Photoaged microplastic composite toxicity detection and risk determination method based on integrated biomarker response index (IBR)

Abstract

The invention belongs to the technical field of environmental pollutant toxicity detection and risk assessment, and particularly relates to a photoaged microplastic composite toxicity detection and risk judgment method based on a comprehensive biomarker response index (IBR), and the method comprises the following steps: S1, preparing photoaged microplastic; s2, performing a biological exposure experiment; s3, multi-dimensional biological response detection; s4, calculating a comprehensive biomarker response index (IBR); s5, composite toxicity risk judgment. According to the method, an IBR method is introduced to integrate multiple biological responses, a single comprehensive index is formed, the objectivity and comparability of toxicity judgment are improved, and the method is suitable for environmental pollutants such as pesticides, flame retardants and drugs. The method is short in experimental period, high in sensitivity and visual in result, can truly reflect the biological influence under the coexistence condition of the light aging micro-plastics and the pollutants, and has good application and popularization values.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollutant toxicity detection and risk assessment technology, specifically involving a method for detecting and determining the toxicity and risk of photo-aged microplastic composites based on the Integrated Biomarker Response Index (IBR). Background Technology

[0002] With the long-term accumulation and photoaging of plastics in the environment, microplastics have become an important source of new pollutants. Photoaging alters the surface structure and chemical composition of microplastics, giving them a stronger ability to adsorb pollutants and interact with organisms. Polystyrene microplastics (PS-MPs) produce a large number of oxidized functional groups (such as carbonyl and hydroxyl groups) after aging, resulting in a significant enhancement of their adsorption capacity. When coexisting with persistent organic pollutants (such as hexabromocyclododecane, HBCD), they can produce significant combined toxic effects, posing a potential threat to the neurodevelopment and behavioral functions of aquatic organisms.

[0003] Current research and risk management of microplastic toxicity mainly focus on virgin microplastics. However, in the real environment, a large number of microplastics undergo aging processes such as light exposure and oxidation, resulting in significant changes in their surface structure, electrical properties, and active components. These changes may significantly enhance toxicity and produce a combined toxicity-enhancing effect with environmental pollutants. Existing detection methods cannot: ① systematically quantify the toxicity changes caused by photoaging; ② determine the "amplification" effect of photoaged microplastics on pollutant toxicity; ③ provide objective and quantitative indicators for determining combined toxicity; ④ be used for the regulation of new pollutants and rapid assessment of ecological safety.

[0004] In existing technologies, microplastic toxicity assessments often rely on single indicators (such as swimming speed or ROS levels), lacking systematic integration and risk classification methods, making it difficult to objectively characterize the combined pollution effects.

[0005] Therefore, there is an urgent need to establish a comprehensive judgment method based on multidimensional biological response signals to quantitatively reveal the degree and risk level of photo-aged microplastic composite toxicity. Summary of the Invention

[0006] The purpose of this invention is to address existing problems by providing a method for detecting and assessing the toxicity and risk of photo-aged microplastic composites based on the Integrated Biomarker Response Index (IBR).

[0007] This invention is achieved through the following technical solution: A method for detecting and assessing the toxicity and risk of photo-aged microplastic composites based on the Integrated Biomarker Response Index (IBR) includes the following steps: S1. Preparation of photo-aged microplastics: The original microplastics were subjected to ultraviolet light aging treatment to obtain photo-aged microplastics; S2, Biological Exposure Experiment: Zebrafish embryos or juveniles were divided into at least four exposure groups, including a blank control group, a pollutant single exposure group, a group exposed to a combination of original microplastics and pollutants, and a group exposed to a combination of photo-aged microplastics and pollutants, and were then subjected to exposure culture. S3, Multidimensional Biological Response Detection: After exposure, multiple biological indicators related to neurotoxicity were measured in each group of zebrafish. S4. Calculate the Integrated Biomarker Response Index (IBR): The biological indicators measured in step S3 are standardized, and the Integrated Biomarker Response Index (IBR) for each exposure group is calculated based on the standardized values. S5. Assessment of Complex Toxicity Risk: The influence of photo-aged microplastics on pollutant toxicity was determined by comparing the IBR values ​​of each exposure group and the IBR values ​​of the combined exposure group of photo-aged microplastics and pollutants with those of the pollutant single exposure group and the original microplastics and pollutant combined exposure group.

[0008] Further, the microplastic mentioned in step S1 is a polystyrene microplastic with a particle size of 1.0 ± 0.05 μm; The conditions for the ultraviolet aging treatment were as follows: continuous irradiation for 14 days at 50°C and 50% humidity under an ultraviolet light source with a wavelength of 254 nm, with a total irradiance of 58 mW·cm. -2 .

[0009] Furthermore, the pollutant mentioned in step S2 is a persistent organic pollutant; The zebrafish embryos were exposed for 2 hours to 5 days after fertilization.

[0010] Furthermore, the aforementioned biological indicators include spontaneous tail movement in embryos, juvenile fish behavior, neurotransmitter content, and neuronal fluorescence imaging.

[0011] Furthermore, the behavioral indicators include the frequency of spontaneous embryonic movement and / or the average swimming speed of juvenile fish; the neurotransmitter content includes acetylcholine (ACh), serotonin (5-HT), and gamma-aminobutyric acid (GABA).

[0012] Furthermore, in step S4, the standardization process employs a formula. In progress, among which... For indicator values, The mean, The standard deviation is denoted as .

[0013] Further, in step S4, the formula for calculating the Integrated Biomarker Response Index (IBR) is as follows: , where ΔSi is the difference or change of the i-th standardized biological indicator relative to the control group.

[0014] Furthermore, in step S5, the determination rule is as follows: (1) Calculate the IBR values ​​for the pollutant-only exposure group, the original microplastic and pollutant combined exposure group, and the photo-aged microplastic and pollutant combined exposure group, respectively; (2) Compare the IBR values ​​of the combined exposure group of photo-aged microplastics and pollutants with the IBR values ​​of the pollutant-only exposure group: When the former is significantly higher than the latter, it is determined that photo-aged microplastics have an amplification effect on the toxicity of pollutants under combined exposure conditions; (3) Further compare the IBR values ​​of the photo-aged microplastics and pollutant combined exposure group with the IBR values ​​of the original microplastics and pollutant combined exposure group: When the two are close or have no significant difference, it is determined that the photoaging process has a limited impact on the toxicity of microplastic composites. (4) When the IBR value of the combined exposure group of photo-aged microplastics and pollutants is lower than the IBR value of the original combined exposure group of microplastics and pollutants, or lower than the IBR value of the pollutant exposure group alone, it is determined that the photo-aged microplastics have a protective or antagonistic effect on the toxicity of pollutants under combined exposure conditions. (5) Based on the above judgment results, classify and output the types of toxic effects of photo-aged microplastics under the combined exposure conditions of pollutants.

[0015] Furthermore, the method can be used for ecotoxicity screening of new or typical pollutants in the environment, environmental risk classification assessment, and regulatory decision support.

[0016] The present invention has the following advantages over the prior art: 1. This invention introduces the IBR method to integrate multiple biological responses into a single comprehensive index, improving the objectivity and comparability of toxicity assessment. This method features a short experimental cycle, high sensitivity, and intuitive results, accurately reflecting the biological impact of photo-aged microplastics coexisting with pollutants, and has significant application and promotion value.

[0017] 2. This invention establishes for the first time a rapid screening and quantitative determination system for the composite toxicity of photo-aged microplastics, unifying different toxicity endpoints into comparable and regulatoryly applicable IBR indicators. It distinguishes between the toxicity of virgin and aged microplastics, revealing the "aging amplification effect," and is applicable to environmental pollutants such as pesticides, flame retardants, and pharmaceuticals. Furthermore, the method of this invention can be used for government supervision of new environmental pollutants, product safety screening, and risk warning. Its operational procedures are standardized, the data is objective, and it is suitable for forming standards and guidelines. Attached Figure Description

[0018] Figure 1 This is a flowchart of the method process; Figure 2 Characterization diagram of aged microplastics; Figure 3 A multidimensional biological response index diagram; Figure 4 This is a graph showing the results of the Integrated Biomarker Response Index (IBR). Detailed Implementation

[0019] To further explain the present invention, the following specific embodiments are described.

[0020] Example 1: A method for detecting and assessing the toxicity and risk of photo-aged microplastic composites based on the Integrated Biomarker Response Index (IBR), comprising the following steps ( Figure 1 ): (1) Preparation of photo-aged microplastics: Polystyrene microspheres (PS) with a particle size of (1.0 ± 0.05 μm) were selected. The microspheres were ultrasonically dispersed in ultrapure water for 10 min, washed three times with pure water to remove surface impurities, and dried for later use. The dried PS samples were spread evenly in a quartz dish and placed in a UV aging chamber (model: Sailham UV-254, China) for continuous irradiation for 14 days at 50°C and 50% humidity. The light source was a 4 × 500 W xenon lamp with a main peak wavelength of 254 nm and a total irradiance of 58 mW·cm². -2 .

[0021] (2) Characterization analysis of photo-aged microplastics: Morphology and structure: The surface morphology before and after aging was observed using a scanning electron microscope (SEM, Hitachi SU8010); Functional group changes: Absorption peak changes (4000–400 cm⁻¹) detected by Fourier transform infrared spectroscopy (FTIR, Bruker Vertex 70) -1 scope); Elemental composition: Energy dispersive spectroscopy (EDS, Oxford X-Max) was used to determine the C and O contents and calculate the O / C ratio.

[0022] Analysis results as follows Figure 2 As shown, the results indicate that the surface morphology and chemical properties of polystyrene microplastics are altered after ultraviolet aging.

[0023] (3) Zebrafish exposure experiment: Exposure methods and sample collection A total of 1200 zebrafish embryos (2 hpf) with normal morphology and synchronous development were selected. The embryos were randomly assigned to 12 sterile culture dishes, with 100 embryos per dish and 50 mL of exposure solution added to each dish. The experiment was divided into four groups (Table 1), with three replicates per group.

[0024] Table 1 ; Note: The particle size of PS and UV-PS is uniformly 1 μm; UV-PS refers to particles after simulated photoaging. Exposure time: 2 hpf — 5 dpf; Exposure system: 50 mL / 100 embryos / 90 mm diameter sterile culture dish; Culture conditions: 28 ± 0.5°C, photoperiod 14 h light / 10 h dark; Exposure fluid replacement: Replace the entire solution every 24 hours to avoid dosage deviation caused by particle sedimentation; Embryo observation: Record embryo survival rate, malformation rate, and hatching rate daily; Non-viable or ruptured embryos should be removed promptly. Sample preservation: At 5 dpf, 24 individuals from each group were randomly selected for behavioral testing; the remaining individuals were anesthetized with ice bath and collected in 1.5 mL EP tubes, stored at -80℃ for later testing.

[0025] (4) Multidimensional biological response detection Embryonic tail-coiling frequency test: This test was used as a sensitive endpoint for early neurotoxicity. The test was conducted 24 hpf post-fertilization. Randomly selected zebrafish embryos from each exposure group were transferred to 96-well plates, one embryo per well. After acclimatization on a microscope platform for 10 min, observation was performed for 1 min using an inverted microscope and its accompanying imaging system. The number of spontaneous tail-coiling cycles per unit time was counted. The embryonic tail-coiling frequency (unit: cycles / min) was calculated and used as an indicator of early neural reflexes and the maturity of primary motor circuits in the spinal cord.

[0026] Testing of zebrafish kinetic behavior: Five-day-fiber (dpf) zebrafish juveniles were placed in 24-well plates, one fish per well. Before the experiment, the 24-well plates were placed in darkness for 20 minutes to acclimate the zebrafish. Then, the plates were placed in a zebrafish behavior tracking and analysis system to monitor the kinetic activity of the juvenile zebrafish under alternating light and dark stimuli for 40 minutes (10 minutes light / 10 minutes darkness). Swimming distance and time data were collected, and the average swimming speed was calculated. Data output was: Raw Data → Preprocessing → Statistical Analysis (ANOVA) Detection of neuronal development in transgenic zebrafish: Embryo collection and exposure methods were the same as above. 5 dpf transgenic zebrafish embryos were hypothermia-anesthetized, and the development of motor neurons in surviving transgenic zebrafish larvae was measured using a stereomicroscope (SMZ25, Japan). Green fluorescent protein (GFP) fluorescence intensity was calculated using ImageJ software. Data processing: ImageJ → ROI setting → Background subtraction → Average pixel intensity statistics. Determination of neurotransmitter levels: The levels of ACh, 5-HT, and GABA in zebrafish were determined using an ELISA kit. The kit employed a one-step sandwich ELISA method with double antibodies. Samples, standards, and HRP-labeled detection antibodies were added sequentially to microwells pre-coated with ACh, 5-HT, and GABA antibodies, followed by incubation and thorough washing. The substrate TMB was used for color development; TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the levels of ACh, 5-HT, and GABA in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the sample concentration was calculated.

[0027] The results of the multidimensional biological response index test are as follows Figure 3 As shown, the differences in typical neurotoxicity-related indicators among the exposure groups are illustrated, including the number of spontaneous tail movements in embryos, the average swimming speed of juvenile fish, changes in neurotransmitter content (ACh, 5-HT, GABA), and neuronal fluorescence intensity.

[0028] The results showed that UV-PS exacerbated the neurotoxic damage caused by HBCD, manifested as decreased motor function, neurotransmitter abnormalities, and significantly enhanced neuronal damage.

[0029] (5) Calculation and risk assessment of Integrated Biomarker Response Index (IBR) Data standardization: All measured biological indicators (swimming speed, neurofluorescence intensity, ACh, 5-HT, GABA, etc.) were standardized using the following formula: ; in, For indicator values, The mean, The standard deviation is denoted as .

[0030] IBR index calculation: The comprehensive response area (ΔA) is calculated based on the standardized values ​​of each indicator, and then summed to obtain the comprehensive response index: ; A higher IBR value indicates a higher overall toxicity level.

[0031] The decision table is shown in Table 2 below.

[0032] Table 2 ; The overall toxic response of zebrafish in different exposure groups (control, HBCD, PS+HBCD, UV-PS+HBCD) is presented in the form of IBR index.

[0033] The results showed that ( Figure 4 The IBR of the UV-PS+HBCD group was significantly higher than that of the PS+HBCD group and the single HBCD group, which verifies that photo-aged microplastics have an enhancing effect on the neurotoxicity of pollutants.

[0034] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting and assessing the toxicity and risk of photo-aged microplastic composites based on the Integrated Biomarker Response Index (IBR), characterized in that, Includes the following steps: S1. Preparation of photo-aged microplastics: The original microplastics were subjected to ultraviolet light aging treatment to obtain photo-aged microplastics; S2, Biological Exposure Experiment: Zebrafish embryos or juveniles were divided into at least four exposure groups, including a blank control group, a pollutant single exposure group, a group exposed to a combination of original microplastics and pollutants, and a group exposed to a combination of photo-aged microplastics and pollutants, and were then subjected to exposure culture. S3, Multidimensional Biological Response Detection: After exposure, multiple biological indicators related to neurotoxicity were measured in each group of zebrafish. S4. Calculate the Integrated Biomarker Response Index (IBR): The biological indicators measured in step S3 are standardized, and the Integrated Biomarker Response Index (IBR) for each exposure group is calculated based on the standardized values. S5. Assessment of Complex Toxicity Risk: The influence of photo-aged microplastics on pollutant toxicity was determined by comparing the IBR values ​​of each exposure group and the IBR values ​​of the combined exposure group of photo-aged microplastics and pollutants with those of the pollutant single exposure group and the original microplastics and pollutant combined exposure group.

2. The method according to claim 1, characterized in that, The microplastics mentioned in step S1 are polystyrene microplastics with a particle size of 1.0 ± 0.05 μm; The conditions for the ultraviolet aging treatment were as follows: continuous irradiation for 14 days at 50°C and 50% humidity under an ultraviolet light source with a wavelength of 254 nm, with a total irradiance of 58 mW·cm. -2 .

3. The method according to claim 1, characterized in that, The pollutant mentioned in step S2 is a persistent organic pollutant; The zebrafish embryos were exposed for 2 hours to 5 days after fertilization.

4. The method according to claim 1, characterized in that, In step S3, the multiple biological indicators include spontaneous tail movement of the embryo, juvenile fish behavior, neurotransmitter content, and neuronal fluorescence imaging.

5. The method according to claim 4, characterized in that, The behavioral indicators include the frequency of spontaneous embryonic movement and / or the average swimming speed of juvenile fish; the neurotransmitter content includes acetylcholine (ACh), serotonin (5-HT) and gamma-aminobutyric acid (GABA).

6. The method according to claim 1, characterized in that, In step S4, the standardization process uses the formula In progress, among which... For indicator values, The mean, The standard deviation is denoted as .

7. The method according to claim 6, characterized in that, In step S4, the formula for calculating the Integrated Biomarker Response Index (IBR) is as follows: , where ΔSi is the difference or change of the i-th standardized biological indicator relative to the control group.

8. The method according to claim 1, characterized in that, In step S5, the determination rule is as follows: (1) Calculate the IBR values ​​for the pollutant-only exposure group, the original microplastic and pollutant combined exposure group, and the photo-aged microplastic and pollutant combined exposure group, respectively; (2) Compare the IBR values ​​of the combined exposure group of photo-aged microplastics and pollutants with the IBR values ​​of the pollutant-only exposure group: When the former is significantly higher than the latter, it is determined that photo-aged microplastics have an amplification effect on the toxicity of pollutants under combined exposure conditions; (3) Further compare the IBR values ​​of the photo-aged microplastics and pollutant combined exposure group with the IBR values ​​of the original microplastics and pollutant combined exposure group: When the two are close or have no significant difference, it is determined that the photoaging process has a limited impact on the toxicity of microplastic composites. (4) When the IBR value of the combined exposure group of photo-aged microplastics and pollutants is lower than the IBR value of the original combined exposure group of microplastics and pollutants, or lower than the IBR value of the pollutant exposure group alone, it is determined that the photo-aged microplastics have a protective or antagonistic effect on the toxicity of pollutants under combined exposure conditions. (5) Based on the above judgment results, classify and output the types of toxic effects of photo-aged microplastics under the combined exposure conditions of pollutants.

9. The method according to claim 1, characterized in that, The method can be used for ecotoxicity screening of new or typical pollutants in the environment, environmental risk classification assessment, and regulatory decision support.

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