A method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans

Abstract

The present invention discloses a method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans, comprising the following steps: (1) filtering the sewage, adjusting the pH, extracting, drying, re-dissolving with an organic solvent aqueous solution, and then diluting with K buffer solution to obtain a contaminated liquid; (2) adding C. elegans at the L1 stage into the K buffer solution to obtain a worm liquid; (3) contaminated liquid exposure group: mixing and exposing the contaminated liquid, the worm liquid, and the OP50 concentrate; blank control group: replacing the contaminated liquid in the contaminated liquid exposure group with the K buffer solution; (4) after exposure, transferring the nematodes to a well plate containing an agar gasket and shooting the movement videos of the nematodes; (5) analyzing and characterizing to obtain the indexes of the nematodes, and calculating the inhibition rate of each index or the value of each group of indexes relative to the blank control group. The method provided by this application shortens the exposure time to 50 h, improves the detection throughput while saving the sample usage amount and experimental time, is simple to operate, and the analysis result is reliable.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental pollutant toxicity detection, and specifically provides a method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans. Background Art

[0002] Water quality toxicity assessment technology can reflect the overall toxicity effect of water quality and is widely used in the evaluation of sewage treatment technologies. Some studies [1] have shown that new sewage treatment technologies may also cause potential ecological risk hazards while reducing conventional physical and chemical index parameters. At present, most sewage evaluation methods mainly rely on chemical detection. Due to the complexity of sewage water quality, it is difficult to evaluate the actual harm of sewage through single chemical detection. Therefore, it is urgent to introduce biological toxicity detection methods to evaluate the overall toxicity effect of sewage. Therefore, carrying out water quality biological toxicity monitoring on municipal sewage can play a guiding role in evaluating the effectiveness of treatment technologies and is of great significance for protecting human health and maintaining the stability of the ecosystem.

[0003] At present, many scholars have applied water quality toxicity assessment to study the reduction of water quality ecological toxicity by sewage treatment processes. The biological test endpoints used include zebrafish genotoxicity [2] , the number of generations of Caenorhabditis elegans [3] , survival rate [4,5] , the acute toxicity of Daphnia magna [6] , the acute toxicity of Photobacterium phosphoreum [1] etc. Among them, Caenorhabditis elegans is a commonly used model organism in environmental exposure and ecological toxicology research. Its characteristics such as short life cycle and reproductive cycle make it have the potential for high-throughput evaluation of municipal sewage toxicity. There is an ISO-10872 standard for the water environment toxicity assessment technology of Caenorhabditis elegans internationally, which is to detect body length and the number of generations after 96 hours of exposure in a 12-well plate. However, there are problems such as low experimental throughput, manual measurement or counting, long detection time for a single sample, and generally single-dimensional detection indicators, making it difficult to comprehensively present the interference effect of the detected sample on Caenorhabditis elegans from multiple angles; moreover, the credibility of manual counting data is difficult to guarantee. For example, the counting results of the nematode swing frequency detected by different people vary greatly, and it is not suitable for carrying out large-sample toxicity detection. Therefore, the present invention provides a method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans to effectively solve the above technical problems. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans in view of the deficiencies of the prior art.

[0005] To solve the above technical problems, the present invention discloses a method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans, including the following steps:

[0006] (1) Adjust the pH of the sewage after passing through the membrane, extract it, dry it, redissolve it with an aqueous organic solvent solution, and then dilute it with K buffer solution (K Medium solution) to obtain the contaminated liquid.

[0007] (2) Add Caenorhabditis elegans at the L1 stage to the K buffer solution to obtain the worm liquid; the number of worms in the worm liquid is 20 - 30 worms / 20 μL.

[0008] (3) Contaminated liquid exposure group: Mix and expose the contaminated liquid obtained in step (1), the worm liquid obtained in step (2), and OP50 concentrate (concentrated bacteria of E. coli OP50); blank control group: Replace the contaminated liquid in the contaminated liquid exposure group with K buffer solution.

[0009] (4) After exposure, transfer the worms to a well plate containing an agar gasket and take a motion video of the worms.

[0010] (5) Use Wormlab software to analyze the video taken in step (4), obtain the indicators of the worms through analysis and characterization, and calculate the inhibition rate I of each indicator or the value I' of each indicator relative to the blank control group through the following formula; the indicators of the worms are any one or several of the body length, body width, body area of the worms, the wavelength, amplitude, maximum amplitude of the worm movement trajectory, the swing frequency, swimming speed and vitality of the worm activity.

[0011]

[0012] I' = I1 / I0

[0013] Wherein, I is the inhibition rate of each indicator, I' is the value of each indicator relative to the blank control group, I0 is the indicator value of the blank control group, and I1 is the indicator value of the contaminated liquid exposure group.

[0014] In step (1), the membrane passing is through a 0.45 μm cellulose acetate filter membrane to remove large particle impurities, and the filtrate is collected; the pH is 2 - 3, preferably pH 2.

[0015] In step (1), the extraction is to enrich and extract organic pollutants in the water sample using an OASIS HLB (waters) solid phase extraction column, which specifically includes the following steps:

[0016] S0: Activation: Activate the HLB column with methanol / ethyl acetate (50:50, V:V), and then activate the HLB column with ultrapure water acidified to pH = 2 ± 0.5 with hydrochloric acid.

[0017] S1: Loading and adsorption: Load the sewage with adjusted pH onto the OASIS HLB solid phase extraction column.

[0018] S2: Rinse and dry: Rinse with ultrapure water at pH 2 ± 0.5 and then dry.

[0019] S3: Gradient elution: First, perform the first elution with a mixed solution of methanol and ethyl acetate, then perform the second elution with a mixed solution of methanol and ethyl acetate containing 2% ammonia water, and finally perform the third elution with a mixed solution of methanol and ethyl acetate containing 1.7% formic acid. Collect the eluate.

[0020] In step S0, the flow rate of the activation solvent is 10 mL / min and the volume of the activation solvent is 6 mL for both.

[0021] In step S1, the sample loading flow rate is 10 mL / min and the sample loading volume is 3 L.

[0022] In step S2, the rinsing flow rate is 10 mL / min and the rinsing volume is 6 mL.

[0023] In step S2, the judgment criterion for drying is whether a dry white line appears; the drying is either pumping dry, which takes about 0.5 h, or drying with nitrogen, which takes about 5 min.

[0024] In step S3, during the first elution process, the elution flow rate is 0.9 mL / min and the elution volume is 6 mL; during the second elution process, the elution flow rate is 0.9 mL / min and the elution volume is 6 mL; during the third elution process, the elution flow rate is 0.9 mL / min and the elution volume is 6 mL.

[0025] In step S3, in the mixed solution, the volume ratio of methanol, or methanol containing ammonia water, or methanol containing formic acid, to ethyl acetate is 1:1.

[0026] In step (1), pass the sewage through a membrane, adjust the pH, then extract, gently dry with nitrogen, re-dissolve with a 10% aqueous solution of dimethyl sulfoxide (DMSO), and then dilute with K buffer (K Medium solution) to obtain a toxicant solution with a relative enrichment multiple of 30, thus obtaining the toxicant solution.

[0027] In step (2), synchronize the nematodes at the spawning stage one day before poisoning for 17 - 24 h to obtain Caenorhabditis elegans at the L1 stage.

[0028] In step (2), add the L1-stage Caenorhabditis elegans to the K buffer, observe the number of L1-stage nematodes contained in 20 μL of the worm solution under a microscope, and ensure that the number of nematodes in 20 μL of the worm solution is between 20 and 30 by adding K Medium again or centrifuging and sucking away a little supernatant to avoid too high nematode density in the same well affecting parallelism, thus obtaining the worm solution.

[0029] In step (3), the preparation method of the OP50 concentrate (concentrated bacteria of E. coli OP50) is as follows: (i) Streak E. coli OP50 on an LB plate and culture it in a 37°C constant temperature incubator for 48 h. Pick a single colony from the solid medium and place it into a 200 mL conical flask containing LB liquid medium. Incubate it overnight with shaking at 220 rpm at 37°C for 17 h until the OD 600 is between 0.4 and 0.6, and dispense it into 50 mL centrifuge tubes; (ii) Centrifuge the bacterial liquid obtained in step (i) at 7000 rpm for 5 min, then pour off the supernatant. Add K Medium under a sterile environment, shake well and then centrifuge again. Repeat this process 3 - 5 times to thoroughly wash away the LB medium, and then add K Medium to make the volume up to 25 mL to obtain concentrated E. coli OP50 bacteria.

[0030] In step (3), the volume ratio of the contaminated liquid, the worm liquid, and the OP50 concentrate is 100:10 - 30:90 - 70.

[0031] In step (3), the exposure is to shake at 12 - 25°C for 40 - 55 h, preferably to expose in a 20°C constant temperature shaker for 50 h, and set the shaking speed to 200 rpm.

[0032] In step (4), transfer the nematodes to a well plate containing an agar gasket and let them adapt for 30 s - 2 min, and shoot a 10 s - 30 s movement video of the nematodes. Preferably, let them adapt for 1 - 2 min and shoot a 20 s movement video of the nematodes.

[0033] In step (5), the nematode vitality is the number of pixels swept between every two strokes of the nematode per unit time, which can characterize the activity and vigor of the body bending during swimming.

[0034] In summary, based on the ISO - 10872 standard, the present invention has established a high - throughput water quality toxicity detection system based on Caenorhabditis elegans: The present invention uses a 96 - well plate to expose nematodes, improving the exposure throughput of toxicity experiments. It abandons the detection of the offspring number index, adds a total of 9 indexes characterizing the development, movement, and activity of wild - type nematodes, and advances the detection node to 50 h after exposure, that is, advances the detection of various physiological indexes from the original time point when growing from the L1 stage to the egg - laying stage to the time point when growing from the L1 stage to the L4 stage, shortening the experimental period. The present invention also combines the Wormlab image recognition software to expand the category of detection indexes and improve the accuracy of the measured index data. The method provided by the present invention lays a foundation for completing the toxicity screening work of a large number of sewage samples within a certain time.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0036] The present invention uses Caenorhabditis elegans as a model organism to establish a method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans. First, the nematodes are synchronized to obtain nematodes at the L1 stage, and a 96-well plate is used as the exposure carrier. Then, the exposure solution and Escherichia coli OP50 are added as food, and the exposure time is shortened to 50 h. Nine indicators in three categories, namely growth and development, movement, and activity, are used as toxicity endpoints, which not only improves the detection throughput but also saves the sample usage and experimental time. The operation is simple and the analysis results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0038] Figure 1 It is a high-throughput detection flow chart for the toxicity of municipal sewage based on Caenorhabditis elegans.

[0039] Figure 2 It is a diagram of Caenorhabditis elegans at the egg-laying stage (A) and eggs obtained by synchronization (B).

[0040] Figure 3 It is a diagram of the influence results of the nematode development, movement behavior, and activity in the along-flow treatment section of a certain municipal sewage treatment plant.

[0041] Figure 4 It is a diagram of the interference influence results of the nematode growth and development, movement, and vitality in the along-flow treatment section of a certain municipal sewage treatment plant, REF = 15 (*: p < 0.05, **: p < 0.01). SPECIFIC EMBODIMENTS

[0042] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0043] The materials used in the following examples are as follows:

[0044] Caenorhabditis elegans (purchased from the Caenorhabditis Genetics Center).

[0045] Uracil auxotrophic Escherichia coli OP50 (purchased from the Caenorhabditis Genetics Center).

[0046] NGM Solid Medium: 1.2 g of NaCl, 1.0 g of peptone, 6.8 g of agar, dissolved in 400 mL of MilliQ Water. After autoclaving at 121 °C for 20 min, it is cooled to 55 °C in a water bath. Then, under sterile conditions, 400 μL of 1 M CaCl2, 400 μL of 1 M MgSO4, 400 μL of 5 mg / L cholesterol solution, and 10 mL of 1 M phosphate buffer are added. After thorough mixing, it is poured into a 60-mm diameter petri dish in a laminar flow hood, about 10 mL of NGM medium is poured into each dish, allowed to solidify at room temperature, and stored at 4 °C for later use. Among them, 1 M CaCl2 solution: Weigh 4.44 g of CaCl2, add deionized water to 40 mL, and ultrasonically dissolve it until fully dissolved. After passing through a membrane, it is reserved for use. 1 M MgSO4 solution: Weigh 9.8592 g of MgSO4·7H2O, add deionized water to 40 mL, and ultrasonically dissolve it until fully dissolved. After passing through a membrane, it is reserved for use. 5 mg / L cholesterol solution: Weigh 0.2 g of cholesterol, add 40 mL of absolute ethanol, and ultrasonically dissolve it until fully dissolved. After passing through a membrane, it is reserved for use. 1 M potassium phosphate buffer: Weigh 108.3 g of KH2PO4 and 46.648 g of K2HPO4·3H2O, add deionized water to 1 L, adjust the pH to 6.0, stir well, and autoclave at 121 °C for 20 min.

[0047] K Medium (K buffer): 2.386 g of KCl, 2.98 g of NaCl, add deionized water to 1 L, stir well, and autoclave at 121 °C for 20 min.

[0048] Lysis solution: Mix 700 μL of K-Medium, 200 μL of 5% NaClO, and 100 μL of 5 M NaOH to prepare a lysis solution in a 1 mL system, which is prepared immediately before use. Among them, K Medium: 3.8 g of NaCl, 2.975 g of KCl, added to 1 L of MilliQ Water and dissolved, autoclaved at 121 °C, and cooled to room temperature.

[0049] Agar gasket: Weigh 4 g of agarose and dissolve it in 200 mL of MilliQ Water, heat it to boiling, and transfer it to a six-well plate while it is hot, 2.5 mL of agar liquid in each well, try to make the surface smooth and flat, cool it at room temperature, and store it refrigerated at 4 °C after solidification. This agar gasket is used as a carrier for nematode observation.

[0050] Cultivation of E. coli OP50: Take out the frozen tube of E. coli OP50 from the -20°C refrigerator. Wait until it starts to melt slightly. Under sterile conditions, use an inoculation loop to pick a small amount and streak it on an LB plate. Incubate it in a 37°C constant temperature incubator for 48 h. Pick a single colony from the solid medium and put it into a conical flask containing 200 mL of LB liquid medium. Prepare another conical flask with 200 mL of LB liquid medium as a blank control. Incubate it overnight with shaking at 220 rpm at 37°C for 17 h until OD 600 is between 0.4 and 0.6. Among them, LB liquid medium: 10 g of peptone, 5 g of yeast extract, 5 g of NaCl, add deionized water to 1 L, adjust the pH of the medium to 7.0 with NaOH solution, stir well and sterilize at 121°C for 20 min.

[0051] Cultivation and passage of nematodes: Pipette 100 μL of E. coli OP50 bacterial solution and drop it on the surface of a 60-mm NGM solid medium. Burn the spreader with an alcohol lamp, and after cooling, gently spread the bacterial solution evenly on the surface of the NGM solid medium. Let it stand at room temperature for the bacterial solution to be absorbed, and transfer the petri dish to a 20°C sterile biochemical incubator for about 48 h. When a thin layer of bacterial surface is formed on the medium by Escherichia coli without contamination by other bacteria, it can be used to culture Caenorhabditis elegans. For a small amount of passage, use a worm pick to pick a single hermaphrodite nematode in the oviposition period under a microscope and transfer it to the OP50-coated medium for culture (burn the worm pick with an alcohol lamp before each use). For a large amount of passage, first sterilize the scalpel with an alcohol lamp, and after it cools slightly, cut a piece of medium containing more nematodes, invert it and place it on a new NGM medium coated with E. coli OP50. The nematodes will automatically crawl to the surface of the new medium. When there is no Escherichia coli OP50 on the surface of the medium, passage the nematodes again and transfer them to a new OP50-coated medium. All Caenorhabditis elegans are cultured in a 20°C incubator.

[0052] Preparation of concentrated E. coli OP50 bacteria: To make the OD of the OP50 concentrated solution in the experimental exposure system 600Maintain it between 0.4 and 0.6. Prepare concentrated E. coli OP50 in advance as the food supply source in the Caenorhabditis elegans experiment exposure. Aliquot the OP50 bacterial solution obtained after shaking at 37°C and 220 rpm for 17 h into 50 mL centrifuge tubes under sterile conditions, centrifuge at 7000 rpm for 5 min, pour off the supernatant, add K Medium under sterile conditions, shake well and then centrifuge again. Repeat this process 3 - 5 times to thoroughly wash away the LB medium, and then add K Medium to make up to 25 mL to obtain concentrated E. coli OP50. To exclude the decomposition of toxic substances in the exposure system by OP50, which may affect the toxicity characterization of Caenorhabditis elegans samples and cause experimental errors, place the concentrated E. coli OP50 in an oven at 65°C for 30 min to inactivate it before the experiment. [7] 。

[0053] The detection equipment used in each example is an inverted fluorescence microscope (Nikon, TS2-FL, Japan).

[0054] Example 1: A high-throughput detection method for the toxicity of municipal sewage based on Caenorhabditis elegans ( Figure 1 )

[0055] (1) After transporting 3 L of a certain municipal sewage water sample to the laboratory at low temperature, immediately filter it through a 0.45 μm cellulose acetate filter membrane to remove large particle impurities.

[0056] Among them, the municipal sewage water sample includes the influent water, effluent water from the anoxic tank, effluent water from the aerobic tank, effluent water from the secondary sedimentation tank, and the final effluent water sample of the sewage treatment plant.

[0057] (2) Add hydrochloric acid to adjust the pH of the filtered water sample to approximately 2.0.

[0058] (3) Use an OASIS HLB (waters) solid-phase extraction column to enrich and extract organic pollutants in the water sample. The specific steps are as follows:

[0059] ① Activation: Activate the HLB small column with 6 mL of methanol / ethyl acetate (50:50, V:V), and then activate the HLB small column with 6 mL of ultrapure water acidified with hydrochloric acid to pH = 2 ± 0.5. Among them, the flow rate of the activation solvent is 10 mL / min for both;

[0060] ② Loading: The loading volume is 3000 mL, and the flow rate is 10 mL / min;

[0061] ③ Elution: 6 mL of ultrapure water acidified with hydrochloric acid to pH = 2 ± 0.5; the flow rate is 10 mL / min, and dry for 1 h;

[0062] (4) Elution: The eluent for the first elution process is methanol / ethyl acetate (50:50, V:V), with a total volume of 6 mL and a flow rate of 0.9 mL / min. The eluent for the second elution process is methanol / ethyl acetate (50:50, V:V) containing 2% v / v ammonia water, with a total volume of 6 mL and a flow rate of 0.9 mL / min. The eluent for the third elution process is methanol / ethyl acetate (50:50, V:V) containing 1.7% v / v formic acid, with a total volume of 6 mL and a flow rate of 0.9 mL / min. The eluents from the three elution processes are collected in one test tube.

[0063] (4) Gently blow-dry the eluate obtained in step (3) with nitrogen, then add 1 mL of an aqueous solution of dimethyl sulfoxide (DMSO) with a volume ratio of 10% to re-dissolve the dried eluate. After vortex oscillation, filter it through a 0.45 μm organic phase filter head, transfer the filtrate to a 1 mL chromatographic vial, and store it in a -20 °C refrigerator for future use; the sample enrichment factor at this time is 3000 times.

[0064] (5) Use K Medium to dilute an appropriately amount of the 3000-fold concentrated municipal sewage sample prepared in step (4) to a contaminated solution with a relative enrichment factor of 30, that is, dilute it 100 times. Specifically, in a 1.5 mL centrifuge tube, add 396 μL of K Medium and 4 μL of the sample concentrate prepared in step (4), and pipette and mix well.

[0065] (6) Synchronize the nematodes during the egg-laying period one day before the contamination experiment:

[0066] Select NGM culture dishes with most Caenorhabditis elegans in the egg-laying period. Use 1.5 mL of K Medium to rinse a large number of Caenorhabditis elegans in the egg-laying period from the surface of the NGM solid medium and transfer them to a 1.5 mL centrifuge tube. After standing for 1 - 2 min, the Caenorhabditis elegans in the egg-laying period naturally sink to the bottom. Aspirate the supernatant, add fresh K Medium and shake well, re-precipitate and discard the supernatant, then add 1 mL of lysis solution and shake it once every 30 s until the nematodes are lysed into 2 - 3 segments to release the eggs. Then centrifuge at 2500 rpm for 2 min, discard the supernatant, add 1 mL of K Medium and shake well, and then centrifuge at 2500 rpm for 2 min and discard the supernatant again. This is the washing step, and this washing step is repeated 4 times. After the eggs are washed clean and the supernatant is discarded, add 0.5 mL of K Medium to the centrifuge tube, pipette and mix the eggs well, transfer them to a 35 mm culture dish (there is 3 mL of K Medium in the 35 mm culture dish), pipette and mix again, and place it in a 20 ± 1 °C incubator for 17 h. On the day of the experiment, nematodes all in the L1 stage are obtained.

[0067] (7) Transfer the hatched L1-stage nematodes together with K Medium into a 1.5 mL centrifuge tube. Pipette 20 μL of the worm solution and drop it onto a transparent glass slide. Observe the number of L1-stage nematodes contained in the 20 μL worm solution under a microscope. By adding K Medium again or aspirating a small amount of the supernatant after centrifugation, ensure that the number of nematodes in the 20 μL worm solution is between 20 and 30, and avoid excessive nematode density in the same well, which may affect parallelism.

[0068] (8) Municipal sewage contaminated liquid exposure group: Use a 96-well plate as the carrier. The exposure system for each well is to add 100 μL of the municipal wastewater contaminated liquid prepared in step (5) + 80 μL of OP50 concentrate (see E. coli OP50 concentrated bacteria in the materials of the example) + 20 μL of the worm solution prepared in step (7). At this time, the relative enrichment multiple of the contaminated liquid in the exposure group system is 15 times that of the raw water. The exposure method is to expose in a 20 °C constant temperature shaker for 50 h, and the shaking speed is set at 200 rpm. There are 3 parallel wells in each group.

[0069] Blank control group: Replace the contaminated liquid in the municipal sewage contaminated liquid exposure group with 100 μL of K Medium, and the rest is the same.

[0070] (9) After 50 h of exposure, transfer the nematodes in the exposure group and the blank control group to a 6-well plate containing agar pads to adapt for 1 min. Randomly select 20 nematodes for photography, and then take a 20 s movement video of each nematode.

[0071] (10) Use Wormlab software to analyze the videos taken in step (9). The body length, body width, and body area representing the growth and development of nematodes, the wavelength, amplitude, and maximum amplitude representing the movement trajectory of nematodes, and the swing frequency, swimming speed, and inhibition rate of vitality representing the activity of nematodes are obtained. Calculate the inhibition rate I of each index or the value I' of each group of indexes relative to the blank control group through the following formula:

[0072]

[0073] I' = I1 / I0

[0074] Among them, I is the inhibition rate of each index, I' is the value of each group of indexes relative to the blank control group, I0 is the index value of the blank control group, and I1 is the index value of the municipal sewage contaminated liquid exposure group.

[0075] The results of the toxicity detection of Caenorhabditis elegans in the egg-laying period and the synchronized eggs obtained therefrom, and a certain section of municipal sewage along the way are shown in Figures 2-3 .

[0076] Figure 2This is a picture of Caenorhabditis elegans in the egg-laying period and the actual eggs obtained through synchronization. It shows the condition of the eggs after the selection and synchronization operation of the nematodes in the egg-laying period.

[0077] Figure 3 This is a result diagram of the effect of the treatment section along the municipal sewage plant on the development, movement and activity of nematodes. Figure A is the ratio of each index to the blank control after exposure to 15-fold concentrated samples of each section along the process, and Figure B is a visual effect diagram of the effect of the section along the process on the development of nematodes. In the figure, 1-Inf, 2-A2, 3-O, 4-Sed, 5-Eff, and CK respectively represent the 15-fold concentrated sample exposure group and the blank control group of the sewage treatment plant's influent, anoxic tank effluent, aerobic tank effluent, secondary sedimentation tank effluent, and final effluent of the sewage treatment plant. For developmental indicators, the relative body length of the nematode increased from 0.54±0.02 at the water inlet to 0.89±0.02 at the water outlet, and the changes in body width and body area were similar to those of body length. For the characteristics of the movement trajectory, the relative wavelength of the nematode's movement trajectory gradually increased from 0.51±0.04 at the water inlet to 0.92±0.07 at the water outlet, and the relative average amplitude decreased from 0.54±0.07 at the water inlet to 0.45±0.08 in the anoxic section, and then gradually recovered to 0.77±0.02 in the water outlet. For the characteristics of the movement state, the relative swing frequency of the nematode at the water inlet was 0.74±0.18, and the head swing was most active in the second sedimentation tank section, rising to 1.22±0.14, and then falling to 1.07±0.37 in the water outlet. The relative swimming speed of the nematode was 0.49±0.08 in the water inlet section, and rose to 1.19±0.17 in the water outlet section, the highest in the entire process section. In general, the inhibition rate of each index shows a trend of gradually decreasing from inlet to outlet, and the ratio of each index in the outlet section compared with the blank control is close to 1. In summary, it can be seen that the sample concentration multiple and each detection index of the present invention can better distinguish the toxicity of growth and movement behavior along the process of municipal sewage treatment plants.

[0078] Example 2

[0079] Same as Example 1, only the sewage was replaced with sewage from the inlet, anaerobic effluent, anoxic effluent, aerobic effluent, secondary sedimentation tank effluent, filter tank effluent, and final effluent of a municipal sewage plant in Jiangsu Province. The results are as follows: Figure 4 shown.

[0080] Figure 4 This is a result graph of 9 index data of water samples from a treatment section along the municipal sewage treatment plant in Jiangsu Province obtained by using the high-throughput detection method of the present invention based on wild-type Caenorhabditis elegans in the embodiment, with a relative enrichment multiple of 15. The horizontal axes Inf, A1, A2, O, Sed, Filter, Eff, and Ctrl of each small graph in the figure represent the influent, anaerobic section, anoxic section, aerobic section, secondary sedimentation tank, filter, effluent, and blank control exposure groups of the sewage treatment plant, respectively.

[0081] Body width, body length, and body area are all indicators characterizing the growth and development of nematodes. The influent of this municipal sewage caused 100% lethality to nematodes upon exposure. The water samples from all sections except the anoxic section significantly inhibited the body length of nematodes. When exposed to water samples from the anaerobic, aerobic, secondary sedimentation, filter, and effluent sections at a 15-fold concentration factor, the relative inhibition rates of nematode body length were 36.86±3.12%, 9.18±6.74%, 23.29±7.31%, 21.55±7.34%, and 18.52±5.78%, respectively. For the body width of nematodes, the relative body width inhibition rates in the anaerobic and anoxic sections were 20.88±4.32% and -6.74±5.32%, respectively, with the anoxic section promoting body width development. For the body area of nematodes, the relative inhibition rates in the anaerobic, secondary sedimentation, and filter sections were 54.18±3.23%, 31.11±4.19%, and 20.76±3.32%, respectively.

[0082] When nematodes move naturally, their movement trajectories are sinusoidal waveforms. Three indicators, namely the average wavelength, average amplitude, and maximum amplitude of the sine wave, are selected to describe the changes in the characteristics of nematode movement trajectories. For the average wavelength, the wavelength inhibition rates in the anaerobic, secondary sedimentation, and filter sections were 35.38±2.43%, 23.09±2.32%, and 28.15±2.43%, respectively. For the average amplitude, the average amplitude inhibition rates in the anaerobic, filter, and effluent sections were 30.68±2.32%, 24.69±5.43%, and 18.62±3.11%, respectively. For the maximum amplitude, the maximum amplitude inhibition rates in the anaerobic and filter sections were 19.77±5.21% and 13.47±2.31%, respectively.

[0083] The head swing frequency, swimming speed, and vitality of nematodes can characterize the specific movement states of nematodes and reflect their movement abilities. Among them, only the vitality index showed a significant difference between the anaerobic section at a 15-fold exposure and the control group.

[0084] Based on the above results, the lethality rate of Caenorhabditis elegans exposed to concentrated water samples from each section of the municipal sewage at a 15-fold concentration was moderate. Most of the indicators selected in this invention showed significant differences from the blank control at the experimental concentration factor, which could reflect the toxicity differences among different sections. For this sewage treatment plant, the indicators related to the development and movement trajectories of nematodes can better distinguish the toxicity differences among different sections than the movement indicators.

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[0093] The present invention provides an idea and method for a method of high-throughput detecting the toxicity of sewage based on Caenorhabditis elegans. There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A method for high-throughput detection of sewage toxicity based on Caenorhabditis elegans, characterized in that, It includes the following steps: (1) Filter the sewage through a membrane, adjust the pH, then extract it. After drying, redissolve it in an aqueous organic solvent solution, and then dilute it with K buffer solution to obtain a toxicant solution; (2) Add Caenorhabditis elegans at the L1 stage to K buffer solution to obtain a worm solution; the number of worms in the worm solution is 20 - 30 worms / 20 μL; (3) Toxicant solution exposure group: Mix and expose the toxicant solution obtained in step (1), the worm solution obtained in step (2), and OP50 concentrated solution; the relative enrichment multiple of the toxicant solution in the toxicant solution exposure group is 15 times that of the raw water; Blank control group: Replace the toxicant solution in the toxicant solution exposure group with K buffer solution; The exposure is to shake at 12 - 25 °C for 40 - 55 h; A 96-well plate is used as the exposure carrier; (4) After exposure, transfer the worms to a well plate containing an agar gasket, and take a motion video of the worms through an inverted fluorescence microscope; (5) Use Wormlab software to analyze the video taken in step (4), obtain the indicators of the worms through analysis and characterization, and calculate the inhibition rate I of each indicator or the value I' of each indicator relative to the blank control group through the following formula; the indicators of the worms are the body length, body width, body area of the worms, the wavelength, amplitude, and maximum amplitude of the movement trajectory of the worms, and the swing frequency, swimming speed, and vitality of the worm activity; I' = I1 / I0 where I is the inhibition rate of each indicator, I' is the value of each indicator relative to the blank control group, I0 is the indicator value of the blank control group, and I1 is the indicator value of the toxicant solution exposure group; The K buffer solution: 2.386 g of KCl, 2.98 g of NaCl, add deionized water to 1 L, stir well, and sterilize at 121 °C for 20 min.

2. The method according to claim 1, wherein In step (1), the membrane filtration is through a 0.45 μm cellulose acetate filter membrane, and the filtrate is collected; the pH is 2 - 3.

3. The method according to claim 1, wherein In step (1), the extraction includes the following steps: S1: Loading and adsorption: Load the sewage with adjusted pH onto an OASIS HLB solid-phase extraction column; S2: Washing and drying: Wash with ultrapure water with a pH of 2 ± 0.5 and dry; S3: Gradient elution: First, perform the first elution with a mixed solution of methanol and ethyl acetate, then perform the second elution with a mixed solution of methanol and ethyl acetate containing 2% ammonia water, and finally perform the third elution with a mixed solution of methanol and ethyl acetate containing 1.7% formic acid, and collect the eluate.

4. The method according to claim 3, wherein In step S1, the loading flow rate is 10 mL / min, and the loading volume is 3 L.

5. The method according to claim 3, characterized in that, In step S2, the washing flow rate is 10 mL / min, and the washing volume is 6 mL.

6. The method according to claim 3, wherein In step S3, during the first elution process, the elution flow rate is 0.9 mL / min, and the elution dose is 6 mL; during the second elution process, the elution flow rate is 0.9 mL / min, and the elution dose is 6 mL; during the third elution process, the elution flow rate is 0.9 mL / min, and the elution dose is 6 mL.

7. The method according to claim 3, characterized in that In step S3, in the mixed solution, the volume ratio of methanol, or methanol containing ammonia water, or methanol containing formic acid, to ethyl acetate is 1:

1.

8. The method according to claim 1, characterized in that In step (3), the volume ratio of the toxicant solution, the worm solution, and the OP50 concentrated solution is 100:10 - 30:90 - 70.

9. The method according to claim 1, characterized in that In step (4), transfer the nematodes to a well plate containing an agar gasket and let them adapt for 30 s to 2 min, and shoot a 10-s to 30-s movement video of the nematodes.

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