FM550 in-vivo enrichment detection method and risk monitoring method for tilapia mossambica
By combining solvent extraction and gas chromatography-mass spectrometry with detection methods for tilapia liver and muscle samples, the accuracy of FM550 accumulation in aquatic organisms has been solved, enabling efficient risk assessment and monitoring, which is applicable to aquaculture and ecological protection.
Patent Information
- Application Number
- CN202511563717.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-30
- Publication Date
- 2026-01-13
AI Technical Summary
Existing technologies are insufficient to accurately reflect the actual accumulation of FM550 in aquatic organisms, lack suitable biological monitoring index species, and have low sensitivity and efficiency in detection technologies, thus failing to meet the needs of large-scale environmental monitoring.
Tilapia was used as an indicator organism. Its liver and muscle samples were obtained, freeze-dried, pulverized and homogenized, and then subjected to solvent extraction, QuEChERS purification and gas chromatography-mass spectrometry detection to obtain the in vivo enrichment results of FM550. The enrichment factor was calculated and the enrichment rule was established for risk assessment.
It enables accurate detection and risk assessment of FM550 in aquatic organisms. It is easy to operate, highly sensitive, suitable for large-scale applications, and can truly reflect the actual exposure level of organisms, providing a scientific risk assessment tool.
Smart Images
Figure CN121324538A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of ecological monitoring, and particularly relates to a FM550 in vivo enrichment detection method and a risk monitoring method for tilapia. BACKGROUND
[0002] As a new type of bromine flame retardant widely used in the industrial field, FM550 mainly contains TBB (2-ethylhexyl-2,3,4,5-tetrabromobenzoate) and TBPH (tetrabromophthalic acid bis (2-ethylhexyl) ester), two important organic bromine compounds. A large number of environmental chemistry studies have shown that this type of brominated flame retardant has significant hydrophobic properties, strong environmental persistence and obvious biological accumulation effect. More importantly, a number of toxicology studies have confirmed that these compounds can cause persistent harm to the growth and reproductive capacity of aquatic organisms through various pathways such as interfering with the function of the endocrine system and affecting metabolic processes.
[0003] At present, the environmental monitoring methods for FM550 can be summarized into two major technical routes: the first is the traditional physicochemical analysis technology, such as using gas chromatography-mass spectrometry (GC-MS) or high performance liquid chromatography (HPLC) to detect the content of TBB / TBPH in environmental media such as water and sediments; the second is an indirect method based on risk assessment, which mainly compares the environmental monitoring concentration with the toxicity threshold value measured in the laboratory to calculate the potential environmental risk degree. However, through in-depth analysis, it can be found that the existing detection method system has the following obvious technical shortcomings: Firstly, the existing method cannot accurately reflect the actual accumulation in the organism. These technologies are limited to the determination of pollutant concentration in environmental media, and cannot directly quantify the actual exposure level in aquatic organisms, which may cause a large deviation between the risk assessment result and the actual situation.
[0004] Secondly, there is a lack of suitable biological monitoring indicator species. In the current environmental monitoring system, there is no standard method for using common economic fish (such as carp, crucian carp, etc.) in aquaculture as an indicator organism, which leads to the current situation that the detection result is difficult to directly serve the risk early warning work of the actual aquaculture environment.
[0005] Thirdly, the sensitivity and efficiency of the existing detection technology need to be improved. The traditional sample pretreatment process usually needs to go through complex and tedious extraction, purification and concentration steps, which not only consumes time and effort, but also has unstable purification effect, which seriously restricts the efficiency of large-scale environmental monitoring work.
[0006] In view of the above problems, in the current environmental monitoring and ecological protection work, it is urgent to develop a new FM550 detection method system based on the enrichment dynamic law in aquatic organisms. This method should be able to accurately reflect the accumulation process of pollutants in aquatic organisms, while having the technical advantages of simple operation, high sensitivity, and suitable for large-scale application. SUMMARY
[0007] The application provides a FM550 in vivo enrichment detection method and risk monitoring method of tilapia, which can reflect the actual risk of flame retardants in the environment and is convenient for application in aquaculture and ecological risk assessment.
[0008] To solve the above technical problems, the embodiments of the present application provide a detection method for FM550 in vivo enrichment of tilapia, comprising the following steps: Obtain liver samples and muscle samples of target tilapia, freeze dry for 72 hours, take 1g, crush and homogenize, and reserve; the target tilapia refers to tilapia exposed to FM550 environment for 56 days; After solvent extraction, QuEChERS purification treatment and solvent conversion treatment of the crushed and homogenized sample, gas chromatography mass spectrometry detection is performed to obtain the detection result of FM550 in vivo enrichment.
[0009] As some optional embodiments of the present application, the solvent extraction, QuEChERS purification treatment and solvent conversion treatment of the crushed and homogenized sample comprise the following steps: Acetone / n-hexane / NaCl extraction is adopted, 13C-BDE209 internal standard solution is added, and after mixing, centrifugal treatment and extraction treatment, nitrogen blowing is performed to constant volume; QuEChERS purification, centrifugation and filtration are performed; after nitrogen blowing drying, isooctane is used for constant volume.
[0010] As some optional embodiments of the present application, the volume ratio of acetone:n-hexane:NaCl:13C-BDE209 internal standard solution is 2mL:2mL:1mL:10 μL; The concentration of the 13C-BDE209 internal standard solution is 0.5 mg / L.
[0011] As some optional embodiments of the present application, after mixing, centrifugal treatment and extraction treatment, nitrogen blowing is performed to constant volume, comprising the following steps: Vortex oscillation for 10 min, centrifugation at 7500 r / min for 10 min, collection of supernatant, repeated extraction for 2 times, nitrogen blowing to constant volume to 1 mL to obtain the extraction solution.
[0012] As some optional embodiments of the present application, the QuEChERS purification, centrifugation and filtration are performed; after nitrogen blowing drying, isooctane is used for constant volume, comprising the following steps: The extraction solution was transferred to a centrifuge tube containing 50 mg of anhydrous magnesium sulfate and 50 mg of C18, shaken for 10 min, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane; Blown dry under gentle nitrogen, and isooctane was added to 200 μL.
[0013] As some optional embodiments of the present application, the parameters of the gas chromatography mass spectrometry detection are as follows: Chromatographic column: Agilent DB-5MS (15 m x 0.25 mm x 0.1 μm); Carrier gas: high-purity helium; Temperature program: 110℃ (5 min) → 200℃ (20℃ / min, 4.5 min) → 310℃ (10℃ / min, 15 min) → 350℃ (4 min); Injection mode: splitless, 1 μL; Ion source temperature 150℃, quadrupole temperature 150℃, transfer line 280℃; SIM mode quantification.
[0014] In addition, the present application also provides a risk monitoring method for FM550 in vivo enrichment based on tilapia, comprising the following steps: Based on the detection result of FM550 in vivo enrichment obtained by the detection method, an enrichment factor is calculated; Based on the enrichment factor, an enrichment rule is established; Based on the enrichment rule, risk judgment is performed.
[0015] As some optional embodiments of the present application, the enrichment factor is calculated based on the following formula: BCF=C fish / C feed In the formula, BCF is the enrichment factor; C fish is the concentration of the target substance in the liver or muscle of tilapia, ng·g - ¹; C feed is the average concentration of the target substance in the feed, ng·g - ¹.
[0016] As some optional embodiments of the present application, based on the enrichment factor, the enrichment rule is established, comprising the following steps: Corresponding the BCF results of different lengths of time with the feed concentration, an enrichment curve is drawn, and the enrichment rule with exposure time is obtained.
[0017] As some optional embodiments of the present application, the risk judgment based on the enrichment rule comprises the following steps: If BCF long-term >1, it indicates that the tilapia has significant enrichment of the target substance, suggesting that there is a cumulative risk in the environment or feed.
[0018] For the environmental risk research of the new flame retardant FM550, scholars at home and abroad mainly focus on the detection and analysis of target pollutants in water and sediments, but for a long time, there is a lack of effective monitoring means that can accurately reflect the actual exposure condition of aquatic organisms. The present application innovatively proposes a new method for detecting FM550 exposure based on the model organism tilapia, and the specific implementation scheme comprises the following key steps: first, obtain the liver tissue samples and muscle tissue samples of the target tilapia, which are experimental samples exposed to water environment containing FM550 for 56 days under precise control of exposure conditions; then, the collected tissue samples are placed in a freeze dryer for continuous drying treatment for 72 hours, and after the samples are completely dehydrated, 1 gram of dried sample is accurately weighed, ground and homogenized to prepare biological samples meeting the detection requirements. In the sample pretreatment stage, the ground and homogenized biological samples are extracted by using an optimized organic solvent extraction technology, then the improved QuEChERS purification method is used to remove the matrix interference components, and finally the test solution suitable for instrument analysis is obtained through solvent conversion treatment. The samples treated as above are quantitatively detected by using a high-sensitivity gas chromatography-mass spectrometry (GC-MS), so as to obtain accurate data of the spatial and temporal enrichment characteristics of TBB and TBPH, the two main components of FM550, in the key tissues (liver and muscle) of tilapia. The innovation value of the present application lies in that the above method effectively solves the major technical bottleneck that the traditional environmental monitoring technology can only detect the concentration of environmental medium and cannot intuitively reflect the actual accumulation level in the organism, thereby providing a set of scientific and reliable analysis tools and technical support for aquatic product safety monitoring, aquaculture environmental risk assessment and ecosystem health evaluation. BRIEF DESCRIPTION OF DRAWINGS
[0019] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual proportions.
[0020] Figure 1 The enrichment result curve of TBB and TBPH in tilapia is shown in the following figure.
[0021] Figure 2 The BCF result curve of TBB and TBPH in the body of tilapia described in the embodiments of the present application.
[0022] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION
[0023] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0024] As described above, the present application proposes a tilapia FM550 in vivo enrichment detection method and a risk monitoring method to overcome the deficiencies of the prior art. That is: In a first aspect, the tilapia FM550 in vivo enrichment detection method comprises the following steps: (1) Feed preparation and exposure: the main components TBB and TBPH of FM550 are uniformly mixed into feed at different concentrations (0, 10, 100, 400 ng·g - ¹) to prepare experimental feed. The tilapia is randomly divided into groups, and the bait exposure is carried out for 56 days.
[0025] (2) Freeze-drying and homogenization of tilapia samples: take the liver and muscle samples of tilapia at different time points (such as 14d, 28d, 42d, 56d) during the exposure period, freeze-dry for 72 h, accurately weigh 1 g, and homogenize in a pulverizer.
[0026] (3) Solvent extraction: add 4 mL of acetone / n-hexane (volume ratio 1:1) and 1 mL of saturated sodium chloride solution, add 10 μL of 0.5 mg / L ^13C-BDE209 internal standard solution, vortex for 10 min, centrifuge at 7500 r / min for 10 min, collect the supernatant, and repeat the extraction for 2 times. Nitrogen blow constant volume: blow the extraction liquid to 1 mL under nitrogen.
[0027] (4) QuEChERS purification: transfer the extraction liquid to a centrifuge tube containing 50 mg of anhydrous magnesium sulfate and 50 mg of C18, oscillate for 10 min, centrifuge at 12000 r / min for 10 min, take the supernatant, and filter through a 0.22 μm filter membrane.
[0028] (5) Solvent conversion: blow dry under soft nitrogen, and constant volume to 200 μL with isooctane.
[0029] (6) Gas chromatography-mass spectrometry (GC-MS) was used to detect TBB and TBPH in the sample, with 13C-BDE209 as the internal standard. Column: Agilent DB-5MS (15 m × 0.25 mm × 0.1 μm); Carrier gas: high-purity helium; Temperature program: 110℃ (hold for 5 min) → 200℃ (20℃ / min, hold for 4.5 min) → 310℃ (10℃ / min, hold for 15 min) → 350℃ (hold for 4 min); Injection method: splitless, 1 μL; Ion source temperature 150℃, quadrupole temperature 150℃, transfer line 280℃; Quantification in SIM mode.
[0030] Secondly, the method for monitoring the in vivo enrichment risk of FM550 in tilapia includes the following steps: Based on the detection data obtained in the first aspect above (i.e., the concentrations of TBB and TBPH in tilapia liver and muscle at different feed exposure groups and different time points), the following steps are further included: Enrichment factor calculation: based on the concentration C detected in tilapia. fish With the concentration of the target substance C in the feed feed Perform ratio calculation: BCF=C fish / C feed Where: C fish The concentration of the target substance in the liver or muscle of tilapia (ng·g) - ¹)(C feed The average concentration of the target compound in the feed (ng·g) - ¹).
[0031] Establish enrichment patterns: Correspond BCF results for different exposure durations (e.g., 14d, 28d, 42d, 56d) to feed concentrations, plot enrichment curves, and obtain the enrichment trend with exposure time.
[0032] Risk Assessment: Based on the established enrichment patterns, the risk level of FM550 under different exposure scenarios is determined: If BCF is consistently >1, it indicates that tilapia have a significant enrichment of the target substance, suggesting a cumulative risk in the environment or feed. By comparing different feed concentration groups, the dose-response relationship can be obtained to infer the exposure risk trend in the actual environment or farm.
[0033] As shown in Table 1, according to the method described in this application, the actual total contents of TBB and TBPH in the control group, low concentration group (10 ng / g), medium concentration group (100 ng / g) and high concentration group (400 ng / g) feed were measured to be 2.07 ng / g, 13.83 ng / g, 104.23 ng / g and 420.81 ng / g, respectively, which are basically close to the concentration set in this application.
[0034] Table 1. Content of TBB and TBPH in feed (ng / g) ; Throughout the exposure period, both TBB and TBPH were detected to varying degrees in tilapia. Figure 1 As shown, under different doses of TBB and TBPH exposure, the contents of TBB and TBPH in tilapia tissue gradually increased with time during the exposure period, indicating that the two compounds can accumulate in tilapia in a time-dependent manner. Under the same exposure conditions, the concentrations of TBB and TBPH in the liver were consistently significantly higher than those in the muscle. P<0.05 The results indicate that the two flame retardants, TBB and TBPH, are more likely to accumulate in the liver, which is their main target organ for accumulation.
[0035] As shown in Figure 2, the BCF calculated based on the measured concentration showed a continuous upward trend with exposure time, reaching its maximum at 56 days, indicating that the bioaccumulation effect of TBB and TBPH in tilapia has a significant time dependence. Under different exposure doses, BCF also varied significantly with dose, with the low-dose group showing relatively higher BCF values, suggesting a possible low-dose promoting effect. When the BCF value was consistently >1, it indicated that tilapia significantly accumulated the target substances, suggesting a potential accumulation risk in the environment or feed.
[0036] Therefore, the method described in this application can accurately and comprehensively reflect the actual exposure level of organisms, demonstrating significant innovation and application value. Specifically, this application innovatively uses tilapia as an indicator biological model, systematically detecting the concentration levels of two flame retardant components, TBB and TBPH, in its liver and muscle tissue, and accurately calculating the bioaccumulation factor (BCF). This allows for a direct and accurate representation of the actual accumulation of FM550 in aquatic organisms. This method effectively overcomes the limitations of existing technologies that rely solely on water or sediment detection, fundamentally solving the technical challenge of traditional methods failing to reveal the true exposure level within organisms, and providing a new technical pathway for environmental pollutant monitoring.
[0037] It is particularly worth pointing out that the method described in the present application has the remarkable advantages of simple operation and high sensitivity. In the sample pretreatment link, the advanced QuEChERS purification technology is adopted, combined with a high-sensitivity gas chromatography-mass spectrometry (GC-MS) detection system, so that the whole operation process is simple and efficient, and the time consumption is significantly shortened. At the same time, the method has excellent detection sensitivity, and the quantitative results are reliable and stable, which fully meets the rapid analysis and detection needs of large quantities of samples, and provides technical support for practical application.
[0038] Further research shows that the method described in the present application can also reveal the enrichment rules and tissue difference characteristics of pollutants. By accurately calculating the BCF value, the dynamic enrichment rules of TBB and TBPH in different tissue organs of tilapia and under different exposure times are successfully established. The research results first confirmed that the enrichment level of liver tissue is significantly higher than that of muscle tissue, which provides an important theoretical basis and experimental evidence for scientific assessment of pollutant risk.
[0039] In the application layer, the method described in the present application realizes the whole-chain monitoring system from detection to risk assessment. On the basis of accurate detection, further through BCF value calculation and dose-response relationship modeling, the risk level under different exposure intensities can be scientifically judged. This innovative method is especially suitable for long-term environmental monitoring, and can realize early warning of FM550 pollution risk, which has important application value in the fields of environmental protection and aquaculture.
[0040] It needs to be emphasized that the method described in the present application is not only specially applicable to the detection and risk monitoring of FM550, but also has broad application potential. The technical route and principle of the method can be applied to the monitoring of other new flame retardants and various persistent organic pollutants, and provides an innovative technical tool and solution for the fields of aquaculture safety, ecological environment protection and food safety supervision, which has important social benefits and application value.
[0041] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.
Claims
1. A method for detecting the in vivo enrichment of FM550 in tilapia, characterized in that, Includes the following steps: Liver and muscle samples of the target tilapia were obtained, freeze-dried for 72 hours, and 1g of each sample was pulverized, homogenized, and set aside for later use. The target tilapia refers to tilapia exposed to FM550 environment for 56 days. The homogenized sample was subjected to solvent extraction, QuEChERS purification and solvent conversion, and then gas chromatography-mass spectrometry was used to obtain the detection results of FM550 in vivo enrichment.
2. The method for detecting the in vivo enrichment of FM550 in tilapia according to claim 1, characterized in that, The process of solvent extraction, QuEChERS purification, and solvent conversion of the homogenized sample includes the following steps: The extraction was performed using acetone / n-hexane / NaCl, with the addition of ^13C-BDE209 internal standard solution. After mixing and centrifugation, the mixture was purified by nitrogen blowing and brought to a final volume. The mixture was then purified by QuEChERS, centrifuged, filtered, dried by nitrogen blowing, and brought to a final volume with isooctane.
3. The method for detecting the in vivo enrichment of FM550 in tilapia according to claim 2, characterized in that, The volume ratio of the acetone:n-hexane:NaCl:^13C-BDE209 internal standard solution is 2mL:2mL:1mL:10 μL; The concentration of the ^13C-BDE209 internal standard solution was 0.5 mg / L.
4. The method for detecting the in vivo enrichment of FM550 in tilapia according to claim 2, characterized in that, After mixing, centrifugation, and extraction, the mixture is brought to volume under nitrogen blowing, including the following steps: Vortex for 10 min, centrifuge at 7500 r / min for 10 min, collect the supernatant, repeat the extraction twice, and then bring the volume to 1 mL with nitrogen to obtain the extract.
5. The method for detecting the in vivo enrichment of FM550 in tilapia according to claim 2, characterized in that, The process of QuEChERS purification, centrifugation, filtration, nitrogen drying, and final volume adjustment with isooctane includes the following steps: The extract was transferred to a centrifuge tube containing 50 mg of anhydrous magnesium sulfate and 50 mg of C18, shaken for 10 min, centrifuged at 12000 r / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. Dry under a gentle nitrogen atmosphere, then bring the volume to 200 μL with isooctane.
6. The method for detecting the in vivo enrichment of FM550 in tilapia according to claim 1, characterized in that, The parameters for gas chromatography-mass spectrometry detection are: Column: Agilent DB-5MS, 15 m × 0.25 mm × 0.1 μm; Carrier gas: High-purity helium; Temperature program: Hold at 110℃ for 5 min, then increase the temperature to 200℃ at 20℃ / min and hold for 4.5 min, then increase the temperature to 310℃ at 10℃ / min and hold for 15 min, then increase the temperature to 350℃ and hold for 4 min. Injection method: splitless, 1 μL; Ion source temperature 150℃, quadrupole temperature 150℃, transmission line temperature 280℃; Quantitative SIM mode.
7. A risk monitoring method for the in vivo enrichment of FM550 in tilapia, characterized in that, Includes the following steps: Based on the detection results of FM550 in vivo enrichment obtained by the method described in any one of claims 1-6, the enrichment factor is calculated. Based on the enrichment factors, enrichment rules are established; Risk assessment is performed based on the enrichment patterns described above.
8. The risk monitoring method for FM550 in vivo enrichment based on tilapia according to claim 7, characterized in that, The enrichment factor is calculated based on the following formula: BCF=C fish / C feed In the formula, BCF is the enrichment factor; C fish The concentration of the target substance in the liver or muscle of tilapia, in ng·g - ¹; C feed The average concentration of the target compound in the feed, in ng·g - ¹.
9. The risk monitoring method for FM550 in vivo enrichment based on tilapia according to claim 7, characterized in that, The establishment of enrichment rules based on the enrichment factor includes the following steps: By correlating BCF results at different durations with feed concentrations, enrichment curves were plotted to obtain the enrichment pattern over exposure time.
10. The risk monitoring method for FM550 in vivo enrichment based on tilapia according to claim 7, characterized in that, The risk assessment based on the enrichment pattern includes the following steps: If BCF is consistently greater than 1, it indicates that tilapia have a significant enrichment of the target species, suggesting a cumulative risk in the environment or feed.