Ecological risk assessment method for organophosphorus flame retardant based on multi-dimensional toxicity index

Abstract

The invention discloses an ecological risk assessment method for an organophosphorus flame retardant based on multi-dimensional toxicity indexes. The ecological risk assessment method comprises the following steps: culturing microcystis aeruginosa, assessing physiological and biochemical influence, assessing algal toxin synthesis and release influence, assessing a combined toxicity action mode of TCPP and microcystin, and assessing the influence of TCPP on the ecological risk of a water body. According to the method disclosed by the invention, the growth and metabolism change of microcystis aeruginosa and the combined toxicity and ecological risk of algal toxin and the organophosphorus flame retardant under the action of different concentrations of the organophosphorus flame retardant in the water body are evaluated, and the evaluation method gives consideration to the macroscopic and microscopic processes of microalgae in the water body, so that the ecological risk of the organophosphorus flame retardant in the water body can be systematically and comprehensively evaluated; the evaluation method can provide powerful theoretical basis and support for behavior research and use management of the organophosphorus flame retardant in the water environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental risk assessment, and particularly relates to an ecological risk assessment method for organophosphorus flame retardants based on multi-dimensional toxicity indicators. Background Art

[0002] Due to its high-efficiency flame retardant properties, organophosphorus flame retardant TCPP (tris(2-chloropropyl) phosphate) is widely used in electronic appliances, building materials, and plastic products. However, the persistence and bioaccumulation of TCPP in water bodies have raised serious environmental concerns. Research shows that the detected concentration of TCPP in waters in the Yangtze River Delta region of China is as high as 1 - 200 μg / L, and it can cause ecological risks through the food chain. Existing technologies mainly focus on the single toxic effect of TCPP, such as the acute toxicity to zebrafish embryos (96 h LC50 is 14.1 mg / L), or the inhibitory effect on microbial respiration and metabolism, but lack a systematic study on its mechanism of inducing the generation of algal toxins.

[0003] Microcystis aeruginosa, as a typical toxin-producing cyanobacterium, releases microcystin (MC-LR) with strong hepatotoxicity and carcinogenic promotion. Existing research has shown that traditional pollutants (such as heavy metals) can activate algal toxin synthesis genes ( mcy A, mcy H), but the regulatory effect of organophosphorus flame retardants on such genes is not yet clear. In addition, existing ecological risk assessment models are mostly based on single biomass or survival rate indicators, without integrating multi-dimensional data such as toxin generation and oxidative stress, resulting in insufficient sensitivity of risk prediction. For example, traditional biomass detection takes 96 h, while early warning indicators such as cell membrane integrity can reflect toxic effects within 24 h.

[0004] Current environmental supervision standards for flame retardants are mainly based on acute toxicity data, but an assessment system for chronic toxicity and combined pollution effects (such as the synergistic effect of TCPP and MC-LR) under long-term low-dose exposure has not been established. Market analysis indicates that the TCPP industry is facing pressure in the research and development of green alternatives, but its environmental behavior and risk prediction technology still lag behind the needs of industrial development. Therefore, there is an urgent need to develop a multi-dimensional assessment method that integrates the direct toxicity and indirect ecological risks of flame retardants to provide technical support for water environment safety management. Summary of the Invention

[0005] To solve the above problems, the object of the present invention is to provide an ecological risk assessment method for organophosphorus flame retardants based on multi-dimensional toxicity indicators, which evaluates the risk of Microcystis aeruginosa bloom outbreak, the ecological risk of microcystin and the ecological risk of pollutants under the action of organophosphorus flame retardants at different concentrations. The assessment method takes into account both the macroscopic and microscopic processes of microalgae in water bodies, includes the action mechanisms of organophosphorus flame retardants on the growth, synthesis and release of microcystin of Microcystis aeruginosa, and considers the environmental risk of microalgae metabolites, and can systematically and comprehensively evaluate the ecological risk of organophosphorus flame retardants in water bodies. This assessment method can provide a strong theoretical basis and support for the behavior research and use management of organophosphorus flame retardants in the water environment.

[0006] The object of the present invention is achieved as follows, including the following steps: (1) Culturing Microcystis aeruginosa: Expose Microcystis aeruginosa with the same initial algal density to TCPP solutions at different concentrations for cultivation, and the concentrations are 0 μg / L, 0.1 μg / L, 1 μg / L, 10 μg / L, 100 μg / L, 1000 μg / L, 10000 μg / L, 100000 μg / L respectively, and the cultivation conditions are the same; (2) Physiological and biochemical impact assessment, evaluating the macroscopic and microscopic impacts of organophosphorus flame retardants on Microcystis aeruginosa in water bodies: On the 0th, 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, and 19th days of cultivation, measure the density of Microcystis aeruginosa cultured at different concentrations of TCPP; The algal density can be measured by methods such as ultraviolet absorbance method, cell counter, hemocytometer counting method, and flow cytometer. The ultraviolet absorbance method requires establishing a standard curve of the absorbance of the algal solution at 680 nm and the algal density. However, due to the influence of algal cell growth metabolites and possible influence on its accuracy during the decline period due to cell breakage, the cell counter cannot distinguish between intact cells and damaged cells, and the hemocytometer has a large workload and many error factors. Therefore, it is recommended to use a flow cytometer to measure the algal density; The maximum biomass ( K ) and the maximum growth rate ( R max ) of Microcystis aeruginosa growth under different concentrations of TCPP exposure can be analyzed through the logistic growth model, and the formula is as follows: In the formula: N —— the algal density at time t (d), cells / mL; K —— the maximum biomass (the maximum algal density reached during cultivation), cells / mL; a —— a constant in the logistic model, indicating the relative position to the origin; r —— Intrinsic growth rate, / d; R max —— Maximum algal growth rate, cells / mL / d; Take the Microcystis aeruginosa cultured on the 13th day of the logarithmic growth phase, and measure the chlorophyll a content of Microcystis aeruginosa cultured under different concentrations of TCPP. Analyze the photosynthetic activity of algal cells by measuring the chlorophyll content; Take 2 mL of the sample, centrifuge it in a high-speed refrigerated centrifuge for 10 min, with a rotation speed of 10,000 rpm and a temperature of 4°C. After centrifugation, remove the supernatant, add 2 mL of 95% ethanol, then quickly freeze it in liquid nitrogen for 1 min, thaw it naturally, freeze-thaw it repeatedly in liquid nitrogen three times, and then put it into an ultrasonic machine for ultrasonic treatment for 30 min, with a power of 100%. After ultrasonic treatment, repeat the above centrifugation operation. After centrifugation, take the supernatant for measurement. Then use the following formula to calculate the chlorophyll a content (μg / L) of the extract; ; Take the Microcystis aeruginosa cultured on the 13th day of the logarithmic growth phase, and evaluate the metabolic activity, oxidative stress, and cell membrane integrity of Microcystis aeruginosa cultured under different concentrations of TCPP; Among them, the metabolic activity is analyzed by measuring the ATP content to analyze the photosynthetic activity of algal cells. A kit can be purchased and measured according to the kit instructions; The oxidative stress situation is analyzed by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and reduced glutathione (GSH). All three indicators can be purchased with corresponding kits and measured according to the kit instructions; The cell membrane integrity can be measured using SYTOX Green nucleic acid fluorescent dye to measure the membrane permeability. It is recommended to use a flow cytometer or a microplate reader for measurement; (3) Evaluation of the influence of algal toxin synthesis and release: Take the Microcystis aeruginosa cultured on the 13th day of the logarithmic growth phase, and measure the extracellular algal toxin and total algal toxin concentrations of Microcystis aeruginosa cultured under different concentrations of TCPP; For the analysis of extracellular algal toxins, the supernatant needs to be measured after centrifuging the algal solution. For the measurement of total algal toxins, the algal cells need to be broken to release the algal toxins. The algal cells can be broken by methods such as freeze-thaw method and ultrasonic method. When using the freeze-thaw method, the breaking effect needs to be tested first to ensure accuracy. It is recommended to use the ultrasonic method to break the algal cells. After cell breaking, centrifuge and measure the supernatant; The concentration of algal toxins can be measured through the corresponding algal toxin kit; (4) Evaluation of the combined toxic effect mode of TCPP and microcystin: The EC50 of organophosphorus flame retardants and microcystins was obtained through the luminous bacteria exposure experiment. Then, the organophosphorus flame retardants and microcystins were mixed at an equitoxic ratio concentration, and the luminous bacteria exposure experiment was continued to obtain the EC50 of the mixed system. The combined toxicity mode of the two was judged through the isoeffect curve. (5)Evaluate the impact of TCPP on the aquatic ecological risk: Compare the peak concentration of microcystin in the TCPP culture system at different concentrations with the toxicity endpoint value EC50, which characterizes the harm of microcystin, measured from the water samples collected from the water body to be evaluated, to obtain the risk quotient RQ. When RQ≥1, it is a high risk; when 0.1≤RQ<1, it is a medium risk; when 0.01≤RQ<0.1, it is a low risk, so as to make a preliminary judgment on the ecological risk of TPCC. The calculation formula is as follows: In the formula: MEC——Measured environmental concentration of the pollutant, mg / L; PNEC——Predicted no-effect concentration of the pollutant, mg / L; L(E)C50——Concentration of the test substance that causes a certain effect change in 50% of the test organisms, obtained through published literature, existing toxicity databases, and toxicity prediction software, mg / L; AF——Assessment factor for selecting acute toxicity data, set to 1000.

[0007] Among them, the selection of ecological receptors needs to be determined. It is recommended to select relatively representative aquatic biological species as ecological receptors. The toxicity endpoint value L(E)C50 of the organisms can be obtained through self-experiment or from the US ECOTOX database.

[0008] Preferably, the initial biomass of Microcystis aeruginosa under TCPP exposure in step (1) is 1.0~1.5×10 6 cells / mL.

[0009] Preferably, in step (1), 1.7 g of the culture medium is taken into a conical flask, 1000 mL of distilled water is added, and it is sterilized with high-temperature steam at 121 °C for 30 min. After the culture medium is cooled to room temperature, algal seeds are inoculated at an inoculation amount of 10%, and it is used after the number of algal cells is expanded to the required density. 150 μL of the TCPP solution is added to 149.85 mL of the experimental sample respectively, so that the final concentration of TCPP in the sample is 0.1-100000 μg / L. Three parallel samples are set for each concentration of the sample, and all experimental conditions are the same as those for algal seed culture. The algal cells are cultured in a light incubator, and the culture conditions are: temperature 25 °C, light intensity 4000-6000 lx, light-dark ratio 12 h:12 h, and it is continuously shaken at a speed of 120 rpm.

[0010] Preferably, step (4) is specifically to conduct a toxicity experiment using Photobacterium phosphoreum. The TCPP and MC-LR to be tested are diluted to 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, 160 mg / L, 320 mg / L, and 640 mg / L. All solutions are made up to volume with 3% NaCl; then 2 mL of the sample solution is added to 5 mL test tubes respectively, and 2 mL of 3% NaCl is added to the blank tube. A new pipette tip is used for each sample, and three parallel groups are set for each concentration; 10 μL of the bacterial solution is added to each test tube in turn, the bottle cap is covered and inverted more than 5 times to fully mix the bacterial solution, the bottle cap is removed and put back in place, and wait for 15 min; the time should be accurately measured to the second when adding the bacterial solution to each test tube, so that the reaction time of each sample with the bacterial solution is 15 min; immediately after 15 min of reaction in the first test tube with the added bacterial solution, the test tube is put into a portable biotoxicity analyzer to measure the luminescence intensity and record it; For the obtained EC50 of the single substance, in the form of a mixed mode with equal toxicity ratio, that is, the toxicity ratio of each substance in the mixture is equal to 1, a binary mixed system is formed by TCPP and MC-LR (microcystin), and 7 concentration gradients are set according to 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 times of EC50, and the above exposure experiment is repeated.

[0011] Compared with the prior art, the present invention has the following technical effects: 1. Based on the effects of organophosphorus flame retardants at different concentrations on the growth and algal toxin production of Microcystis aeruginosa, the present invention establishes an assessment method for the environmental risk of organophosphorus flame retardants in water bodies, which will supplement the understanding of the interaction between Microcystis aeruginosa and organophosphorus flame retardants in water, and provide theoretical guidance for accurately assessing and controlling the risk levels of biological and toxic pollutants during the utilization of organophosphorus flame retardants, as well as better formulating control measures and controlling Microcystis aeruginosa blooms; 2. The present invention can be used to evaluate the environmental risks and environmental behaviors of organophosphorus flame retardants, providing a strong basis for the research, use, and management of the ecological and environmental risks of organophosphorus flame retardants; 3. The present invention takes into account both the macroscopic and microscopic processes of microalgae in water bodies, encompasses the action mechanisms of organophosphorus flame retardants on the growth, algotoxin synthesis, and release of Microcystis aeruginosa, and considers the environmental risks of microalgae metabolites, enabling a systematic and comprehensive evaluation of the environmental risks of organophosphorus flame retardants in water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Algal density curve (A), maximum biomass, and maximum growth rate (B) of Microcystis aeruginosa under exposure to different concentrations of TCPP in Example 1; Figure 2 Contents of chlorophyll a (A) and ATP (B) in Microcystis aeruginosa under exposure to different concentrations of TCPP in Example 1; Figure 3 Determination results of oxidative stress indexes of Microcystis aeruginosa under exposure to different concentrations of TCPP in Example 1, (A) is ROS, (B) is SOD, and (C) is GSH; Figure 4 Cell membrane integrity of Microcystis aeruginosa under exposure to different concentrations of TCPP in Example 1; Figure 5 Content of algotoxin per cell of Microcystis aeruginosa under exposure to different concentrations of TCPP in Example 1; Figure 6 Judgment diagram of the combined toxic effect mode of TCPP and MC-LR in Example 1; Figure 7 Ecological risks brought by TCPP in actual water bodies in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be further described below in conjunction with the examples and the drawings, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0014] Example 1 The ecological risk assessment method of organophosphorus flame retardants based on multi-dimensional toxicity indexes in this example includes the following steps: (1) Culturing Microcystis aeruginosa: Microcystis aeruginosa was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (FACHB905). 150 mL of experimental algae was incubated in a 250 mL conical flask sterilized by high pressure for the drug exposure experiment; the initial algal density was approximately 1.0×10 6Microcystis aeruginosa cells / mL were exposed to different concentrations of TCPP solution (0 μg / L, 0.1 μg / L, 1 μg / L, 10 μg / L, 100, 1000 μg / L, 10000 μg / L, and 100000 μg / L). The concentration design considered the actual concentration of TCPP in water and the continuously accumulating concentration. The culture conditions were: temperature 25 °C, light intensity 2000 lx, light-dark ratio 12 h:12 h, and humidity 50%; (2)Physiological and biochemical impact assessment: Algal density: On the 0th, 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, and 19th days of culture, the density of Microcystis aeruginosa cultured under different concentrations of TCPP was measured, and a cell counter was used to calculate the algal density. The results are as Figure 1 shown in A. Under the influence of 0.1 - 10000 μg / L of TCPP, the growth of Microcystis aeruginosa was promoted. When the TCPP concentration was 0.1 - 100 μg / L, the promoting effect of TCPP on the growth of Microcystis aeruginosa increased with the increase in TCPP concentration. The promoting effect of 100 μg / L of TCPP on the growth of Microcystis aeruginosa was the best. On the 21st day, the difference in the number of algal cells was the largest, and the biomass of Microcystis aeruginosa in the 100 μg / L group reached 148% of the control group. After entering the growth stable period (27 - 31 days), the promoting phenomenon still existed. However, when the TCPP concentration continued to increase, the biomass of Microcystis aeruginosa began to show a downward trend. Under the influence of the 100 mg / L group, the growth of Microcystis aeruginosa was inhibited compared with the control group; As Figure 1 shown in B, the maximum biomass ( K ) and maximum growth rate ( R max ) of Microcystis aeruginosa growth under different concentrations of TCPP exposure were analyzed through the logistic growth model; the results showed that under the influence of low-concentration (0.1 - 100 μg / L) TCPP, the maximum population density ( K ) and maximum population growth rate ( R max ) of Microcystis aeruginosa were both significantly increased, and both continued to rise with the increase in concentration; among them, under the influence of 100 μg / L TCPP, the K value was the largest, which was 1.41 times that of the control group, R max and K were 1.46 times that of the control group, respectively. With the increase in the TCPP concentration, the R max value and KThe values were lower than those of the control group. This indicates that low concentrations of TCPP in water can promote the growth of Microcystis aeruginosa, making it grow more and in larger quantities, and as TCPP continuously enters the water body, this promoting effect will increase, which may lead to an increased risk of Microcystis aeruginosa blooms in the water body; Metabolic activity: For Microcystis aeruginosa cultured in the logarithmic growth phase on the 13th day, after centrifuging 2 mL of algal solution at different concentrations of TCPP (10000 rpm, 10 min, 4 °C), the supernatant was discarded, 2 mL of 95% ethanol at 80 °C was added, and the mixture was incubated in a water bath at 80 °C for 1 h. Using 95% ethanol as a reference, the OD649 and OD665 of the supernatant were measured, and then calculated according to the formula (μg / L). The results are as Figure 2 shown in A. It can be seen that as the concentration of TCPP increases, the content of chlorophyll a in algal cells shows a trend of first increasing and then decreasing. Under the influence of 0.1 - 100 μg / L of TCPP, chlorophyll a gradually increases and reaches the highest value at 100 μg / L, which is 1.04 times that of the control group. The content of chlorophyll a is an indicator of cell metabolism. The higher the content of chlorophyll a, the more active the metabolic activities of algal cells. Therefore, low concentrations of TCPP can improve the photosynthesis efficiency of algal cells, and the promoting ability of photosynthesis gradually increases with the increase of TCPP concentration. The trend of TCPP promoting chlorophyll a is consistent with the trend of promoting algal growth, indicating that the increase in chlorophyll a is one of the factors promoting the growth of Microcystis aeruginosa, while high concentrations of TCPP inhibit photosynthesis, thus inhibiting the growth of Microcystis aeruginosa; Metabolic activity: Take 2 mL of algal solution of Microcystis aeruginosa cultured in the logarithmic growth phase on the 13th day for centrifugation to obtain a certain number (5 million - 20 million) of algal cells (10000 rpm, 10 min, 4 °C), and then add ATP extract. Freeze the cells in liquid nitrogen, thaw them at ambient temperature and repeat three cycles. Then, measure ATP according to the manufacturer's recommendations of the kit (Grees Biotech, China). The results are as Figure 2 shown in B. Under the influence of 0.1 - 100 μg / L of TCPP, the ATP content in algal cells gradually increases and reaches the highest value at 100 μg / L, which is 1.12 times that of the control group. ATP is the basic carrier of energy conversion in organisms and the most direct energy source in organisms. The increase in ATP indicates that low concentrations of TCPP enhance the metabolic activity of Microcystis aeruginosa. These changes verify the growth trend of Microcystis aeruginosa under low concentrations of TCPP; Oxidative stress indicators: Take about 5 million to 20 million Microcystis aeruginosa algal cells cultured on the 13th day of the logarithmic growth phase, place them in a centrifuge tube and centrifuge (10000 rpm, 10 min, 4 °C). After centrifugation, remove the supernatant, then add 1 mL of DCFH-DA probe with a concentration of 10 μM, shake well, and incubate in the dark at 37 °C for 30 min. After incubation, centrifuge (10000 rpm, 10 min, 4 °C), and also remove the supernatant after centrifugation. Wash twice with 1 mL of phosphate buffer (PBS), and the washing steps are the same as the above steps of centrifuging and removing the supernatant. After washing, add 1 mL of PBS to resuspend; Add 0.2 mL of the resuspended sample to a 96-well plate, and use a microplate reader to measure the fluorescence intensity at Ex / Em: 465 / 525 nm. The measurement results are as Figure 3 shown. It can be seen from Figure 3 that exposure to TCPP led to a significant increase in ROS. ROS is a common by-product of oxidative metabolism, and SOD and GSH are important components of the antioxidant system, which increase due to oxidative stress respectively. These results indicate that low concentrations of TCPP cause mild oxidative stress in Microcystis aeruginosa cells, while high concentrations of TCPP cause strong oxidative stress; Cell membrane integrity: Take 1 mL of algal solution, centrifuge (10000 rpm, 10 min, 4 °C), remove the supernatant, then add 1 mL of SYTOX GREEN stock solution, mix well, and incubate in the dark at 37 °C for 7 min. Measure the fluorescence intensity of the SYTOX GREEN nucleic acid dye using FL1 (530 nm). The cell membrane integrity of Microcystis aeruginosa is as Figure 4 shown. The effect of 0.1 - 100 μg / L of TCPP on cell membrane permeability is not significant. At this concentration, TCPP cannot damage the cell membrane of algal cells. However, with the increase in the concentration of TCPP, the cell membrane permeability has increased significantly, and the highest membrane permeability reaches 1.5 times that of the control group. Therefore, exposure to relatively high concentrations of TCPP (100 mg / L) will damage the cell integrity of Microcystis aeruginosa; (3)Assessment of the impact on microcystin synthesis and release: Take Microcystis aeruginosa cultured on the 13th day of the logarithmic growth phase, and measure the extracellular microcystin and total microcystin concentrations of Microcystis aeruginosa cultured under different concentrations of TCPP; Among them, for the determination of extracellular microcystin: Take 1 mL of the sample, centrifuge at 10000 rpm and 4 °C for 10 min, and take the supernatant for determination; For the determination of total microcystin: First, break the cells to release intracellular microcystin. Take 1 mL of the sample, quickly freeze it in liquid nitrogen for 1 min, thaw naturally, freeze-thaw three times repeatedly, and then centrifuge and take the supernatant for determination (10000 rpm, 10 min, 4 °C); The concentration of microcystin was determined by a microcystin detection kit, ELISA kit (Beacon, USA), with a detection range of 0 - 2.0 ppb. Therefore, a pre-experiment was conducted before each determination to determine the dilution factor, and a relatively large final solution volume was preferably selected during dilution to reduce errors. Note that the kit needs to be warmed to ambient temperature in advance before use, and the determination process is carried out according to the kit instructions. As Figure 5 shown, under the influence of TCPP at 0.1 - 100 μg / L, the total amount of MC-LR produced by algal cells gradually increased and reached the highest at 100 μg / L, which was 1.15 times that of the control group. This indicates that while promoting the growth of algal cells, TCPP also promoted the synthesis of microcystin, increasing the total amount of microcystin. The proportion of intracellular MC-LR decreased continuously, while the proportion of extracellular MC-LR increased continuously, indicating that with the increase of TCPP concentration, more MC-LR was released outside the cells; (4)Evaluation of the combined toxic effect mode of TCPP and microcystin: The toxicity experiment was carried out using Photobacterium phosphoreum. The TCPP and MC-LR to be tested were diluted to 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, 160 mg / L, 320 mg / L, and 640 mg / L, and all solutions were made up to volume with 3% NaCl; then 2 mL of the sample solution was added to 5 mL test tubes respectively, with 2 mL of 3% NaCl added to the blank tube. A new pipette tip was used for each sample, and three parallel groups were set for each concentration; 10 μL of the bacterial solution was added to each test tube in turn, the bottle cap was covered and inverted more than 5 times to mix the bacterial solution thoroughly, the bottle cap was removed and the test tube was placed back in place and waited for 15 min; the time should be accurately measured to the second when adding the bacterial solution to each test tube, so that the reaction time of each sample with the bacterial solution was the same at 15 min; immediately after 15 min of reaction in the first test tube with the added bacterial solution, the test tube was placed in a portable biotoxicity analyzer to measure the luminescence intensity and record it; For the obtained EC50 of single substances, in the form of a mixed mode with equal toxicity ratio, that is, the toxicity ratio of each substance in the mixture was equal to 1, a binary mixed system was composed of TCPP and MC-LR (microcystin), and 7 concentration gradients were set at 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 times the EC50, and the above exposure experiment was repeated; The acute toxicity of the two substances was obtained by exposing Photobacterium phosphoreum. The EC50 values of TCPP and MC-LR were 77 mg / L and 69 mg / L respectively. The mixing of TCPP and MC-LR decreased the luminescence intensity of Photobacterium phosphoreum, indicating that the combined toxicity increased after mixing the two substances. At this time, the EC50 was 58% of that when the single substance acted. The combined toxic effect mode between the two substances can be judged by the isobologram. From Figure 6It can be seen that the combined toxic effect mode of TCPP and MC-LR is additive effect. This indicates that after TCPP enters the water body, in addition to the risk brought by its own toxicity, it will also form a greater combined toxicity with MC-LR, increasing the toxicity of the water body and bringing greater ecological risks; (5)Evaluate the impact of TCPP on the ecological risk of water bodies: Compare the peak concentration of microcystin in the TCPP culture system at different concentrations with the toxicity endpoint value EC50, which characterizes the harm of microcystin, measured from the water samples collected from the water body to be evaluated, to obtain the risk quotient RQ. When RQ≥1, it is a high risk; when 0.1≤RQ<1, it is a medium risk; when 0.01≤RQ<0.1, it is a low risk, so as to make a preliminary judgment on the ecological risk of TPCC. The calculation formula is as follows: In the formula: MEC——Measured environmental concentration of pollutants, mg / L; PNEC——Predicted no-effect concentration of pollutants, mg / L; L(E)C50——Concentration of the test substance that causes a certain effect change in 50% of the test organisms, obtained through published literature, existing toxicity databases, and toxicity prediction software, mg / L; AF——Assessment coefficient for selecting acute toxicity data, set to 1000; Evaluate the ecological risk of TCPP in the water body of Dianchi Lake. The concentrations of TCPP in the water samples at five sampling points in Dianchi Lake are 15.48 ng / L, 6.70 ng / L, 22.39 ng / L, 7.21 ng / L, and 6.60 ng / L respectively; from Figure 7 It can be seen that the ecological risks brought by TCPP at the five sampling points in Dianchi Lake are basically below low risk, and only the TCPP risk level at one sampling point reaches low risk, indicating that the risk of TCPP in Dianchi Lake is relatively low. However, with the continuous reuse of reclaimed water and the non-point source pollution brought by a part of rainwater and domestic sewage, etc., TCPP in the water body will continue to accumulate and the concentration will continue to rise. Therefore, the risks brought by TCPP will also continue to rise. At the same time, although the risks brought by TCPP itself are not high, the harm caused by its stimulation of the growth and metabolism of harmful Microcystis aeruginosa cannot be ignored. On the one hand, the concentration of TCPP in the actual environment will stimulate the growth of algae and exacerbate the outbreak of water blooms. On the other hand, the concentration of TCPP in the actual environment will stimulate the synthesis and release of toxic metabolites of algae and produce combined toxicity with them, increasing the ecological risk of the water body.

Claims

1. An ecological risk assessment method for organophosphorus flame retardants based on multi-dimensional toxicity indicators, characterized by The following steps are involved: (1) Cultivation of Microcystis aeruginosa: Microcystis aeruginosa with the same initial algae density was exposed to TCPP solutions of different concentrations for cultivation. The concentrations were 0 μg / L, 0.1 μg / L, 1 μg / L, 10 μg / L, 100 μg / L, 10000 μg / L, and 100000 μg / L, respectively. The culture conditions were the same. (2) Physiological and biochemical impact assessment: On the 0th, 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, and 19th days of culture, the density of Microcystis aeruginosa cultured with different concentrations of TCPP was measured; The chlorophyll a content of Microcystis aeruginosa cultured at the 13th day of logarithmic growth phase was measured under different concentrations of TCPP. The metabolic activity, oxidative stress, and cell membrane integrity of Microcystis aeruginosa cultured at different concentrations of TCPP were evaluated by taking the Microcystis aeruginosa cultured at day 13 of logarithmic growth phase. (3) Assessment of the impact of algal toxin synthesis and release: The extracellular algal toxins and total algal toxins of Microcystis aeruginosa cultured at the 13th day of logarithmic growth phase were measured under different concentrations of TCPP. (4) Evaluation of the combined toxicity of TCPP and microcystins: The EC50 of organophosphorus flame retardant and microcystin was obtained through luminescent bacteria exposure experiment, and then the organophosphorus flame retardant and microcystin were mixed at equal toxicity ratio concentration, and the luminescent bacteria exposure experiment was continued to obtain the EC50 of the mixed system, and the iso-effect curve was used to determine the combined toxicity mode of the two. (5) Assess the impact of TCPP on water ecological risks: The peak concentration of algae toxins in the TCPP culture system with different concentrations was compared with the toxic endpoint value EC50 of the water sample taken from the water body to be evaluated and sent to the laboratory to measure the hazard of algae toxins, and the risk quotient RQ was obtained. When RQ ≥ 1, it is a high risk; when 0.1 ≤ RQ < 1, it is a medium risk; when 0.01 ≤ RQ < 0.1, it is a low risk, so as to make a preliminary judgment on the ecological risk of TPCC. The calculation formula is as follows: Where: MEC——measured environmental concentration of pollutants, mg / L; PNEC – predicted no effect concentration of a pollutant, mg / L; L(E)C50——the concentration of the test substance that causes a certain effect change in 50% of the test organisms, mg / L; AF - Assessment factor for selection of acute toxicity data.

2. The method for ecological risk assessment of organophosphorus flame retardants based on multi-dimensional toxicity indicators according to claim 1 is characterized in that Step (1) The initial biomass of Microcystis aeruginosa exposed to TCPP was 1.0-1.5×10 6 cells / mL.

3. The method for ecological risk assessment of organophosphorus flame retardants based on multi-dimensional toxicity indicators according to claim 1 is characterized in that In step (1), 1.7 g of culture medium was taken into a conical flask, 1000 mL of distilled water was added, and the mixture was sterilized at 121°C for 30 min. After the culture medium was cooled to room temperature, the algae was inoculated at a 10% inoculation rate, and the algae cells were used after the number of algae cells was expanded to the required density; 150 μL of TCPP solution was added to 149.85 mL of the experimental sample, respectively, so that the final concentration of TCPP in the sample was 0.1-100000 μg / L, and three parallel samples were set for each concentration. All experimental conditions were the same as those for the algae culture; the algae cells were cultured in a light incubator under the following culture conditions: temperature 25°C, light intensity 4000-6000 lx, light-dark ratio 12 h:12 h, and continuous shaking at 120 rpm.

4. The method for ecological risk assessment of organophosphorus flame retardants based on multi-dimensional toxicity indicators according to claim 1 is characterized in that Step (4) specifically uses Photobacterium luminescens to conduct a toxicity experiment, dilutes the TCPP and MC-LR to be tested to 10 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, 160 mg / L, 320 mg / L, and 640 mg / L, and all solutions are fixed to volume with 3% NaCl; then add 2 mL of sample solution to 5 mL test tubes respectively, wherein the blank tube is added with 2 mL of 3% NaCl, a new pipette tip is used for each sample, and three parallel groups are set for each concentration; 10 μL of bacterial solution is added to each test tube in turn, the bottle cap is covered and inverted for more than 5 times to fully mix the bacterial solution, the bottle cap is removed and returned to its original position and waited for 15 minutes; each test tube should be accurately timed when adding bacterial solution, and the time should be recorded to seconds, so that the reaction time of each sample and bacterial solution is the same as 15 minutes; after the first bacterial solution tube reacts for 15 minutes, the test tube is immediately placed in a portable biotoxicity analyzer to measure the luminescence and record it; The EC50 of a single substance was obtained by mixing with equal toxicity ratios, that is, the toxicity ratio of each substance in the mixture was equal to 1. TCPP and MC-LR were combined into a binary mixed system, and 7 concentration gradients were set at 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 times the EC50, and the above exposure experiment was repeated.

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