Method for representing influence of PPCPs on toxicity of activated sludge system by using viable and dead bacteria staining method

Abstract

The invention relates to the field of ecological environment, in particular to a method for characterizing the influence of PPCPs on the toxicity of an activated sludge system by using a viable and dead bacteria staining method, which comprises the following steps of: improving an SYTO 9 dye and PI dye cell staining method, namely quantitatively detecting the fluorescence intensity corresponding to dead bacteria and viable bacteria by using a three-dimensional fluorescence spectrophotometer; a quantitative detection technology of flora activity is established, and a set of method capable of rapidly and accurately quantifying the ratio of dead bacteria to viable bacteria in a sample is constructed; according to the invention, degradation conditions of PPCPs and organic matters in water with different concentrations and different PPCPs under the same operation condition and a change rule of water toxicity are explored, the influence of PPCPs on the toxicity of an activated sludge system is represented by using a viable bacteria and dead bacteria staining method, and the influence degree of PPCPs on a sewage treatment system can be rapidly analyzed through relative fluorescence intensity. Therefore, a rapid and effective detection method is provided for monitoring the influence of the PPCPs in the inlet water of the sewage plant on the treatment process of the water plant, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of ecological environment, and in particular to a method for characterizing the toxicity of PPCPs on an activated sludge system by using a live bacteria and dead bacteria staining method. Background Art

[0002] Pharmaceuticals and personal care products (PPCPs) are a new type of trace organic pollutants that have a long-term potential impact on the ecological environment and pose a threat to human health. Since sewage treatment plants cannot completely remove PPCPs, most sewage discharges become the main pathway for PPCPs to migrate into the environment. PPCPs have varying degrees of toxic effects on the biological environment.

[0003] Since some PPCPs are designed to target microorganisms in humans and animals, they are bound to have certain toxic effects on microorganisms in sewage treatment systems. Long-term exposure will change the community structure of microorganisms, thereby affecting the efficiency of sewage treatment. Biological toxicity testing can intuitively reflect the comprehensive toxicity of pollutants to biological populations and determine the direct relationship between the mass concentration of toxic substances and biological effects. It is an indispensable means to predict and control chemical pollution. Therefore, a detection method is needed that can monitor the toxic effects of PPCPs on the system during the sewage treatment process, which can effectively predict the toxic effects of PPCPs content on the sludge state of the sewage treatment system in advance.

[0004] At present, the toxicity detection methods for activated sludge systems mainly characterize the toxic effects of PPCPs on activated sludge by analyzing nitrification efficiency, electron transfer system activity (TTC-ETS), specific oxygen uptake rate (SOUR), and ammonia uptake rate (AUR). These methods generally have problems such as long reaction time, unstable monitoring data, and complex operation process. As a quick, convenient and accurate toxicity detection method, the live and dead bacteria staining method can be used for toxicity detection and monitoring of PPCPs in sewage. However, the accuracy of conventional toxicity detection using the staining method is low, and the process of analyzing and counting from microscope images is relatively complicated, which is prone to detection deviations. For example, the patent technical document CN118130411A discloses a differential ultraviolet absorption spectrum detection method for PPCPs. On the premise that the ultraviolet-visible absorption spectrum can detect DOM-PPCPs: complex, the ultraviolet-visible absorption spectrum is differentiated to amplify the difference in ultraviolet absorption spectra of PPCPs of different concentrations. Different PPCPs have different characteristic peaks in the differential spectrum, which can accurately realize the concentration of free PPCPs and DOM-PPCPs complex substances in the DOM-PPCPs system. However, this method has the problems of long reaction time and unstable monitoring data.

[0005] Therefore, based on the above-mentioned related technologies, it is urgent to develop a method using live bacteria and dead bacteria staining to characterize the toxic effects of PPCPs on activated sludge systems. Summary of the invention

[0006] In view of this, the purpose of the present invention is to propose a method for characterizing the toxic effects of PPCPs on activated sludge systems using live and dead bacteria staining, so as to provide a method that can quickly and sensitively detect toxic conditions in sewage systems and take timely early warning measures.

[0007] Based on the above purpose, the present invention provides a method for characterizing the toxic effect of PPCPs on an activated sludge system using a live bacteria and dead bacteria staining method.

[0008] A method for characterizing the toxicity of PPCPs on an activated sludge system using a live bacteria and dead bacteria staining method comprises the following steps:

[0009] Step S1. preparing wastewater samples;

[0010] Step S2. inoculating sludge;

[0011] Step S3. preparing PPCPs samples;

[0012] Step S4. operating the reactor;

[0013] Step S5. staining;

[0014] Step S6. Cytotoxicity detection;

[0015] The PPCPs sample in step S3 is any one of trimethoprim, sulfamethoxazole, ibuprofen and diclofenac;

[0016] The reagent used in the dyeing in step S5 is a mixed dye;

[0017] The mixed dye is prepared from SYTO 9 dye and PI dye;

[0018] The instrument used in the cytotoxicity detection in step S6 is a three-dimensional fluorescence spectrometer.

[0019] Preferably, the wastewater sample in step S1 has sodium acetate as a carbon source, ammonium chloride as a nitrogen source and potassium dihydrogen phosphate as a phosphorus source;

[0020] The conditions of the wastewater sample in step S1 are chemical oxygen demand of 270-280 mg / L, NH4 + -N is 36-38 mg / L, total phosphorus is 3.41-3.64 mg / L, and pH is 7.5-7.8.

[0021] Preferably, the steps of inoculating sludge in step S2 are as follows:

[0022] Step A1. After filtering the sludge through a 30-50 mesh sieve, add glucose as a carbon source and expose for 2-5 days, and filter through a 30-50 mesh sieve to obtain a sludge mixture;

[0023] Step A2. Take out 5 portions of the sludge mixture, add the wastewater sample, dilute the sludge concentration to 2000-2100 mg / L, and obtain a sludge mixture.

[0024] Preferably, the mass ratio of the sludge to glucose in step A1 is 10-15:0.1-0.5.

[0025] Preferably, the concentration of the PPCPs sample in step S3 is 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L and 1.5 mg / L;

[0026] The PPCPs sample in step S3 is prepared and added into the reactor.

[0027] Preferably, the reactor in step S4 is a sequencing batch activated sludge reactor;

[0028] The operating conditions of the reactor in step S4 are aeration and stirring for 5 hours and sedimentation for 30-60 minutes;

[0029] The aeration is performed by an air pump, and stirring is performed at 150-180 r / min during aeration, and the dissolved oxygen is maintained at 5-6 mg / L.

[0030] Preferably, the dyeing steps in step S5 are as follows:

[0031] Step B1. Take SYTO 9 dye and PI dye, add enzyme-free water, mix well, and store at 4-8°C to obtain a mixed dye;

[0032] Step B2. During the operation of the reactor, 1 mL of the sludge mixture was extracted every 1 h, 30 μL of the mixed dye was added, and the mixture was reacted in the dark for 15-30 min.

[0033] Preferably, the volume ratio of SYTO 9 dye, PI dye and enzyme-free water in step B1 is 20-40:20-40:360-380.

[0034] Preferably, the experimental conditions of the three-dimensional fluorescence spectrometer in the cytotoxicity detection in step S6 are:

[0035] The excitation wavelength Ex was 480 nm and 490 nm, the emission wavelength Em was 500 nm and 635 nm, the Ex and Em slit widths were both 5 nm, the scanning speed was 60000 nm / min, and the integration time was 1 s.

[0036] Beneficial effects of the present invention:

[0037] The invention provides a method for characterizing the toxic effect of PPCPs on an activated sludge system by using a live bacteria and dead bacteria staining method. By improving the SYTO 9 dye and PI dye cell staining method, that is, using a three-dimensional fluorescence spectrometer to quantitatively detect the fluorescence intensity corresponding to dead bacteria and live bacteria, the quantitative analysis of the ratio of live and dead bacteria in a sample system is realized, a quantitative detection technology for bacterial community activity is established, and a set of methods for quickly and accurately quantifying the ratio of live and dead bacteria in a sample is constructed. The invention explores the degradation of PPCPs and organic matter in water and the changing law of water toxicity under the same operating conditions with different concentrations and different PPCPs, characterizes the toxic effect of PPCPs on an activated sludge system by using a live bacteria and dead bacteria staining method, and can quickly analyze the influence of PPCPs on a sewage treatment system by relative fluorescence intensity, thereby providing a rapid and effective detection method for monitoring the influence of PPCPs in the influent of a sewage plant on the treatment process of a water plant, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or 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 in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 The figure is a diagram showing the removal of PPCPs in the sequencing batch activated sludge system and the effect of adding PPCPs on the removal of organic matter in the sequencing batch activated sludge system;

[0040] Figure 2 The figure is a graph showing the change in the ratio of live bacteria to dead bacteria in the activated sludge at different stimulation times in the reactor after adding different concentrations (0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L) of trimethoprim for stimulation in the present invention;

[0041] Figure 3 The figure is a graph showing the change in the ratio of live bacteria to dead bacteria of activated sludge at different stimulation times in the reactor after different concentrations (0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L) of sulfamethoxazole were added to stimulate the present invention;

[0042] Figure 4 The graph is a change diagram of the ratio of live bacteria to dead bacteria in the activated sludge at different stimulation times in the reactor after different concentrations (0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L) of ibuprofen were added to stimulate the present invention;

[0043] Figure 5 This is a graph showing the change in the ratio of live bacteria to dead bacteria in the activated sludge at different stimulation times in the reactor after different concentrations (0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, and 1.5 mg / L) of diclofenac were added for stimulation in the present invention. DETAILED DESCRIPTION

[0044] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0045] The sources and properties of some of the raw materials used in the present invention are as follows:

[0046] Trimethoprim was purchased from Wuhan Taiyicheng Biochemical Pharmaceutical Co., Ltd.; sulfamethoxazole was purchased from Jiangsu Rongshuo Pharmaceutical Co., Ltd.; ibuprofen was purchased from Jiangsu Rongshuo Pharmaceutical Co., Ltd.; and diclofenac was purchased from Hubei Weideli Chemical Technology Co., Ltd.

[0047] Example 1: A method for characterizing the toxicity of PPCPs on an activated sludge system using a live bacteria and dead bacteria staining method, comprising the following steps:

[0048] S1. Prepare wastewater samples. The wastewater samples use sodium acetate as carbon source, ammonium chloride as nitrogen source and potassium dihydrogen phosphate as phosphorus source. The conditions of the wastewater samples are chemical oxygen demand of 270 mg / L, NH4 + -N is 36 mg / L, total phosphorus is 3.41 mg / L, and pH is 7.5;

[0049] S2. After filtering 10 g of sludge through a 30-mesh sieve, add 0.1 g of glucose as a carbon source and expose for 2 days, filter through a 30-mesh sieve to obtain a sludge mixture;

[0050] S3. Take out 5 portions of the sludge mixture, add the wastewater sample, dilute the sludge concentration to 2000 mg / L, and obtain a sludge mixture;

[0051] S4. Trimethoprim was prepared at concentrations of 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L and 1.5 mg / L and was put into a sequencing batch activated sludge reactor;

[0052] S5. Operate a sequencing batch activated sludge reactor. The operating conditions of the reactor are aeration and stirring for 5 hours and sedimentation for 30 minutes. Aeration is performed by an air pump. During aeration, stirring is performed at 150 r / min. The dissolved oxygen is maintained at 5 mg / L.

[0053] S6. Take 20 μL SYTO 9 dye and 20 μL PI dye, add 360 μL enzyme-free water, mix well and store at 4°C to obtain a mixed dye;

[0054] S7. During the operation of the reactor, 1 mL of the sludge mixture was extracted every 1 h, 30 μL of the mixed dye was added, and the reaction was carried out in the dark for 15 min;

[0055] S8. Cytotoxicity detection was performed using a three-dimensional fluorescence spectrometer. The experimental conditions of the three-dimensional fluorescence spectrometer were as follows: excitation wavelength Ex was 480 nm and 490 nm, emission wavelength Em was 500 nm and 635 nm, Ex and Em slit widths were both 5 nm, scanning speed was 60,000 nm / min, and integration time was 1 s.

[0056] Example 2: A method for characterizing the toxicity of PPCPs on an activated sludge system using live and dead bacteria staining method, comprising the following steps:

[0057] S1. Prepare wastewater samples. The wastewater samples use sodium acetate as carbon source, ammonium chloride as nitrogen source and potassium dihydrogen phosphate as phosphorus source. The conditions of the wastewater samples are chemical oxygen demand of 272 mg / L, NH4 +-N was 37 mg / L, total phosphorus was 3.48 mg / L, and pH was 7.6;

[0058] S2. After filtering 12 g of sludge through a 40-mesh sieve, 0.2 g of glucose was added as a carbon source and exposed for 3 days, and filtered through a 40-mesh sieve to obtain a sludge mixture;

[0059] S3. Take out 5 parts of the sludge mixture, add the wastewater sample, dilute the sludge concentration to 2040 mg / L, and obtain a sludge mixture;

[0060] S4. Prepare sulfamethoxazole at concentrations of 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L and 1.5 mg / L and put them into a sequencing batch activated sludge reactor;

[0061] S5. Operate a sequencing batch activated sludge reactor. The operating conditions of the reactor are aeration and stirring for 5 hours and sedimentation for 40 minutes. Aeration is performed by an air pump. During aeration, stirring is performed at 160 r / min and dissolved oxygen is maintained at 5.4 mg / L.

[0062] S6. Take 30 μL SYTO 9 dye and 30 μL PI dye, add 365 μL enzyme-free water, mix well, and store at 4-8°C to obtain a mixed dye;

[0063] S7. During the operation of the reactor, 1 mL of the sludge mixture was extracted every 1 h, 30 μL of the mixed dye was added, and the reaction was carried out in the dark for 20 min;

[0064] S8. Cytotoxicity detection was performed using a three-dimensional fluorescence spectrometer. The experimental conditions of the three-dimensional fluorescence spectrometer were as follows: excitation wavelength Ex was 480 nm and 490 nm, emission wavelength Em was 500 nm and 635 nm, Ex and Em slit widths were both 5 nm, scanning speed was 60,000 nm / min, and integration time was 1 s.

[0065] Example 3: A method for characterizing the toxicity of PPCPs on an activated sludge system using live and dead bacteria staining method, comprising the following steps:

[0066] S1. Prepare wastewater samples. The wastewater samples use sodium acetate as carbon source, ammonium chloride as nitrogen source and potassium dihydrogen phosphate as phosphorus source. The conditions of the wastewater samples are chemical oxygen demand of 278 mg / L, NH4 + -N is 37.5 mg / L, total phosphorus is 3.56 mg / L, and pH is 7.7;

[0067] S2. After filtering 14 g of sludge through a 40-mesh sieve, 0.4 g of glucose was added as a carbon source and exposed for 4 days, and filtered through a 40-mesh sieve to obtain a sludge mixture;

[0068] S3. Take out 5 portions of the sludge mixture, add the wastewater sample, dilute the sludge concentration to 2080 mg / L, and obtain a sludge mixture;

[0069] S4. Prepare ibuprofen at concentrations of 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L and 1.5 mg / L and put them into a sequencing batch activated sludge reactor;

[0070] S5. Operate a sequencing batch activated sludge reactor. The operating conditions of the reactor are aeration and stirring for 5 hours and sedimentation for 50 minutes. Aeration is performed by an air pump. During aeration, stirring is performed at 170 r / min. The dissolved oxygen is maintained at 5.8 mg / L.

[0071] S6. Take 35 μL SYTO 9 dye and 35 μL PI dye, add 370 μL enzyme-free water, mix well and store at 6°C to obtain a mixed dye;

[0072] S7. During the operation of the reactor, 1 mL of the sludge mixture was extracted every 1 h, 30 μL of the mixed dye was added, and the reaction was carried out in the dark for 25 min;

[0073] S8. Cytotoxicity detection was performed using a three-dimensional fluorescence spectrometer. The experimental conditions of the three-dimensional fluorescence spectrometer were as follows: excitation wavelength Ex was 480 nm and 490 nm, emission wavelength Em was 520 nm and 640 nm, Ex and Em slit widths were both 5 nm, scanning speed was 60,000 nm / min, and integration time was 1 s.

[0074] Example 4: A method for characterizing the toxicity of PPCPs on an activated sludge system using live and dead bacteria staining method, comprising the following steps:

[0075] S1. Prepare wastewater samples. The wastewater samples use sodium acetate as carbon source, ammonium chloride as nitrogen source and potassium dihydrogen phosphate as phosphorus source. The conditions of the wastewater samples are chemical oxygen demand of 280 mg / L, NH4 + -N is 38 mg / L, total phosphorus is 3.64 mg / L, and pH is 7.8;

[0076] S2. After filtering 15 g of sludge through a 50-mesh sieve, add 0.5 g of glucose as a carbon source and expose for 5 days, filter through a 50-mesh sieve to obtain a sludge mixture;

[0077] S3. Take out 5 portions of the sludge mixture, add the wastewater sample, dilute the sludge concentration to 2100 mg / L, and obtain a sludge mixture;

[0078] S4. Diclofenac was prepared at concentrations of 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L and 1.5 mg / L and was put into a sequencing batch activated sludge reactor;

[0079] S5. Operate the sequencing batch activated sludge reactor. The operating conditions of the reactor are aeration and stirring for 5 hours and sedimentation for 60 minutes. Aeration is performed by an air pump. During aeration, stirring is performed at 180 r / min and dissolved oxygen is maintained at 6 mg / L.

[0080] S6. Take 40 μL SYTO 9 dye and 40 μL PI dye, add 380 μL enzyme-free water, mix well and store at 8°C to obtain a mixed dye;

[0081] S7. During the operation of the reactor, 1 mL of the sludge mixture was extracted every 1 h, 30 μL of the mixed dye was added, and the reaction was carried out in the dark for 30 min;

[0082] S8. Cytotoxicity detection was performed using a three-dimensional fluorescence spectrometer. The experimental conditions of the three-dimensional fluorescence spectrometer were as follows: excitation wavelength Ex was 480 nm and 490 nm, emission wavelength Em was 500 nm and 635 nm, Ex and Em slit widths were both 5 nm, scanning speed was 60,000 nm / min, and integration time was 1 s.

[0083] Performance Testing:

[0084] The three-dimensional fluorescence spectrometer was used to analyze Examples 1 to 4, and the ratio of live bacteria to dead bacteria in the sludge mixtures prepared in Examples 1 to 4 was calculated. The calculation formula is as follows:

[0085] The formula for calculating the ratio of live bacteria to dead bacteria is:

[0086] Among them, Ratio G / R is the ratio of live bacteria to dead bacteria in the sample; F cell,green F is the fluorescence intensity detected at excitation / emission wavelengths of 480 / 500 nm; cell,red It is the fluorescence intensity detected at the excitation / emission wavelength of 490 / 635 nm.

[0087] Data Analysis:

[0088] The method provided by the present invention can quickly and effectively detect toxicity in sewage systems;

[0089] from Figure 1 It can be seen that the four different PPCPs will produce a certain removal effect in the sequencing batch activated sludge system, among which the removal effects of trimethoprim and ibuprofen will show a significant downward trend with the increase of the added concentration; Figure 1According to the ammonia nitrogen situation of the inlet and outlet water of the activated sludge reactor, the ammonia nitrogen removal rate of the reactor after being stimulated by PPCPs was obtained. The effects of trimethoprim and sulfamethoxazole on the ammonia nitrogen removal efficiency are more obvious. In the blank sample without the addition of PPCPs, the ammonia nitrogen removal rate was 96.1%. After the addition of TMP and SMX, some samples dropped to 81.5% (when the concentration of trimethoprim was 1.5 mg / L) or 88.7% (when the concentration of sulfamethoxazole was 1.5 mg / L). However, the ammonia nitrogen removal rate was not greatly affected by the addition of ibuprofen and diclofenac, and the removal efficiency could be maintained in a stable state in the domesticated sludge, reaching more than 97%, indicating that the influence of antibiotic PPCPs on the nitrification capacity of the activated sludge system is more prominent.

[0090] Figure 1 It also reflects that the removal efficiency of chemical oxygen demand is significantly affected by antibiotic PPCPs. After adding PPCPs, the co-metabolic degradation process consumes a certain amount of carbon source and energy, and the removal of chemical oxygen demand is also significantly inhibited. When 0.1 mg / L trimethoprim is added, the removal rate of chemical oxygen demand drops from the original 91.1% to 77.5%. The toxic effects of antibiotic PPCPs on heterotrophic bacteria and nitrifying bacteria in activated sludge systems are more prominent.

[0091] from Figure 2 It can be seen that the Ratio G / R The trend of change is similar to that of the blank sample without adding the agent. However, in the fourth hour after trimethoprim stimulation, the samples stimulated by trimethoprim showed a rapid increase in peak value, indicating that the dominant bacteria are in a state of rapid reproduction and growth. At this time, the Ratio of the reactor sample with trimethoprim at 0.1 mg / L G / R The highest was 4.08, which was higher than the sample without the addition of the agent. When the reactor was running for 5 hours and aeration was ended, the Ratio of the sample G / R The results showed that the Ratio of the samples after 5 hours of pressure stimulation with different concentrations of trimethoprim (0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L, and 1.5 mg / L) decreased, probably because the nutrients and carbon sources had been consumed and there was no energy to support the consumption of bacteria in the reactor, resulting in the death of some bacteria. G / R It gradually decreased with the increase of trimethoprim concentration, indicating that with the increase of trimethoprim concentration, the probability of cell death due to toxic stimulation increased. The presence of trimethoprim in the activated sludge system has a certain toxic effect on cell activity.

[0092] from Figure 3It can be seen that the trend of the toxic effect of sulfamethoxazole on the activated sludge system with stimulation time is roughly the same as that of trimethoprim, which also decreases in the first 3 hours and reaches a peak at the 4th hour. Figure 1 Analysis showed that when the concentration of sulfamethoxazole was 0.1 mg / L, the removal rate of sulfamethoxazole reached 100%, indicating that sulfamethoxazole had a high co-metabolism degradation ability at this concentration, and quickly consumed carbon sources and organic matter in the first 2 hours, achieving a state of stable growth of dominant bacteria.

[0093] from Figure 4 It can be seen that the effect of ibuprofen on cell mortality is consistent with that of trimethoprim. The difference is that ibuprofen has a certain promoting effect on cell activity at a concentration of 0.5 mg / L. Three hours before ibuprofen stimulation, the Ratio G / R The decline rate slowed down significantly, and the peak value at the 4th hour was higher than that of samples with other concentrations, indicating that ibuprofen had a relatively weak toxic effect on activated sludge at a concentration of 0.5 mg / L.

[0094] from Figure 5 It can be seen that when different concentrations of diclofenac are added to the activated sludge system, as the stimulation time increases, the Ratio G / R It gradually decreases, and with the increase of addition concentration, the slope of the downward curve will also increase, indicating that the concentration of diclofenac has obvious toxic effect on the nitrifying sludge system, and has a greater inhibitory effect on the nitrification of the activated sludge system. This shows that the rapid rise of diclofenac stimulation in the activated sludge system in the later stage is due to the rapid reproduction of anaerobic bacteria in the sludge system under the condition of excess nutrition, and may even lead to endogenous consumption in the reactor, resulting in a decrease in sludge concentration and affecting the normal operation of the activated sludge system.

[0095] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0096] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for characterizing the toxicity of PPCPs on activated sludge system using live and dead bacteria staining method, characterized in that: The following steps are involved: Step S1. preparing a wastewater sample; Step S2. inoculating sludge; Step S3. preparing PPCPs samples; Step S4. operating the reactor; Step S5. staining; Step S6. Cytotoxicity detection; The PPCPs sample in step S3 is any one of trimethoprim, sulfamethoxazole, ibuprofen and diclofenac; The reagent used in the dyeing in step S5 is a mixed dye; The mixed dye is prepared from SYTO 9 dye and PI dye; The instrument used in the cytotoxicity detection in step S6 is a three-dimensional fluorescence spectrometer.

2. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 1, characterized in that: The wastewater sample in step S1 has sodium acetate as a carbon source, ammonium chloride as a nitrogen source and potassium dihydrogen phosphate as a phosphorus source; The conditions of the wastewater sample in step S1 are chemical oxygen demand of 270-280 mg / L, NH4 + -N is 36-38 mg / L, total phosphorus is 3.41-3.64 mg / L, and pH is 7.5-7.

8.

3. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 1, characterized in that: The steps of inoculating sludge in step S2 are as follows: Step A1. After filtering the sludge through a 30-50 mesh sieve, add glucose as a carbon source and expose for 2-5 days, and filter through a 30-50 mesh sieve to obtain a sludge mixture; Step A2. Take out 5 portions of the sludge mixture, add the wastewater sample, dilute the sludge concentration to 2000-2100 mg / L, and obtain a sludge mixture.

4. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 3, characterized in that: The mass ratio of sludge to glucose in step A1 is 10-15:0.1-0.

5.

5. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 1, characterized in that: The concentrations of the PPCPs sample in step S3 are 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1.0 mg / L and 1.5 mg / L; The PPCPs sample in step S3 is prepared and added into the reactor.

6. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 1, characterized in that: The reactor in step S4 is a sequencing batch activated sludge reactor; The operating conditions of the reactor in step S4 are aeration and stirring for 5 hours and sedimentation for 30-60 minutes; The aeration is performed by an air pump, and stirring is performed at 150-180 r / min during aeration, and the dissolved oxygen is maintained at 5-6 mg / L.

7. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 1, characterized in that: The steps of dyeing described in step S5 are as follows: Step B1. Take SYTO 9 dye and PI dye, add enzyme-free water, mix well, and store at 4-8°C to obtain a mixed dye; Step B2. During the operation of the reactor, 1 mL of the sludge mixture was extracted every 1 h, 30 μL of the mixed dye was added, and the mixture was reacted in the dark for 15-30 min.

8. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 7, characterized in that: The volume ratio of SYTO 9 dye, PI dye and enzyme-free water in step B1 is 20-40:20-40:360-380.

9. The method for characterizing the toxicity of PPCPs on activated sludge system by using live bacteria and dead bacteria staining method according to claim 1, characterized in that: The experimental conditions of the three-dimensional fluorescence spectrometer in the cytotoxicity detection in step S6 are: The excitation wavelength Ex was 480 nm and 490 nm, the emission wavelength Em was 500 nm and 635 nm, the Ex and Em slit widths were both 5 nm, the scanning speed was 60000 nm / min, and the integration time was 1 s.

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