Recycled water environment toxicology detection method adopting biomarker

Abstract

The invention provides a reclaimed water environment toxicology detection method adopting biomarkers, which comprises the following steps: respectively preparing four biological sensing units by using expanded nephroplasmosis, chlorella vulgaris, zebra fish and jonquil; the four biological sensing units comprise an ecological sensing unit, a photosynthetic sensing unit, a developmental toxicity sensing unit and a plant sensing unit; the method comprises the following steps: continuously pumping a water sample to be detected, filtering suspended impurities, introducing the water sample to be detected into four micro-chambers for culturing, detecting toxicological data after the culture is completed, and respectively placing one of four biological sensing units in each of the four micro-chambers; and performing data processing on the toxicology data, and outputting a toxicity monitoring report. Therefore, the scientific and reliable reclaimed water environment toxicology detection early warning method is constructed, the detection precision and the ecological coverage range are effectively balanced, the limitation of existing chemical indexes is made up, and safe recycling of water resources is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of reclaimed water treatment technology, and in particular relates to a method for environmental toxicology detection of reclaimed water using biomarkers. Background Technology

[0002] With the increasing severity of water scarcity, wastewater reuse and resource recovery have become key means to alleviate urban water environment pressures and promote sustainable development. After primary, secondary, and even advanced treatment, urban wastewater produces reclaimed water that can be widely used in non-potable applications such as municipal miscellaneous uses, industrial cooling, greening irrigation, and landscape river replenishment. To ensure the safety of reclaimed water, a series of water quality standards have been formulated both domestically and internationally, such as my country's "Water Quality Standard for Urban Wastewater Reuse for Miscellaneous Uses" (GB / T 18920-2022). These standards mainly set detection limits based on physicochemical indicators, such as pH, BOD5, ammonia nitrogen, total nitrogen, total phosphorus, heavy metal ions, and fluoride, with traditional chemical analysis methods as the primary detection method. While these methods have some guiding significance in assessing conventional pollutants, they cannot fully reflect the biotoxic effects of complex pollutants in reclaimed water, let alone assess potential unknown toxins and their combined effects.

[0003] In reality, even after advanced treatment, when indicators such as COD, nitrogen, and phosphorus meet standards, urban sewage may still retain various trace organic pollutants, such as endocrine disruptors, drug metabolic residues, antibiotics, and pesticide degradation intermediates. These pollutants may exhibit synergistic, cumulative, or non-linear toxic effects, creating "hidden toxicity risks." Therefore, developing scientific and reliable methods for detecting and warning of biotoxicological contamination in reclaimed water to compensate for the limitations of existing chemical indicators is an important direction for ensuring the safe reuse of water resources.

[0004] Currently, chemical analysis remains the most widely used method for water quality testing. Its operational procedures generally include: collecting water samples and performing pretreatment steps such as filtration and solid-phase extraction, followed by qualitative and quantitative analysis of pollutants in the water using chromatography-mass spectrometry instruments such as GC-MS, LC-MS / MS, ICP-MS, or AAS. These methods have the advantages of high sensitivity and high selectivity, but also have significant limitations: the equipment is expensive, maintenance costs are high, pretreatment steps are complex and time-consuming, and it is difficult to achieve rapid online or on-site monitoring; more importantly, they can only detect known target substances and are difficult to reveal unknown toxic factors or the combined toxic effects between pollutants.

[0005] To overcome the aforementioned problems, a series of biochemical and cell / molecular biological methods have been developed in recent years for the toxicological assessment of reclaimed water. For example, in vitro cell models are used for MTT cell activity detection, DNA damage analysis, and determination of specific gene expression levels, which can indirectly reflect the interference of pollutants on biological metabolic processes and molecular mechanisms. These methods have certain value in the study of toxicity mechanisms, but due to the species limitations of cell lines and the sensitivity of experimental conditions to components such as turbidity and organic matter in water samples, the results have weak correlation with the toxic effects on actual ecosystems, and the reproducibility is not high, making it difficult to extend to routine monitoring and on-site early warning systems.

[0006] In summary, current toxicological testing of reclaimed water still mainly relies on chemical analysis methods. Although these methods play an important role in assessing compliance with physicochemical indicators, they have significant shortcomings in identifying ecological risks, responding to complex pollution, and providing rapid on-site early warning. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a method for environmental toxicology detection of reclaimed water using biomarkers. This method aims to address the problem that existing methods for detecting reclaimed water quality cannot effectively and comprehensively assess biological toxicity effects, unknown toxins, and their combined toxicity effects. In this way, an efficient early warning method can be constructed that can accurately identify the comprehensive biological toxicity of reclaimed water and is suitable for on-site monitoring, thereby making up for the shortcomings of existing physicochemical indicators in ecological risk assessment when ensuring the safe reuse of reclaimed water.

[0008] To solve the above-mentioned technical problems, the present invention proposes a method for environmental toxicology detection of reclaimed water using biomarkers, the steps of which include: S1. Four types of biosensory units were prepared using *Nematocystis glomeratus*, *Chlorella vulgaris*, zebrafish, and *Kalanchoe blossfeldiana*. The four types of biosensory units include an ecosensory unit, a photosynthetic sensory unit, a developmental toxicity sensory unit, and a plant sensory unit. S2. The water sample to be tested is extracted by continuous pump suction, suspended impurities are filtered out, and the water sample to be tested is introduced into four micro chambers for cultivation. After cultivation, the toxicological data are detected. Each of the four micro chambers contains one of four biosensor units. S3. Process the toxicological data and output a toxicity monitoring report.

[0009] In some embodiments, step S1 includes: S1.1 In a six-well plate, add two autoclaved wheat grains and 10 mL of sterile distilled water to each well, and inoculate with 10 cells that have undergone expansion culture. 4 Ind. / mL of *Bombyx mori* were cultured at 25°C under normal light and dark conditions, gently shaken once daily, until the density stabilized at 10. 4After ind. / mL, collect 5mL of culture medium, centrifuge to remove the culture medium, and then suspend in the same volume of sterile water to obtain the ecological sensing unit; S1.2, The logarithmic growth phase seeds of common Chlorella are seeded at 10... 5 Cells / mL were seeded in culture medium and cultured in shake flasks at 25°C with a light-dark cycle every 12 hours until OD reached. 680 =0.8, take 10 mL of algal solution, centrifuge for 3 minutes, discard the supernatant to obtain algal cells, resuspend the algal cells in 10 mL of fresh culture medium, and adjust the concentration to 10. 5 cells / mL, to obtain photosynthetic sensing units; S1.3. Zebrafish were placed in a fully automated recirculating aquaculture system for 7 days for acclimatization, fed once a day. The water quality in the recirculating aquaculture system was maintained at a temperature of 28±0.5℃ and a dissolved oxygen level of 7.0 mg·L⁻¹. -1 Light: The photocycle of darkness is 14 hours: 10 hours. After one week of cultivation, the population of zebrafish tends to stabilize and developmental toxicity sensory units are obtained. S1.4 Select healthy Kalanchoe plants without disease spots from the middle and lower parts of the plant. Irrigate every 3 days using drip irrigation with 300 mL per plant. Cultivate the plants under natural light and darkness or light and darkness cycles of 12 hours to obtain plant sensory units.

[0010] In some embodiments, step S2 includes: S2.1. A peristaltic pump is used to extract the water sample to be tested at a constant rate of 0.5 mL / min. A 20 μm stainless steel microporous filter screen is connected in series to filter the water sample to be tested. An online pressure sensor is set to monitor the pressure drop of the stainless steel microporous filter screen. When the pressure drop exceeds the set threshold, it prompts to clean or replace the stainless steel microporous filter screen. S2.2 The filtered water sample enters the multi-way distribution valve and is divided into four channels. Each channel is separately delivered to four micro-chambers. All micro-chambers are placed in a constant temperature chamber at 25±0.5℃. The algae, zebrafish and plant chambers are equipped with independent LED light sources to simulate the day and night cycle. S2.3. The ecological sensing units, photosynthetic sensing units, developmental toxicity sensing units, and plant sensing units were placed in their respective microchambers for cultivation. S2.4 After cultivation, the ecological sensing unit, photosynthetic sensing unit, developmental toxicity sensing unit, and plant sensing unit were tested and treated to obtain toxicological data. The toxicological data included data on multiple biomarkers, including total protein, five antioxidant system markers (including total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, and malondialdehyde content), five energy metabolism system markers (including triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity), and chlorophyll content.

[0011] In some embodiments, step S2.4 includes: S2.4.1 After the end of the culture of the ecological sensing unit, the community in the microcell was transferred to a pre-cooled centrifuge tube, centrifuged and the supernatant was discarded. The cell slurry was washed with phosphate buffer and lysed by shaking at 4°C for 15 minutes. After centrifugation, the crude enzyme solution of the supernatant was collected. The total protein content was detected by a kit. The total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the ecological sensing unit were detected by an enzyme-linked immunosorbent assay kit at 37°C, reaction volume of 100µL, and incubation for 30 minutes. S2.4.2. Take 50 mL of photosynthetic sensing unit, centrifuge for 15 minutes, and wash three times with phosphate buffer until the final concentration of photosynthetic sensing unit is 10. 7 Cells / mL, add 1mL phosphate buffer and lysis buffer, perform low-temperature lysis using a high-performance tissue homogenizer, centrifuge for 10 minutes, collect the supernatant, collect the crude enzyme solution, and use a kit to detect the total protein content. Use an enzyme-linked immunosorbent assay (ELISA) kit at 37℃, 100µL reaction volume, and incubate for 30 minutes to detect the total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, and other parameters of the photosynthetic sensing unit. Ketokinase activity, glutathione reductase activity, and glutamate dehydrogenase activity were measured. An additional 8 mL of photosynthetic sensing unit was centrifuged at 1000 rpm for 5 minutes and then at 3000 rpm for 10 minutes. 5 mL of anhydrous ethanol was added, and the mixture was shaken for 1 minute. After storage at 4°C for 24 hours, the mixture was centrifuged for 10 minutes, and the supernatant was collected. Using anhydrous ethanol as a reference, the absorbance was measured at wavelengths of 470, 646, 663, and 750 nm using a spectrophotometer to determine the chlorophyll content. S2.4.3. Five developmental toxicity receptor units of similar developmental size were randomly selected from the microchamber, anesthetized, weighed, and then placed in centrifuge tubes. They were stored at -80℃ for later use. The developmental toxicity receptor unit tissues were added to pre-cooled phosphate buffer at a ratio of 1g:5ml, homogenized with lysis buffer in a biological sample homogenizer, centrifuged, and the supernatant crude enzyme solution was collected. The total protein content was detected using a kit. The total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the developmental toxicity receptor units were detected using an enzyme-linked immunosorbent assay kit at 37℃, a reaction volume of 100µL, and an incubation period of 30 minutes. S2.4.4 Weigh and chop the leaves below the middle of the plant's sensory unit, or leaves with similar leaf areas at the same position. Mix them thoroughly, quickly dispense them into cryovials, and store them at -80℃. Add phosphate buffer, homogenize the tissue with lysis buffer in a biological sample homogenizer, centrifuge, and collect the crude enzyme solution from the supernatant. Use a kit to detect the total protein content. Use an enzyme-linked immunosorbent assay (ELISA) kit at 37℃, 100µL reaction volume, and incubation for 30 minutes to detect the total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the plant sensory unit. Then, take leaves below the middle of the plant sensory unit, or leaves with similar leaf areas at the same position, and use a TYS-N instrument to determine the chlorophyll content.

[0012] In some embodiments, step S3 includes: S3.1 Calculate the mean and standard deviation of each biomarker data in the toxicological data of one of the four biological sensory units, and obtain the standardized results of each biomarker data through the mean and standard deviation; S3.2 Calculate the non-negative score for each biomarker data using the standardized results of each biomarker data; S3.3 Sum the absolute values ​​of the non-negative scores of each biomarker data to obtain the risk index of each biosensory unit and output a toxicity monitoring report.

[0013] In some embodiments, step S3.1 includes: S3.1.1. Take the average of the data for each biomarker obtained from one of the four biosensor units on the same sampling day to obtain the daily mean X of each biomarker data. The formula for calculating the daily mean X of each biomarker data is as follows: in, The number of biological sensory units, For the biomarker data of the g-th biosensory unit; S3.1.2 Summarize the X values ​​for each sampling day and calculate the mean value of each biomarker data. with standard deviation The calculation formula is: in, The time of the sampling date. For each biomarker data point sampled at time t, the daily mean value for that biomarker. S3.1.3, the daily average of each biomarker data on the sampling day at time t. The standardization process is performed using the following formula: The standardized results for each biomarker data sampled at time t.

[0014] In some embodiments, step S3.2 includes: S3.2.1 Determine the direction of response based on the changes in data of each biomarker over culture time; S3.2.2 If the malondialdehyde content increases with incubation time, then the response direction is determined to be activating. ,in, As a response value, if any one of the following decreases with culture time: total protein content, total antioxidant activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, or glutamate dehydrogenase activity, then... ; S3.2.3, Minimum standardized value for all time points The absolute value is used as the offset to calculate the non-negative score for each biomarker data point, using the following formula: in, The non-negative score is the score of the i-th biomarker data from the sampling day at time t.

[0015] In some embodiments, step S3.3 includes: S3.3.1 Summing up the absolute values ​​of the non-negative scores of all biomarker data for each biosensor unit on the same sampling day, using the following formula: in, The risk index is n, where n is the total number of biomarker data. S3.3.2, if If the value is less than or equal to the first threshold, then output a low-risk result; otherwise... If the value is greater than the first threshold and less than or equal to the second threshold, then output a medium-risk result. If the result exceeds the second threshold, a high-risk result will be output.

[0016] Compared with existing technologies, the environmental toxicology detection method for reclaimed water using biomarkers in this invention has the following advantages: This comprehensive assessment covers multiple levels and endpoints. From protozoa (Diffuse-like organisms), primary producers (Chlorella), vertebrates (zebrafish), to terrestrial plants (Kalanchoe), four biosensory units provide multiple perspectives including ecology, photosynthesis, and development, truly achieving a holistic toxicity fingerprint from molecular enzyme activity to individual growth, making the identification of mixed pollution and synergistic effects more reliable. It considers both acute and chronic toxicity; Diffuse-like organisms and Chlorella show developmental and photosynthetic damage within days, while zebrafish and Kalanchoe can capture chronic, cumulative toxicity over a longer period. The superposition of multiple timescales forms a toxicity panorama, enabling both rapid early warning and long-term risk assessment. Data processing and model output offer high automation and visualization advantages. Through standardization, non-negative scoring, and risk index calculation, toxicity monitoring reports are generated, comprehensively improving decision-making efficiency and control effectiveness. This effectively and comprehensively assesses biotoxicity effects, unknown toxins, and their combined toxicity effects, constructing a highly efficient early warning method that can accurately identify the comprehensive biotoxicity of reclaimed water and is suitable for on-site monitoring, thus compensating for the shortcomings of existing physicochemical indicators in ecological risk assessment to ensure the safe reuse of reclaimed water. Attached Figure Description

[0017] Figure 1 This is a graph showing the total protein content of *Desmodium styracifolium* in one embodiment of the present invention; Figure 2 This is a partial biomarker data diagram of *Desmodium styracifolium* in one embodiment of the present invention; Figure 3 This is a data graph of another part of the biomarkers of *Desmodium styracifolium* in one embodiment of the present invention; Figure 4 This is a graph showing the total protein content of common Chlorella in one embodiment of the present invention; Figure 5 This is a partial biomarker data diagram of common Chlorella in one embodiment of the present invention; Figure 6 This is a data graph of another part of the biomarkers of Chlorella vulgaris in one embodiment of the present invention; Figure 7 This is a graph showing the chlorophyll content of common Chlorella in one embodiment of the present invention; Figure 8 This is a graph showing the total protein content of zebrafish in one embodiment of the present invention; Figure 9 This is a partial biomarker data diagram of zebrafish in one embodiment of the present invention; Figure 10 This is a data graph of another portion of the biomarkers of zebrafish in one embodiment of the present invention; Figure 11 This is a graph showing the total protein content of Kalanchoe blossfeldiana in one embodiment of the present invention; Figure 12 This is a partial biomarker data diagram of Kalanchoe blossfeldiana in one embodiment of the present invention; Figure 13 This is a data graph of another part of the biomarkers of Kalanchoe blossfeldiana in one embodiment of the present invention; Figure 14 This is a graph showing the chlorophyll content of Kalanchoe blossfeldiana in one embodiment of the present invention; Figure 15 This is a risk index diagram of *Desmodium styracifolium* in one embodiment of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] This invention proposes a method for environmental toxicology detection of reclaimed water using biomarkers, the steps of which include: S1. Four types of biosensory units were prepared using *Desmodium styracifolium*, *Chlorella vulgaris*, zebrafish, and *Kalanchoe blossfeldiana*. The four types of biosensory units include an ecosensory unit, a photosynthetic sensory unit, a developmental toxicity sensory unit, and a plant sensory unit.

[0020] Step S1 includes: S1.1 In a six-well plate, add two autoclaved wheat grains and 10 mL of sterile distilled water to each well, and inoculate with 10 cells that have undergone expansion culture. 4 Ind. / mL of *Bombyx mori* were cultured at 25°C under normal light and dark conditions, gently shaken once daily, until the density stabilized at 10. 4 After ind. / mL, collect 5mL of culture medium, centrifuge to remove the culture medium, and then suspend in the same volume of sterile water to obtain the ecological sensing unit.

[0021] A stable and homogeneous protozoan community can be obtained in a short time by inoculating high-density *Odontozoa* into six-well plates and culturing them in purified wheat grains and sterile distilled water. This method utilizes the solid carrier and slow-release effect of wheat grains to ensure the attachment, growth, and long-term survival of *Odontozoa* in the microchamber; simultaneously, gentle daily shaking prevents colony aggregation and maintains the homeostasis of dissolved oxygen and metabolic waste in the water. Technically, this culture method provides a highly reproducible ecological response baseline, which is beneficial for the accurate comparison and sensitive identification of subsequent toxicity signals.

[0022] S1.2, The logarithmic growth phase seeds of common Chlorella are seeded at 10... 5 Cells / mL were seeded in culture medium and cultured in shake flasks at 25°C with a light-dark cycle every 12 hours until OD reached. 680 =0.8, take 10 mL of algal solution, centrifuge for 3 minutes, discard the supernatant to obtain algal cells, resuspend the algal cells in 10 mL of fresh culture medium, and adjust the concentration to 10. 5 The photosynthetic sensing units were obtained by dividing the cells / mL.

[0023] Common Chlorella cells in their logarithmic growth phase were precisely seeded and cultured in shake flasks under controlled light and dark conditions to ensure the cells remained in a state of optimal growth and photosynthetic efficiency. This step involved pre-calibrating the OD using spectrophotometry. 680 This ensures a high degree of consistency between algal biomass and chlorophyll content, thereby enabling online measurement of chlorophyll fluorescence and OD. 680 The changes laid the foundation for accuracy. This move highlights the rapid response capability of the photosynthetic system and improves the detection sensitivity of photoinhibitory pollutants in water samples.

[0024] S1.3. Zebrafish were placed in a fully automatic recirculating aquaculture system for 7 days of acclimatization. They were fed once a day. The water quality of the recirculating aquaculture system was maintained at a water temperature of 28±0.5℃ and a dissolved oxygen level of 7.0 mg·L-1. The photoperiod was 14 hours:10 hours. After one week of cultivation, the population of zebrafish tended to stabilize and developmental toxicity-sensing units were obtained.

[0025] By precisely controlling the temperature, regularly feeding, and circulating fluids in zebrafish, the embryos or larvae are ensured to grow in a consistent developmental environment, reducing physiological background noise caused by environmental fluctuations. A 14h:10h light-dark cycle simulates the natural day-night rhythm, keeping the zebrafish in a normal diurnal physiological state and enabling stable measurements of developmental indicators such as heart rate and tail wagging. This measure ensures the highest detection sensitivity and repeatability for chronic developmental toxicity effects during the critical developmental window.

[0026] S1.4 Select healthy petunia plants with 4-6 true leaves. Move each plant, along with its roots, into a well-ventilated but not drained sealed environment. Irrigate with purified water every 3 days. The ambient temperature is controlled at 25±2℃, the relative humidity is controlled at 60%, and the conditions are natural light and darkness or light and darkness cycled every 12 hours to obtain plant sensory units.

[0027] S1.4 Select healthy Kalanchoe plants without disease spots from the middle and lower parts of the plant. Irrigate every 3 days using drip irrigation with 300 mL per plant. Cultivate the plants under natural light and darkness or light and darkness cycles of 12 hours to obtain plant sensory units.

[0028] Cultivating Kalanchoe under natural light-dark conditions or a 12-hour light-dark cycle provides a stable photosynthetic environment, allowing changes in chlorophyll fluorescence and morphology to more accurately reflect the chronic toxic effects of water samples on higher terrestrial plants. This approach combines photosynthetic measurement with growth maintenance, significantly improving the reliability of monitoring the accumulation of environmental toxins and damage to photosynthetic function at the plant level.

[0029] S2. The water sample to be tested is continuously pumped and the suspended impurities are filtered out. The water sample is then introduced into four micro-chambers for incubation. After incubation, the toxicological data are detected. Each of the four micro-chambers contains one of four biosensor units.

[0030] Step S2 includes: S2.1. A peristaltic pump is used to extract the water sample at a constant rate of 0.5 mL / min. A 20 μm stainless steel microporous filter is connected in series to filter the water sample. An online pressure sensor is set to monitor the pressure drop of the stainless steel microporous filter. When the pressure drop exceeds the set threshold, it prompts to clean or replace the stainless steel microporous filter.

[0031] This step uses a peristaltic pump to extract in-situ water samples at a constant rate, and then removes suspended particles and impurities through a 20μm stainless steel microporous filter. This design ensures that only soluble contaminants and colloidal substances remain in the water sample entering the microchamber, preventing large particles from clogging or disturbing the biosensing unit and affecting the accuracy of subsequent detection. An online pressure sensor monitors the pressure drop across the filter, and provides real-time maintenance alerts once the threshold is exceeded, ensuring the stability and reliability of the system's long-term continuous operation.

[0032] S2.2 The filtered water sample enters the multi-way distribution valve and is divided into four channels. Each channel is separately delivered to four micro-chambers. All micro-chambers are placed in a constant temperature chamber at 25±0.5℃. The algae, zebrafish and plant chambers are equipped with independent LED light sources to simulate the day and night cycle.

[0033] After filtration, the water sample is precisely distributed to four microchambers via a multi-way distribution valve. All microchambers are placed in a constant temperature chamber at 25±0.5℃. The algae, zebrafish, and plant chambers are also equipped with programmable LED light sources to simulate day and night cycles. This strictly controlled temperature and light environment ensures that different biological sensory units are exposed to the same conditions under optimal physiological conditions, improving the comparability and reproducibility of the data.

[0034] In one embodiment, when conducting a reclaimed water toxicity assessment experiment based on multiple biosensor units, to ensure the representativeness and statistical reliability of the data, it is necessary to reasonably estimate the amount of reclaimed water sample required for each biological model. According to ISO / OECD standards and laboratory practices, the water sample volume for each biosensor unit can be estimated as follows: For *Desmodium styracifolium*, a 50mL beaker is typically used, with an exposure volume of 30mL per group, setting 4 concentration gradients × 3 replicates, requiring only approximately 0.36L; for *Chlorella vulgaris*, a 250mL Erlenmeyer flask is used, with 100mL per flask, setting 5 gradients × 3 replicates, requiring approximately 1.5L; for zebrafish experiments, 2 mL / well is prepared using 24-well plates, with 3 plates per gradient, and the solution needs to be changed twice during the exposure period, requiring a total of approximately 2.16L. The water sample should be fully aerated before use to ensure dissolved oxygen reaches saturation, and the temperature should be maintained at 26±1°C throughout the process. The pH should be controlled within the range of 6.5–8.5, and the fluctuation should not exceed 1.5 units during the entire exposure period to avoid affecting normal embryonic development and toxic response. Kalanchoe is propagated by soil cuttings. Each plant is irrigated with approximately 400–800 mL. There are 3–4 gradients and 3 replicates. Under long-term exposure or every other day water change conditions, each experiment requires approximately 7.2 L.

[0035] In designing water sample gradients, a doubling decrease method (e.g., 100%, 50%, 25%, 6.25%) can effectively capture non-linear toxicity trends. For animal models such as zebrafish, the number of embryos and the frequency of fluid changes should be determined in accordance with ethical requirements. When allocating water samples, 10–20% of the volume should be reserved for blank controls, chemical detection, and quality control. If limited by water sample volume or experimental throughput, initial screening can be performed using methods such as algal microplate assays (200 µL / well) or low-volume embryotoxicity assays (1 mL / well), followed by detailed testing at different gradients after any abnormal responses are detected. Overall, a complete exposure experiment for each biological unit requires at least 18–20 L of reclaimed water sample to meet the needs of all concentration gradients, replicates, and quality control.

[0036] S2.3. Place the ecological sensing units, photosynthetic sensing units, developmental toxicity sensing units, and plant sensing units in their respective microchambers for cultivation.

[0037] Dilatatus nephrolepis, Chlorella vulgaris, zebrafish, and Kalanchoe blossfeldiana plants were placed in their respective microchambers and simultaneously exposed online using their optimal culture conditions (community density, light-dark cycle, flow rate, etc.). This multi-level, parallel culture strategy not only preserves the sensitive responses of each organism to specific pollutants but also reveals the comprehensive impact of pollutants on different ecological levels through horizontal comparison, providing a solid foundation for constructing a comprehensive toxicity fingerprint.

[0038] S2.4 After cultivation, the ecological sensing unit, photosynthetic sensing unit, developmental toxicity sensing unit, and plant sensing unit were tested and treated to obtain toxicological data. The toxicological data included data on multiple biomarkers, including total protein, total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, glutamate dehydrogenase activity, and chlorophyll content.

[0039] After cultivation, following a standardized procedure of lysis, centrifugation, and enzyme-linked immunosorbent assay (ELISA), 11 enzyme activities and content indicators of each biosensor unit were quantitatively obtained. This procedure is highly standardized and has strong batch processing capabilities, enabling high-throughput assays at 37°C, a 100µL reaction volume, and a 30-minute incubation period. It also balances the precision and sensitivity of both chemical (spectrophotometric) and biological methods, significantly improving the efficiency and reliability of toxicity detection.

[0040] Step S2.4 includes: S2.4.1 After 5 days of culture, the community in the microcell was transferred to pre-cooled centrifuge tubes. The supernatant was discarded after centrifugation, and the cell suspension was washed with phosphate-buffered saline (PFS). The cells were lysed by shaking at 4°C for 15 minutes, and the crude enzyme solution in the supernatant was collected after centrifugation. The total protein content was determined using a Bicinchoninic Acid (BCA) protein quantification kit. Using a mlbio ELISA kit at 37°C, a reaction volume of 100 µL, and an incubation time of 30 minutes, the total antioxidant capacity, superoxide dismutase (SOD), catalase, glutathione peroxidase (GAP), malondialdehyde (MDA) content, triphosphate kinase (TPK) activity, adenosine triphosphate (ATP) activity, pyruvate kinase (PkT) activity, glutathione reductase (GRU) activity, and glutamate dehydrogenase (GDH) activity of the ecological sensing units were measured.

[0041] After 5 days of exposure, the *Desmodium styracifolium* colonies were centrifuged and enriched, washed with PBS, lysed by shaking at 4°C for 15 min, and then centrifuged again to obtain a high-purity crude enzyme solution. This method maximizes the preservation of intracellular enzyme activity while removing extracellular interfering substances. Subsequent BCA protein quantification and ELISA enzyme activity assays were performed at 37°C in a 100 µL system, ensuring not only the accuracy of total protein data but also good reproducibility in the determination of the activities of sensitive antioxidant markers such as total antioxidant capacity, superoxide dismutase, and catalase.

[0042] S2.4.2. Take 50 mL of photosynthetic sensing unit, centrifuge for 15 minutes, and wash three times with phosphate buffer until the final concentration of photosynthetic sensing unit is 10. 7 Cells / mL were added to 1 mL of phosphate buffer and lysis buffer, and the cells were lysed at low temperature using a high-performance tissue homogenizer. After centrifugation for 10 minutes, the supernatant was collected, and the crude enzyme solution was analyzed using a kit to determine the total protein content. Using an enzyme-linked immunosorbent assay (ELISA) kit at 37°C, 100 µL reaction volume, and 30 minutes, the total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the photosynthetic sensing unit were measured. Take another 8 mL photosynthetic sensing unit and centrifuge it sequentially at 1000 r / min for 5 minutes and at 3000 r / min for 10 minutes. Add 5 mL of anhydrous ethanol, shake for 1 minute, store in a 4℃ refrigerator for 24 hours, then take it out and centrifuge for 10 minutes. Take the supernatant and use anhydrous ethanol as a reference to measure the absorbance at wavelengths of 470, 646, 663, and 750 nm using a spectrophotometer to detect the chlorophyll content.

[0043] Take 50 mL of algal solution and centrifuge to concentrate to 10. 7 Cells / mL, washed with phosphate buffer, mechanically disrupted at low temperature, and centrifuged again to obtain crude enzyme solution, which was used for the detection of 11 biochemical indicators; 8 mL of algal solution was extracted with anhydrous ethanol for 24 hours, and the absorbance at 470 / 646 / 663 / 750 nm was measured to rapidly obtain chlorophyll content. This dual-channel extraction method takes into account both enzyme activity and pigment detection, eliminates component loss and mutual interference caused by single extraction methods, and improves the sensitivity and accuracy of photosynthetic system toxicity effects.

[0044] S2.4.3. Five developmental toxicity receptor units of similar developmental size were randomly selected from the microchamber, anesthetized, weighed, and then placed in centrifuge tubes. The tubes were stored at -80℃ for later use. The tissues of the developmental toxicity receptor units were added to pre-cooled phosphate buffer at a ratio of 1g:5ml, homogenized using a biological sample homogenizer with lysis buffer, centrifuged, and the supernatant was collected. The total protein content was determined using a kit. Using an enzyme-linked immunosorbent assay (ELISA) kit at 37℃, 100µL reaction volume, and incubation for 30 minutes, the total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the developmental toxicity receptor units were measured.

[0045] Five zebrafish of similar developmental size were randomly selected, anesthetized, weighed, and frozen at -80°C. The resulting homogenate was then centrifuged at a ratio of 1 g: 5 mL, and the supernatant was collected. This whole-fish homogenization method avoids local biases associated with single-tissue extraction and simultaneously reflects systemic metabolic and antioxidant status. ELISA was used to detect enzyme activity under standard conditions, yielding data that are both systematic and representative, effectively revealing the interference of developmental toxicity on the overall body's energy and antioxidant network.

[0046] S2.4.4 Weigh and chop the leaves below the middle of the plant's sensory unit, or leaves with similar leaf areas at the same position. Mix them thoroughly, quickly dispense them into cryovials, and store them at -80℃. Add phosphate buffer, homogenize the tissue with lysis buffer in a biological sample homogenizer, centrifuge, and collect the crude enzyme solution from the supernatant. Use a kit to detect the total protein content. Use an enzyme-linked immunosorbent assay (ELISA) kit at 37℃, 100µL reaction volume, and incubation for 30 minutes to detect the total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the plant sensory unit. Then, take leaves below the middle of the plant sensory unit, or leaves with similar leaf areas at the same position, and use a TYS-N instrument to determine the chlorophyll content.

[0047] Standardized sampling of the middle and lower leaves was performed, followed by storage at -80℃ and homogenization lysis to obtain crude enzyme solutions for the determination of 11 biochemical indicators. Total chlorophyll content was rapidly quantified using a TYS-N chlorophyll meter. This procedure considers both the biochemical quality and pigment content of the leaves, utilizing heat-stable plant tissue to ensure sample consistency, while the online measurement of the TYS-N provides real-time evaluation of photosynthetic function, comprehensively reflecting the cumulative effects of chronic toxicity at the plant level.

[0048] S3. Process the toxicological data and output a toxicity monitoring report.

[0049] Step S3 includes: S3.1 Calculate the mean and standard deviation of each biomarker data in the toxicological data of one of the four biological sensory units, and obtain the standardized result of each biomarker data through the mean and standard deviation.

[0050] Step S3.1 includes: S3.1.1. Take the average of the data for each biomarker obtained from one of the four biosensor units on the same sampling day to obtain the daily mean X of each biomarker data. The formula for calculating the daily mean X of each biomarker data is as follows: in, The number of biological sensory units, This represents the biomarker data for the g-th biosensor unit. On the same sampling day, the average of single-point observations from each treatment group is used to obtain the daily mean X. This smooths out single-measurement errors, ensures that intraday physiological differences are diluted by multiple data sets, and provides consistent representative values ​​for multiple batches of parallel samples, greatly enhancing the reliability of time series data.

[0051] S3.1.2 Summarize the X values ​​for each sampling day and calculate the mean value of each biomarker data. with standard deviation The calculation formula is: in, The time of the sampling date. Let t be the daily mean of each biomarker data on the sampling day. By using the daily mean series of all sampling days, the overall mean m and standard deviation s can be calculated, which can accurately characterize the baseline level and natural fluctuations of the biomarker throughout the monitoring period, and realize real-time correction of baseline drift in long-term online monitoring.

[0052] S3.1.3, the daily average of each biomarker data on the sampling day at time t. The standardization process is performed using the following formula: This represents the standardized results of each biomarker data sampled at time t. The daily average value at each time point is standardized according to a formula, which preserves the dynamic characteristics of the indicators in response to pollution and suppresses the interference of differences in the dimensions and amplitudes of different indicators on the comprehensive model, thus laying a solid statistical foundation for non-negative scoring and fingerprint fusion.

[0053] S3.2 Calculate the non-negative score for each biomarker data using the standardized results of each biomarker data.

[0054] Step S3.2 includes: S3.2.1 Determine the direction of response based on the changes in data of each biomarker over culture time.

[0055] S3.2.2 If the malondialdehyde content increases with incubation time, then the response direction is determined to be activating. ,in, As a response value, if any one of the following decreases with culture time: total protein content, total antioxidant activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, or glutamate dehydrogenase activity, then... .

[0056] Based on the increasing or decreasing trend of malondialdehyde (MDA) activation indicators and other inhibition indicators over time, the system automatically classifies the dynamic responses at each endpoint into two categories: increase or decrease. This ensures that subsequent scoring truly reflects the direction of biological effects and enhances the biological significance of score interpretation.

[0057] S3.2.3, Minimum standardized value for all time points The absolute value is used as the offset to calculate the non-negative score for each biomarker data point, using the following formula: in, The non-negative score is the score of the i-th biomarker data from the sampling day at time t.

[0058] Using the absolute value of the minimum standardized value across all sampling points as the offset effectively suppresses the stretching of the scoring scale by occasional outliers. This ensures that the generated non-negative scores remain stable across multiple batches of samples and long-term monitoring, preventing misleading risk assessments due to single extreme fluctuations. A formula is used to correct the standardized values ​​at all time points, avoiding negative scores caused by extremely small values. This not only maintains numerical monotonicity but also enhances the detection sensitivity to minute changes by utilizing the minimum value offset, making it particularly suitable for the early capture of subtle toxic signals in real-time monitoring.

[0059] S3.3 Sum the absolute values ​​of the non-negative scores of each biomarker data to obtain the risk index of each biosensory unit and output a toxicity monitoring report.

[0060] Step S3.3 includes: S3.3.1 Summing up the absolute values ​​of the non-negative scores of all biomarker data for each biosensor unit on the same sampling day, using the following formula: in, , where n is the risk index and n is the total number of biomarker data.

[0061] The summation of scores from each endpoint into a comprehensive risk index not only simplifies the interpretation of multidimensional data, but also accurately reflects the overall ecological damage intensity through a one-dimensional value, providing decision-makers with an intuitive and easily comparable quantitative risk indicator.

[0062] S3.3.2, if If the value is less than or equal to the first threshold, then output a low-risk result; otherwise... If the value is greater than the first threshold and less than or equal to the second threshold, then output a medium-risk result. If the result exceeds the second threshold, a high-risk result will be output.

[0063] By setting three thresholds—low, medium, and high—the continuous risk index is mapped to discrete risk levels. Subsequently, standardized reports and visualization charts are generated, ensuring that the monitoring results have a scientific and rigorous quantitative basis and can quickly convey key early warning information to managers, significantly improving the efficiency of emergency response and the practicality of environmental risk management.

[0064] Each microcompartment was exposed to a series of concentration gradients (e.g., 0.1, 0.5, 1, 2, 5 mg / L) of typical toxins (such as Cd²⁺ and DDVP), and the concentrations at each concentration were recorded. - Concentration curves. Extract key concentrations from the curves: the concentration with no observed effect and the lowest observed effect concentration, where the concentration with no observed effect represents the lowest concentration. Still less than or equal to the first value, the lowest observed effect concentration is indicated at the lowest concentration. The threshold value is greater than the first value, which is the first threshold used to identify the boundary where a detectable biological effect just appears. The second threshold value can be set to... The value corresponding to half of the maximum concentration is used to identify the boundary where serious biological effects occur.

[0065] In this embodiment, the first test reclaimed water sample is named RW1, the second test reclaimed water sample is named RW2, and the tap water is named TW as a control.

[0066] Please refer to Figure 1The effects of reclaimed water samples on the total protein content of *Desmodium styracifolium* were tested, and the overall effect was mainly inhibition. In the RW1 experiment, the total protein content on day 1 was not significantly different from the control group, but was significantly lower than the control group from day 2 to day 4. In the RW2 experiment, the total protein content on days 1-3 was significantly lower than the control group; however, it was significantly higher than the control group on day 4.

[0067] Please refer to Figure 2 and Figure 3 The test results showed that the reclaimed water samples affected the antioxidant and energy metabolism markers of *Desmodium dilatatum* to some extent. Ten markers exhibited both inducing and inhibiting effects, showing different temporal dynamics: the inducing effect was dominant on days 1-2, while the inhibitory effect gradually increased on days 3-4.

[0068] Please refer to Figure 4 The test showed that the reclaimed water samples significantly increased the total protein content of common Chlorella. The total protein content of the RW1 experimental group was significantly higher than that of the control group in the first 4 days. The total protein content of the RW2 experimental group was significantly lower than that of the control group on the 1st day, and significantly higher than that of the control group on the 2nd and 4th days. The changes were not significant compared with the control group at other time points.

[0069] Please refer to Figure 5 , Figure 6 and Figure 7 The test showed that reclaimed water samples caused changes in the antioxidant system, energy metabolism level and chlorophyll content in Chlorella cells. Antioxidant and energy metabolism markers showed a predominantly inducing effect on day 1 and a predominantly inhibitory effect on days 2-4. The response patterns of chlorophyll a and b were basically consistent, showing an overall inhibitory effect.

[0070] Please refer to Figure 8 The study showed that reclaimed water samples had a significant impact on the total protein content of zebrafish. On days 2-3 of the RW1 and RW2 treatment experiments, the total protein content of zebrafish in both treatments increased synchronously compared to the control group. On days 4, 9, and 14 of the RW1 and RW2 experiments, the total protein content of zebrafish decreased synchronously compared to the control group. Overall, protein synthesis was induced within 3 days and inhibited after 3 days.

[0071] Please refer to Figure 9 and Figure 10 Reclaimed water induced oxidative stress in zebrafish, showing short-term inhibition and long-term induction effects, as represented by total antioxidant capacity (T-AOC) and malondialdehyde (MDA). Reclaimed water samples caused changes in intracellular energy metabolism levels in zebrafish, but the response patterns of various markers were inconsistent, generally exhibiting a recurring process of induction and inhibition. In the long term, pyruvate kinase (PK) was significantly induced on day 14, while glycerol triphosphate kinase (PGK) was significantly inhibited.

[0072] Please refer to Figure 11 The reclaimed water sample showed a significant inhibitory effect on the total protein content of Kalanchoe blossfeldiana, with dilution providing some relief. In the short to medium term, on days 2 and 9, the total protein content of the four experimental groups (RW1, 1 / 2RW1 (dilution water), RW2, and 1 / 2RW2 (dilution water)) was significantly lower than that of the control group. After long-term treatment, on day 15, the total protein content of the RW2 and 1 / 2RW1 experimental groups was lower than that of the control group, while the total protein content of the 1 / 2RW2 and RW1 groups was higher than that of the control group.

[0073] Please refer to Figure 12 and Figure 13 The study tested the effects of reclaimed water samples on oxidative stress and energy metabolism levels in Kalanchoe blossfeldiana. Overall, in the short to medium term (days 2 and 9), the 10 biomarkers showed a predominantly inducing effect, while after long-term treatment, an inhibitory effect began to appear on day 15.

[0074] Please refer to Figure 14 The effect of reclaimed water on the chlorophyll content of Kalanchoe blossfeldiana was mainly a medium- to long-term inhibitory effect, with some mitigation effect after dilution. In the short term, on day 2, the chlorophyll content of RW1, 1 / 2RW2, and RW2 was higher than the control group, while 1 / 2RW1 showed no significant difference compared to the control group. In the medium to long term, on days 9 and 15, the chlorophyll content of all four experimental groups was significantly lower than the control group, and the chlorophyll content of the diluted reclaimed water was higher than that of the original water.

[0075] Please refer to Figure 15 Taking *Desmodium styracifolium* as an example, the data of various biomarkers were analyzed and processed to calculate the risk index of each biosensory unit in order to assess the environmental toxicological characteristics of reclaimed water. For example... Figure 15 As shown, the pressure effect of reclaimed water RW2 (I(t) = 1.40) on *Odontospira edodes* was higher than that of RW1 (I(t) = 1.11). The maximum effect of RW1 on *Odontospira edodes* occurred on day 3 (I(t) = 1.53), and the minimum effect occurred on day 1 (I(t) = 0.78); the maximum effect of RW2 on *Odontospira edodes* occurred on day 1 (I(t) = 1.61), and the minimum effect occurred on day 4 (I(t) = 1.19). Based on the consistency of the response of each marker I(t) value to the two reclaimed water samples (… Figure 15 C), the optimal combination of biomarkers for risk warning is SOD and GR, with the best response time being day 1-2. An upward adjustment indicates increased risk. In this range, the environmental risks of the two types of reclaimed water are: RW2 > RW1. Figure 15 D).

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for environmental toxicology detection of reclaimed water using biomarkers, characterized in that the steps include... include: S1. Four types of biosensory units were prepared using *Nematocystis glomeratus*, *Chlorella vulgaris*, zebrafish, and *Kalanchoe blossfeldiana*. The four types of biosensory units include an ecosensory unit, a photosynthetic sensory unit, a developmental toxicity sensory unit, and a plant sensory unit. S2. The water sample to be tested is extracted by continuous pump suction, suspended impurities are filtered out, and the water sample to be tested is introduced into four micro chambers for cultivation. After cultivation, the toxicological data are detected. Each of the four micro chambers contains one of four biosensor units. S3. Process the toxicological data and output a toxicity monitoring report.

2. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 1, characterized in that, Step S1 includes: S1.1 In a six-well plate, add two autoclaved wheat grains and 10 mL of sterile distilled water to each well, and inoculate with 10 cells that have undergone expansion culture. 4 Ind. / mL of *Bombyx mori* were cultured at 25°C under normal light and dark conditions, gently shaken once daily, until the density stabilized at 10. 4 After ind. / mL, collect 5mL of culture medium, centrifuge to remove the culture medium, and then suspend in the same volume of sterile water to obtain the ecological sensing unit; S1.2, The logarithmic growth phase seeds of common Chlorella are seeded at 10... 5 Cells / mL were seeded in culture medium and cultured in shake flasks at 25°C with a light-dark cycle every 12 hours until OD reached. 680 =0.8, take 10 mL of algal solution, centrifuge for 3 minutes, discard the supernatant to obtain algal cells, resuspend the algal cells in 10 mL of fresh culture medium, and adjust the concentration to 10. 5 cells / mL, to obtain photosynthetic sensing units; S1.

3. Zebrafish were placed in a fully automatic recirculating aquaculture system for 7 days of acclimatization. They were fed once a day. The water quality of the recirculating aquaculture system was maintained at a water temperature of 28±0.5℃ and a dissolved oxygen level of 7.0 mg·L-1. The photoperiod of light:dark was 14 hours:10 hours. After one week of cultivation, the population of zebrafish tended to stabilize and developmental toxicity sensing units were obtained. S1.4 Select healthy Kalanchoe plants without disease spots from the middle and lower parts of the plant. Irrigate every 3 days using drip irrigation with 300 mL per plant. Cultivate the plants under natural light and darkness or light and darkness cycles of 12 hours to obtain plant sensory units.

3. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 1, characterized in that, Step S2 includes: S2.

1. A peristaltic pump is used to extract the water sample to be tested at a constant rate of 0.5 mL / min. A 20 μm stainless steel microporous filter screen is connected in series to filter the water sample to be tested. An online pressure sensor is set to monitor the pressure drop of the stainless steel microporous filter screen. When the pressure drop exceeds the set threshold, it prompts to clean or replace the stainless steel microporous filter screen. S2.2 The filtered water sample enters the multi-way distribution valve and is divided into four channels. Each channel is separately delivered to four micro-chambers. All micro-chambers are placed in a constant temperature chamber at 25±0.5℃. The algae, zebrafish and plant chambers are equipped with independent LED light sources to simulate the day and night cycle. S2.

3. The ecological sensing units, photosynthetic sensing units, developmental toxicity sensing units, and plant sensing units were placed in their respective microchambers for cultivation. S2.4 After cultivation, the ecological sensing unit, photosynthetic sensing unit, developmental toxicity sensing unit, and plant sensing unit were tested and treated to obtain toxicological data. The toxicological data included data on multiple biomarkers, including total protein, five antioxidant system markers (including total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, and malondialdehyde content), five energy metabolism system markers (including triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity), and chlorophyll content.

4. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 3, characterized in that, Step S2.4 includes: S2.4.1 After 5 days of culture, the community in the microcell was transferred to a pre-cooled centrifuge tube. The supernatant was discarded after centrifugation, and the cell lysate was washed with phosphate buffer. The cells were lysed by shaking at 4°C for 15 minutes. After centrifugation, the crude enzyme solution in the supernatant was collected. The total protein content was detected using a kit. The total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the ecological sensing unit were detected using an enzyme-linked immunosorbent assay (ELISA) kit at 37°C, 100µL reaction volume, and 30 minutes. S2.4.

2. Take 50 mL of photosynthetic sensing unit, centrifuge for 15 minutes, and wash three times with phosphate buffer until the final concentration of photosynthetic sensing unit is 10. 7 Cells / mL, add 1mL phosphate buffer and lysis buffer, perform low-temperature lysis using a high-performance tissue homogenizer, centrifuge for 10 minutes, collect the supernatant, collect the crude enzyme solution, and determine the total protein content using a kit. Then, use an enzyme-linked immunosorbent assay (ELISA) kit at 37℃, 100µL reaction volume, and incubation for 30 minutes to detect the total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, and other parameters of the ecological sensing unit. Ketokinase activity, glutathione reductase activity, and glutamate dehydrogenase activity were measured. An additional 8 mL of photosynthetic sensing unit was centrifuged at 1000 rpm for 5 minutes and then at 3000 rpm for 10 minutes. 5 mL of anhydrous ethanol was added, and the mixture was shaken for 1 minute. After storage at 4°C for 24 hours, the mixture was centrifuged for 10 minutes, and the supernatant was collected. Using anhydrous ethanol as a reference, the absorbance was measured at wavelengths of 470, 646, 663, and 750 nm using a spectrophotometer to determine the chlorophyll content. S2.4.

3. Five developmental toxicity receptor units of similar developmental size were randomly selected from the microchamber, anesthetized, weighed, and then placed in centrifuge tubes. They were stored at -80℃ for later use. The developmental toxicity receptor unit tissues were added to pre-cooled phosphate buffer at a ratio of 1g:5ml, homogenized with lysis buffer in a biological sample homogenizer, centrifuged, and the crude enzyme solution of the supernatant was collected. The total protein content was detected using a kit. The total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the ecological receptor units were detected using an enzyme-linked immunosorbent assay kit at 37℃, reaction volume of 100µL, and incubation for 30 minutes. S2.4.4 Weigh and chop the leaves below the middle of the plant's sensory unit, or leaves with similar leaf areas at the same position. Mix them thoroughly, quickly dispense them into cryovials, and store them at -80℃. Add phosphate buffer, homogenize the tissue with lysis buffer in a biological sample homogenizer, centrifuge, and collect the crude enzyme solution from the supernatant. Use a kit to detect the total protein content. Use an enzyme-linked immunosorbent assay (ELISA) kit at 37℃, 100µL reaction volume, and incubation for 30 minutes to detect the total antioxidant capacity activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, malondialdehyde content, triphosphate glycerol kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, and glutamate dehydrogenase activity of the plant sensory unit. Then, take leaves below the middle of the plant sensory unit, or leaves with similar leaf areas at the same position, and use a TYS-N instrument to determine the chlorophyll content.

5. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 1, characterized in that, Step S3 includes: S3.1 Calculate the mean and standard deviation of each biomarker data in the toxicological data of one of the four biological sensory units, and obtain the standardized results of each biomarker data through the mean and standard deviation; S3.2 Calculate the non-negative score for each biomarker data using the standardized results of each biomarker data; S3.3 Sum the absolute values ​​of the non-negative scores of each biomarker data to obtain the risk index of each biosensory unit and output a toxicity monitoring report.

6. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 5, characterized in that, Step S3.1 includes: S3.1.

1. Take the average of the data for each biomarker obtained from one of the four biosensor units on the same sampling day to obtain the daily mean X of each biomarker data. The formula for calculating the daily mean X of each biomarker data is as follows: in, The number of biological sensory units, For the biomarker data of the g-th biosensory unit; S3.1.2 Summarize the X values ​​for each sampling day and calculate the mean value of each biomarker data. with standard deviation The calculation formula is: in, The time of the sampling date. For each biomarker data point sampled at time t, the daily mean value for that biomarker. S3.1.3, the daily average of each biomarker data on the sampling day at time t. The standardization process is performed using the following formula: The standardized results for each biomarker data sampled at time t.

7. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 5, characterized in that, Step S3.2 includes: S3.2.1 Determine the direction of response based on the changes in data of each biomarker over culture time; S3.2.2 If the malondialdehyde content increases with incubation time, then the response direction is determined to be activating. ,in, As a response value, if any one of the following decreases with culture time: total protein content, total antioxidant activity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity, triphosphate kinase activity, adenosine triphosphate kinase activity, pyruvate kinase activity, glutathione reductase activity, or glutamate dehydrogenase activity, then... ; S3.2.3, Minimum standardized value for all time points The absolute value is used as the offset to calculate the non-negative score for each biomarker data point, using the following formula: in, The non-negative score is the score of the i-th biomarker data from the sampling day at time t.

8. The method for environmental toxicology detection of reclaimed water using biomarkers according to claim 5, characterized in that, Step S3.3 includes: S3.3.1 Summing up the absolute values ​​of the non-negative scores of all biomarker data for each biosensor unit on the same sampling day, using the following formula: in, The risk index is n, where n is the total number of biomarker data. S3.3.2, if If the value is less than or equal to the first threshold, then output a low-risk result; otherwise... If the value is greater than the first threshold and less than or equal to the second threshold, then output a medium-risk result. If the result exceeds the second threshold, a high-risk result will be output.

Images