μg / L did not significantly affect the chlorophyll level of autotrophic Euglena. Under the mixotrophic mode, chlorophyll a continued to increase significantly with the increase of PFOS concentration, and other pigments only showed significant changes in the high concentration group, which is presumably due to the short mixotrophic exposure time.

[0053] Example 2

[0054] This example provides a study of the organelle distribution of PFOS in Euglena gracilis cells under exposure to different environmental concentrations of PFOS. The specific experimental process is as follows:

[0055] (1) Prepare autotrophic culture medium and mixotrophic culture medium containing 50 μg / L PFOS, and the control group is autotrophic culture medium and mixotrophic culture medium without PFOS. Euglena gracilis was inoculated into the above culture medium and cultured for 7 days. After inoculation, the cell density of Euglena gracilis was 10 4 pieces / mL.

[0056] (2) The exposed and cultured Euglena gracilis cells were placed in a 2.5% volume fraction glutaraldehyde solution and fixed for 2 days; the fixed Euglena gracilis cells were suspended in 2.5% warm agar.

[0057] (3) After the agar solidifies, cut the sample into cubes of appropriate size; place the cube sample in a 1% by mass osmium tetroxide solution and fix it for 1 hour. The fixation process uses a Na2HPO4-KH2PO4 buffer solution with a pH value of 7.2 (concentration of 0.07M).

[0058] (4) Dehydrating the sample fixed in step (3) in an aqueous solution with gradually increasing ethanol concentration; embedding the dehydrated sample in Durcupan resin, and then sectioning it using a microtome (Reichert Jung Ultracut M) and mounting it on a copper grid.

[0059] (5) Sections mounted on copper grids were contrast stained with lead citrate solution and 5% uranyl acetate, respectively; the stained samples were observed using a scanning transmission electron microscope to observe the accumulation of PFOS in organelles such as chloroplasts, mitochondria, and eyespots. Figure 4 shown.

[0060] In this example, energy spectrum scanning technology was used to observe sliced ​​euglena cells. The results showed that a clear fluorine-specific enrichment signal could be detected in the euglena cells after PFOS exposure, confirming the significant bioaccumulation of PFOS in the euglena cells. Combined S-TEM and EDS image analysis revealed that the accumulation and distribution of PFOS in the euglena cells exhibited distinct selectivity. Specifically, organelles such as chloroplasts, mitochondria, and eyespots showed a high affinity for PFOS accumulation, making PFOS more likely to accumulate in these areas. In contrast, significant PFOS accumulation was almost undetectable in locations such as the vacuole and paramyloliquefaciens of the euglena cells.

[0061] Example 3

[0062] This example provides a method for measuring the horizontal and vertical light-driven movement speeds of Euglena gracilis cells under exposure to different environmental concentrations of PFOS. The specific experimental process is as follows:

[0063] (1) Prepare autotrophic culture medium and mixotrophic culture medium containing 0.005, 0.05, 0.5, 5, 50, and 500 μg / L PFOS, respectively. The control group is an autotrophic culture medium and a mixotrophic culture medium without PFOS. Euglena gracilis was inoculated into the above culture medium. The cell density of Euglena gracilis after inoculation was 10 4 pieces / mL.

[0064] (2) The vertical driving light motion speed is detected using a cylindrical micro-scale experimental device, such as Figure 1 (a) The cylindrical device has an inner diameter of 4.4 cm and a height of 10.0 cm. An LED light source (blue-white light, 3500 lux) illuminates from the top of the device, while the side walls are opaque. Euglena gracilis, exposed to ambient concentrations, was placed in the cylindrical microscale experimental device. After 20 minutes of irradiation, algal liquid was sampled at 3 cm intervals along the height of the cylindrical microscale experimental device for later use.

[0065] (3) The horizontal driving light motion speed is detected using a cuboid micro-scale experimental device, such as Figure 1 (b) The rectangular device measured 4 × 4 × 9 cm. An LED light source (blue-white light, 3500 lux) illuminated the device from one side, with light only passing through the side closest to the light source. After 20 minutes of irradiation, algal liquid was sampled at 3 cm intervals along the length of the rectangular microscale experimental device for later use.

[0066] (4) Calculate the horizontal light-driving speed or the vertical light-driving speed using the following formula:

[0067]

[0068] Where: V is the horizontal or vertical light-driven movement speed of Euglena gracilis, μm / s; n is the number of samples; Ci is the cell density of Euglena gracilis in the algae solution, cells / mL; Vi is the volume of the algae solution, mL; t is the irradiation time, s; Si is the movement distance, μm.

[0069] The results are as follows Figure 5 As shown. It can be seen in this example that PFOS significantly inhibits the vertical and horizontal movement speed of Euglena. 50.0ng / L is the critical threshold concentration. When this concentration is exceeded, the movement ability is significantly impaired, and the vertical movement is more strongly inhibited than the horizontal direction. Figure 6 The phenomenon of increased flagellar shedding rate in mice confirms that flagellar damage is directly related to decreased motility.

[0070] Example 4

[0071] This example provides a method for determining the flagella shedding rate of Euglena gracilis cells under exposure to different environmental concentrations of PFOS. The specific experimental process is as follows:

[0072] (1) Prepare autotrophic culture medium containing 0.5, 5, and 50 μg / L PFOS, and inoculate Euglena gracilis into the above culture medium. After inoculation, the cell density of Euglena gracilis is 10 4 After inoculation, the cells were exposed and cultured for 7 days.

[0073] (2) Take 20 μL of the Euglena gracilis liquid in the above culture medium, fix it in a blood cell counting chamber for 5 minutes, and randomly count 300 Euglena cells under an optical microscope and calculate the flagella shedding rate. Repeat 3 times for each sample. The flagella shedding rate is calculated as follows: Shedding rate (%) = (number of cells without flagella / total number of cells) × 100. The results are shown in Figure 2. Figure 6 shown.

[0074] The results of this example show that the shedding rate of Euglena flagella is significantly positively correlated with the PFOS exposure concentration. Although the difference in shedding rate between the low-concentration groups was not significant, when the PFOS concentration increased from 5.0 μg / L to 50.0 μg / L, the shedding rate rose sharply from 37.3% to 48.7%. Flagella are core organs that regulate Euglena's diurnal vertical migration, phototaxis, and heterotrophic feeding. Large-scale shedding of flagella will directly weaken the cells' photosynthetic positioning ability and nutrient acquisition efficiency. This damage not only leads to a decline in individual motor function but is also likely to cause a decrease in Euglena population density in natural water bodies, ultimately disturbing the ecological balance of aquatic microbial communities.

[0075] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis, characterized in that: In situ testing of photosynthetic activity and motility of Euglena gracilis exposed to environmental concentrations of PFOS, as well as the organelle distribution of PFOS in Euglena gracilis cells; The photosynthetic activity includes in-situ monitoring of chlorophyll fluorescence parameters and maximum non-photochemical quenching coefficient of Euglena gracilis, and quantitative analysis of chloroplast pigment content; the movement ability includes the measurement of horizontal light-driven movement speed and vertical light-driven movement speed of Euglena gracilis, and calculation of flagella shedding rate.

2. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The Euglena gracilis is deposited in the Freshwater Algae Seed Bank of the Chinese Academy of Sciences with the deposit number FACHB-848. The deposit address is the Freshwater Algae Seed Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, No. 7, Shandong Hunan Road, Luojia Mountain, Wuhan.

3. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The environmental concentration of the perfluorooctane sulfonic acid is selected to be 0.005 to 500 μg / L.

4. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The horizontal light-driven motion speed was measured using a cuboid microscale experimental device. The specific method was as follows: Euglena gracilis, exposed to ambient concentrations, was placed in the cuboid microscale experimental device. An LED light source was used to illuminate the device from one side, with light only passing through the device on the side closest to the light source. After 20 minutes of irradiation, algal liquid was sampled at 3 cm intervals along the length of the cuboid microscale experimental device. The vertical light-driven motion speed was measured using a cylindrical microscale experimental device. The specific method was as follows: Euglena gracilis, exposed to ambient concentrations, was placed in the cylindrical microscale experimental device and illuminated from the top of the device using an LED light source, with the side walls of the device opaque. After 20 minutes of irradiation, algal liquid was sampled at 3 cm intervals along the height of the cylindrical microscale experimental device. The calculation formula of the horizontal light driving motion speed or the vertical light driving motion speed is as follows: Where: V is the horizontal or vertical light-driven movement speed of Euglena gracilis, μm / s; n is the number of samples; Ci is the cell density of Euglena gracilis in the algae solution, cells / mL; Vi is the volume of the algae solution, mL; t is the irradiation time, s; Si is the movement distance, μm.

5. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 4, characterized in that: The dimensions of the rectangular micro-scale experimental device are 4×4×9 cm; the inner diameter of the cylindrical micro-scale experimental device is 4.4 cm and the height is 10.0 cm; the illumination intensity of the LED light source is 3500 lux.

6. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The organelle distribution of PFOS in Euglena gracilis cells was observed using scanning transmission electron microscopy, as shown below: S1: Euglena gracilis cells exposed to environmental concentrations of PFOS were fixed in a 2.5% glutaraldehyde solution for 2 days; the fixed Euglena gracilis cells were suspended in 2.5% agar. S2: After the agar solidifies, cut the sample into cubes of appropriate size; fix the cubes in a 1% osmium tetroxide solution for 1 hour using a Na2HPO4-KH2PO4 buffer solution with a pH of 7.2; S3: Dehydrating the sample fixed in step S2 in an aqueous solution with increasing ethanol concentrations; embedding the dehydrated sample in Durcupan resin, then sectioning the sample using a microtome and mounting it on a copper grid; S4: Sections mounted on copper grids were contrast-stained with lead citrate solution and 5% uranyl acetate solution, respectively; the stained samples were observed using a scanning transmission electron microscope to observe the accumulation of PFOS in organelles such as chloroplasts, mitochondria, and eyespots.

7. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The Euglena gracilis is cultured autotrophically using EM autotrophic medium, or mixotrophically using HUT medium; The EM autotrophic medium includes: 1.6 g / L NH4Cl, 60 mg / L CO(NH2)2, 1.0 g / L KH2PO4, 0.6 g / L MgSO4, 0.02 g / L CaCl2, 3 mg / L Fe2(SO4)3, 0.48 mg / L Na2EDTA, 1 mg / L Vitamin B1, 5 μg / L Vitamin B 12 , 1mL / L of first mother liquor and 1mL / L of second mother liquor; the first mother liquor includes 0.01mol HCl, 1.8g / L of MnCl2 and 1.6g / L of CoSO4; the second mother liquor includes 0.5g / L of ZnSO4, 0.4g / L of Na2MoO4 and 0.4g / L of CuSO4.

8. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The chlorophyll fluorescence parameters and the maximum non-photochemical quenching coefficient are detected in situ using a handheld chlorophyll fluorescence instrument.

9. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The quantitative analysis of chloroplast pigments is specifically as follows: after extracting the Euglena gracilis cells exposed to environmental concentrations of perfluorooctane sulfonic acid using 80% by volume acetone, the absorbance at 470 nm, 646 nm, and 663 nm is measured using a UV-visible spectrophotometer to calculate the contents of chlorophyll a, chlorophyll b, and carotenoids.

10. The method for evaluating the environmental risk of perfluorooctane sulfonate using Euglena gracilis according to claim 1, characterized in that: The cell density of the algae exposed to the environmental concentration of PFOS was 10 4 -10 5 pieces / mL.

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