Method for rapidly detecting toxicity of microplastics and antibiotic mixture and application thereof

Abstract

This invention provides a method for rapid detection of the toxicity of a mixture of microplastics and antibiotics, and its application, belonging to the field of environmental ecotoxicology. This invention uses *Chlorella vulgaris* as a model organism, culturing *Chlorella vulgaris* in its logarithmic growth phase under low-dose stress with an optimized culture medium and a mixture of microplastics and antibiotics to obtain a *Chlorella vulgaris* stock solution that responds more rapidly and has a more stable signal to pollutants. Then, through grouped experiments, the Fv / Fm and OD values ​​of the control and experimental groups were measured. 681 The method described in this invention calculates the relative change rate of algae in response to complex pollutants, and determines toxicity or toxicity response thresholds based on the relative change rate. This significantly improves the sensitivity and accuracy of algal responses to complex pollutants and can be effectively used for aquatic environment monitoring / assessment and / or aquatic organism risk early warning.

Description

Technical Field

[0001] This invention belongs to the field of environmental ecotoxicology technology, specifically relating to a method for rapid detection of the toxicity of mixtures of microplastics and antibiotics and its application. Background Technology

[0002] Plastic debris smaller than 5 mm is generally defined as microplastics. Microplastics have a large specific surface area and are highly hydrophobic, easily adsorbing antibiotics from the aquatic environment, thus forming complex pollutants. The toxicity and complexity of treating complex pollutants are greater than those of single pollutants, causing greater harm to the ecological environment. The combined pollution of microplastics and antibiotics in the aquatic environment has become a global environmental problem. Their synergistic effect exacerbates the toxic effects on aquatic organisms; therefore, rapid and accurate detection of such mixtures is crucial for ecological and environmental risk assessment.

[0003] Currently, existing technologies exist for detecting the toxicity of microplastics and antibiotics combined pollutants based on algae. However, most of these technologies utilize *Scenedesmus obliquus*, and their solutions all have certain limitations, particularly regarding the culture medium and algal sensitivity. For example, existing technologies often use conventional BG11 medium, whose nutrient composition is not optimized for microplastic-antibiotic combined pollution responses, failing to provide the algae with a suitable physiological environment to maximize their sensitivity to the combined pollutants. Furthermore, some components in conventional culture media (such as high-concentration phosphates) can bind to antibiotics and adsorb microplastics, reducing the effective interaction concentration between the pollutants and the algae, thus interfering with the accuracy of the response results. Simultaneously, the algae exhibit a high response threshold to low concentrations of the combined pollutants, resulting in insufficient response sensitivity and making it difficult to provide early warning of the risk of microplastic-antibiotic combined pollution in the environment. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide a method for rapidly detecting the toxicity of mixtures of microplastics and antibiotics. Through customized optimization of the culture medium and regulation of algal physiological state, the sensitivity and accuracy of algal response to complex pollutants are significantly improved.

[0005] A second objective of this invention is to provide the application of the method in aquatic environment monitoring / assessment and / or aquatic organism risk early warning.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for rapidly detecting the toxicity of a mixture of microplastics and antibiotics, comprising the following steps: Chlorella in its logarithmic growth phase was cultured in an optimized medium for 20–25 h, then microplastics and antibiotics were added to a final concentration of 0.002 mg / L, and the culture was continued for another 8–12 h. After centrifugation, washing, and resuspending, the culture was brought to a concentration of 1 × 10⁻⁶. 5 cells·mL -1 ~9×10 5 cells·mL -1 Chlorella stock solution; A blank control group and a pollutant experimental group were set up. The optimized culture medium and pollutant solution were mixed with equal volumes of Chlorella stock solution and cultured for 20-25 hours. The Fv / Fm and OD of each group were then measured. 681 And / or ROS content; calculate the relative change rate of the index, and determine toxicity or toxicity response threshold based on the relative change rate of the index; The optimized culture medium was based on BG11 medium, with dipotassium hydrogen phosphate and ferric ammonium citrate removed, sodium nitrate concentration adjusted to 0.8 g / L, and 0.05 mmol / L sodium pyrophosphate, 0.1 g / L urea, 0.08 mmol / L EDTA-sodium iron and 0.02 mmol / L salicylic acid added.

[0007] Preferably, the optimized culture medium comprises: sodium nitrate 0.8 g / L, urea 0.1 g / L, sodium pyrophosphate 0.05 mmol / L, magnesium sulfate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, EDTA-iron sodium 0.08 mmol / L, disodium ethylenediaminetetraacetate 0.001 g / L, salicylic acid 0.02 mmol / L, and 1000× trace element solution A5 1 mL / L.

[0008] Preferably, the microplastics include polystyrene microplastics, aminopolystyrene microplastics, polyvinyl chloride microplastics and / or carboxylated polystyrene microplastics; the antibiotics include ciprofloxacin, sulfapyridine, sulfamethazine, sulfadiazine, sulfamethoxazole and / or levofloxacin.

[0009] Preferably, the cultivation conditions are: 22~26℃, light intensity 3000~3500 lux, light-dark ratio 12h:12h.

[0010] Preferably, the centrifugation and washing are repeated 2 to 4 times, with each centrifugation being performed at 4000 to 6000 r / min for 3 to 5 minutes, and the washing medium being an optimized culture medium.

[0011] Preferably, the resuspension medium is an optimized culture medium.

[0012] Preferably, the concentration of microplastics in the pollutant solution is 0.002~10 mg / L, and the concentration of antibiotics is 0.0002~1 mg / L.

[0013] Preferably, dark adaptation is performed for 10-12 minutes before Fv / Fm detection; the conditions for Fv / Fm detection include: excitation wavelength 440nm and emission wavelength 685nm.

[0014] This invention also provides the application of the above method in water environment monitoring and assessment.

[0015] This invention also provides the application of the above method in aquatic organism risk early warning.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention uses common Chlorella as a model organism and makes breakthroughs from the perspectives of optimizing culture medium formulation and enhancing algal sensitivity. By adjusting the nutrient composition of the culture medium, removing interfering components, and pre-activating the algal stress response system, the toxicity response sensitivity to microplastic-antibiotic mixtures can be significantly improved, with a low response threshold and accurate and stable results. At the same time, the single-cell characteristics of common Chlorella make the response signal more uniform and more stable, effectively solving the core pain points of high toxicity response threshold and unstable results in existing technologies.

[0017] The method described in this invention significantly improves the sensitivity of toxicity response to microplastic-antibiotic mixtures and has strong universality. It not only breaks through the bottleneck of existing technology in toxicity testing of low-concentration pollutants, but also can stably adapt to the complex pollution of microplastics with different functional groups (amino and carboxyl modified) and different types of antibiotics (sulfonamides and quinolones), thus having higher universality.

[0018] The method described in this invention shortens the pollutant response time to 20-25 hours, effectively improving the overall detection efficiency of the toxicity of microplastics and antibiotic mixtures. It is fully adaptable to the emergency response needs of sudden pollution events, and has low application cost, requires no special equipment, and is easy to operate. Detailed Implementation

[0019] This invention provides a method for rapidly detecting the toxicity of a mixture of microplastics and antibiotics, comprising the following steps: Chlorella in its logarithmic growth phase was cultured in an optimized medium for 20–25 h, then microplastics and antibiotics were added to a final concentration of 0.002 mg / L, and the culture was continued for another 8–12 h. After centrifugation, washing, and resuspending, the culture was brought to a concentration of 1 × 10⁻⁶. 5 cells·mL -1 ~9×10 5 cells·mL -1 Chlorella stock solution; A blank control group and a pollutant experimental group were set up. The optimized culture medium and pollutant solution were mixed with equal volumes of Chlorella stock solution and cultured for 20-25 hours. The Fv / Fm and OD of each group were then measured. 681 And / or ROS content; calculate the relative change rate of the index, and determine toxicity or toxicity response threshold based on the relative change rate of the index.

[0020] This invention uses common Chlorella as a model organism. Chlorella has a single-celled structure, and its cell membrane has a larger contact area with pollutants. Compared with the multicellular aggregated growth of Scenedesmus oblique, it responds to pollutants more quickly and the signal is more stable.

[0021] The optimized culture medium of this invention uses BG11 medium as the base medium, removing dipotassium hydrogen phosphate and ferric ammonium citrate, adjusting the sodium nitrate concentration to 0.8 g / L, and adding 0.05 mmol / L sodium pyrophosphate, 0.1 g / L urea, 0.08 mmol / L EDTA-sodium iron, and 0.02 mmol / L salicylic acid. Preferably, the components of the optimized culture medium are: sodium nitrate 0.8 g / L, urea 0.1 g / L, sodium pyrophosphate 0.05 mmol / L, magnesium sulfate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, EDTA-sodium iron 0.08 mmol / L, disodium ethylenediaminetetraacetate 0.001 g / L, salicylic acid 0.02 mmol / L, and 1 mL / L of 1000× trace element solution A5. This invention effectively reduces the adsorption interference of phosphorus on pollutants by using sodium pyrophosphate as the phosphorus source; it also enhances the metabolic activity of Chlorella by reducing the concentration of sodium nitrate as the nitrogen source and adding urea to create a mixed nitrogen source; and it promotes chlorophyll synthesis in Chlorella by adding EDTA-sodium iron to the adjusted basal medium, thereby enhancing the sensitivity of photosynthesis to pollutants. Furthermore, it activates the antioxidant stress system of Chlorella by adding salicylic acid. Based on conventional BG11 medium, this invention eliminates high concentrations of phosphate, adjusts the form and concentration of nitrogen source, and adds sensitivity enhancers. This eliminates the adsorption interference of components on pollutants while providing a suitable nutrient environment for Chlorella, thus strengthening its stress response to pollutants.

[0022] In this invention, the components of the 1000× trace element solution A5 include 2.86 g / L boric acid, 1.81 g / L manganese chloride tetrahydrate, 0.222 g / L zinc sulfate heptahydrate, 0.39 g / L sodium molybdate dihydrate, 0.079 g / L copper sulfate pentahydrate, and 0.049 g / L cobalt nitrate hexahydrate. As an optional embodiment, the preparation method of the optimized culture medium of this invention includes: dissolving the basic salt components sequentially in approximately 800 mL of deionized water, adding 1 mL of trace element solution A5, and bringing the volume to 1 L; autoclaving at 121°C for 15-20 min.

[0023] This invention involves pre-culturing Chlorella in an optimized culture medium, followed by the addition of microplastics and antibiotics at a final concentration of 0.002 mg / L to the medium for further cultivation, thereby pre-activating the algal stress system. By pre-culturing Chlorella with low doses of microplastics and antibiotics, and employing a low-dose stress pre-culturing method, combined with the rapid reproduction characteristics of common Chlorella, this invention can quickly activate its antioxidant stress system and cell membrane signaling pathways, reducing the algal body's toxic response threshold to pollutants.

[0024] In this invention, the microplastics include polystyrene microplastics, aminopolystyrene microplastics, polyvinyl chloride microplastics, and / or carboxylated polystyrene microplastics; the antibiotics include ciprofloxacin, sulfapyridine, sulfamethazine, sulfadiazine, sulfamethoxazole, and / or levofloxacin. Preferably, this invention selects any one of the microplastics and antibiotics as the type of microplastic and antibiotic added in the low-dose stress pre-culture.

[0025] In this invention, the culture conditions for Chlorella are: 22~26℃, light intensity 3000~3500 lux, light-dark ratio 12h:12h; wherein the temperature is preferably 23~25℃, more preferably 24℃; and the light intensity is preferably 3100~3400 lux, more preferably 3200~3300 lux.

[0026] This invention involves centrifuging and washing the pre-activated Chlorella solution, repeating the centrifugation and washing 2-4 times, preferably 3 times. Each centrifugation is performed at 4000-6000 r / min for 3-5 min, with a preferred centrifugation speed of 5000 r / min and a preferred time of 4 min. The washing medium and resuspension medium are both optimized. This invention purifies Chlorella by centrifugation and washing to remove residual mixed stress agents, resuspends it in optimized medium, and adjusts the concentration to obtain a sensitivity-enhanced ordinary Chlorella stock solution.

[0027] The contaminant solution described in this invention is the test solution. In this contaminant solution, the concentration of microplastics is 0.002~10 mg / L, and the concentration of antibiotics is 0.0002~1 mg / L. In the microplastic-antibiotic mixed contaminant solution of this invention, the mixing concentration ratio of microplastics and antibiotics can be selected as 1:10~10:1. In this invention, the contaminant solution is mixed with an equal volume of Chlorella stock solution and then cultured. The culture conditions for Chlorella are: 22~26℃, light intensity 3000~3500 lux, light-dark ratio 12h:12h; wherein the preferred temperature is 23~25℃, more preferably 24℃; and the preferred light intensity is 3100~3400 lux, more preferably 3200~3300 lux.

[0028] The Fv / Fm detection described in this invention is a maximum photochemical quantum yield detection method. It calculates Fv / Fm = (Fm - Fo) / Fm by measuring the minimum fluorescence (Fo) of algal cells after dark adaptation and the maximum fluorescence (Fm) under saturated pulsed light excitation. Preferably, dark adaptation is performed for 10-12 minutes before Fv / Fm detection. The conditions for Fv / Fm detection include: excitation wavelength 440 nm and emission wavelength 685 nm.

[0029] The OD of the present invention 681 The detection method is optical density detection, which utilizes the characteristic that chlorophyll a has a maximum absorption peak at a wavelength of 680 nm to reflect the algal cell concentration by measuring the absorbance.

[0030] The ROS content detection method described in this invention is a reactive oxygen species content detection method, which can be selected as the DCFH-DA fluorescent probe method, in conjunction with a fluorescent microplate reader or flow cytometer to detect reactive oxygen species content; after the DCFH-DA probe enters the cell, it is hydrolyzed into DCFH by esterase, and DCFH reacts with ROS to generate fluorescent DCF, and the fluorescence intensity is proportional to the ROS content.

[0031] This invention detects Fv / Fm and OD in each group after culture. 681 And / or ROS content; calculate the relative change rate of the index, and determine toxicity or toxicity response threshold based on the relative change rate of the index.

[0032] The relative change rate of the indicators described in this invention includes photosynthetic indicators (Fv / Fm, OD). 681 The relative inhibition rate of photosynthesis and the relative increase rate of oxidative stress indicators (ROS). Relative inhibition rate of photosynthesis indicators (Fv / Fm, OD) 681 The relative increase rate of oxidative stress index (ROS) is calculated as follows: (Experimental group test value - Experimental group test value) / Blank control group test value × 100%; (Experimental group test value - Experimental group test value) / Blank control group test value × 100%. The blank control group consists of groups that only receive optimized culture medium and Chlorella stock solution, without any contaminants, and its index value is the baseline reference value.

[0033] As an optional implementation, the present invention is based on the photosynthetic efficiency index (OD). 681 The relative inhibition rate was calculated by fitting the inhibition rate data using a custom-written Logit function in Matlab and then calculating EC. 50 Value (half-maximal effect concentration), and converted to pEC. 50 (-lgEC) 50 ), using pEC 50 Toxicity is determined by pEC values. 50 The higher the value, the stronger the toxicity.

[0034] As another optional implementation, the present invention sets Fv / Fm and OD respectively. 681 The weighting of ROS content, based on the weighting of the relative change rates of each indicator, uniformly characterizes the overall response intensity of algae to pollutants. Preferably, this invention sets the weighting of Fv / Fm (core parameter of photosynthesis) to 0.4 and OD... 681 (Chlorophyll content characterization) weight 0.3, ROS (oxidative stress level) weight 0.3; Overall response value = Fv / Fm relative inhibition rate × 0.4 + OD 681 The relative inhibition rate is multiplied by 0.3, and the relative increase rate of ROS is multiplied by 0.3. This invention can determine toxicity based on the magnitude of the comprehensive response value, such as <20% non-toxic or almost non-toxic, 20%~50% low toxicity, 50%~80% moderate toxicity, and >80% high toxicity.

[0035] This invention also provides the application of the above method in water environment monitoring / assessment and / or aquatic organism risk early warning.

[0036] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] Unless otherwise specified, the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1 A rapid method for detecting the toxicity of a mixture of microplastics and antibiotics: The optimized culture medium composition was as follows: sodium nitrate 0.8 g / L, urea 0.1 g / L, sodium pyrophosphate 0.05 mmol / L, magnesium sulfate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, EDTA-iron sodium 0.08 mmol / L, disodium ethylenediaminetetraacetate 0.001 g / L, salicylic acid 0.02 mmol / L, and 1000× trace element solution A5 1 mL / L. 1000× trace element solution A5 consisted of: boric acid 2.86 g / L, manganese chloride tetrahydrate 1.81 g / L, zinc sulfate heptahydrate 0.222 g / L, sodium molybdate dihydrate 0.39 g / L, copper sulfate pentahydrate 0.079 g / L, and cobalt nitrate hexahydrate 0.049 g / L. Optimize the culture medium preparation: Dissolve the basic salt components sequentially in about 800 mL of deionized water, add 1 mL of trace element solution A5, and bring the volume to 1 L; autoclave at 121℃ for 20 min.

[0040] Chlorella vulgaris (FACHB-8) in the logarithmic growth phase was selected and diluted to a concentration of 4 × 10⁻⁶ using optimized culture medium. 5 cells·mL -1 The cells were pre-cultured in a light incubator for 24 hours under the following conditions: 25℃, light intensity 3000 lux, and light-dark ratio 12h:12h. A low dose of mixed stress agent (0.002 mg / L polystyrene microplastics + 0.0002 mg / L ciprofloxacin) was added to the pre-cultured Chlorella solution, and the culture was continued for another 10 hours. The Chlorella solution was then centrifuged and washed three times (5000 rpm, 5 min, with optimized culture medium) to remove residual mixed stress agent. The solution was then resuspended in optimized culture medium and the concentration adjusted to 5 × 10⁻⁶. 5 cells·mL -1 Chlorella stock solution was obtained.

[0041] An exposure system was constructed using 96-well colorless transparent microplates, with a blank control group and an experimental group, each with three replicates. Ultrapure water was added to the outer wells of the microplates to create a constant temperature and humidity barrier to avoid edge effect interference. The sample preparation for each well was as follows: the blank control group was prepared with 100 μL of optimized culture medium + 100 μL of Chlorella stock solution; the experimental group was prepared with 100 μL of a microplastic-antibiotic mixed solution (the test solution, 0.01 mg / L polystyrene microplastic + 0.001 mg / L sulfamethoxazole mixed solution) + 100 μL of Chlorella stock solution, with a total volume of 200 μL for each well. The microplates were then incubated in an intelligent light incubator for 24 h under the following conditions: 25 °C, light intensity 3000 lux, and light-dark ratio 12 h:12 h.

[0042] After cultivation, the core indicators (Fv / Fm, OD) of each group of Chlorella solutions were simultaneously detected using a multi-functional microplate reader. 681 (ROS content); among which, Fv / Fm requires dark adaptation for 12 minutes before detection, excitation wavelength is 440nm and emission wavelength is 685nm; OD 681 The results were obtained using an enzyme-linked immunosorbent assay (ELISA) reader; the ROS content was detected using the DCFH-DA fluorescent probe method, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0043] Calculate the relative inhibition rate / relative increase rate of each indicator: relative inhibition rate of photosynthetic indicators (Fv / Fm, OD) 681 Calculate separately) = (Blank control group test value - Experimental group test value) / Blank control group test value × 100%; Relative increase rate of oxidative stress index (ROS) = (Experimental group test value - Blank control group test value) / Blank control group test value × 100%.

[0044] According to the photosynthetic index (OD) 681 Toxicity assessment or toxicity response threshold determination is performed based on the relative inhibition rate: the inhibition rate data is fitted using a custom-written Logit function in Matlab, and EC is calculated. 50 Value (half-maximal effect concentration), and converted to pEC. 50 (-lgEC) 50 ), using pEC 50 Toxicity is determined by pEC values. 50 The higher the value, the stronger the toxicity.

[0045] Based on the relative change rates of the three sets of indicators, combined with the indicator weights (Fv / Fm weight 0.4, OD...), 681 Calculate the overall response value (overall response value = Fv / Fm relative inhibition rate × 0.4 + OD) with weights of 0.3 and ROS weights of 0.3. 681 The relative inhibition rate × 0.3 + the relative increase rate of ROS × 0.3 is used to determine the toxicity of the mixture or the toxicity response threshold.

[0046] Example 2 The difference between this embodiment and Embodiment 1 is that the microplastic is amino polystyrene microplastic.

[0047] Example 3 The difference between this embodiment and Embodiment 1 is that the microplastic is polyvinyl chloride microplastic.

[0048] Example 4 The difference between this embodiment and Embodiment 1 is that the microplastic is a carboxylated polystyrene microplastic.

[0049] Example 5 The difference between this embodiment and Embodiment 1 is that the antibiotic is sulfapyridine.

[0050] Example 6 The difference between this embodiment and Embodiment 1 is that the antibiotic is sulfamethazine.

[0051] Example 7 The difference between this embodiment and Embodiment 1 is that the antibiotic is sulfadiazine.

[0052] Example 8 The difference between this embodiment and Embodiment 1 is that the antibiotic is sulfamethoxypyridazine.

[0053] Example 9 The difference between this embodiment and Embodiment 1 is that the antibiotic is levofloxacin.

[0054] Experimental Example 1 Validation of the effects of optimizing culture medium and enhancing the sensitivity of Chlorella 1. Group Setup: Four experimental groups were set up: "Conventional Culture Medium + Common Chlorella" group (control group 1), "Conventional Culture Medium + Enhanced Chlorella" group (experimental group 1), "Optimized Culture Medium + Common Chlorella" group (experimental group 2), and "Optimized Culture Medium + Enhanced Chlorella" group (experimental group 3). The optimized culture medium was the same as in Example 1, the conventional culture medium was BG11 medium, the enhanced Chlorella was the Chlorella stock solution prepared in Example 1, and the common Chlorella was the common Chlorella in the logarithmic growth phase used in Example 1 (the density of the common and enhanced Chlorella used was the same).

[0055] 2. Exposure Treatment: The treatment method was the same as in Example 1. The test solution was a mixture of 0.01 mg / L polystyrene microplastics and 0.001 mg / L sulfamethoxazole, and the mixture was incubated for 24 hours. The relative inhibition rate of photosynthesis (Fv / Fm, OD) was detected. 681 The relative increase rates of oxidative stress (ROS) and other indicators are shown in the table below.

[0056] Table 1 Comparison of index data of the experimental groups

[0057] The results showed that the synergistic effect of optimizing the culture medium and enhancing sensitivity, combined with the single-cell advantage of Chlorella vulgaris, could significantly improve the algal body's sensitivity to the toxicity response of compound pollutants.

[0058] Experimental Example 2 Comparison of detection data for different types of microplastics / antibiotics under the optimized system of this invention: The relative inhibition rates (Fv / Fm, OD) of photosynthetic indicators in Examples 1-9 were statistically analyzed respectively. 681 The relative increase rate of oxidative stress index (ROS) and the overall response value were calculated based on the results shown in the table below.

[0059] Table 2 Comparison of index data for different types of microplastics / antibiotics under optimized system

[0060] As shown in the table above, the optimized system of this invention is applicable to the toxicity detection of various types of microplastics and antibiotic mixtures.

[0061] Experimental Example 3 Detection of toxicity gradients and pEC of microplastic-antibiotic mixtures with different functional groups 50 Quantitative analysis: The experiment was conducted using the optimized culture medium and Chlorella stock solution from Example 1. The experimental steps are as follows: Preparation of mixed stock solutions: Microplastics and antibiotics were mixed at a ratio of 10:1 (mass ratio) to prepare a mixed stock solution with an initial concentration of 10 mg / L (10 mg / L microplastics + 1 mg / L antibiotics). Specific combinations were as follows: PS-CIP, COOH-PS-LVX, PS-LVX, NH2-PS-CIP, COOH-PS-CIP, and NH2-PS-LVX. High and low ratio combinations were also prepared: PS-LVX (high ratio 20:1), NH2-PS-CIP (high ratio 20:1), PS-CIP+LVX (low ratio 1:1), and COOH-PS-CIP+LVX (low ratio 1:1). A total of 10 microplastic-antibiotic combinations were prepared. PS: Polystyrene microplastics; COOH-PS: Carboxyl-modified polystyrene microplastics; NH2-PS: Amino-modified polystyrene microplastics; CIP: Ciprofloxacin; LVX: Levofloxacin.

[0062] Concentration gradient dilution: Twelve concentration gradients were designed for the 10 mixed stock solutions with a dilution factor F=0.7. The solutions were then diluted stepwise with optimized culture medium, resulting in a concentration range of 0.0005~8.23 mg / L (covering the entire range of toxic effects from no to significant inhibition).

[0063] Microplate system construction: Microplate pretreatment: 300 μL of ultrapure water was added to the outer 36 wells (rows 1 / 12 and columns 1 / 12) to prevent edge effects and water evaporation; Blank control group setting: columns 2, 6, 7, and 11 served as blank control groups, with 100 μL of ultrapure water added to each well; Experimental group setting: 10 groups of mixed drugs with 12 concentration gradients were added to columns 3, 4, 5 and 8, 9, and 10, with 100 μL of mixed drug solution added to each well; Algal solution addition: 100 μL of Chlorella stock solution was added to both the blank control group and the experimental group, with a total volume of 200 μL per well; Replication setting: All treatments were replicated on 3 microplates.

[0064] Culture and Detection: Culture conditions: The microplates were placed in an artificial light incubator at 25℃, light intensity of 3500 lux, and a light-dark ratio of 12h:12h, with exposure times of 0h and 24h. Detection parameters: The optical density (OD) at 681nm was measured using an Infinite 200 Pro multi-mode microplate reader. 681 Data calculation: Inhibition rate = (OD value of blank control group - OD value of experimental group) / OD value of blank control group × 100%; The inhibition rate data were fitted using a self-written Logit function in Matlab, and EC was calculated. 50 Value (half-maximal effect concentration), and converted to pEC. 50 (-lgEC) 50 pEC 50 The higher the value, the stronger the toxicity, as shown in the table below.

[0065] Table 3 pEC of different experimental groups 50 value

[0066] The order of toxicity of the 10 mixtures was as follows: PS-CIP < PS-LVX < PS-CIP+LVX (low ratio 1:1) < PS-LVX (high ratio 20:1) < COOH-PS-CIP < COOH-PS-LVX < COOH-PS-CIP+LVX (low ratio 1:1) < NH2-PS-CIP < NH2-PS-LVX < NH2-PS-CIP (high ratio 20:1). The results showed that the pEC of the mixture of NH2-PS, COOH-PS, and quinolone antibiotics was... 50 The value was higher than that of unmodified PS, indicating that amino / carboxyl modification significantly enhances the toxicity of microplastic-antibiotic mixtures, and the effect of amino modification is greater than that of carboxyl modification; pEC of LVX and microplastic mixtures 50 The value is higher than CIP, indicating that levofloxacin is slightly more toxic than ciprofloxacin; the pEC value of the microplastics:antibiotic combination of 20:1 50 A value higher than 10:1 indicates that an increased proportion of microplastics enhances synergistic toxicity; the CIP+LVX bisquinolone combination pEC 50 The value is higher than that of a single antibiotic, indicating that the combination of multiple quinolones can have cumulative toxicity, making it a high-risk combination of pollutants that should be of greater concern.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid detection of the toxicity of a mixture of microplastics and antibiotics, characterized in that, Includes the following steps: Chlorella in its logarithmic growth phase was cultured in an optimized medium for 20–25 h, then microplastics and antibiotics were added to a final concentration of 0.002 mg / L, and the culture was continued for another 8–12 h. After centrifugation, washing, and resuspending, the culture was brought to a concentration of 1 × 10⁻⁶. 5 cells·mL -1 ~9×10 5 cells·mL -1 Chlorella stock solution; A blank control group and a pollutant experimental group were set up. The optimized culture medium and pollutant solution were mixed with equal volumes of Chlorella stock solution and cultured for 20-25 hours. The Fv / Fm and OD of each group were then measured. 681 And / or ROS content; calculate the relative change rate of the index, and determine toxicity or toxicity response threshold based on the relative change rate of the index; The optimized culture medium was based on BG11 medium, with dipotassium hydrogen phosphate and ferric ammonium citrate removed, sodium nitrate concentration adjusted to 0.8 g / L, and 0.05 mmol / L sodium pyrophosphate, 0.1 g / L urea, 0.08 mmol / L EDTA-sodium iron and 0.02 mmol / L salicylic acid added.

2. The method according to claim 1, characterized in that, The optimized culture medium consists of: sodium nitrate 0.8 g / L, urea 0.1 g / L, sodium pyrophosphate 0.05 mmol / L, magnesium sulfate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, sodium carbonate 0.02 g / L, citric acid 0.006 g / L, EDTA-iron sodium 0.08 mmol / L, disodium ethylenediaminetetraacetate 0.001 g / L, salicylic acid 0.02 mmol / L, and 1000× trace element solution A5 1 mL / L.

3. The method according to claim 1, characterized in that, The microplastics include polystyrene microplastics, aminopolystyrene microplastics, polyvinyl chloride microplastics and / or carboxylated polystyrene microplastics; the antibiotics include ciprofloxacin, sulfapyridine, sulfamethazine, sulfadiazine, sulfamethoxazole and / or levofloxacin.

4. The method according to claim 1, characterized in that, The cultivation conditions are: 22~26℃, light intensity 3000~3500 lux, light-dark ratio 12h:12h.

5. The method according to claim 1, characterized in that, The centrifugation and washing were repeated 2 to 4 times, with each centrifugation performed at 4000 to 6000 r / min for 3 to 5 minutes, and the washing medium being an optimized culture medium.

6. The method according to claim 1, characterized in that, The resuspension medium is an optimized culture medium.

7. The method according to claim 1, characterized in that, The concentration of microplastics in the pollutant solution is 0.002~10 mg / L, and the concentration of antibiotics is 0.0002~1 mg / L.

8. The method according to claim 1, characterized in that, Before Fv / Fm detection, dark adaptation is performed for 10-12 minutes; the conditions for Fv / Fm detection include: excitation wavelength 440nm and emission wavelength 685nm.

9. The application of the method according to any one of claims 1 to 8 in water environment monitoring and assessment.

10. The application of the method according to any one of claims 1 to 8 in aquatic organism risk early warning.

Images