Method for rapidly detecting composite toxicity of micro-nano plastic and coexisting pollutants

Abstract

The invention discloses a method for rapidly detecting the composite toxicity of micro-nano plastic and coexisting pollutants, which comprises the following steps of: preparing a working bacterial suspension, namely culturing bacteria and separating and resuspending thalli; poison exposure: sequentially adding the pollutant solution, the micro-nano plastic dispersion liquid and the working bacterium suspension into each hole of a light-proof 96-hole plate to form an exposure solution; detecting the luminous intensity: detecting the luminous intensity of each hole by using a microplate reader at high flux, and calculating the luminous inhibition ratio; and compound toxicity judgment: judging the action type and the action intensity of the compound toxicity by using an independent action model. By combining a Q67 toxicity test and a microwell plate high-throughput detection technology, the action type and the action strength of the composite toxicity are judged by utilizing an independent action model, and the toxicity effects of dozens of composite exposure groups can be synchronously evaluated in a single exposure experiment of 60 minutes, so that the composite pollution risk evaluation efficiency is remarkably improved, and the evaluation cost is reduced.

Description

Technical Field

[0001] This invention belongs to the field of environmental risk assessment technology for pollutants, specifically relating to a method for rapid detection of the combined toxicity of micro-nanoplastics and coexisting pollutants. Background Technology

[0002] Microplastics and nanoplastics have become ubiquitous pollutants in the global aquatic environment, often coexisting with various other environmental pollutants such as heavy metals, antibiotics, plastic additives, and pesticides. Due to their large specific surface area and high hydrophobicity, microplastics and nanoplastics readily interact with coexisting pollutants in the aquatic environment, producing synergistic, antagonistic, and additive complex toxic effects on aquatic organisms, threatening ecological security and human health. Therefore, assessing the complex toxic effects of microplastics and coexisting pollutants is crucial for accurately evaluating the environmental risks posed by microplastics and nanoplastics.

[0003] Currently, the assessment of the combined toxicity of microplastics and coexisting pollutants mainly relies on traditional biotoxicity tests. These methods are typically time-consuming, labor-intensive, and costly. For example, assessing the combined toxicity of a pollutant and microplastics requires 2-3 days for a standard bacterial toxicity test and even up to 30 days for a fish toxicity test. Therefore, there is an urgent need to develop rapid and inexpensive new methods to assess the combined toxicity of microplastics and coexisting pollutants.

[0004] Vibrio qinghaiensis sp.-Q67 is a typical freshwater bioluminescent bacterium capable of rapidly responding to pollutants within tens of minutes, thus it is widely used in environmental monitoring. Combining high-throughput microplate detection technology with Q67 toxicity testing holds promise for constructing a high-throughput testing platform for the combined toxicity of micro- and nano-plastics and coexisting pollutants, enabling rapid assessment of the environmental risks of large-scale combined pollutants. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapidly detecting the combined toxicity of micro / nanoplastics and coexisting contaminants, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting contaminants, comprising the following steps:

[0007] (1) Preparation of working bacterial suspension:

[0008] a) Bacterial culture: After thawing Vibrio qinghaiensis Q67 glycerol bacteria frozen at -80℃ in a 37℃ water bath, 100μL of glycerol bacteria was transferred to 100mL of liquid culture medium and shaken overnight at 22℃ and 180r / min. The bacterial culture was harvested during the logarithmic growth phase. The culture medium consisted of 13.6mg KH2PO4, 35.8mg Na2HPO4·12H2O, 0.25g MgSO4·7H2O, 0.61g MgCl2·6H2O, 33.0mg CaCl2, 1.34g NaHCO3, 1.54g NaCl, 5.0g yeast extract, 5.0g tryptone, 3.0g glycerol, and 1000mL deionized water.

[0009] b) Cell isolation and resuspension: The bacterial suspension was centrifuged at 3000g and 4℃ for 8 min, and the supernatant was discarded. The bacterial cells were resuspended in 0.85% NaCl solution (containing 0.75% supernatant) to prepare the Q67 bacterial suspension, which was stored at 0℃ for later use. The Q67 bacterial suspension was diluted 5.25 times with 0.85% NaCl solution at room temperature to obtain the working bacterial suspension for subsequent toxicity testing.

[0010] (2) Exposure to toxic substances: 75 μL of contaminant solution, 75 μL of micro-nano plastic dispersion and 50 μL of working bacterial suspension were added to each well of an opaque 96-well plate to form an exposure solution of 200 μL.

[0011] (3) Detection of luminescence intensity: The luminescence intensity of each well was detected by high-throughput microplate reader, and the luminescence inhibition rate was calculated;

[0012] (4) Assessment of combined toxicity: The type and intensity of combined toxicity are determined using the independent action model (IA).

[0013] Preferably, the test bacteria is Vibrio qinghaiensis Q67, and the final bacterial concentration is 4–5 × 10⁻⁶. 5 CFU / mL.

[0014] In any of the above embodiments, it is preferred that the exposure time of the pollutant, micro-nanoplastics and test bacteria is 60 minutes.

[0015] Preferably, in any of the above embodiments, the concentration of sodium chloride in the exposure solution is 0.85%, the concentration of bacterial culture supernatant is 0.035%, and the bacteria can luminesce normally in this solution, and the micro-nanoplastics can be uniformly dispersed.

[0016] Preferably, in any of the above schemes, the combined exposure experiment is a 4×4 orthogonal design, and the concentration of the pollutant is its respective EC50. 50 The concentrations of micro- and nano-plastics were 1 / 50, 1 / 5, 1, and 2 times that of the standard; the concentrations of micro- and nano-plastics were 0.1, 1, 10, and 100 mg / L.

[0017] Preferably, in any of the above schemes, the combined toxicity effects include three types: synergistic, antagonistic, and additive; by comparing the model prediction values ​​(E0) of the combined toxicity... IA The type of combined toxicity is determined by E and measured values: IA Between the upper limit of the 95% confidence interval of the measured value (E) UCL ) and lower limit (E) LCL When E is positive, it is an additive effect; when E is negative, it is an additive effect. IA Greater than E UCL When E is present, it is an antagonistic effect; when E is present, it is an antagonistic effect. IA Less than E LCL At that time, it is a synergistic effect.

[0018] Preferably, in any of the above schemes, the composite toxicity intensity (V) is... cg V represents the deviation between the model predictions and measured values ​​of the composite toxicity. cg Positive and negative values ​​represent the strength of antagonistic and synergistic effects, respectively. V cg The larger the absolute value, the stronger the antagonistic or synergistic effect.

[0019] The technical effects and advantages of this invention are as follows: This method for rapid detection of the combined toxicity of micro-nanoplastics and coexisting pollutants combines Q67 toxicity testing and microplate high-throughput detection technology. It uses an independent action model to determine the type and intensity of combined toxicity, and can simultaneously assess the toxic effects of dozens of combined exposure groups in a single 60-minute exposure experiment. This significantly improves the efficiency of combined pollution risk assessment and reduces assessment costs. It has the advantages of high efficiency, simple operation, and low cost, and can rapidly assess the combined toxicity of 750 groups of micro-nanoplastics and coexisting pollutants within one day. Attached Figure Description

[0020] Figure 1 This refers to the arrangement of the samples on a 96-well plate.

[0021] Figure 2 The types and intensity of the toxic effects of heavy metal cations (a), anions (b) combined with polystyrene micro / nanoplastics;

[0022] Figure 3 The types and intensity of the toxic effects of antibiotics combined with polystyrene micro / nanoplastics. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.

[0025] Example 1: Rapid assessment of the combined toxicity of various micro / nanoplastics and heavy metals

[0026] This embodiment evaluated the combined toxicity of 28 groups of four polystyrene micro / nanoplastics and seven typical heavy metals. The four polystyrene micro / nanoplastics were unmodified polystyrene particles with particle sizes of 50 and 100 nanometers (denoted as PS, respectively). 50 and PS 100 ), 100 nm carboxyl-modified polystyrene particles (COOH-PS) 100 ) and amino-modified polystyrene particles (NH2-PS) 100 The seven heavy metals are Zn 2+ Cu 2+ Pb 2+ Cd 2+ Cr 3+ , and All 28 groups underwent a 4×4 orthogonal experiment, with a total of 16 concentration groups. The four exposure concentrations for micro / nanoplastics were 0.1, 1, 10, and 100 mg / L, and the four exposure concentrations for heavy metals were their respective half-maximal effective concentrations (EC50). 50 1 / 50, 1 / 5, 1, 2 times.

[0027] The distribution of the sample on the 96-well plate is as follows Figure 1 As shown. The positive control was 0.17 mg / L Zn. 2+ Its luminescence inhibition rate should be around 50%; the negative control (i.e., blank control) is 0.85% NaCl. In a white, opaque 96-well plate, 75 μL of heavy metal solution (diluted with 1.7% NaCl solution) and 75 μL of micro / nanoplastics suspension (diluted with deionized water) were added to each well sequentially. After equilibration at 22±1℃ for 1 h, 50 μL of Q67 bacterial suspension was added to each well using a 12-channel pipette. The final exposed system was 0.85% sodium chloride solution (containing 0.035% bacterial culture supernatant), and the final bacterial concentration was 4–5 × 10⁻⁶. 5 CFU / mL. After 60 min of exposure, the luminescence intensity of each microwell was measured using an ELISA reader. The average relative luminescence unit (I0) of the negative control group and the average relative luminescence unit (I) of each treatment group were calculated. The luminescence inhibition effect (E) of the treatment group on Q67 was calculated according to formula (1).

[0028] E=(I0-I) / I0 (1)

[0029] The type of action of the combined toxicity effect was evaluated using an independent-action model. The model-predicted value E of the combined toxicity effect was calculated according to formula (2). IA By comparing the predicted value E of the combined toxicity effect. IA The type of combined toxic effects is determined by combining measured values ​​with E. IA Between the upper limit of the 95% confidence interval (OCIs) of the measured value (E) UCL ) and lower limit (E) LCL When E is positive, it is an additive effect; when E is negative, it is an additive effect. IA Greater than E UCL When E is present, it is an antagonistic effect; when E is present, it is an antagonistic effect. IA Less than E LCL At that time, it is a synergistic effect.

[0030] E IA =E(c mix )=1-[1-E(c1)]×[1-E(c2)] (2)

[0031] Where c1 and c2 represent the concentrations of micro-nanoplastics and coexisting pollutants in the mixture, respectively. mix E(c1) represents the total concentration of the mixture, and E(c2) and E(c2) represent the inhibitory effects of micro / nanoplastics and coexisting pollutants when c1 and c2 concentrations act individually, respectively. mix ) is a mixture in c mix The combined effect at different concentrations.

[0032] Using a synergistic-antagonistic heatmap based on an independent action model (SAHmap) IA Quantify and visualize the type and intensity of combined toxicity. Calculate SAHmap using formula (3). IA The values ​​corresponding to each color level (V) cg ), where positive and negative values ​​represent the strength of antagonistic and synergistic effects, respectively. V cg It characterizes the degree of deviation between the predicted values ​​of the IA model and the experimental observations, and its absolute value reflects the strength of the antagonistic or synergistic effect.

[0033]

[0034] Experimental results are as follows Figure 2 As shown, heavy metal cations (Zn) 2+ Cu 2+ Pb 2+ Cd 2+ Cr 3+ When exposed to polystyrene nanoplastic composites, heavy metal anions ( ) mainly exhibit antagonistic or additive effects; and When combined with polystyrene nanoplastics, a synergistic or additive effect is observed. The strength of this synergistic or antagonistic effect depends on various factors, including the particle size and surface modification groups of the nanoplastics, the nature of the heavy metals, and the exposure concentration. Small-particle-size (50 nm) nanoplastics exhibit a stronger antagonistic / synergistic effect with heavy metals than larger (100 nm) particles. Carboxylation modification enhances the antagonistic effect of nanoplastics with metal cations, while amination modification enhances their antagonistic effect with... The synergistic effect; metal cations with smaller hydrated ionic radii and higher charge numbers exhibit higher antagonistic strength; high-concentration nanoplastics exhibit a stronger complexing effect, while heavy metal cations in EC 50 The most pronounced antagonistic effect was observed at a concentration of [specific concentration not specified]. Anions are at 1 / 5 EC 50 The synergistic effect was most pronounced at the concentration of [specific concentration not specified].

[0035] Example 2: Rapid assessment of the combined toxicity of various micro / nanoplastics and organic pollutants

[0036] In this example, antibiotics were used as representative organic pollutants to evaluate the combined toxicity of 20 groups of four polystyrene-based nanoplastics and five antibiotics. The five antibiotics were ciprofloxacin (CIP), norfloxacin (NOR), chlortetracycline (CTC), oxytetracycline (OTC), and tetracycline (TC). The types and experimental methods of the four polystyrene-based nanoplastics were the same as in Example 1.

[0037] Experimental results are as follows Figure 3 As shown, CIP, NOR, OTC, and TC, when combined with polystyrene nanoplastics, mainly exhibit synergistic or additive effects; while the combination of CTC and polystyrene nanoplastics shows antagonistic or additive effects. The strength of the synergistic or antagonistic effects is closely related to the particle size and surface modification groups of the nanoplastics, the properties of the antibiotics, and the exposure concentration. The synergistic effect of small-particle nanoplastics (50 nm) with antibiotics is more significant than that of large-particle nanoplastics (100 nm); CTC shows an antagonistic effect with aminated nanoplastics, but an additive effect with other nanoplastics; highly water-soluble antibiotics show a stronger synergistic effect with nanoplastics; high-concentration nanoplastics show a stronger composite effect, while CIP, NOR, and CTC show a stronger composite effect in EC. 50 The most significant synergistic or antagonistic effects were observed at concentrations of 1 / 5 EC, while TC and OTC showed the most pronounced synergistic or antagonistic effects. 50 The synergistic effect was most pronounced at the concentration of [specific concentration not specified].

[0038] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting contaminants, characterized in that: Includes the following steps: S1: Preparation of working bacterial suspension, which includes bacterial culture and cell isolation and resuspension; S2: Toxic exposure: Add the contaminant solution, micro-nano plastic dispersion and working bacterial suspension sequentially to each well of an opaque 96-well plate to form the exposure solution; S3: Luminescence intensity detection: The luminescence intensity of each well is detected in high throughput using an ELISA reader, and the luminescence inhibition rate is calculated; S4: Assessment of combined toxicity: Use the independent action model to determine the type and intensity of combined toxicity.

2. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting pollutants according to claim 1, characterized in that: The bacteria were Vibrio qinghaiensis Q67 at a final concentration of 4–5 × 10⁻⁶. 5 CFU / mL.

3. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting pollutants according to claim 2, characterized in that: The bacterial culture process involves thawing the frozen Vibrio qinghaiensis Q67 glycerol bacteria in a water bath, transferring them to a liquid culture medium, shaking overnight, and harvesting the bacterial solution during the logarithmic growth phase.

4. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting pollutants according to claim 3, characterized in that: The steps of bacterial cell isolation and resuspension are as follows: centrifuge the bacterial solution, discard the supernatant, resuspend the bacterial cells in NaCl solution to prepare Q67 bacterial suspension, store it at 0℃ for later use, and dilute the Q67 bacterial suspension 5.25 times with room temperature NaCl solution to obtain working bacterial suspension for subsequent toxicity testing.

5. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting pollutants according to claim 1, characterized in that: In step S2, the combined exposure time of the pollutants, micro-nanoplastics, and bacteria is 60 minutes.

6. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting contaminants according to claim 5, characterized in that: The concentration of sodium chloride in the exposure solution is 0.85%, and the concentration of bacterial culture supernatant is 0.035%. In this solution, the bacteria can emit light normally, and the micro-nanoplastics can be uniformly dispersed.

7. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting pollutants according to claim 1, characterized in that: In step S4, the method for determining the type of combined toxicity is as follows: First, calculate the luminescence suppression effect: E = (I0 - I) / I0 Where E represents the luminescence inhibition effect, I0 represents the average relative luminescence units of the negative control group, and I represents the average relative luminescence units of each treatment group; Then, the model prediction value E of the combined toxicity effect is calculated using the following formula. IA By comparing the predicted value E of the combined toxicity effect IA The type of combined toxic effects is determined by combining measured values ​​with E. IA When E is between the upper and lower limits of the 95% confidence interval of the measured value, it is an additive effect; when E IA Greater than E UCL When E is present, it is an antagonistic effect; when E is present, it is an antagonistic effect. IA Less than E LCL At that time, it is a synergistic effect; E IA =E(c mix )=1-[1-E(c1)]×[1-E(c2)] Where c1 and c2 represent the concentrations of micro-nanoplastics and coexisting pollutants in the mixture, respectively. mix E(c1) represents the total concentration of the mixture, and E(c2) and E(c2) represent the inhibitory effects of micro / nanoplastics and coexisting pollutants when c1 and c2 concentrations act individually, respectively. mix ) is a mixture in c mix The combined effect at different concentrations.

8. The method for rapid detection of the combined toxicity of micro / nanoplastics and coexisting contaminants according to claim 7, characterized in that: In step S4, the method for determining the intensity of the combined toxicity is as follows: SAHmap is calculated using the following formula. IA The values ​​corresponding to each color level, with positive and negative values ​​representing the intensity of antagonistic and synergistic effects, respectively; Where V cg For SAHmap IA The values ​​corresponding to each color level, E IA E is the predicted value for combined toxic effects. UCL E represents the upper limit of the 95% confidence interval for the measured value. LCL This is the lower limit of the 95% confidence interval for the measured value.

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