Method for detecting biotoxicity of wastewater

Abstract

The invention relates to the technical field of wastewater detection, and discloses a wastewater biotoxicity detection method which comprises the following steps: (1) adding an organic solvent into to-be-detected wastewater, centrifuging, and taking supernate; (2) dispersing the freeze-dried photogenic bacteria in a 2% sodium chloride solution, and resuscitating at normal temperature to obtain resuscitated bacteria liquid with activity; (3) diluting the to-be-tested wastewater according to a preset dilution ratio, and then carrying out a luminous bacterium method biotoxicity test to obtain a light inhibition rate gamma value; and (4) making a double logarithmic line graph by using the light suppression rate gamma value and the dilution ratio, and calculating to obtain an EC50 value through an interpolation method. According to the present invention, the biological toxicity test is performed by using the luminous bacteria method, the biological toxicity condition of the to-be-detected wastewater can be rapidly determined, and the dimethyl sulfoxide is added to the to-be-detected wastewater to assist the dissolution of the indissolvable organic matter before the detection, such that the solubility of the organic matter in the to-be-detected wastewater can be effectively improved, and the accuracy of the determination result can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of wastewater detection, and in particular to a method for detecting biological toxicity of wastewater. Background Art

[0002] Industrial wastewater contains various pollutants such as heavy metals and toxic insoluble chemicals, and has a complex composition. If it is discharged directly without proper treatment, it will cause serious pollution to the water body. Therefore, it is particularly important to conduct biological toxicity determination of wastewater and choose an appropriate treatment method. Although there are many conventional detection methods for wastewater, they lack specificity, especially when wastewater containing insoluble organic matter has poor solubility when detected using conventional methods, resulting in incomplete wastewater toxicity detection and large deviation during determination, affecting the accuracy of the determination results. Summary of the invention

[0003] In view of this, the present invention provides a wastewater biological toxicity detection method to solve the problem that the existing wastewater detection method has poor solubility for wastewater containing insoluble organic matter, resulting in large deviations in the measurement results.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] A method for detecting biological toxicity of wastewater comprises the following steps:

[0006] (1) Add an organic solvent to the wastewater to be tested, centrifuge and take the supernatant;

[0007] (2) dispersing the freeze-dried luminescent bacteria in a 2% sodium chloride solution and resuscitating the bacteria at room temperature to obtain an active resuscitated bacterial solution;

[0008] (3) Using 2% sodium chloride solution as a dilution solvent, diluting the supernatant into a series of concentrations of water samples to be tested according to a preset dilution ratio, mixing the series of concentrations of water samples to be tested with the resuscitated bacterial solution, reading the initial luminescence, and reading the luminescence again after a preset reaction time to obtain the inhibition rate of the water sample to be tested, and then obtaining the inhibition rate gamma value according to the inhibition rate of the water sample to be tested; wherein the inhibition rate gamma value = inhibition rate of the water sample to be tested / (1-inhibition rate of the water sample to be tested);

[0009] (4) Plot the inhibition rate gamma value and the dilution ratio in a double logarithmic line graph, and calculate the EC by interpolation. 50 value.

[0010] Preferably, the pH value of the wastewater to be tested in step (1) is 6.5-7.5.

[0011] Preferably, the dissolved oxygen concentration in the supernatant is 3-4 mg / L and the salinity is 2%.

[0012] Preferably, the organic solvent in step (1) is dimethyl sulfoxide, and the volume concentration of the dimethyl sulfoxide is 0.01-0.1%.

[0013] Preferably, the centrifugation in step (1) is performed at 5000-8000 g for 5-10 min.

[0014] Preferably, the freeze-dried luminescent bacteria in step (2) is freeze-dried Vibrio fischeri powder.

[0015] Preferably, the resuscitation time is 15 minutes.

[0016] Preferably, the active resuscitation bacterial solution meets the following conditions: after resuscitation at room temperature, the ratio of the luminescence value of the luminescent bacteria after surviving in a 2% sodium chloride solution for 15 minutes to the initial luminescence value of the luminescent bacteria is 0.6-1.8, and the light inhibition rate of the luminescent bacteria after reacting in a standard substance solution for 15 minutes is 20-80%.

[0017] Preferably, the standard substance solution is 20 mg / L zinc sulfate heptahydrate.

[0018] Preferably, in step (3), the supernatant is diluted into water samples of a series of concentrations to be tested according to a two-fold dilution method.

[0019] Preferably, in step (3), the mixing reaction temperature is 15° C., and the preset reaction time is 15 min.

[0020] Preferably, the step (1) further includes the following steps: collecting the wastewater to be tested, sealing it and storing it in a dark environment at 2-5°C for ≤48h.

[0021] The present invention provides a method for detecting biological toxicity of wastewater. Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention adopts the luminescent bacteria method for biological toxicity testing, which can quickly determine the biological toxicity of the wastewater to be tested. In addition, since an organic solvent is added to the wastewater to be tested before testing to assist in the dissolution of insoluble organic matter, the solubility of the organic matter in the wastewater to be tested can be effectively improved, thereby improving the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0024] Figure 1It is a double logarithmic line graph of the gamma value of the light suppression rate and the dilution rate of the water sample to be tested in Example 1;

[0025] Figure 2 It is a double logarithmic line graph of the gamma value of the light suppression rate and the dilution rate of the water sample to be tested in Example 2;

[0026] Figure 3 It is a double logarithmic line graph of the gamma value of the light suppression rate in Example 1 and the dilution rate of the water sample to be tested. DETAILED DESCRIPTION

[0027] The present invention will be described below by specific examples, and it will be appreciated by those skilled in the art that the following specific examples are only for illustrative purposes, and do not limit the scope of the present invention in any way. In addition, in the following examples, unless otherwise specified, the reagents and equipment used are all commercially available. If in the following examples, specific treatment conditions and treatment methods are not clearly described, then conditions and methods known in the art can be used to process.

[0028] The embodiment of the present invention provides a method for detecting biological toxicity of wastewater, comprising the following steps:

[0029] (1) Collect the wastewater to be tested, seal it and store it in a dark environment at 2-5℃ for ≤48h.

[0030] In the present invention, the wastewater to be tested is collected in a clean and dry container to avoid secondary contamination of the wastewater to be tested. Preferably, the storage temperature of the wastewater to be tested can be 2°C, 3°C, 4°C or 5°C, etc. The collected wastewater to be tested is stored in a sealed, low-temperature and light-proof environment to ensure the stability of the water quality of the wastewater to be tested.

[0031] (2) The wastewater to be tested should be pretreated before testing.

[0032] In some embodiments of the present invention, the pH value of the wastewater to be tested is adjusted to maintain the pH value at 6.5-7.5, for example, 6.5, 6.8, 7.0, 7.2, 7.5, etc., to meet the pH environment required for the normal survival of luminous bacteria. The pH value can be adjusted using 1-5 mol / L sodium hydroxide solution and 1-5 mol / L hydrochloric acid, which is not particularly limited.

[0033] In some embodiments of the present invention, an organic solvent is added to the wastewater to be tested after adjusting the pH value, and the non-toxic / low-toxic organic solvent is added to assist in the dissolution of insoluble organic matter in the wastewater to be tested, thereby improving the accuracy of the test results. The organic solvent may be dimethyl sulfoxide, and the volume concentration of dimethyl sulfoxide in the water sample to be tested after adding dimethyl sulfoxide is preferably 0.01-0.1%, for example, 0.01%, 0.02%, 0.05%, 0.06%, 0.08%, 0.1%, etc.

[0034] In some embodiments of the present invention, after using an organic solvent to assist in dissolving the organic matter in the wastewater to be tested, centrifugation is performed at 5000-8000g for 5-10 minutes to obtain a supernatant. Centrifugation can prevent the solid particles in the wastewater to be tested from affecting the absorbance value of the subsequent detection process. Among them, the centrifugation conditions can be centrifugation at 5000g for 10 minutes, centrifugation at 6500g for 8 minutes, centrifugation at 8000g for 5 minutes, etc., which can be adjusted according to actual conditions.

[0035] In some embodiments of the present invention, the supernatant is taken after centrifugation and the dissolved oxygen concentration of the supernatant is measured. If the dissolved oxygen concentration is low, the dissolved oxygen concentration can be adjusted to 3-4 mg / L by micro-oxygenation, for example, 3 mg / L, 3.2 mg / L, 3.4 mg / L, 3.6 mg / L, 3.8 mg / L, 4.0 mg / L, etc., to ensure the normal metabolic activity of the luminous bacteria.

[0036] In some embodiments of the present invention, it is also necessary to measure the salinity of the supernatant after oxygenation. If the salinity is low, the salinity can be adjusted to 2% by adding sodium chloride to meet the living environment of luminescent bacteria.

[0037] (3) The freeze-dried luminescent bacteria are dispersed in a 2% sodium chloride solution and revived at room temperature to obtain an active revived bacterial solution.

[0038] In some embodiments of the present invention, the freeze-dried luminescent bacteria can be Vibrio fischeri freeze-dried powder, which can be quickly revived, thus reducing the overall measurement time. The resuscitation time of Vibrio fischeri freeze-dried powder at room temperature is preferably 15 minutes, so that the test result is more accurate. After 15 minutes, the revived bacterial solution is tested for activity. If the activity test result is qualified, it is confirmed that the Vibrio fischeri freeze-dried powder has been successfully revived and can be used subsequently.

[0039] Specifically, the activity test results need to meet two of the following conditions at the same time: (1) The ratio of the luminescence value of the revived luminescent bacteria (i.e., revived bacterial solution) after surviving for 15 minutes in a 2% sodium chloride solution to the initial luminescence value of the luminescent bacteria is 0.6-1.8; (2) The light inhibition rate of the luminescent bacteria after reacting in the standard substance solution for 15 minutes is 20-80%, wherein the standard substance solution is 20 mg / L zinc sulfate heptahydrate. Only the revived bacterial solution that meets these two conditions can effectively determine the toxicity of the wastewater. Among them, condition (1) is used as a negative control for the blank group, and condition (2) is used as a positive control for quality control to verify the accuracy of the experimental operation process, reagent quality, and instrument performance.

[0040] During the activity test, the test principle is the comparison of luminescence intensity. Therefore, it is sufficient to ensure that the amount of resuscitated bacterial solution in each group is the same during the measurement process. For example, if the volume of the measurement system solution is set to 10 ml, the blank group is 1 ml of resuscitated bacterial solution + 9 ml of ultrapure water, the positive control group is 1 ml of bacterial solution + 9 ml of zinc sulfate heptahydrate, and the sample group is 1 ml of bacterial solution + 9 ml of water sample to be tested. The luminescence intensity of the toxic water sample to be tested is lower than that of the blank group.

[0041] It should be noted that the freeze-dried powder of Vibrio fischeri is preferably stored at -20°C, cannot be passaged, and must be used immediately after recovery.

[0042] (4) Using 2% sodium chloride solution as a dilution solvent, the supernatant is diluted into a series of concentrations of water samples to be tested according to a preset dilution ratio, and a biotoxicity test is performed using a luminescent bacteria method. The water samples to be tested with the series of concentrations are mixed and reacted with the resuscitated bacterial solution, and the initial luminescence amount L1 is read. After a preset reaction time, the luminescence amount L2 is read again to obtain the inhibition rate of the water sample to be tested, and then the inhibition rate gamma value is obtained based on the inhibition rate of the water sample to be tested.

[0043] Among them, the light suppression rate of the water sample to be tested = (L2 / L1-1) × 100%;

[0044] The gamma value of the inhibition rate = the inhibition rate of the water sample to be tested / (1-the inhibition rate of the water sample to be tested).

[0045] In some embodiments of the present invention, the supernatant is diluted into a series of concentrations of water samples to be tested according to the two-fold dilution method, and each concentration of the water samples to be tested is repeated at least three times to improve the accuracy of the measurement results, wherein the two-fold dilution method can be replaced by any multiple dilution method, such as five-fold, ten-fold, etc., without special limitation.

[0046] In some embodiments of the present invention, the temperature at which the water samples of a series of concentrations to be tested are mixed with the resuscitated bacterial solution is 15° C., and the preset reaction time is 15 min to ensure the accuracy of the test results.

[0047] In some embodiments of the present invention, the luminescent bacteria method biological toxicity test uses a concentric double-layer detection glass tube to reduce the influence of the chromaticity of the wastewater to be tested.

[0048] (5) Plot the inhibition rate gamma value and the dilution ratio in a double logarithmic line graph, and calculate the EC by interpolation. 50 value.

[0049] In the present invention, EC 50 The value refers to the dilution multiple that can cause 50% inhibition rate. The larger the value, the stronger the toxicity, and the smaller the value, the weaker the toxicity. If the water sample to be tested has no inhibition at all or the inhibition rate is a negative value, it means it is non-toxic.

[0050] In some embodiments of the present invention, the supernatant is diluted into a series of concentrations of water samples to be tested according to the two-fold dilution method, and the dilution multiple is set as the horizontal coordinate X, and the suppression rate gamma value is set as the vertical coordinate Y. After the supernatant is diluted 2 times, the logarithm is taken, and the horizontal coordinate is determined to be lg2. Assuming that the suppression rate is 90% after dilution 2 times, the suppression rate gamma value is 90% / (1-90%)=9, and point 1 (2,9) is obtained. After taking the logarithm, point (lg2, lg9) is obtained; after further dilution 4 times, the suppression rate is 80%, and point (4,4) is also obtained. After taking the logarithm, point (lg4, lg4) is obtained. Points are made in sequence, and then each point after taking the logarithm is linearly correlated to obtain a linear function lgy=klgx+b. When the suppression rate gamma value = 1, the suppression rate is 50%, which is substituted into the calculation to obtain X, which is the dilution multiple. The larger the dilution multiple, the stronger the toxicity of the measured water sample.

[0051] The wastewater biological toxicity detection method of the present invention is described below through specific examples.

[0052] Example 1

[0053] Dyeing wastewater from a leather factory was collected, sealed and stored in a light-proof environment at 5°C, and pretreatment was started immediately after returning to the laboratory.

[0054] The pH value of the dyeing wastewater from the leather factory to be tested was 8.2. The pH value of the wastewater to be tested was adjusted to 7.5 by dropping 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid. 0.05% (v / v) dimethyl sulfoxide was added to the wastewater to be tested, and the mixture was placed in a centrifuge. After centrifugation at 8000 g for 10 min, the supernatant was taken and the dissolved oxygen concentration was controlled to be 4 mg / L by micro-oxygenation. The salinity of the supernatant after oxygenation was measured to be 0.5%. Sodium chloride was added to adjust the salinity to 2%.

[0055] The freeze-dried powder of Vibrio fischeri was dissolved in 2% sodium chloride solution and revived at room temperature for 15 minutes. The revived bacterial solution was tested for activity. The ratio of the luminescence value of the luminescent bacteria after surviving in 2% sodium chloride solution for 15 minutes to the initial luminescence value was 1.8. After reacting in 20 mg / L zinc sulfate heptahydrate for 15 minutes, the photosensitivity was 80%, and the activity test was qualified.

[0056] The wastewater to be tested was diluted using a 2% sodium chloride solution as the dilution solvent according to the two-fold dilution method, and then a biotoxicity test using the luminescent bacteria method was performed. The water sample to be tested was added to the bacterial solution and placed in a concentric double-layer detection glass tube. The initial luminescence was read, and the reaction time was 15 minutes. The reaction temperature was controlled at 15°C, and the luminescence was read again. Each dilution multiple was repeated three times to obtain the inhibition rate gamma value and the dilution rate of the water sample to be tested.

[0057] Make a double logarithmic line graph of the inhibition rate gamma value and the dilution rate of the water sample to be tested, such as Figure 1 As shown, EC is calculated by interpolation 50 The value is 1016 times.

[0058] Therefore, it can be judged that the dyeing wastewater from the leather factory has a high biological toxicity. It is recommended that after advanced oxidation pretreatment, the biological toxicity be reduced, and then it be mixed and diluted with non-toxic or low-toxic wastewater in a ratio of 1:2000 and then enter the biological section of the sewage treatment plant for disposal.

[0059] Example 2

[0060] The filtrate from the hydrothermal carbonization of sludge from a sewage treatment plant was collected, sealed and stored in a dark place at 2°C, and pretreated after 2 days.

[0061] The pH value of the hydrothermal carbonization filtrate of the sludge to be tested is 4.8. The pH value of the wastewater to be tested is adjusted to 6.5 by dropping 3 mol / L sodium hydroxide and 3 mol / L hydrochloric acid. 0.01% (v / v) dimethyl sulfoxide is added to the wastewater to be tested, and the mixture is placed in a centrifuge. After centrifugation at 5000 g for 5 minutes, the supernatant is taken and the dissolved oxygen concentration is controlled to be 3 mg / L by micro-oxygenation. The salinity of the supernatant after oxygenation is measured to be 0.2%, and the salinity is adjusted to 2% by adding sodium chloride.

[0062] The freeze-dried powder of Vibrio fischeri was dissolved in 2% sodium chloride solution and revived at room temperature for 15 minutes. The revived bacterial solution was tested for activity. The ratio of the luminescence value of the luminescent bacteria after surviving in 2% sodium chloride solution for 15 minutes to the initial luminescence value was 0.6. After reacting in 20 mg / L zinc sulfate heptahydrate for 15 minutes, the photosensitivity was 20%, and the activity test was qualified.

[0063] The wastewater to be tested was diluted using a 2% sodium chloride solution as the dilution solvent according to the two-fold dilution method, and then a biotoxicity test using the luminescent bacteria method was performed. The water sample to be tested was added to the bacterial solution and placed in a concentric double-layer detection glass tube. The initial luminescence was read, and the reaction time was 15 minutes. The reaction temperature was controlled at 15°C, and the luminescence was read again. Each dilution multiple was repeated three times to obtain the inhibition rate gamma value and the dilution rate of the water sample to be tested.

[0064] Make a double logarithmic line graph of the inhibition rate gamma value and the dilution rate of the water sample to be tested, such as Figure 2 As shown, EC is calculated by interpolation 50 The value is 294 times.

[0065] Therefore, it can be judged that the biological toxicity of the plant's sludge hydrothermal carbonization filtrate is relatively low. It is recommended that the sludge filtrate and the sewage treatment plant's influent water be returned to the front end of the sewage treatment plant's process section for disposal at a ratio of 1:600.

[0066] Example 3

[0067] Wastewater from a coking plant was collected, sealed and stored in a dark place at 2°C, and pretreated after 2 days.

[0068] The pH value of the coking wastewater to be tested was 6.2. The pH value of the wastewater to be tested was adjusted to 7.0 by dropping 5 mol / L sodium hydroxide and 5 mol / L hydrochloric acid. 0.1% (v / v) dimethyl sulfoxide was added to the wastewater to be tested, and the mixture was placed in a centrifuge. After centrifugation at 6000 g for 6 minutes, the supernatant was taken and the dissolved oxygen concentration was controlled to be 3.6 mg / L by micro-oxygenation. The salinity of the supernatant after oxygenation was measured to be 0.6%. Sodium chloride was added to adjust the salinity to 2%.

[0069] The freeze-dried powder of Vibrio fischeri was dissolved in 2% sodium chloride solution and revived at room temperature for 15 minutes. The revived bacterial solution was tested for activity. After the luminous bacteria survived in 2% sodium chloride solution for 15 minutes, the ratio of the luminescence value to the initial luminescence value was 1.2. After reacting in 20 mg / L zinc sulfate heptahydrate for 15 minutes, the photosensitivity was 60%, and the activity test was qualified.

[0070] The wastewater to be tested was diluted using a 2% sodium chloride solution as the dilution solvent according to the two-fold dilution method, and then a biotoxicity test using the luminescent bacteria method was performed. The water sample to be tested was added to the bacterial solution and placed in a concentric double-layer detection glass tube. The initial luminescence was read, and the reaction time was 15 minutes. The reaction temperature was controlled at 15°C, and the luminescence was read again. Each dilution multiple was repeated three times to obtain the inhibition rate gamma value and the dilution rate of the water sample to be tested.

[0071] Make a double logarithmic line graph of the inhibition rate gamma value and the dilution rate of the water sample to be tested, such as Figure 3 As shown, EC is calculated by interpolation 50 The value is 695 times.

[0072] Therefore, it can be judged that the biological toxicity of the coking wastewater of this plant is average. It is recommended that after anaerobic hydrolysis pretreatment and the biological toxicity is reduced, it should be mixed and diluted with non-toxic or low-toxic wastewater in a ratio of 1:1400 and then enter the biological section of the sewage treatment plant for disposal.

[0073] In summary, the wastewater biological toxicity detection method of the present invention has high detection integrity and rapid result response. According to the toxicity of the wastewater, an appropriate treatment method can be selected to reduce the impact of biological toxicity on the sewage treatment process and protect the health of the water environment.

[0074] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting biological toxicity of wastewater, characterized in that: The following steps are involved: (1) Add an organic solvent to the wastewater to be tested, centrifuge and take the supernatant; (2) dispersing the freeze-dried luminescent bacteria in a 2% sodium chloride solution and resuscitating the bacteria at room temperature to obtain an active resuscitated bacterial solution; (3) Using 2% sodium chloride solution as a dilution solvent, diluting the supernatant into a series of concentrations of water samples to be tested according to a preset dilution ratio, mixing the series of concentrations of water samples to be tested with the resuscitated bacterial solution, reading the initial luminescence, and reading the luminescence again after a preset reaction time to obtain the inhibition rate of the water sample to be tested, and then obtaining the inhibition rate gamma value according to the inhibition rate of the water sample to be tested; wherein the inhibition rate gamma value = inhibition rate of the water sample to be tested / (1-inhibition rate of the water sample to be tested); (4) Plot the inhibition rate gamma value and the dilution ratio in a double logarithmic line graph, and calculate the EC by interpolation. 50 value.

2. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: The pH value of the wastewater to be tested in step (1) is 6.5-7.5; The dissolved oxygen concentration in the supernatant is 3-4 mg / L, and the salinity is 2%.

3. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: In step (1), the organic solvent is dimethyl sulfoxide, and the volume concentration of the dimethyl sulfoxide is 0.01-0.1%.

4. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: The centrifugation in step (1) is carried out at 5000-8000g for 5-10 min.

5. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: The freeze-dried luminescent bacteria in step (2) is freeze-dried Vibrio fischeri powder; The resuscitation time is 15 minutes.

6. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: The active resuscitation bacterial solution meets the following conditions: After recovery at room temperature, the ratio of the luminescence value of the luminescent bacteria after surviving in a 2% sodium chloride solution for 15 minutes to the initial luminescence value of the luminescent bacteria is 0.6-1.8, and the light inhibition rate of the luminescent bacteria after reacting in a standard substance solution for 15 minutes is 20-80%.

7. The method for detecting biological toxicity of wastewater according to claim 6, characterized in that: The standard substance solution is 20 mg / L zinc sulfate heptahydrate.

8. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: In step (3), the supernatant is diluted into water samples of a series of concentrations according to a two-fold dilution method.

9. The method for detecting biological toxicity of wastewater according to claim 1, characterized in that: The mixed reaction temperature in step (3) is 15° C., and the preset reaction time is 15 min.

10. The method for detecting biological toxicity of wastewater according to any one of claims 1 to 9, characterized in that: The method also includes the following steps before step (1): collecting the wastewater to be tested, sealing it and storing it in a light-proof environment at 2-5°C for a storage time of ≤48h.

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