Plastic foam toxicity evaluation method combining light aging simulation and biological detection

Abstract

The invention discloses a plastic foam toxicity evaluation method combining light aging simulation and biological detection, and belongs to the technical field of environmental health and microplastic toxicology. The method comprises the following steps: firstly, carrying out accelerated light aging on plastic foam particles under a simulated sunlight condition, and characterizing physicochemical properties (including surface morphology, particle size distribution and functional group change) of the plastic foam particles; then, carrying out toxicity detection by utilizing a model organism caenorhabditis elegans, and systematically evaluating the biological effect of the light aging EPS through indexes such as egg laying amount, movement behavior and body length. According to the method, the light aging process of the plastic foam in the environment can be truly represented, the incidence relation between the physicochemical change and the toxicity effect can be established, and an effective experimental basis and methodological support are provided for scientific evaluation of the environmental health risk of the plastic foam and product safety supervision.

Description

Technical Field

[0001] This invention belongs to the field of environmental health and microplastic toxicology technology, specifically relating to a method for evaluating the toxicity of plastic foam that combines simulated photoaging and biological detection. Background Technology

[0002] Expanded polystyrene (EPS) is widely used in packaging, construction, and electronics due to its lightweight, cushioning, and thermal insulation properties. However, due to its low recycling rate, EPS has become a significant source of microplastics in the environment. Studies show that EPS has been detected in oceans, freshwater, and wastewater discharges at concentrations reaching thousands of micrograms per liter, potentially posing a threat to ecosystems and human health.

[0003] EPS that enters the environment inevitably undergoes photoaging due to exposure to sunlight. The photoaging process causes surface roughening, changes in particle size distribution, and an increase in functional groups, accompanied by the release of harmful substances such as organic additives and heavy metals. These changes may lead to increased toxicity of EPS, particularly its reproductive, developmental, and neurotoxic effects. However, current research primarily focuses on unaged plastic particles, lacking a systematic evaluation method for the toxicity of EPS under photoaging conditions. Summary of the Invention

[0004] The purpose of this invention is to provide a method for evaluating the toxicity of plastic foam that combines simulated photoaging with biological detection. This aims to offer a scientific and practical approach for systematically evaluating the toxic risks of photo-aged plastic foam. By simulating common photoaging processes in the environment and combining this with toxicity testing using model organisms, the method can accurately reflect the changes in the physicochemical properties and potential toxic effects of plastic foam after photoaging, providing technical support for environmental health risk assessment of plastic products.

[0005] A method for evaluating the toxicity of plastic foam that combines simulated photoaging and biological testing includes the following steps: (1) Aging treatment: Plastic foam is subjected to freeze-pulverization and then placed in a photochemical reactor for simulated aging treatment; (2) Physicochemical property testing: The physicochemical properties of the plastic foam treated in step (1) were detected by scanning electron microscopy, X-ray diffraction, contact angle measurement, dynamic light scattering, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy. (3) Biotoxicity testing: Caenorhabditis elegans was bred and exposed to an environment containing aged plastic. Toxicity data were then automatically recorded and analyzed using the WormLab nematode behavior imaging analyzer.

[0006] Furthermore, the cryogenic pulverization process described in step (1) specifically involves first cutting the plastic foam into 5-10 mm blocks, then pre-cooling them in liquid nitrogen, and finally pulverizing and sieving them.

[0007] Furthermore, the pre-cooling treatment involves placing the block of plastic foam in liquid nitrogen for 4-5 minutes, followed by crushing. During crushing, the duration of each crushing session is controlled to be 30-120 seconds, and then supplemental cooling is performed at 30-60-second intervals to prevent softening upon rewarming.

[0008] Furthermore, the particle size of the particles after the crushing and sieving process does not exceed 800 μm.

[0009] Furthermore, the photochemical reaction device described in step (1) is equipped with a xenon lamp to simulate sunlight, during which the temperature in the photochemical reaction device is controlled at 25°C, the humidity at 50%, the power at 500 W, and the radiation intensity at 58 mW / cm². 2 .

[0010] Furthermore, the specific cultivation method of Caenorhabditis elegans described in step (3) is as follows: using Escherichia coli OP50 as food, the wild-type Caenorhabditis elegans model organism placed on the nematode growth medium plate is cultured to the gestation period, and then lysed using a lysis buffer composed of 0.45 M NaOH and 2% HOCl to obtain eggs; in order to obtain fourth-instar larvae of synchronized age, the eggs are placed on a new nematode growth medium plate and cultured for 48 h.

[0011] Furthermore, the WormLab nematode behavior imaging analyzer described in step (3) specifically counts the head swings and body bends of Caenorhabditis elegans. To measure head swings, with the nematode head as the reference, a movement in which the head deflects to the left or right sides at an angle ≥ 10° and returns to its original position is counted as one effective swing. To measure body bends, with the nematode's body axis as the reference, a movement in which the body bends to the ventral or dorsal side at an angle ≥ 30° and forms a "C" or "S" shape is counted as one effective bend.

[0012] Furthermore, in addition to counting head movements and body bending, the nematode's body length and width are automatically recorded and analyzed.

[0013] The beneficial effects of this invention are: This invention first accelerates the photoaging of polystyrene foam particles under simulated sunlight conditions and characterizes their physicochemical properties (including surface morphology, particle size distribution, and functional group changes). Then, using the model organism *Caenorhabditis elegans*, toxicity testing is conducted. The biological effects of photoaged EPS are systematically evaluated through indicators such as oviposition, motility, and body length. This method not only realistically reproduces the photoaging process of polystyrene foam in the environment but also establishes the correlation between physicochemical changes and toxic effects, providing effective experimental evidence and methodological support for the scientific assessment of the environmental health risks of polystyrene foam and the supervision of product safety. The method is simple, scientific, and easy to operate, facilitating large-scale application. Attached Figure Description

[0014] Figure 1 Experimental tests corresponding to Embodiment 2 of the present invention. Figure 1 ; Figure 2 Experimental tests corresponding to Embodiment 2 of the present invention. Figure 2 . Detailed Implementation

[0015] The specific embodiments of the present invention will be further described in detail with reference to the accompanying drawings, examples, and comparative examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0016] Example 1: A method for evaluating the toxicity of plastic foam by combining simulated photoaging and biological testing, comprising the following steps: (1) Aging treatment: First, cut the polystyrene foam into 5-10 mm pieces, then pre-cool them in liquid nitrogen for 4-5 minutes (extend the cooling time by 1-2 minutes after the vigorous bubbling stops). Next, pulverize the foam, controlling each pulverization session to be 30-120 seconds, followed by a 30-60 second cooling interval to prevent softening upon rewarming. Pulverize for a total of 2-8 minutes, ensuring the final particle size after sieving does not exceed 800 μm. Then, place the foam in a photochemical reactor for simulated aging. The reactor is equipped with a xenon lamp to simulate sunlight, maintaining a temperature of 25°C, humidity of 50%, power of 500 W, and radiation intensity of 58 mW / cm². 2 .

[0017] (2) Physicochemical property testing: The physicochemical properties of the plastic foam treated in step (1) were detected by scanning electron microscopy (SEM), X-ray diffraction (XRD), contact angle measurement, dynamic light scattering, Fourier transform infrared spectroscopy (FTIR), and energy dispersive X-ray spectroscopy (XPS). (3) Biotoxicity testing: To cultivate *C. elegans*, *Escherichia coli* OP50 was used as food. Wild-type *C. elegans*, placed on a nematode growth medium plate, were cultured to gestation. Eggs were then obtained by lysis using a lysis buffer consisting of 0.45 M NaOH and 2% HOCl. To obtain age-synchronized fourth-instar larvae, the eggs were cultured on a new nematode growth medium plate for 48 hours. They were then exposed to an environment containing aged plastic, and toxicity data were automatically recorded and analyzed using the WormLab nematode behavior imaging analyzer. Specifically, the WormLab analyzer counted head movements and body bending of *C. elegans*. To measure head movements, a movement with a deflection angle ≥ 10° to the left or right and a return to the original position was counted as one effective movement. To measure body bending, a bending angle ≥ 30° towards the ventral or dorsal side, forming a "C" or "S" shape, was counted as one effective bending. In addition to counting by head swaying and body bending, the nematode's body length and width are automatically recorded and analyzed.

[0018] Example 2: To further test and verify the effectiveness of the present invention, expanded polystyrene (EPS) plastic was selected for testing, including the following steps: (1) Aging treatment: First, cut the EPS into 5-10 mm blocks, then pre-cool them in liquid nitrogen for 4-5 minutes (extend the cooling time by 1-2 minutes after the vigorous bubbling stops). Next, pulverize the EPS, controlling each pulverization session to be 30-120 seconds, followed by a 30-60 second cooling interval to prevent softening upon rewarming. Pulverize for a total of 2-8 minutes. The final particle size after sieving should not exceed 800 μm. The pulverized EPS particles, without subsequent aging treatment, are designated EPS-0. Then, place them in a photochemical reactor for simulated aging treatment. The reactor is equipped with a xenon lamp to simulate sunlight, and the temperature, humidity, power, and radiation intensity are controlled at 25℃, 50%, 500 W, and 58 mW / cm². 2 Specifically, it undergoes a 60-day aging process, and the resulting particles are designated as EPS-60.

[0019] (2) Physicochemical property testing: The physicochemical properties of the EPS processed in step (1) were detected using scanning electron microscopy (SEM), X-ray diffraction (XRD), contact angle measurement, dynamic light scattering, Fourier transform infrared spectroscopy (FTIR), and energy dispersive X-ray spectroscopy (XPS); specifically: EPS was coated with a thin gold film, and images were captured at an accelerating voltage of 20 kV and a working distance of 10.3 mm. FTIR: 10 mg EPS and 200 mg pure potassium bromide were finely ground and transferred to a mold. The mixture was pressed into a thin sheet on a tablet press and then placed in the sample chamber for measurement. XPS analysis used monochromatic Al Kα rays as the excitation source to determine the elemental composition and chemical valence state of the sample surface under high vacuum conditions. Contact angle measurement used the static droplet method. The sample sheet was laid flat on a glass slide, deionized water or other test liquid was added, and the droplet profile was automatically captured and the contact angle value was calculated using a contact angle meter. XRD: 20 mg EPS was detected under conditions of a measurement angle range of 5-90 degrees and a scanning rate of 10 degrees / min.

[0020] (3) Biotoxicity testing: To cultivate *C. elegans*, *Escherichia coli* OP50 was used as food. Wild-type *C. elegans*, placed on a nematode growth medium plate, were cultured to gestation. Eggs were then obtained by lysis using a lysis buffer consisting of 0.45 M NaOH and 2% HOCl. To obtain age-synchronized fourth-instar larvae, the eggs were cultured on a new nematode growth medium plate for 48 hours. The larvae were then exposed to aging-treated EPS (expanded EPS) at concentrations of 0.1 μg / L, 1 μg / L, 10 μg / L, and 100 μg / L. The exposed nematodes were collected for toxicity testing, and the toxicity data were automatically recorded and analyzed using a WormLab nematode behavior imaging analyzer. The WormLab nematode behavior imaging analyzer specifically counts the head swings and body bends of *C. elegans*. To measure head swings, a valid swing is defined as a head rotation angle ≥ 10° to the left or right and a return to the original position. To measure body bends, a valid bend is defined as a body bend angle ≥ 30° towards the ventral or dorsal side, forming a "C" or "S" shape. In addition to counting head swings and body bends, the analyzer also automatically records and analyzes the nematode's body length and width.

[0021] Compared to EPS-0, an EPS-60 exposure group with the same dose was established. The oviposition rate, motility, and body length of *C. elegans* were cultured and measured under the same conditions. Synchronized individuals from the same batch were used, with fixed temperature and food sources, and the exposure time was controlled to be consistent with the vector. Each group had ≥3 biological replicates, and each replicate contained ≥40 nematodes. Intergroup means were compared and statistical tests were performed to quantify the differences in the effects of EPS-60 compared to EPS-0 on nematode reproductive, neural, and developmental indicators. All experiments were conducted under the same temperature and humidity conditions, and at least 30 nematodes were measured in each group to ensure the statistical reliability of the results.

[0022] Specific tests are attached. Figure 1 and 2 As shown. (The attached text is incomplete and cannot be translated.) Figure 1 Figure A in the image is a scanning electron microscope (SEM) image, showing that EPS-0 has a smooth surface, while EPS-60 exhibits obvious surface cracks and wrinkles; (Attached) Figure 1 Figure B in the figure is the XRD spectrum, which shows that the crystallinity of EPS-60 is improved; (See attached image) Figure 1 Figure C in the diagram is the water contact angle diagram. The results show that the contact angle of EPS-60 is slightly lower than that of EPS-0. Figure 1 Figure D in the diagram shows the particle size distribution. The results indicate that the average particle size of EPS-60 decreased from 566.9 μm to 566.9 μm, suggesting that ultraviolet irradiation caused particle fragmentation. Figure 1 Figure E in the figure shows the FTIR spectrum, revealing the compositional changes after photoaging; (See attached image) Figure 1 Figure F in the figure shows the XPS spectrum, where the oxygen content of EPS-60 is significantly increased, with its O / C ratio increasing from 0.084 to 0.30.

[0023] Appendix Figure 2 Figure A shows the oviposition rate data, Figure B shows the body length data, Figure C shows the body bending frequency data, and Figure D shows the head wagging frequency data. It can be seen that *C. elegans* exposed to EPS-60 in the concentration range of 10-100 μg / L exhibited significantly lower oviposition rate, body length, body bending frequency, and head wagging frequency compared to the control group. Furthermore, compared to EPS-0, nematodes exposed to 100 μg / L EPS-60 showed significantly lower oviposition rate, body length, body bending frequency, and head wagging frequency. This indicates that photoaging enhances its biotoxic effects.

[0024] 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 the evaluation of the toxicity of plastic foams combining simulated photodamage with biological detection, characterized in that, It comprises the following steps: (1) aging treatment: The plastic foam is subjected to frozen crushing treatment, and then is placed in a photochemical reaction device for simulated aging treatment; (2) physical and chemical property detection: The plastic foam treated in step (1) is subjected to physical and chemical property detection by using a scanning electron microscope, X-ray diffraction method, contact angle measuring instrument, dynamic light scattering, Fourier transform infrared spectroscopy and energy dispersive X-ray spectroscopy; (3) biological toxicity detection: Caenorhabditis elegans is cultivated, is exposed to an environment containing the plastic after aging treatment, and then is subjected to automatic recording and analysis of toxicity data by using a WormLab nematode behavior imaging analyzer.

2. A method for evaluating the toxicity of plastic foams in combination with the simulation of photoaging and biological detection according to claim 1, characterized in that, The frozen crushing treatment in step (1) is specifically that the plastic foam is cut into blocks with a size of 5-10 mm, is subjected to precooling treatment in liquid nitrogen, and is then crushed and sieved.

3. A method for evaluating the toxicity of plastic foams in combination with the simulation of photoaging and biological detection according to claim 2, characterized in that, The precooling treatment is that the block-shaped plastic foam is placed in liquid nitrogen for 4-5 min, and then is crushed, wherein the crushing time is controlled to be 30-120 s each time, and then is subjected to cold supplement treatment every 30-60 s to prevent softening due to temperature rise.

4. A method for evaluating the toxicity of plastic foams in combination with the simulation of photoaging and biological detection according to claim 2, characterized in that, The particle size of the plastic foam after the crushing and sieving treatment is not more than 800 μm.

5. The method for evaluating the toxicity of plastic foams by combining the photoaging simulation with biological detection according to claim 1, characterized in that, The photochemical reaction device in step (1) is provided with a xenon lamp to simulate sunlight, and the temperature in the photochemical reaction device is controlled at 25°C, the humidity is 50%, the power is 500 W, and the radiation intensity is 58 mW / cm 2 .

6. The method for evaluating the toxicity of plastic bubbles by combining the photoaging simulation with biological detection according to claim 1, characterized in that, The specific cultivation method of Caenorhabditis elegans in step (3) is that Escherichia coli OP50 is used as food, and the model organism wild-type Caenorhabditis elegans placed on a nematode growth medium plate is cultured to the pregnant stage, and then is lysed by using a lysate mixed by 0.45 M NaOH and 2% HOCl to obtain eggs; in order to obtain age-synchronized fourth instar larvae, the eggs are placed on a new nematode growth medium plate and cultured for 48 h.

7. The method for evaluating the toxicity of plastic foams by combining the photoaging simulation with biological detection according to claim 1, characterized in that, The WormLab nematode behavior imaging analyzer in step (3) specifically counts the head swing and body bending of Caenorhabditis elegans; in order to measure the head swing, the deflection angle of the head to the left and right sides is ≥ 10°, and the movement returning to the original position is recorded as 1 effective swing; in order to measure the body bending, the bending angle of the body to the ventral or dorsal side is ≥ 30°, and the movement forming a "C" shape or "S" shape is recorded as 1 effective bending.

8. A method for evaluating the toxicity of plastic foams in combination with the simulation of photoaging and biological detection according to claim 7, characterized in that, In addition to counting the head swing and body bending, the body length and body width of the nematode are also automatically recorded and analyzed.

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