A method for evaluating the bioavailability of bound organic pollutants

Abstract

The present invention provides a method for evaluating the bioeffectiveness of bound organic pollutants, including: determining the concentration range of water environment occurrence; using an exposure device to expose aquatic organisms to the organic pollutants to be evaluated to obtain a first exposed organism and a second exposed organism; determining a first cumulative organ and a second cumulative organ; analyzing the first cumulative organ and the second cumulative organ to obtain the metabolic pathway of the first cumulative organ, the key metabolites of the first cumulative organ, the metabolic pathway of the second cumulative organ, and the key metabolites of the second cumulative organ; calculating the concentration of the first key metabolite and the concentration of the second key metabolite; obtaining the change trend of the concentration of the second metabolite according to the concentration of the first key metabolite and the concentration of the second key metabolite; and evaluating the bioeffectiveness of the organic pollutants to be evaluated according to the change trend of the concentration of the second metabolite. The present invention solves the problem that the bioeffectiveness of bound organic pollutants cannot be accurately evaluated.

Description

Technical Field

[0001] The invention relates to the technical field of environmental science and engineering and aquatic toxicology, and in particular to a method for evaluating the biological effectiveness of bound organic pollutants. Background Art

[0002] Organic pollutants are widely present in the aquatic environment and can quickly accumulate in different aquatic organisms through bioaccumulation. They also cause endocrine disruption, behavioral abnormalities, metabolic disorders and other hazards to aquatic organisms through the endocrine, nervous and metabolic systems, seriously affecting the stability and health of aquatic ecosystems. Assessing and predicting the bioavailability of organic pollutants is a key step in understanding their ecological risks to the aquatic environment, and is also an important basis for implementing organic pollution management and control.

[0003] The composition of natural water environment is complex, and organic pollutants do not exist alone. Sediment, humus, metal hydrate oxides, and artificially synthesized particles generally coexist. Since these substances generally have the characteristics of small particle size, large specific surface area, high surface activity, and strong adsorption, organic pollutants will inevitably interact with them, thereby generating bound organic pollutants with significantly different bioavailability, which affects the accurate assessment of the ecological and environmental risks of organic pollutants.

[0004] For example, nanoparticles can effectively promote the bioaccumulation of perfluorooctane sulfonic acid in zebrafish (Danio rerio), but significantly reduce the biological effect of benzo(a)pyrene in mussels. The bioavailability of bound organic pollutants is affected by multiple factors such as the type of coexisting substances, particle size, and morphology. Accurately assessing the bioavailability of bound organic pollution is a key node for understanding the ecological risks of organic pollutants in the natural environment and implementing risk assessment.

[0005] At present, the ecological risk assessment of organic pollutants usually uses typical model organisms (zebrafish, Daphnia magna, etc.) to expose them to dilute pollutants, selects appropriate methods to extract and analyze their concentrations in the organism, and uses bioaccumulation methods to quantitatively evaluate their bioavailability. However, this method includes all organic pollutants, and the use of organic solvents will separate the bound organic pollutants, ignoring the changes in their bioavailability, resulting in excessively high bioaccumulation concentrations of organic pollutants, making it impossible to truly and effectively assess their actual risks in the aquatic environment. In addition, there are also risk assessments based on biomarker methods, but due to the large amount of biomass required, insensitive biological endpoints, and non-linear dose-effect relationships, assessments based on biomarker methods are also unable to accurately assess the effective bioavailability of bound organic pollutants. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for evaluating the bioeffectiveness of bound organic pollutants. The present invention solves the problem in the prior art that the bioeffectiveness of bound organic pollutants cannot be accurately evaluated.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] A method for evaluating the bioavailability of bound organic pollutants, comprising:

[0009] Determine the concentration range of the organic pollutants to be evaluated in the water environment;

[0010] Based on the concentration range of the water environment, the test aquatic organism is exposed to the organic pollutant to be evaluated by using an exposure device to obtain a first exposed organism and a second exposed organism; wherein an exposure baffle is provided in the exposure device;

[0011] Determine the first cumulative organ and the second cumulative organ according to the accumulation of the organic pollutant to be evaluated in each organ of the first exposed organism and the accumulation of the organic pollutant in each organ of the second exposed organism; analyze the first cumulative organ and the second cumulative organ respectively to obtain the metabolic pathway of the first cumulative organ, the key metabolites of the first cumulative organ, the metabolic pathway of the second cumulative organ, and the key metabolites of the second cumulative organ;

[0012] Based on the metabolic pathway of the first cumulative organ, a first key metabolite concentration is calculated according to the key metabolites of the first cumulative organ;

[0013] Based on the metabolic pathway of the second cumulative organ, a second key metabolite concentration is calculated according to the key metabolites of the second cumulative organ;

[0014] Obtaining a change trend of a second metabolite concentration according to the first key metabolite concentration and the second key metabolite concentration;

[0015] The bioavailability of the organic pollutant to be evaluated is evaluated based on the change trend of the concentration of the second metabolite.

[0016] Preferably, the exposure process of exposing the aquatic organisms to the organic pollutants to be evaluated by using an exposure device to obtain the exposed organisms includes:

[0017] Monitoring and updating the solution in the exposure device to ensure that the concentration of the pollutant to be tested is within a preset concentration range;

[0018] The behavioral changes of the test aquatic organisms in the exposure device are monitored to ensure the stability of the test aquatic organisms.

[0019] Preferably, obtaining the change trend of the second metabolite concentration according to the first key metabolite concentration and the second key metabolite concentration includes:

[0020] Obtaining a difference between the concentration of the first key metabolite and the concentration of the second key metabolite;

[0021] The concentration change trend of the second metabolite is obtained according to the difference.

[0022] Preferably, monitoring and updating the solution in the exposure device to ensure that the concentration of the pollutant to be detected is within a preset concentration range includes:

[0023] Liquid chromatography-mass spectrometry is used to regularly test the unbound target pollutants to obtain the exposure solution concentration test results;

[0024] If the exposure solution concentration test results vary by more than 10% of the target concentration, refill the solution immediately. If it does not exceed 10%, replace 50% of the exposure solution every day.

[0025] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0026] The present invention provides a method for evaluating the bioeffectiveness of bound organic pollutants. The present invention analyzes and evaluates the bioeffectiveness of bound organic pollutants by detecting the concentration of metabolites in the main accumulation organs of organisms, which can reduce the consumption of 3 / 4 of organic reagents and is more environmentally friendly and clean. It can more effectively evaluate the bioeffectiveness of organic pollutants in organisms in real water environments and improve the accuracy of evaluating the bioeffectiveness of bound organic pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 A flow chart of a method for evaluating the bioeffectiveness of bound organic pollutants provided in an embodiment of the present invention;

[0029] Figure 2 A technical roadmap for analyzing and evaluating the bioavailability of bound organic pollutants provided by embodiments of the present invention;

[0030] Figure 3 A schematic diagram of the structure of the experimental exposure device and the partition provided in the embodiment of the present invention;

[0031] Figure 4 Metabolites and transformation pathways of the target substance provided in the embodiments of the present invention in typical aquatic organisms;

[0032] Figure 5 A schematic diagram showing a comparison of the cumulative concentrations of the target substance and its key metabolites in a living body provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The purpose of the present invention is to provide a method for evaluating the bioeffectiveness of bound organic pollutants, which solves the problem in the prior art that the bioeffectiveness of bound organic pollutants cannot be accurately evaluated.

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] like Figure 1 As shown, the present invention provides a method for evaluating the bioeffectiveness of bound organic pollutants, comprising:

[0037] Step 100: Determine the water environment occurrence concentration range of the organic pollutant to be evaluated;

[0038] Specifically, by consulting relevant literature and collecting data, the concentration range of organic pollutants in the water environment is determined;

[0039] Step 200: Based on the concentration range of the water environment, using an exposure device to expose the test aquatic organism to the organic pollutant to be evaluated, to obtain a first exposed organism and a second exposed organism; wherein the exposure device is provided with an exposure baffle;

[0040] Specifically, a biological exposure device for bound organic pollutants is constructed, wherein the key component is an exposure device partition with different pore sizes and materials;

[0041] Specifically, the material of the partition of the exposure device is determined according to the adsorption characteristics of the target pollutants, and generally polytetrafluoroethylene (PTFE) material is used, and different pore sizes are selected according to the particle size of natural sediment or artificial particles in the water environment or the type and hydrated particle size of humic acid;

[0042] Step 300: determining a first cumulative organ and a second cumulative organ according to the cumulative situation of the organic pollutant to be evaluated in each organ of the first exposed organism and the cumulative situation of the organic pollutant in each organ of the second exposed organism; Step 400: analyzing the first cumulative organ and the second cumulative organ respectively to obtain the metabolic pathway of the first cumulative organ, the key metabolites of the first cumulative organ, the metabolic pathway of the second cumulative organ and the key metabolites of the second cumulative organ;

[0043] Step 500: Based on the metabolic pathway of the first cumulative organ, calculate the first key metabolite concentration according to the key metabolites of the first cumulative organ;

[0044] Step 600: Based on the metabolic pathway of the second cumulative organ, calculate the second key metabolite concentration according to the key metabolites of the second cumulative organ;

[0045] Step 700: Obtaining a change trend of a second metabolite concentration according to the first key metabolite concentration and the second key metabolite concentration;

[0046] Step 800: Evaluate the bioavailability of the organic pollutant to be evaluated according to the variation trend of the concentration of the second metabolite.

[0047] Furthermore, the exposure process of exposing the aquatic organisms to the organic pollutants to be evaluated by using an exposure device to obtain the exposed organisms includes:

[0048] Monitoring and updating the solution in the exposure device to ensure that the concentration of the pollutant to be tested is within a preset concentration range;

[0049] The behavioral changes of the test aquatic organisms in the exposure device are monitored to ensure the stability of the test aquatic organisms.

[0050] Specifically, the exposure process is as follows: based on the exposure device, the test aquatic organisms are exposed to the target organic pollutants in combination with the environmentally relevant concentrations; the exposure period of different test organisms is adjusted and determined according to the incubation time, and the exposure solution is regularly monitored and updated to ensure the stability of the target pollutant concentration; various behavioral changes of the test organisms are regularly monitored to ensure the stability of the test organisms during the exposure period; after the exposure, the test organisms are collected, and the accumulation of target pollutants in different organs is extracted and analyzed by appropriate methods to determine the main accumulation organs; the metabolites in the main accumulation organs are fully scanned by a high-resolution mass spectrometer, and the biological metabolic pathways and key metabolites of the target pollutants in the main accumulation organs are determined in combination with literature data, and the key metabolites used to indicate the biological effectiveness of the target pollutants are determined in combination with the changes in the accumulation of the two. Figure 2 shown.

[0051] Furthermore, obtaining the change trend of the second metabolite concentration according to the first key metabolite concentration and the second key metabolite concentration includes:

[0052] Obtaining a difference between the concentration of the first key metabolite and the concentration of the second key metabolite;

[0053] The concentration change trend of the second metabolite is obtained according to the difference.

[0054] Further, monitoring and updating the solution in the exposure device to ensure that the concentration of the pollutant to be tested is within a preset concentration range includes:

[0055] Liquid chromatography-mass spectrometry is used to regularly test the unbound target pollutants to obtain the exposure solution concentration test results;

[0056] If the exposure solution concentration test results vary by more than 10% of the target concentration, refill the solution immediately. If it does not exceed 10%, replace 50% of the exposure solution every day.

[0057] Specifically, a biological exposure device for bound organic pollutants is constructed to conduct exposure experiments:

[0058] Add the target organic pollutant to one side of the partition of the exposure device, add the coexisting substances such as silt, artificial particles or humic acid to the other side of the partition of the exposure device, and stir evenly to form a stable combined organic pollutant exposure solution;

[0059] The test organisms are added to both sides of the partition of the exposure device for exposure experiments. The non-bound target pollutants are regularly detected by liquid chromatography-mass spectrometry. If the detected concentration range exceeds 10% of the target concentration, the solution is immediately replenished. If it does not exceed 10%, 50% of the exposure solution is replaced every day to maintain the stability of the exposure solution concentration.

[0060] After the exposure, the test organisms on both sides of the partition of the exposure device were collected respectively, the concentrations of the target substances and their key metabolites in the main accumulation organs were extracted and tested, the bioaccumulation levels of the bound target pollutants and their key metabolites were calculated, and the difference between the two in the bound and unbound exposure groups was obtained by comparison. The concentration change trend of the key metabolites in the bound organic matter treatment group was determined to evaluate the biological effectiveness of the bound organic pollutants.

[0061] Specifically, in the exposure device of the present invention, the container support component is made of glass or polytetrafluoroethylene, which will not adsorb organic pollutants. If different types of collected organisms need to be exposed, the container model can be adjusted. According to the particle parameters and the type of tested organisms, the ultrafiltration baffle can be replaced with a dialysis bag, such as Figure 3 .

[0062] The present invention also provides the following another embodiment:

[0063] Testing the bioavailability of the antidepressant fluoxetine in zebrafish:

[0064] 1) Determination of the concentration of the antidepressant fluoxetine (FLX):

[0065] The early life zebrafish exposure test was designed according to the OECD Guideline 203 Operational Guidelines. Based on the therapeutic plasma concentration of FLX in humans (91 μg / L), the exposure concentration of FLX to early life zebrafish was determined to be 100 μg / L. In addition, in this embodiment, the coexisting particulate matter selected commercially manufactured carbon nanomaterials (CNMs, 10-30 μm) at a concentration of 1 mg / L, which would not significantly affect the growth and development of zebrafish embryos.

[0066] 2) Determination of main metabolites and pathways

[0067] Based on literature search, the metabolic pathway of FLX in zebrafish was determined as follows Figure 4 The metabolites of FLX in zebrafish mainly include demethylation, N-methylation, hydroxylation, N-acetylation, N-formylation, N-propionylation, and conjugation with L-valine. The demethylation process is the main degradation pathway, and the generated demethylfluoxetine, or norfluoxetine, is the main and most critical metabolite.

[0068] 3) Exposure device operation

[0069] The high-concentration FLX solution in the drug storage system was transported to the drug reserve system by a metering pump, and the heating device and aeration device were adjusted to adjust the water quality parameters to 28°C, a dissolved oxygen concentration of not less than 6 mg / L, and a pH of 7.2±0.1. The exposure solution was fully mixed using a magnetic stirring system, and the solution was transported to the exposure device by a peristaltic pump after its water quality parameters were stable. When the volume of the exposure solution met the experimental requirements, CNMs were added to the other side of the container, and the magnetic stirring system was operated to mix the exposure solution. When the water quality indicators of the exposure solution met the exposure requirements, early-life zebrafish were exposed for 144 hours, and a blank control group and a solvent control group were set up. The zebrafish fry were not fed during the exposure process.

[0070] 4) Experimental exposure

[0071] The exposure device was connected to the sensor and computer control unit to monitor the water quality parameters of the exposure solution in real time. Before the experiment, the main metabolite of FLX in the organism was identified as norfluoxetine (NFLX) based on the metabolite pathway, and then the liquid chromatography-mass spectrometry detection system was set to determine the qualitative and quantitative analysis methods of FLX and its metabolite NFLX. During the exposure period, the exposure solution was quantitatively monitored every 2 hours. If the concentration was 10% lower than the target exposure concentration, the system issued an alarm signal and supplemented the target pollutant dose through the dosing system so that the exposure concentration was basically consistent with the target setting concentration. During the exposure period, zebrafish larvae were collected every 12 hours, and FLX and its main metabolite NFLX in their bodies were concentrated and enriched to obtain the temporal dynamic accumulation effect of the drug and its metabolites.

[0072] 5) End of exposure

[0073] After the 144-h exposure, the collected zebrafish larvae were concentrated and enriched for pollutants to obtain the cumulative concentrations of the target pollutant FLX and its metabolite NFLX in zebrafish larvae.

[0074] 6) Bioaccumulation dynamics of drugs and metabolites and effect responses

[0075] In order to better evaluate the effect of CNMs on the bioavailability of FLX in early zebrafish, this paper evaluated the effect of CNMs on the bioavailability of FLX by calculating the bioaccumulation concentrations of the target compound FLX and its main metabolite NFLX in zebrafish. The results are as follows: Figure 5 shown.

[0076] 7) Result evaluation

[0077] When FLX was exposed alone, the accumulation trends of the pollutant FLX and its metabolite NFLX in the organism were consistent, indicating that FLX could be effectively utilized in the organism and converted into the metabolite NFLX; the generation and accumulation of NFLX could be used to indicate the bioavailability of FLX in zebrafish. When CNMs coexisted, the bioaccumulation concentration of FLX in zebrafish larvae increased significantly, and CNMs changed the accumulation of FLX in the organism. At the same time, the accumulation trend of NFLX in the organism was consistent with that of FLX, indicating that FLX bound to CNMs was biologically active in the organism and could be further bioavailable. The generation and accumulation of NFLX could be further used to indicate the bioavailability of bound FLX in zebrafish. Therefore, under the influence of the coexistence of CNMs, the bioavailability of the bound antidepressant drug FLX in zebrafish larvae can be indicated by the generated and accumulated metabolite NFLX, and the main metabolite is an effective method for evaluating the bioavailability of the bound state.

[0078] The beneficial effects of the present invention are as follows:

[0079] The present invention provides a method for evaluating the bioeffectiveness of bound organic pollutants. The present invention analyzes and evaluates the bioeffectiveness of bound organic pollutants by detecting the concentration of metabolites in the main accumulation organs of organisms, which can reduce the consumption of 3 / 4 of organic reagents and is more environmentally friendly and clean. It can more effectively evaluate the bioeffectiveness of organic pollutants in organisms in real water environments and improve the accuracy of evaluating the bioeffectiveness of bound organic pollutants.

[0080] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the system disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0081] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for evaluating the bioavailability of bound organic pollutants, characterized in that: include: Determine the concentration range of the organic pollutants to be evaluated in the water environment; Based on the concentration range of the water environment, the test aquatic organism is exposed to the organic pollutant to be evaluated by using an exposure device to obtain a first exposed organism and a second exposed organism; wherein an exposure baffle is provided in the exposure device; Determine the first cumulative organ and the second cumulative organ according to the accumulation of the organic pollutant to be evaluated in each organ of the first exposed organism and the accumulation of the organic pollutant in each organ of the second exposed organism; analyze the first cumulative organ and the second cumulative organ respectively to obtain the metabolic pathway of the first cumulative organ, the key metabolites of the first cumulative organ, the metabolic pathway of the second cumulative organ, and the key metabolites of the second cumulative organ; Based on the metabolic pathway of the first cumulative organ, a first key metabolite concentration is calculated according to the key metabolites of the first cumulative organ; Based on the metabolic pathway of the second cumulative organ, a second key metabolite concentration is calculated according to the key metabolites of the second cumulative organ; Obtaining a change trend of a second metabolite concentration according to the first key metabolite concentration and the second key metabolite concentration; evaluating the bioavailability of the organic pollutant to be evaluated according to the concentration variation trend of the second metabolite; The specific steps of using the exposure device to expose the test aquatic organism to the organic pollutant to be evaluated to obtain the first exposed organism and the second exposed organism are: Add the organic pollutants to be evaluated to one side of the partition of the exposure device, add the sediment, artificial particles or humic acid coexisting substances to the other side of the partition of the exposure device, and stir evenly to form a stable combined organic pollutant exposure solution; The test organisms were added to both sides of the partition of the exposure device to conduct exposure experiments; After the exposure, the test organisms on both sides of the partition of the exposure device were collected separately.

2. The method for evaluating the bioeffectiveness of bound organic pollutants according to claim 1, characterized in that: The exposure process of using the exposure device to expose the test aquatic organism to the organic pollutant to be evaluated to obtain the exposed organism includes: Monitoring and updating the solution in the exposure device to ensure that the concentration of the pollutant to be tested is within a preset concentration range; The behavioral changes of the test aquatic organisms in the exposure device are monitored to ensure the stability of the test aquatic organisms.

3. The method for evaluating the bioavailability of bound organic pollutants according to claim 1, characterized in that: The obtaining of the change trend of the second metabolite concentration according to the first key metabolite concentration and the second key metabolite concentration comprises: Obtaining a difference between the concentration of the first key metabolite and the concentration of the second key metabolite; The concentration change trend of the second metabolite is obtained according to the difference.

4. The method for evaluating the bioavailability of bound organic pollutants according to claim 1, characterized in that: Monitoring and updating the solution in the exposure device to ensure that the concentration of the pollutant to be tested is within a preset concentration range includes: Liquid chromatography-mass spectrometry is used to regularly test the non-bound organic pollutants to be evaluated and obtain the exposure solution concentration test results; If the exposure solution concentration test results vary by more than 10% of the target concentration, refill the solution immediately; if it does not exceed 10%, replace 50% of the exposure solution every day.

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