A scientific method for assessing the ecological and environmental risks of combined pollution of microplastics and antibiotics

Abstract

The present invention relates to a method for scientifically assessing the ecological and environmental risks of combined pollution by microplastics and antibiotics. The method comprises: preparing a tetracycline (TC) solution with a concentration gradient, mixing it with small and large particle sizes of polystyrene plastic (PS-NPs), and then stirring it evenly with soil; burying soaked woad seeds in the soil to subject them to combined stress; and stressing the woad again after the woad has grown four true leaves, sampling, and measuring physiological changes in the woad leaves to reflect the impact of combined pollution on the woad. This method studies the physiological effects of combined stress on woad leaves from three perspectives, and through data, reflects the ecological and environmental risks of combined pollution of polystyrene microplastics and tetracycline at different concentrations, providing a basis for the assessment of the ecological and environmental risks of combined pollution.

Description

Technical Field

[0001] The present invention belongs to the field of ecotoxicology, and particularly relates to a method for scientifically assessing the ecological environmental risk of combined pollution by microplastics and antibiotics. Background Art

[0002] The growth of terrestrial plants will be affected by microplastics. Some people have studied the effects of plastic film residues on plant fertilizer utilization efficiency, yield, and root growth. [1] There are also studies on the effects of microplastics on lettuce growth, photosynthesis and antioxidant defense systems. [2] Studies have shown that polystyrene microplastics adsorb tetracycline, with adsorption performance varying depending on the properties and polymer type. While there are numerous studies on the adsorption and desorption of antibiotics in various soils and soil media, there is limited research on their effects on crops grown in the soil. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for scientifically evaluating the ecological environmental risk of combined pollution of microplastics and antibiotics, so as to fill the gap in the existing technology.

[0004] The present invention provides a method for scientifically assessing the ecological environmental risk of combined microplastic and antibiotic pollution, comprising:

[0005] (1) Set up polystyrene plastic PS-NPs with small and large particle sizes;

[0006] (2) preparing a tetracycline TC solution with a concentration gradient, mixing it with the small and large particle sizes of polystyrene plastic PS-NPs prepared in step (1), and then stirring it evenly with soil;

[0007] (3) selecting soil-cultured Isatis indigotica as the test plant, burying the soaked Isatis indigotica seeds in the soil stirred evenly in step (2), and subjecting them to combined stress;

[0008] (4) After the Isatis indigotica has grown four true leaves, stress is applied again, samples are taken, and the physiological changes of the Isatis indigotica leaves are measured to reflect the impact of the combined pollution on Isatis indigotica.

[0009] Preferably, the particle sizes of the small and large polystyrene plastics in step (1) are 775.90±61.66 nm and 50.07 μm±1.29 μm, respectively.

[0010] Preferably, the polystyrene plastics of small and large micron-sized particles in step (1) are ultrasonically dispersed before use.

[0011] Preferably, in step (2), ethanol is selected as the cosolvent for the tetracycline TC solution.

[0012] Preferably, the concentration of tetracycline TC solution in step (2) is 50-500 mg·kg -1 .

[0013] Preferably, the concentration of the small and large particle sizes of polystyrene plastics in step (2) is 80-500 mg·kg -1 .

[0014] Preferably, the mass of the soil in step (2) is 400-600 g.

[0015] Preferably, the soaked Isatis indigotica seeds in step (3) are obtained by soaking plump and uniform Isatis indigotica seeds in ultrapure water for half an hour.

[0016] Preferably, the burial depth in step (3) is 1.5-2.5 cm.

[0017] Preferably, during the combined stress process in step (3), Hoagland nutrient solution is used to maintain the soil moisture content at 55-65%, simulating room temperature conditions of 30±2°C.

[0018] Preferably, the re-stress time in step (4) is 13-15 days.

[0019] Preferably, the indicators for measuring physiological changes in the Isatis indigotica leaves in step (4) are: cell membrane permeability, malondialdehyde content, soluble protein content, superoxide anion content, and antioxidant enzyme activity of the Isatis indigotica leaves. Cell membrane permeability is an investigation at the cellular level, malondialdehyde content, soluble protein content, and superoxide anion content are the effects of the plant's resistance to external stress mechanisms, and antioxidant enzyme activity is the effect of stress on the antioxidant enzyme system.

[0020] Preferably, the determination of the antioxidant enzyme activity includes the determination of superoxide dismutase activity, catalase activity, peroxidase activity and glutathione content.

[0021] Beneficial effects

[0022] (1) The present invention selected Isatis indigotica as the test plant. Isatis indigotica is a biennial plant of the Cruciferae family with a wide planting area. It is the basal plant of the traditional Chinese medicinal herbs Radix Isatidis and Folium Isatidis. Currently, most research on Isatis indigotica focuses on the composition and medicinal properties of Radix Isatidis. However, there are few reports on the growth of Isatis indigotica seedlings under combined stress.

[0023] (2) The present invention studied the physiological effects of combined stress on Isatis indigotica leaves from three perspectives. Among them, cell membrane permeability reflects the difficulty of substances passing through biological semipermeable membranes, soluble protein, superoxide anion, and malondialdehyde (MDA) content reflect the physiological changes of plants under external stress, and the degree of influence on the antioxidant enzyme system is reflected from the changes in four different enzyme activities. The data reflect the ecological and environmental risks of combined pollution of polystyrene microplastics and tetracycline at different concentrations, providing a basis for the ecological and environmental risk assessment of combined pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Scanning electron micrographs (ad) of the polystyrene microparticles used in the present invention.

[0025] Figure 2 The effects of PS-NPs with different particle sizes and tetracycline combined stress on the cell membrane permeability of Isatis indigotica seedling leaves: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0026] Figure 3 The effect of PS-NPs with different particle sizes and tetracycline combined stress on the soluble protein content in the leaves of Isatis indigotica seedlings: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0027] Figure 4 Effects of PS-NPs with different particle sizes and tetracycline combined stress on the superoxide anion content in the leaves of Isatis indigotica seedlings: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0028] Figure 5 Effects of combined stress of PS-NPs with different particle sizes and tetracycline on malondialdehyde in the leaves of Isatis indigotica seedlings: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0029] Figure 6 Effects of combined stress of PS-NPs with different particle sizes and tetracycline on superoxide dismutase activity in the leaves of Isatis indigotica seedlings: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0030] Figure 7 Effects of PS-NPs with different particle sizes and tetracycline combined stress on catalase activity in the leaves of Isatis indigotica seedlings: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0031] Figure 8 Effects of PS-NPs with different particle sizes and tetracycline combined stress on the peroxidase activity of Isatis indigotica seedling leaves: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC.

[0032] Figure 9 Effects of PS-NPs with different particle sizes and tetracycline combined stress on glutathione content in the leaves of Isatis indigotica seedlings: (a) 100 mg·kg -1 PS-NPs+TC; (b) 500 mg·kg -1 PS-NPs+TC. DETAILED DESCRIPTION

[0033] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0034] Example 1

[0035] This embodiment provides a method for scientifically assessing the ecological and environmental risks of combined microplastic and antibiotic pollution, including:

[0036] (1) Small and large polystyrene plastic PS-NPs were prepared and ultrasonically dispersed before use. The particle sizes of the two microplastics were 775.90±61.66nm (Group S) and 50.07±1.29μm (Group B), respectively.

[0037] (2) Ethanol was selected as the cosolvent to prepare tetracycline TC solutions with concentration gradients of 50, 100, 200, and 500 mg kg -1 500 g of soil was set in each pot. The tetracycline TC solution was mixed with the small and large micron-sized polystyrene plastic PS-NPs in step (1) and then stirred evenly with the soil. The concentrations of the small and large polystyrene plastic PS-NPs were 100 and 500 mg kg, respectively.-1 ;

[0038] (3) Soil-grown Isatis indigotica was selected as the test plant. Plump and uniform Isatis indigotica seeds were soaked in ultrapure water for half an hour and then evenly placed in the soil sample at a depth of about 2 cm. Combined stress was applied. During the combined stress process, Hoagland nutrient solution was used to maintain the soil moisture content at about 60%, simulating room temperature conditions of 30 ± 2 °C.

[0039] (4) After the Isatis indigotica had grown to four true leaves, the stress was continued for 14 days, samples were taken, and the physiological changes of Isatis indigotica leaves were measured to reflect the effects of the combined pollution on Isatis indigotica;

[0040] The indicators for measuring physiological changes in Isatis indigotica leaves are:

[0041] (a) Cell membrane permeability: Rinse the fresh sample twice with deionized water, then use a disc to remove 0.5 g of the sample. Place the disc into a stoppered test tube containing 20 mL of distilled water, shake thoroughly, and measure the conductivity (S1). Place the stopper in a water bath for 10 minutes, then cool and allow to stand for 10 minutes. Measure the conductivity (S2). The conductivity of distilled water is used as the blank (S0). The relative conductivity is calculated as follows:

[0042] The results are as follows Figure 2 shown.

[0043] (b) Malondialdehyde content: 2 mL of the extracted crude enzyme solution was added to 2 mL of 0.5% thiobarbituric acid solution, shaken well, and the mixture was reacted in a 95°C water bath for 30 min. After removal, it was immediately cooled in an ice bath and heated at 4000 rpm. -1 After centrifugation for 10 min, the absorbance of the supernatant was measured by spectrophotometer at wavelengths of 450 nm, 532 nm, and 600 nm, with a blank of 0.5% thiobarbituric acid. The calculation formula is as follows: MDA (mmol·g -1 FW) = [6.452 × (A 532 -A 600 )-0.559×A 450 ]×V t / V s ×FW, where V t is the total volume of the extract (unit: mL), V s is the volume of supernatant (unit: mL), FW is the fresh weight of plant (unit: g), and the results are as follows Figure 5 shown.

[0044] (c) Soluble protein content: Accurately weigh 25 mg of bovine serum albumin, dissolve it in distilled water to 100 mL, and then dilute it 2.5 times to obtain a concentration of 100 μg mL -1 Prepare the bovine serum albumin standard solution in 6 15 mL stoppered test tubes and add reagents in the order shown in Table 1 to draw a bovine serum albumin standard curve.

[0045] Table 1 Reagents and dosages for soluble protein standard curve determination

[0046]

[0047] Soluble protein was extracted and the absorbance was measured at 595 nm after adding Coomassie brilliant blue dye. The soluble protein content was calculated as follows: protein content in the sample (mg g -1 FW) = C × V t / FW×Vs×1000, where C is the standard curve value (unit: μg), V t is the total volume of the extract (unit: mL), V s is the amount of sample added during the measurement (unit: mL), FW is the fresh weight of the plant (unit: g), and the results are as follows Figure 3 shown.

[0048] The method for extracting soluble protein is as follows: take 0.2 g of the sample to be tested, add a small amount of quartz sand and distilled water to grind into a slurry, dilute to 10 mL, take 2-3 mL of the slurry and homogenize at 10000 r·min -1 , centrifuged at 4℃ for 10min, and the supernatant was the protein extract.

[0049] (d) Superoxide anion content: Accurately weigh 0.100 g of NaNO2, dilute to 100 mL with distilled water, shake well, take 5 mL and dilute to 1000 mL with distilled water to obtain a concentration of 5 μg·L -1 NO2 - Standard solution. Add the reagents to the corresponding stoppered test tubes according to the order in Table 2 and draw a NaNO2 standard curve.

[0050] Table 2 Reagents and dosages for soluble protein standard curve determination

[0051]

[0052] Extraction of O2 - , add p-aminobenzenesulfonic acid and α-naphthylamine and heat in a water bath, measure the absorbance at a wavelength of 530nm, O2 - The calculation formula of O2 content is: - Content (μg·g -1 FW)=2×X×V t / FW×Vs, where X is the NO2 corresponding to the sample solution found from the standard curve. - The O2 concentration is converted to - Concentration, V t is the volume of sample extract (unit: mL), V sis the amount of sample solution taken for color reaction (unit: mL), FW is the fresh weight of the plant (unit: g), and the results are as follows Figure 4 shown.

[0053] Among them, extract O2 - The method is as follows: take 0.4 g of the sample to be tested, grind it and dilute it to 10 mL with PBS, filter it through four layers of gauze, and spin the filtrate at 10,000 r / min. -1 Centrifuge for 15 minutes and take the supernatant for later use.

[0054] (e) Superoxide dismutase activity: The enzyme kit (96T) from Nanjing Jiancheng Biological Co., Ltd. was used. The results were as follows: Figure 6 shown.

[0055] (f) Catalase activity: CAT activity assay: Add the reaction system according to the table below to a 5 mL centrifuge tube. Immediately after the final addition of H2O2, measure the absorbance at a wavelength of 240 nm. Read the absorbance every 30 seconds for a total of 3 minutes. The results are as follows: Figure 7 shown.

[0056] Table 3 Reagents and dosages for CAT activity assay

[0057]

[0058] The calculation formula is as follows: Where ΔA 240 is the absorbance value of the sample at a wavelength of 240 nm, V t is the volume of enzyme extract (unit: mL), V s is the volume of the enzyme solution (unit: mL), t is the reading time (unit: min), and FW is the fresh weight of the plant (unit: g).

[0059] (7) Peroxidase activity: POD activity assay reaction system was added to a 5 mL centrifuge tube according to the table below. After H2O2 was added last, the absorbance was immediately measured at a wavelength of 470 nm. The reading was taken every 30 seconds for a total of 2 minutes. The results were as follows: Figure 8 shown.

[0060] Table 4 Reagents and dosages related to POD activity determination

[0061]

[0062] The calculation formula is as follows: Where ΔA 470 is the absorbance value of the sample at a wavelength of 470 nm, V t is the volume of enzyme extract (unit: mL), V sis the volume of the enzyme solution (unit: mL), t is the reading time (unit: min), and FW is the fresh weight of the plant (unit: g).

[0063] (8) Glutathione content: The glutathione content was determined using an enzyme kit (96T) from Nanjing Jiancheng Biological Co., Ltd. The results were as follows: Figure 9 shown.

[0064] Figure 2 Studies have shown that when plants are exposed to adverse environmental conditions, their cell membranes are damaged, increasing membrane permeability and leading to extravasation of electrolytes within the cells, which in turn increases the conductivity of plant cell extracts. The degree of increased membrane permeability is related to the intensity of the stress and the plant's resistance to stress. The present study found that the concentration of tetracycline in combined stress conditions had no significant effect on leaf cell membrane permeability, maintaining cell membrane conductivity between approximately 40% and 60%.

[0065] Figure 3 The results show that most soluble proteins in plants are enzymes involved in various metabolisms, and changes in their content can be used to understand plant metabolism. The present invention found that combined pollution can reduce the content of soluble protein in Isatis indigotica leaves by about 16.67% to 33.3%.

[0066] Figure 4 It shows that some molecular oxygen (O2) entering the body is reduced to superoxide anion radical (O2 - ), so the determination of O2 in plant tissues under adverse conditions - The production and clearance rates can indirectly reveal the damage status of tissue cells and the strength of their resistance. Among the combined stresses, the stress of smaller microplastic particles caused Isatis indigotica leaves to produce more superoxide anions, which was twice as much as that of the blank group.

[0067] Figure 5 The results showed that MDA is one of the main products of lipid peroxidation in cells. Changes in MDA content in plants can reflect the degree of cell membrane delipidation and the severity of tissue damage caused by superoxide free radicals under adverse conditions. Polystyrene microplastics are the main factor affecting MDA under combined stress, and larger particle size is associated with higher malondialdehyde content.

[0068] Figure 6 The results showed that superoxide dismutase (SOD) is a ubiquitous metalloenzyme in living organisms. It participates in oxidative metabolism and is closely associated with plant resistance to aging and stress, making it a key protective enzyme in plants. At low tetracycline concentrations, SOD activity in leaves was positively correlated with TC concentration. At high tetracycline concentrations, SOD activity decreased and tended to fall below CK, indicating that the threshold for SOD resistance to oxidative stress in Isatis indigotica leaves had been exceeded.

[0069] Figure 7The results show that catalase (CAT) is an enzyme with an iron porphyrin as a cofactor. SOD dismutates oxygen free radicals to produce H2O2 and O2. H2O2 is still oxidatively toxic in the body. Catalase's function is to decompose hydrogen peroxide into molecular oxygen and water, protecting cells from H2O2 toxicity. Combined stress, small- and medium-sized microplastics have a greater impact on CAT activity. At the same tetracycline concentration, small-sized microplastics increase CAT activity more than large-sized microplastics.

[0070] Figure 8 The results show that peroxidase (POD), a marker enzyme of peroxisomes, has multifaceted effects in plant defense responses, converting certain carbohydrates in tissues into lignin and promoting lignification of affected tissues. POD activity in leaves of plants exposed to high-concentration combined microplastic stress was higher than that in plants exposed to low concentrations, indicating that microplastic concentration is the most influential factor on POD activity under combined stress.

[0071] Figure 9 The results indicate that glutathione (GSH) helps maintain normal immune system function and has antioxidant and detoxification effects. The data showed that microplastic particle size had no significant effect on GSH. The GSH content in the groups exposed to the combined stress of high-concentration PS-NPs and TC was not significantly different from that in the control group. In this case, glutathione is not the primary protective substance for plants.

[0072] Comparative Example 1

[0073] Jiang Shiqi [3] The toxic effects of combined microplastic and tetracycline pollution on zoanthids were studied, and a long-term toxic exposure experiment was conducted on zoanthids. This experiment measured the tetracycline content and microplastic content, and explored the adsorption kinetics and isothermal adsorption characteristics of tetracycline on three types of microplastics. However, there is little research on the physical and chemical properties of stressed zoanthids, which is limited to the adsorption relationship between the two pollutants. In contrast, the present invention places greater emphasis on the changes in the physiological characteristics of the test plants themselves, and describes these changes with specific data, more intuitively reflecting the degree of damage caused by stress to Isatis indigotica.

[0074] In the present invention, the cell membrane permeability of the Isatis indigotica leaves in the treatment group was increased compared with the blank (30.4%). The cell membrane permeability of the Isatis indigotica leaves was as high as 67.8% under the combined stress of low concentration of PS-NPs and TC, and as high as 73.5% under the combined stress of high concentration of PS-NPs and TC. The soluble protein content in the Isatis indigotica leaves in the treatment group was significantly increased compared with the blank (6.18 mg·g -1 ) were all reduced. The lowest soluble protein content in leaves was 3.96 mg·g under the combined stress of low concentration PS-NPs and TC. -1The lowest soluble protein content in leaves was 3.22 mg·g under the combined stress of high concentration PS-NPs and TC. -1 The superoxide anion content in the leaves of Isatis indigotica in the treatment group was higher than that in the blank group (0.736 μg·g -1 ), however 500 mg kg -1 The PS-NPs of group B were respectively -1 When the TC was combined with the superoxide anion content in the plant leaves, it dropped to near the blank, which was 0.834 μg·g -1 , 0.893μg·g -1 The combined stress of PS-NPs and TC in group B had a greater effect on MDA than the combined stress of PS-NPs and TC in group S. The MDA content in the leaves of Isatis indigotica reached a maximum of 6.90 mmol·g under the combined stress of low-concentration PS-NPs and TC in group B. -1 When the high concentration of PS-NPs in group B was combined with TC, the MDA content in the leaves of Isatis indigotica reached a maximum of 5.95 mmol·g -1 ; 500mg·kg of different particle sizes -1 PS-NPs and 100 mg·kg -1 Under the combined stress of TC, the SOD activity of Isatis indigotica leaves was the highest, which was 306.12 U·g -1 (Group S), 269.36 U·g -1 (Group B); 100 mg·kg- 1 PS-NPs in group S were compared with 100 mg·kg- 1 The CAT activity of Isatis indigotica leaves was the highest under TC compound treatment, reaching 4.97 U·g -1 , about blank (1.75U·g -1 ) 2.84 times; 100 mg·kg -1 The highest POD activity was 50.65 U·g when the PS-NPs were combined with TC. -1 When 500 mg·kg-1 PS-NPs were combined with TC, the highest POD activity was 139.37 U·g -1 , for the blank group (33.54 U·g -1 ) is 4.22 times.

[0075] References:

[0076] [1]Mga B, Yu LA, Zsb C. Effects of polyethylene microplastic on thephytotoxicity of di-n-butyl phthalate in lettuce(Lactuca sativa L.var.ramosaHort)[J]. Chemosphere, 237(12):44-48.

[0077] [2]Bosker T, Bouwman LJ, Brun NR, et al. Microplastics accumulate onpores in seed capsule and delay germination and root growth of theterrestrial vascular plant Lepidium sativum[J]. Chemosphere, 2019, 226(7):774-781.

[0078] [3] Jiang Shiqi. Study on the toxic effects of microplastics / tetracycline combined pollutants on zoanthids[D]; Guangdong Ocean University, 2021.

Claims

1. A scientific method for assessing the ecological and environmental risks of combined microplastic and antibiotic pollution, including: (1) setting polystyrene plastics of small and large particle sizes; wherein the particle sizes of the polystyrene plastics of small and large particle sizes in step (1) are 775.90±61.66nm and 50.07μm±1.29μm, respectively; (2) preparing a tetracycline solution with a concentration gradient, mixing it with the small and large micron-sized polystyrene plastics prepared in step (1), and then stirring it evenly with the soil; wherein the tetracycline solution concentration is 50-500 mg·kg -1 ; (3) selecting soil-cultured Isatis indigotica as the test plant, burying the soaked Isatis indigotica seeds in the soil stirred evenly in step (2), and subjecting them to combined stress; (4) After the woad has grown four true leaves, stress is applied again, samples are taken, and physiological changes of the woad leaves are measured to reflect the impact of the combined pollution on the woad; The indicators for measuring the physiological changes of Isatis indigotica leaves are: cell membrane permeability, malondialdehyde content, soluble protein content, superoxide anion content, and antioxidant enzyme activity.

2. The method according to claim 1, characterized in that In the step (1), the polystyrene plastics of small and large particle sizes are ultrasonically dispersed before use.

3. The method according to claim 1, characterized in that In the step (2), ethanol is selected as a cosolvent for the tetracycline solution.

4. The method according to claim 1, wherein In step (2), the concentrations of the small and large particle sizes of polystyrene plastics are 80-500 mg·kg -1 ;Soil mass is 400-600g.

5. The method according to claim 1, wherein The soaked Isatis indigotica seeds in step (3) are obtained by soaking plump and uniform Isatis indigotica seeds in ultrapure water for half an hour; the burying depth is 1.5-2.5 cm.

6. The method according to claim 1, wherein During the combined stress process in step (3), the soil moisture content is maintained at 55-65% using Hoagland nutrient solution, simulating room temperature conditions of 30±2°C.

7. The method according to claim 1, characterized in that The re-stress time in step (4) is 13-15 days.

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