Method for testing high-flux biodegradability based on optical oxygen sensor

Abstract

The invention discloses a high-flux biodegradability testing method based on an optical oxygen sensor, which comprises the following steps: collecting and treating an inoculum, and measuring the living cell concentration of the inoculum; preparing a mineral culture medium, and adding an inoculum; setting different test groups, and adding test chemicals and / or reference chemicals; placing the test bottles on a magnetic stirrer, carrying out dark culture at a certain test temperature for a certain time, and regularly detecting or continuously monitoring the dissolved oxygen concentration of each test bottle; and calculating the biodegradation rate according to the dissolved oxygen concentration. According to the application, the micro optical oxygen sensor fixed on the inner surface of the test container is utilized, and the concentration of oxygen in the container can be measured in a non-intrusive manner through the optical fiber receiver, so that in-situ monitoring and real-time data analysis of dynamic change of dissolved oxygen in a chemical degradation process are realized; the oxygen amount consumed by the microbial community during biodegradation of the sample is corrected by the vacant control of the inoculum in the parallel test, so that the biodegradation rate of the chemical can be accurately calculated.

Description

Technical Field

[0001] This application belongs to the field of environmental technology, specifically relating to a high-throughput biodegradability testing method based on optical sensors, mainly used to determine the biodegradability of chemicals, and can provide reliable data for the durability assessment of chemicals. Background Technology

[0002] In global chemical management, biodegradability is a crucial component of environmental risk assessment. Biodegradability refers to the potential for a chemical substance to be utilized by organisms upon contact with an inoculum. It is a fundamental indicator for identifying the environmental hazards and persistence of chemical substances, classifying and labeling them, and assessing and controlling environmental risks. my country's "Guidelines for Environmental Management Registration of New Chemical Substances" explicitly stipulates that biodegradability data must be provided for the registration of new chemicals as a key basis for determining their "persistence."

[0003] Currently, there are well-defined standard methods for grading and evaluating biodegradability both domestically and internationally, such as the OECD 301 series and my country's environmental industry standard HJ 153, the Guidelines for Chemical Testing. Among these, the closed-bottle test (OECD 301D) is a classic method for rapid biodegradability evaluation. Typically, after inoculating the test culture medium containing the test chemical, the sample solution is kept completely filling the test bottle in the dark at a constant temperature for a period of time. The dissolved oxygen content of the test system is measured periodically, and the biodegradability rate of the chemical is calculated based on the sample consumption to evaluate its rapid biodegradability. This method is applicable not only to test chemicals with high solubility, low volatility, and low adsorption, but also to chemicals that are poorly soluble in water, volatile, and adsorbent. Due to the low concentration of the test chemical in the test system (2~10 mg / L), it is also particularly suitable for chemicals with potential microbial inhibitory effects.

[0004] While the closed-bottle test has the advantage of wide applicability, it has significant limitations in practical applications. Traditional apparatus for this test primarily relies on Winkler titration or Clarke electrode methods to determine the dissolved oxygen concentration in the test bottles. For each measurement, at least two reaction bottles are randomly selected from each test group, the stopper is opened, and the dissolved oxygen probe is inserted into the reaction bottle for measurement. Because the measurement process disrupts the airtightness of the reaction bottle, it cannot be used for subsequent biodegradation tests. This "sacrificial" sampling method requires a large number of reaction bottles for each test, with 60-100 bottles needed for a single sample and test, resulting in solution volumes as high as 15-25L. This is labor-intensive, costly, and requires a large experimental space or floor space, making it difficult to conduct parallel testing under multiple conditions. The test throughput and efficiency are extremely low, failing to meet the needs for rapid screening and timely assessment of the persistence of large quantities of existing chemicals, new chemical substances, and new pollutants with unknown persistence and characteristics. Furthermore, the large quantities of waste solutions containing hazardous chemicals increase the difficulty and cost of environmental treatment. Furthermore, the aforementioned closed-bottle-based biodegradability assays have the following drawbacks in accurately assessing biodegradability: they lack real-time dynamic monitoring capabilities, making it difficult to fully capture the kinetic characteristics of the entire degradation process, and may miss important kinetic parameters related to the degradation potential and biodegradability of chemicals, such as the lag period and the 10-day window; they typically employ static culture systems, which are not conducive to sufficient contact between the test chemical (especially poorly soluble substances) and the inoculum within a limited experimental period, easily leading to false negative results. Therefore, there is an urgent need to develop a simple and easy-to-use high-throughput biodegradability assessment method as an effective supplement to existing standard methods, meeting the demand for timely and effective environmental persistence risk assessment of emerging new chemicals.

[0005] Currently, the mainstream international strategies for rapid, accurate, and high-throughput chemical durability assessment are primarily based on microplates. A common high-throughput biodegradability screening technique is an alternative to colorimetry. This method uses the change in absorbance of the test system resulting from the characteristic color reaction of specific degradation products with a chromogenic agent as the detection indicator. For example, aromatic compounds react with azo dyes (such as 4-nitrobenzene diazonium tetrafluoroborate) under alkaline conditions to form stable colored products, or the color of dyes (such as resazurite) changes from blue to pink during microbial degradation via redox reactions. These methods quantitatively assess the metabolic activity of the tested chemicals. Although this method can perform over 20,000 tests within 28 days, its applicability is limited. It is only suitable for chemicals with aromatic groups, the test volume is very small (0.3 mL or 3 mL), and the amount of test chemicals and inoculated microorganisms (especially specific degrading bacteria) added is limited. It is unsuitable for chemicals with low water solubility and is prone to false negative results. Furthermore, colorimetric methods cannot reflect changes in key endpoints (i.e., DOC, O2, or CO2) that characterize the final biodegradation of chemicals. They can only be used as primary biodegradation screening methods and cannot assess the rapid biodegradability of substances.

[0006] The innovation in dissolved oxygen detection technology, from traditional optical methods to optical fluorescence technology, has supported the development of high-throughput, rapid biodegradability assessment methods. This application, through technology integration and process optimization, fills the gap in high-throughput rapid biodegradability assessment, providing an innovative tool for chemical science, efficient safety evaluation, environmental risk management, and new chemical substance registration. Summary of the Invention

[0007] To address the shortcomings or deficiencies of the existing technologies, this application aims to solve the technical problem of a high-throughput biodegradability testing method based on an optical oxygen sensor. By utilizing a miniature optical oxygen sensor fixed to the inner surface of the test container and a fiber optic receiver, the oxygen concentration inside the container can be measured non-invasively, enabling in-situ monitoring and real-time data analysis of the dynamic changes in dissolved oxygen during the degradation of chemicals. After correction by a blank control of the inoculum in parallel experiments, the amount of oxygen consumed by the microbial community during sample biodegradation can be accurately calculated.

[0008] This application proposes a high-throughput biodegradability testing method based on an optical oxygen sensor, the testing method comprising: Collect and process inoculum, aerate until use, and determine the viable cell concentration of inoculum before use; Prepare a mineral culture medium by adding inoculum to achieve a concentration of 10 mg / L of solution. 4 ~10 6 Each cell is aerated for at least 20 minutes and then allowed to stand until it reaches oxygen saturation. Set up different test groups, add 2-5 mg / L of test chemical and / or reference chemical, add magnetic stir bar, stopper the bottle, and ensure that the liquid level is higher than the sealing opening; Place the test bottles on a magnetic stirrer and incubate them in the dark at a certain test temperature for a certain period of time. Regularly or continuously monitor the dissolved oxygen concentration of each test bottle. Specifically, for example, incubate them in the dark at (20 test) ℃ for 28 days and regularly or continuously monitor the dissolved oxygen concentration of each test bottle. Calculate the biodegradation rate based on the dissolved oxygen concentration.

[0009] Further optionally, the collection and treatment of inoculum described above includes: the inoculum includes secondary effluent from a domestic sewage treatment plant or surface water (such as river water or lake water).

[0010] Further optionally, the above-described collection and processing of inoculum includes: the processing method of the inoculum is precipitation treatment or filtration with coarse filter paper.

[0011] Further, optionally, the determination of the viable cell concentration of the inoculum described above includes: The standard curve was determined using ATP standard substance (rATP); Measure the ATP concentration of the inoculum; The number of viable cells in the inoculum is calculated based on the relationship between the number of viable cells and the concentration of the inoculum.

[0012] Further optionally, the relationship between the number of live cells and the inoculum concentration is approximately 5 × 10⁻⁶ ATP content per cell. -16 g~5×10 -15 g, equivalent to 10 -9 nmol~10 -8 nmol.

[0013] Further optionally, in setting up different test groups and adding test chemicals or (and) reference chemicals as described above, the test groups include a blank control group, a procedure control group, a test group and a toxicity control group, and each group includes at least two replicates; Alternatively, the experimental system components of the blank control group may consist only of the inoculum; Further, optionally, the test system components of the control group mainly include reference chemicals and inoculum; Further, optionally, the test system components of the test group mainly include test chemicals and inoculum; Further optionally, the test system components of the toxicity control group mainly include inoculum, as well as a reference substance in the same amount as the procedural control group and a test chemical in the same amount as the test group.

[0014] Further optionally, in setting up different test groups and adding test chemicals and / or reference chemicals as described above, the method of adding the test chemicals varies depending on their water solubility; for chemicals with high water solubility, a high-concentration stock solution is prepared and then added to the test system; for slightly soluble or insoluble test chemicals, they are added to the test system by direct weighing or with the assistance of organic solvents. In some embodiments, the chemicals may be volatile because the test system is completely sealed.

[0015] Further, optionally, the method of adding the slightly soluble or insoluble test chemical includes: Add directly; Add the solution after preparing a suspension by using ultrasonic dispersion or emulsifiers to assist dispersion; Organic solvent-assisted addition: Prepare a mother liquor of organic solvent for the test chemical, add a certain volume of the mother liquor to the test bottle, and obtain a test bottle containing the test chemical after the solvent has completely evaporated; Add the following after preparing the test chemical – an inert substance / filler mixture: directly adsorb (liquid compound) onto the surface of an inert substance (such as silica gel) or filler (such as a glass fiber membrane), or mix the organic solvent stock solution of the test chemical with the inert substance or filler and then completely evaporate the solvent.

[0016] Alternatively, in the above-mentioned organic solvent-assisted addition and the addition after preparation of the test chemical-inert substance / filler mixture, the reagents involved must not have a microbial promoting or inhibiting effect.

[0017] The aforementioned periodic testing or continuous monitoring of dissolved oxygen concentration in each test bottle includes: the periodic testing is to measure the dissolved oxygen concentration of each test system at fixed time intervals during the 28-day incubation period (once every 3-4 days for the first two weeks, and at least once a week thereafter). During the measurement, an optical fiber receiver is used to align with the oxygen sensor inside the test bottle to read the value.

[0018] The aforementioned periodic or continuous monitoring of dissolved oxygen concentration in each test bottle also includes: the continuous monitoring involves using a fixer to align and fix the fiber optic receiver in the sealed bottle system of the optical oxygen sensor with the oxygen sensor, and automatically reading the dissolved oxygen value of each reaction bottle at fixed time intervals (such as 6h, 12h, or 24h) within 28 days after the start of the test.

[0019] The above calculation of biodegradation rate based on dissolved oxygen concentration includes:

[0020] .

[0021] Compared with the prior art, this application has achieved the following beneficial effects: This application enables in-situ real-time monitoring of dissolved oxygen concentration, avoiding sample destruction in traditional testing. The dissolved oxygen data for each group comes from the same reaction flask, resulting in higher data continuity and accuracy.

[0022] This application breaks through the spatial and temporal limitations of traditional detection methods, enabling the capture of oxygen consumption throughout the entire process and revealing the kinetic mechanism of chemical degradation. This allows for timely adjustment of monitoring frequency and extension or shortening of the test cycle, significantly improving the accuracy and efficiency of chemical durability assessment.

[0023] This application sets up 2-3 parallels for each group, requiring only 6-12 reaction flasks for a single sample and a single test, with a solution volume of only 1.5-3L, which is nearly 10 times less than the traditional method (15-25L). The amount of sample required for the experiment is small, the area occupied is small, the operation is simpler, and the workload and experimental cost are greatly reduced. It solves the problem that the traditional biodegradation method is limited by the inability to expand the experimental throughput.

[0024] The main detection indicators of this application are consistent with traditional methods, ensuring data comparability, simplifying result mutual recognition, reducing the cost of repeated verification, and facilitating standardized application.

[0025] In summary, the method of this application makes up for the shortcomings of existing technologies in terms of throughput and standardization in biodegradability assessment, and can serve as an effective supplement to existing standard methods for biodegradability grading assessment. Attached Figure Description

[0026] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of a sealed bottle system (single channel) based on an optical oxygen sensor according to one embodiment of this application.

[0027] Figure 2 This is a schematic diagram of a sealed bottle system (multi-channel) based on an optical oxygen sensor according to one embodiment of this application.

[0028] Figure 3 This is a comparison of the biodegradation rates of a high-throughput method and a conventional method in one embodiment of this application, where "N" represents the high-throughput method and "O" represents the conventional method; Among them, 10-oxygen sensor, 20-reaction flask, 21-stirrer, 30-fiber optic receiver, 40-magnetic stirrer, 50-oxygen analyzer, and 60-computer. Detailed Implementation

[0029] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Example 1 Acetone glyceryl acrylate is one of the "advanced functional raw materials" for manufacturing "high-performance, environmentally friendly, and fast-curing" coatings, inks, and adhesives. Taking acetone glyceryl acrylate as an example, an embodiment of an evaluation method for determining the biodegradability of chemicals in a closed container based on an optical oxygen sensor is as follows: 1. Collection and processing of inoculum River water was collected on the day of the experiment, and the concentration of viable cells in the water was measured. The standard curve for rATP was y = 19821x + 266325 (R² = 0.993). The average fluorescence value of the river water measured six times was 358603 RLU. The calculated ATP concentration of the river water was 4.7 nM, and the viable cell concentration was 4.66 × 10⁻⁶. 8 Units / L. Aerate river water until use.

[0031] 2. Preparation of oxygen-saturated culture medium Prepare the mineral culture medium according to Table 1, aerate it thoroughly for 2 hours, and then let it stand until it reaches oxygen saturation. Add 0.5 mL of freshly collected river water to each liter of mineral culture medium to achieve a viable cell concentration of 2.33 × 10⁻⁶ cells / mL. 4 per L.

[0032] Table 1. Preparation of Mineral Culture Medium

[0033] 3. Preparation of the experimental system The experiment included a blank control group (IB), a procedural control group (PC), a test group (TS), and a toxicity control group (TC), with different test components added: (1) Blank control group (IB): The total volume of the experimental system is 1L, containing only mineral culture medium and inoculum.

[0034] (2) Program control group (PC): 2 mL of 1 g / L sodium benzoate solution was added to the mineral culture medium containing the inoculum. The total volume of the test system was 1 L, and the final concentration of the reference sample in the test system was 2 mg / L.

[0035] (3) Test group (TS): 2 mL of 1 g / L acetone glycerol acrylate solution was added to the mineral culture medium containing the inoculum. The total volume of the test system was 1 L and the final concentration of acetone glycerol acrylate in the test system was 2 mg / L.

[0036] (4) Toxicity control group (TC): 2 mL of 1 g / L acetone glycerol acrylate solution and 2 mL of 1 g / L sodium benzoate solution were added to the mineral culture medium containing the inoculum. The total volume of the test system was 1 L, and the final concentration of acetone glycerol acrylate in the test system was 2 mg / L.

[0037] After thoroughly mixing the test components from the above groups, the mixture was siphoned into 250mL BOD bottles containing an oxygen sensor and a stir bar. Three replicates were prepared for each of the blank control group and test group, and two replicates were prepared for each of the programmed control group and toxicity control group. No air bubbles were generated during the entire mixing and distribution process. The bottles were then capped, ensuring the liquid level was above the seal.

[0038] 4. Cultivation and Dissolved Oxygen Detection All test bottles 20 were placed on a magnetic stirrer 40, with a stir bar 21 inside each bottle. They were then incubated in the dark at 20°C. After 1 hour, the dissolved oxygen concentration of each bottle was measured as the dissolved oxygen value for day 0. Subsequently, the dissolved oxygen concentration of each test system was measured on days 1, 2, 3, 7, 10, 14, 17, 21, 24, and 28 of the experiment. During measurement, the fiber optic receiver 30 was aligned with the oxygen sensor 10, and after standing for 4-5 seconds, the dissolved oxygen value was read through the oxygen analyzer 50. (See Appendix) Figure 1 As shown, in the appendix Figure 2 In this process, the oxygen analyzer 50 is also connected to a computer 60.

[0039] (5) Calculation of biodegradation rate Based on the dissolved oxygen concentration of each test group, the biochemical oxygen demand (BOD) and biodegradation rate were calculated. At the end of the 28-day experiment, the average BOD of the blank control group was 0.77 mg / L, which was less than 1.5 mg / L; during the experiment, the residual dissolved oxygen concentration in the BOD bottle was not less than 0.5 mg / L; the average biodegradation rate of the programmed control group on day 14 was 73%, which was greater than the 60% effectiveness requirement; the average biodegradation rate of the toxicity control group on day 14 was 43%, which was greater than the 25% effectiveness requirement; the difference in biodegradation rate between parallel groups during the experiment did not exceed 2%; the experiment was valid. The average biodegradation rate of the test group on day 20 (10-day window period) was 16%, and the average biodegradation rate on day 28 was 32%, which did not reach the pass level for rapid biodegradation. The test chemical, acetone glyceryl acrylate, does not have rapid biodegradability. The final results of this example are shown in Tables 2, 3, and 4.

[0040] Table 2 Dissolved oxygen values ​​(mg / L) for each experimental group

[0041] Table 3 Biochemical oxygen demand (BOD, mg / L) values ​​for each experimental group

[0042] Table 4 Biodegradation rate (%) of each experimental group

[0043] 5. Comparison of the method in this application with traditional methods: Concurrently with Example 1, the biodegradability of acetone glyceryl acrylate was tested using conventional methods. The total volume of the experimental system was 10 L. After thorough mixing of the test components for each group, they were siphoned into 250 mL BOD bottles (excluding oxygen sensors and stir bar), with 30 replicates for each group. Similarly, the dissolved oxygen concentration of each experimental system was measured on days 1, 2, 3, 7, 10, 14, 17, 21, 24, and 28 of the experiment. Two BOD bottles were randomly selected from each experimental group each time; the stopper was opened, the dissolved oxygen probe was placed into the test bottle, and the bottle was discarded after testing. The biodegradability of acetone glyceryl acrylate determined by conventional methods is shown in Table 5. Figure 3 As shown, the average biodegradation rate was 16% on day 20 (10-day window) and 29% at the end of day 28.

[0044] Table 5. Biodegradation rate (%) determined by traditional methods

[0045] A search of the ECHA database revealed that the closed-bottle carbon dioxide method (headspace test) showed a biodegradation rate of acetone glyceryl acrylate of 33±5%, which is close to the results obtained by the method in this application and the conventional method. Pearson correlation analysis of the biodegradation rates of the method in this application and the conventional method showed correlation coefficients of 0.989, 0.997, and 0.995 for the test group, the procedure control group, and the toxicity control group, respectively, indicating no significant difference in the test results between the two methods. These results demonstrate that the method in this application is suitable for testing the biodegradation rate of chemicals.

[0046] The method described in this application requires a total volume of 1L for each group of experiments, with 2-3 replicates, which is only 1 / 10 of the volume required by traditional methods. Therefore, it requires less incubator space or floor space, significantly increasing experimental throughput, especially advantageous when experimental conditions are limited. Secondly, the dissolved oxygen concentration of the experimental system is non-destructively measured using an oxygen sensor-fiber optic receiver, enabling multiple measurements anytime and anywhere. The frequency of detection can be increased according to experimental needs, ensuring the determination of key parameters such as the 10-day window period. Furthermore, the experimental period can be extended during the experiment based on the degree of chemical biodegradation (e.g., for enhanced biodegradation testing, it is recommended to extend to 60 days) without the need for additional test bottles.

[0047] In summary, the method proposed in this application for determining the biodegradability of chemicals in closed containers based on optical oxygen sensors provides objective and comparable test data. Compared to traditional methods, it significantly increases throughput, greatly reduces workload, and substantially improves efficiency. This application presents a high-throughput method with standardization potential, providing a time- and cost-effective persistence assessment system for a wide variety of emerging pollutants. It serves as an effective supplement to existing persistence assessment guidelines, providing a basis for the identification, classification, labeling, evaluation, and control of chemical environmental hazards and persistence.

[0048] The above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit it. The preferred embodiments have been described in detail. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the spirit and scope of the technical solutions of this application, and all such modifications and substitutions should be covered within the scope of the claims of this application.

Claims

1. A method for testing high-throughput biodegradability based on optical oxygen sensors, characterized in that, The test method comprises: collecting and treating inoculum, determining the viable cell concentration of the inoculum; preparing mineral medium, adding inoculum; setting different test groups, adding test chemicals or (and) reference chemicals; placing test bottles on a magnetic stirrer, culturing in the dark at a certain test temperature for a certain time, periodically or continuously monitoring the dissolved oxygen concentration of each test bottle; calculating the biodegradation rate according to the dissolved oxygen concentration.

2. The test method of claim 1, wherein, In the above-mentioned collecting and treating inoculum, the inoculum comprises secondary effluent of a domestic wastewater treatment plant or surface water; and / or, the treatment method of the inoculum is sedimentation treatment or filtration with coarse filter paper.

3. The test method of claim 1, wherein, In the above-mentioned determining the viable cell concentration of the inoculum, it comprises: determining the standard curve by using ATP standard substance (rATP); determining the ATP concentration of the inoculum; calculating the viable cell number of the inoculum according to the relationship value between the viable cell number and the inoculum concentration.

4. The test method of claim 3, wherein, In the above-mentioned setting different test groups, adding test chemicals or (and) reference chemicals, the test groups comprise blank control group, procedure control group, test group and toxicity control group, each group comprises at least 2 parallel tests; The relationship between the viable cell count and the inoculum concentration is that the ATP content in each cell is about 5 x 10 -16 g~5 x 10 -15 g, which corresponds to 10 -9 nmol~10 -8 nmol.

5. The test method of claim 1, wherein, and / or, the test system components of the blank control group only include inoculum; and / or, the test system components of the procedure control group mainly include reference chemicals and inoculum; and / or, the test system components of the test group mainly include test chemicals and inoculum; and / or, the test system components of the toxicity control group mainly include inoculum, as well as reference substances equivalent to the procedure control group and test chemicals equivalent to the test group. In the above-mentioned setting different test groups, adding test chemicals and / or reference chemicals, the addition method of the test chemicals varies according to water solubility; for chemicals with high water solubility, high-concentration mother liquor is prepared and added to the test system; for slightly soluble or insoluble test chemicals, they are added to the test system by direct weighing or organic solvent-assisted method.

6. The test method of claim 1, wherein, The addition method of the slightly soluble or insoluble test chemicals comprises:

7. The test method of claim 6, wherein, direct addition; dispersion with ultrasonic wave or emulsifier, preparation of suspension liquid and then addition; organic solvent-assisted addition: preparation of test chemical organic solvent mother liquor, addition of a certain volume of mother liquor to the test bottle, and then complete evaporation of the solvent to obtain a test bottle containing test chemicals; preparation of test chemical-inert material / filler mixture and then addition: direct adsorption on the surface of inert material or filler, or preparation of test chemical organic solvent mother liquor, mixing with inert material or filler, and then complete evaporation of the solvent. In the above-mentioned periodically or continuously monitoring the dissolved oxygen concentration of each test bottle, the periodic detection is to determine the dissolved oxygen concentration of each test system at fixed time intervals during the 28d culture period, and the value is read by using a fiber optic receiver to aim at the oxygen sensor in the test bottle.

8. The test method of claim 1, wherein, ​ 9. The test method of claim 1, wherein, The periodic detection or continuous monitoring of the dissolved oxygen concentration of each test bottle includes: the continuous monitoring is to use a fixer to align the optical fiber receiver of the optical oxygen sensor with the oxygen sensor in the sealed bottle system and fix, and the dissolved oxygen value of each reaction bottle is automatically read at a fixed time interval within 28 days of the test.

10. The test method according to any one of claims 1 to 9, characterized in that, The calculation of the biodegradation rate according to the dissolved oxygen concentration includes: 。

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