Method for detecting concentration of microplastics in water body based on advanced oxidation and image recognition technology

Abstract

The application discloses a water micro-plastic concentration detection method based on advanced oxidation and image recognition technology, and comprises the following steps: S1, dyeing micro-plastics or filter membranes; S2, weighing the micro-plastics in step S1 to prepare a micro-plastic water suspension solution with a standard concentration; S3, filtering the micro-plastics in the series of micro-plastic water suspension standard solutions prepared in step S2 onto the filter membranes; S4, taking photos of the micro-plastic standard filter membranes prepared in step S3; S5, removing the picture background and performing binaryzation processing, measuring the area of the micro-plastics by using Image J software, and obtaining the corresponding relationship between the micro-plastic water suspension concentration and the area; and S6, performing the same dyeing, filtering, picture collection and image processing on the micro-plastics in the water sample, and then obtaining the micro-plastic concentration of the micro-plastic water suspension sample to be detected according to the corresponding relationship between the micro-plastic water suspension concentration and the area. The detection method is simple and efficient, and can quickly and accurately detect the micro-plastic concentration in water.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of water quality detection, and relates to water microplastic concentration, in particular to a water microplastic concentration detection method based on advanced oxidation and image recognition technology. BACKGROUND

[0002] Microplastics, as a new pollutant, are ubiquitous in water environment. The particle size of microplastics ranges from several microns to several millimeters, and they are a mixture of non-uniform plastic particles of various shapes, which are often difficult to distinguish with the naked eye. It is generally believed that the size range of microplastics is between 1 μm and 5 mm. Plastic products are indispensable in life, and microplastic particles will inevitably be generated during their processing and use. For example, microplastics generated during production and processing will migrate under the action of wind and eventually fall into water bodies. Functional toothpaste, shower gel and facial cleanser contain microplastics added during use, which flow into urban pipe networks with water, and are discharged into rivers after treatment in sewage treatment plants. A study in Dongting Lake showed that the microplastic abundance of surface water on the shore of South Dongting Lake was 716.67-2316.67 items / m 3 Water microplastics are of various types, high abundance and wide influence range. Polyethylene, polypropylene, polystyrene, polyethylene terephthalate and rubber are common types of microplastics in water bodies. The first four types of microplastic fragments are white before aging, and turn into light yellow or light green after aging under the action of ultraviolet light and microbial predation in natural water bodies. Rubber tire microplastics produced by tire wear are black.

[0003] Measuring the concentration of microplastics is of great significance for analyzing their environmental impact. The currently widely used methods for detecting water microplastics mainly include fluorescence counting method and spectroscopy. The fluorescence counting method refers to using a filtering device to intercept microplastics in water, then using fluorescent dye to stain and count under a microscope to determine the degree of water pollution by microplastics. The spectroscopy refers to drying and digesting the filter membrane with microplastics after filtering the water sample, and then measuring the infrared spectrum. The results are compared with the standard spectrum of plastics to determine whether the water body is contaminated by microplastics. In addition, the latest pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) technology can also be used for qualitative / quantitative detection of microplastics in water. However, this method requires high-performance instruments and is difficult to apply to on-site determination.

[0004] CN108254284A discloses a method for detecting the content of microplastics in water, comprising the following method steps: a, first, scientific sampling of water is carried out by using direct selection method, large sample method or concentrated sample method, and then the water is loaded into a 500 mL solvent bottle; b, impurities in the sampled water are manually removed; c, silver filter foil is processed and then curved into a cylindrical shape, a glass stirring rod is selected, and the filter foil is completely and seamlessly adhered to the stirring rod by using active carbon powder material magnetic control sputtering; d, a precipitation catalytic reaction solution is configured, and then the configured purification solution is poured into the solvent bottle to be detected for sufficient reaction; e, in step d, filtration is carried out by using a nanometer filter screen, and then microplastics are collected. The method can make microplastics float based on a saturated stable solution, so that the separation of microplastics from the water sample is simple and the detection cost is low, and a new idea is provided for microplastic environmental pollution detection. The method mainly separates microplastic particles by precipitation catalytic reaction and then uses a filter screen, and then the concentration of microplastics is determined by weight determination. However, the method has high requirements for equipment, and the detection process involves precipitation reaction and filtration operation, so the process is long and complex and time-consuming.

[0005] CN107966393A discloses a method for determining the content of microplastics in seawater and the absorption of microplastics by organisms, comprising the following steps: collecting seawater in the environment in a container; placing a fluorescent powder material with adhesion in the container and stirring to fluorescently label the microplastics in the seawater; adding a certain amount of seawater density solution and stirring; removing the static precipitate at the bottom of the container, and then using a fluorescence spectrometer to determine the concentration of fluorescent microplastics in the seawater; placing live marine organisms in the container for a period of time; removing the live marine organisms, and then using a fluorescence spectrometer to determine the concentration of fluorescent microplastics in the seawater in the container; dissecting the live marine organisms, collecting target tissues and organs, and determining the concentration of fluorescent microplastics in each target tissue and organ. The method can accurately detect the content of microplastics in the seawater environment and the absorption of microplastics by organisms, and has positive significance for environmental protection and microplastic management. This method requires fluorescent labeling and fluorescence detection, has high requirements for equipment, and the process is long and complex, which is time-consuming.

[0006] The above methods can accurately detect the degree of water pollution by microplastics, but they require a long detection time, so it is necessary to develop a method for rapidly detecting the concentration of microplastics in water. SUMMARY

[0007] In view of the problems of high equipment requirements, long and complex process, and long time consumption in the existing microplastic detection technology for water, the purpose of the present application is to solve the above problems, and to provide a method for detecting the concentration of microplastics in water based on advanced oxidation and image recognition technology. The detection method is simple and efficient, and can accurately detect the concentration of microplastics in water in a short time.

[0008] To achieve the above object, the application provides a water micro-plastic concentration detection method based on advanced oxidation and image recognition technology, comprising the following steps:

[0009] S1, dyeing

[0010] The micro-plastics or filter membranes are dyed to obtain dyed micro-plastics or filter membranes;

[0011] S2, preparing standard solution

[0012] The micro-plastics in the micro-plastic water suspension prepared in step S1 are weighed to prepare n portions of micro-plastic water suspension standard solutions with different concentrations, and the concentrations of the micro-plastic water suspension standard solutions are distributed in the range of 1-10 mg / L;

[0013] S3, filtering

[0014] The micro-plastics in the micro-plastic water suspension standard solutions prepared in step S2 are filtered onto the filter membranes respectively, and dried to obtain n portions of micro-plastic standard filter membranes;

[0015] S4, picture acquisition

[0016] The micro-plastic standard filter membranes prepared in step S3 are photographed to obtain n portions of micro-plastic standard filter membrane pictures;

[0017] S5, image processing

[0018] The micro-plastic standard filter membrane pictures obtained in step S4 are subjected to background removal processing by using image processing software, and the pictures after background removal are subjected to binarization processing, the area of the micro-plastics is measured by using the image processing software, and the corresponding relationship between the micro-plastic water suspension concentration and the area is obtained;

[0019] S6, detection of the concentration of the sample to be detected

[0020] The sample to be detected is pretreated, and the micro-plastics in the sample are obtained, the obtained micro-plastics are subjected to the same dyeing, filtering, picture acquisition and image processing, and then the concentration of the micro-plastics in the sample to be detected is obtained according to the corresponding relationship between the micro-plastic water suspension concentration and the area.

[0021] The microplastics in the water body can be roughly divided into two categories: light color and dark color. The purpose of dyeing is to increase the contrast between the microplastics and the filter membrane, so that the microplastics are easy to identify. It is worth noting that when the microplastics are white or light-colored, they are subjected to the dyeing process of step S1, and then the microplastics are filtered onto the filter membrane in step S3. If it is difficult to dye the microplastics, the filter membrane is dyed, and the light-colored microplastics are filtered onto the dyed filter membrane. If the microplastics are dark, they can not be dyed and can be directly filtered onto the filter membrane.

[0022] The dyeing of the microplastics or the filter membrane can be carried out by conventional dyeing methods in the art, and there is no special limitation. In the present application, Nile red dyeing agent is used to dye light-colored microplastics or methylene blue dyeing agent is used to dye white filter membrane to dark blue. Specifically, in step S1, the microplastics dyeing step is as follows: dissolve Nile red in an organic solvent to prepare a dyeing working solution, dye the microplastics in the dyeing working solution, heat to a predetermined temperature, filter and dry for storage; the filter membrane dyeing step is as follows: prepare a methylene blue solution, immerse the filter membrane in the methylene blue solution for dyeing, and take out and dry for storage.

[0023] Further, the specific reagent dosage parameters and process parameters such as temperature and time involved in dyeing microplastics with Nile red dyeing agent can refer to conventional parameters in the art, and there is no special limitation. In the present application, the concentration of Nile red is preferably 5-10 mg / L; the organic solvent is selected from acetone, methanol, n-hexane or chloroform. The microplastics can be configured into a suspension with ultrapure water, and then the solution is added to the dyeing working solution to make the microplastics uniformly suspended in the dyeing agent, so that the dyeing is more complete. The volume ratio of Nile red dyeing working solution to microplastics water suspension is preferably 100:2-10.

[0024] Further, in the microplastics dyeing process, it is preferred to heat in water bath at 30-60°C for 30-60 minutes to achieve the effect of strengthening dyeing.

[0025] The filter membrane is selected according to conventional operation in the art. In the dyeing step, an organic filter membrane is used to contact the organic dyeing agent. After dyeing, both the microplastics and the filter membrane are considered stable, so a water-based filter membrane can be used for subsequent filtration. The filter membrane is usually white, and the pore size is preferably 0.22-0.45 μm.

[0026] In step S2 of the method for detecting the concentration of microplastics in water body based on advanced oxidation and image recognition technology, a standard concentration of microplastics water suspension is prepared, and an appropriate amount of sodium hexametaphosphate solution is added to make the microplastics uniformly distributed in the solution to avoid clumping and aggregation. The amount of sodium hexametaphosphate solution can be used according to the conventional proportion in the art.

[0027] In step S3 of the water microplastic concentration detection method based on advanced oxidation and image recognition technology, vacuum filtration is used to filter the microplastic suspension. The filtration device can be a conventional device in the field. In the present application, the filtration device is composed of a receiving bottle, a sand core filter head, a filter membrane, a filter cup and a clamp, which are assembled from bottom to top, and the sand core filter head and the vacuum pump are connected by a silica gel tube.

[0028] In step S4 of the water microplastic concentration detection method based on advanced oxidation and image recognition technology, a high-definition camera is used to take pictures of the microplastic standard filter membrane, and a ruler is added to the pictures of the microplastic standard filter membrane. In the present application, an industrial CCD with a resolution of 3840*2160 is used as the camera, equipped with a 0.35X objective lens with a field of view range (length x width) of 44x35mm; the CCD lens precision parameters are calibrated using software; the distance between the lens and the filter membrane surface is adjusted, the filter membrane is complete during shooting, and the filter membrane is in focus in the whole area; and a ruler is set on the photographed picture using software.

[0029] In step S5 of the water microplastic concentration detection method based on advanced oxidation and image recognition technology, the picture processing software used for removing the picture background, binary processing and microplastic area measurement can be the same software or different software, as long as the corresponding processing effect is achieved. The picture background processing can be performed using conventional image processing software in the field. In the present application, the background is preferably eliminated using Photoshop software. The specific steps of eliminating the background using Photoshop can be performed in a conventional manner. In the present application, the steps are preferably as follows:

[0030] (1) Form a selected area: import the picture, click the "pen tool" in the left menu bar, and draw points along the filter membrane edge at intervals. After closing, select the closed area and press "Ctrl+Enter" to form a selected area;

[0031] (2) Export the picture: click "Select" in the upper menu bar, then click "Invert", and then press "Delete" to delete the background pattern and complete the picture cutting. Finally, export the picture in jpg format.

[0032] The binaryzation processing can adopt an image processing software in the art. In the present application, the binaryzation processing is preferably performed by using Image J software. The microplastic area measurement can be performed by using Imaris, Nano Measurer, Image Pro Plus, Image J and the like, and in the present application, the Image J software is preferably used for microplastic area measurement. The Image J software is simple to operate, can be batched for statistics, and the required data can be exported by simple steps. The operation steps of binaryzation processing and microplastic area measurement by using Image J software are as follows:

[0033] (1) The microplastic standard filter membrane picture after removing the background is imported into the Image J software;

[0034] (2) The scale parameter is set by using the Set Scale tool of the software, the pixel points of the picture itself are matched with the actual length, and the actual length of the picture is obtained;

[0035] (3) The type tool of the software is used to convert the picture type to an 8-bit format gray scale picture;

[0036] (4) The image threshold is adjusted by using the adjust-Threshold tool of the software, and the image is converted into a binary image after application. At this time, the microplastics are black or white, and the background between the microplastics is white or black;

[0037] (5) The Set measurements measurement tool of the software is used to measure the area of the color area of the microplastics in the selected region, that is, the total area S of the microplastics is obtained.

[0038] Further, the background of the picture is removed by using Photoshop software, the binaryzation processing of the picture after removing the background is performed by using Image J software, and the area of the microplastics is measured, so that the corresponding relationship between the microplastic water suspension concentration and the area is obtained. The specific steps are as follows:

[0039] S51, removing the background

[0040] S511, forming a selected area: importing the picture, clicking the "pen tool" in the left menu bar, and forming a closed area by intermittently drawing points along the filter membrane edge. At the same time, the "Ctrl+Enter" key is pressed to form a selected area.

[0041] S512, exporting the picture: clicking "selection" in the upper menu bar, and then clicking "inverted selection", and then pressing the "Delete" key to delete the background pattern to complete the picture cutting. Finally, the jpg format picture is exported.

[0042] S52, binaryzation processing

[0043] S521, setting the scale: open the filter membrane picture in S5 with Image J, use the Straight line tool to trace the scale, click Analyze-Set scale, fill in the length of the scale in Known distance, fill in the unit of length in Unit of Length, check Global, and determine the relationship between pixels and units;

[0044] S522, gray picture: click Image-Type-8bit;

[0045] S523, binarization: click Image-Adjust-Threshold, and select B&W;

[0046] S524, adjusting the threshold: click Set, and set Lower threshold level and Upper threshold level according to the light and dark situation of the picture, ensure that the filter membrane is complete after adjustment and no other shadow area outside the microplastic particles appears, and apply the Lower threshold level and Upper threshold level set this time to each picture;

[0047] S53, measuring the area

[0048] S531, click Analyze-Set measurements, check Area and Limit to threshold; click Analyze-Analyze Particles, set Size and Circularity, check Clear results, Exclude on edges and Summarize; click OK.

[0049] S532, export data: the dialog box popped up is the statistical data, click File-Save As to save.

[0050] The above water microplastic concentration detection method based on advanced oxidation and image recognition technology, in step S5, a standard curve is drawn according to the corresponding relationship between the microplastic water suspension concentration and the area, specifically a scatter plot is drawn by using Origin software, a trend line is added, and the coefficient of determination R 2 is not less than 0.99.

[0051] The microplastic water suspension sample in step S6 is pretreated mainly to obtain microplastics in the water sample. The water sample can be collected from surface water or underground water. The extraction of microplastics in the water body can be performed in a conventional manner in the art. The extraction step is preferably as follows:

[0052] (1) A mesh screen with a pore size of 300-600 pm is used to filter the microplastic water suspension sample to remove large particle impurities.

[0053] (2) A 30% mass concentration H2O2 solution is added to the filtered water sample to form a mixed solution. The mixed solution is placed in a constant temperature shaking box to complete the digestion and purification.

[0054] (3) A saturated sodium chloride solution is added to the digested water sample. After standing for 24-48 h, the supernatant floating with microplastics is removed and filtered to obtain microplastics.

[0055] It is worth noting that if the microplastics contained in the collected sample are white or light-colored, the staining treatment in step S1 can be performed first, and then the microplastics are filtered onto the filter membrane in step S3. Alternatively, the filter membrane can be dyed, and the supernatant floating with microplastics can be directly filtered onto the dyed filter membrane in step S3. If the microplastics are dark-colored, the staining treatment can be omitted, and the supernatant floating with microplastics can be directly filtered onto the filter membrane in step S3.

[0056] The microplastic concentration detection method based on advanced oxidation and image recognition technology provided by the present application has the following beneficial effects:

[0057] (1) The microplastics or filter membranes are dyed and photographed. The image processing software is used to perform background subtraction and binaryzation processing on the pictures. The Image J software is used to process and analyze the pictures. The method can automatically count all microplastics in the selected area and statistically analyze the area. The microplastic concentration can be quickly and accurately calculated. The method effectively improves the efficiency of microplastic content determination in water, and the results are accurate and reliable. The operation is simple and easy to implement.

[0058] (2) The method uses binaryzation to convert the measurement of microplastic area into the statistics of pixel area size. Through the correlation between the pixel area size and the microplastic particle concentration, the concentration of microplastic particles in the solution can be quickly measured. The method can realize rapid batch measurement of microplastic water sample concentration.

[0059] (3) The microplastic sample or filter membrane is subjected to dyeing treatment by the dyeing working solution, the dyeing effect is stable and fast, subsequent image processing software (such as Image J) identification processing is facilitated, the dyed microplastic or filter membrane can be accurately converted into black in the binary graph conversion process, errors can be effectively reduced, and the result is more accurate.

[0060] (4) The background in the image is removed by software, the influence of the image background color on the microplastic area measurement is greatly reduced, the measurement result is more accurate, the present application has the advantages of simple operation, reliable result, accurate objectivity, batch processing of multiple pictures, and greatly improved measurement efficiency of the microplastic content in the water body. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 The device structure for taking filter membrane photos;

[0062] Figure 2 The scale picture is set;

[0063] Figure 3 The filtered photos of Nile red dyed microplastics;

[0064] Figure 4 The filtered photos of methylene blue dyed filter membranes;

[0065] Figure 5 The filter membrane pictures after binaryzation of dyed microplastics;

[0066] Figure 6 The filter membrane pictures after binaryzation of dyed filter membranes;

[0067] Figure 7 The concentration-area standard curve. DETAILED DESCRIPTION

[0068] The technical solutions of the embodiments of the present application will be described clearly and completely in combination with the drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the present application.

[0069] In the following embodiments, the industrial CCD shown in Figure 1 is used to take filter membrane photos, the resolution is 3840*2160, a 0.35X objective lens is equipped, and the field of view range (length x width) is 44*35mm.

[0070] All filter membranes are white microporous filter membranes with a diameter of 25mm and a pore size of 0.22um.

[0071] In the following examples, the original microplastics (PE) are all purchased from the market.

[0072] A self-assembled filtration device is used: from bottom to top, a 250 ml receiving bottle, a sand core filter head suitable for a 25 mm filter membrane, a filter membrane, a 15 ml filter cup, a clamp, and a silica gel tube connecting the sand core filter head and the vacuum pump are assembled.

[0073] Example 1

[0074] In this example, the water sample to be tested is collected from surface water, and PE in the original PE and supernatant after digestion is dyed with Nile red dye. The specific steps of detecting the concentration of microplastics in the water sample by the method for detecting the concentration of microplastics in water provided in this example based on advanced oxidation and image recognition technology are as follows:

[0075] S1, dyeing

[0076] The original microplastics (PE) are white or light-colored. Dyeing them increases the contrast between the microplastics and the filter membrane, making it easier to distinguish them from the filter membrane in subsequent processing. The specific steps are as follows: dissolve Nile red in 0.05 g / L acetone solution, then dilute it 10 times with n-hexane solution to obtain a working solution 1 with a concentration of 5 mg / L. Dissolve the white or light-colored microplastics in ultrapure water to obtain a working solution 2. Mix the working solution 1 and the working solution 2 in a volume ratio of 100:2 to obtain a working solution 3. Heat in a water bath at 30-60°C for 30-60 minutes. After heating, filter the dyed microplastics using a white microporous organic filter membrane, and dry them in an oven at 50-55°C for standby.

[0077] S2, preparation of standard solution

[0078] Weigh 3 portions of 2, 3, 4, 5, and 6 mg of dyed PE from step S1, respectively, and dissolve them in 50 ml of ultrapure water to prepare a standard concentration of microplastic water suspension. This gives us 5 groups of different concentrations of serial microplastic water suspension standard solutions. We can add 0.25 ml of sodium hexametaphosphate solution to each standard solution to make the microplastics evenly distributed in the solution and prevent them from clumping and aggregating.

[0079] S3, filtration

[0080] Filter the serial microplastic water suspension standard solutions prepared in S2 through a white microporous water filter membrane. Rinse the beaker and filter cup walls with ultrapure water during the filtration process until there is no residual microplastic. Place the filter membrane flat on a petri dish for storage and dry it to obtain a series of microplastic standard filters.

[0081] S4, picture collection

[0082] The microplastic standard filter membrane prepared in step S3 is photographed using an industrial CCD to take a filter membrane photo, and a microplastic standard filter membrane picture is obtained as shown in FIG. 2. Figure 3 The specific steps are as follows:

[0083] S41, calibration: the CCD lens precision parameters are calibrated using software;

[0084] S42, photographing: the objective magnification and height are adjusted until the filter membrane surface is clear and complete, and the filter membrane entire area is focused

[0085] S43, setting standard: a ruler is set in the format shown in FIG. 3 using the camera built-in software at the lower right of the picture, and attention is paid to avoiding the filter membrane area. Figure 2

[0086] S5, image processing

[0087] The background of the picture is removed using Photoshop software, the binary processing of the picture after removing the background is performed using Image J software, and the area of the microplastic is measured to obtain the corresponding relationship between the microplastic water suspension concentration and the area. The specific steps are as follows:

[0088] S51, background removal

[0089] S511, forming a selection area: the picture is imported, the "pen tool" in the left menu bar is clicked, the filter membrane edge is intermittently dotted, the closed area is selected after closing, and the "Ctrl+Enter" key is pressed to form a selection area.

[0090] S512, exporting the picture: "select" in the upper menu bar is clicked, and then "inverted selection" is selected, and then the "Delete" key is pressed to delete the background pattern to complete the cutout, and finally the jpg format picture is exported.

[0091] S52, binary processing

[0092] S521, setting the ruler: the filter membrane picture is opened using Image J, the ruler is traced using the Straight straight line tool, Analyze-Set scale is clicked, the length 446 is filled in the Known distance column, the length unit is filled in the Unit of Length column, and Global is checked;

[0093] S522, gray picture: Image-Type-8bit is clicked;

[0094] S523, binary: Image-Adjust-Threshold is clicked, and B&W is selected;

[0095] ​S524, adjust threshold: click Set, set Lower threshold level to 0 and Upper threshold level to 138, apply the Lower threshold level and Upper threshold level set this time to each picture, as shown in Figure 5 ;

[0096] S53, measure area

[0097] S531, click Analyze-Set measurements, check Area and Limit to threshold; click Analyze-Analyze Particles, set Size and Circularity, check Clear results, Exclude on edges and Summarize; click OK.

[0098] S532, export data: the dialog box popped up is the statistical data, click File-Save As to save.

[0099] S54, plot: open origin, set concentration and area size as x-axis and y-axis data respectively, draw scatter plot, add trend line, and make sure R 2 is not less than 0.99, as shown in Figure 7 .

[0100] S6, concentration detection of sample to be tested

[0101] The microplastic water suspension sample to be tested is pretreated and microplastics in the sample liquid are obtained, the obtained microplastics are subjected to the same staining, filtering, picture collection and image processing, and then the microplastic concentration of the microplastic water suspension sample to be tested is obtained according to the corresponding relationship between the microplastic water suspension concentration and the area.

[0102] S61, filtering: shake the collected water sample thoroughly, pass through a 50-mesh (0.300mm) stainless steel screen to remove large particle impurities;

[0103] S62, digestion: transfer the filtered water sample to a conical flask, add 30% H2O2, with a volume ratio of 1:10 to the water sample, and filter the H2O2 with a 0.45um filter membrane before use to avoid bringing other microplastics into the sample, and place the conical flask in a constant temperature shaking incubator for 1 hour;

[0104] S63, density separation: pour the digested water sample into a graduated cylinder, rinse the conical flask with ultrapure water to ensure that there is no residue on the bottle wall, add saturated sodium chloride solution, and stand for 24 hours;

[0105] S64: Aspirate the supernatant: The supernatant floating on the upper part of the measuring cylinder with microplastics is removed and the microplastics are aspirated. After staining in the same way as step S1, the microplastics in the staining solution are directly aspirated onto the filter membrane using the same method as step S3 after the staining is completed;

[0106] S65: Take a picture: Take a picture of the surface of the filter membrane in the same way as step S4;

[0107] S66: Image processing: Remove the background in the same way as step S5, and measure the area of the microplastics;

[0108] S67: Calculate the concentration: The total area of the microplastics on the filter membrane is measured to be 327849.556 μm 2 The concentration calculated according to the standard curve in S7 shows that the concentration of PE in the sample is 0.744 mg.

[0109] Example 2

[0110] In this example, the microplastic water sample to be tested is collected from surface water, and the filter membrane is stained with methylene blue staining agent. The standard curve is drawn according to the standard curve in Example 1, so the standard curve drawing step is omitted. The specific steps of the microplastic concentration detection method based on advanced oxidation and image recognition technology provided in this example for detecting the microplastic concentration of the water sample are as follows:

[0111] S1, Staining

[0112] A methylene blue solution with an original concentration of 500 mg / L is prepared to obtain a staining working solution. The working solution completely covers the white microporous organic filter membrane, and after soaking for 24 hours, the filter membrane is taken out with tweezers and placed in an oven at 50-55°C for drying.

[0113] S6, Detection of sample concentration

[0114] The microplastic water suspension sample to be tested is pretreated and the microplastics in the sample solution are obtained. The obtained microplastics are subjected to the same staining, filtering, picture collection and image processing. According to the corresponding relationship between the microplastic water suspension concentration and the area, the microplastic concentration of the microplastic water suspension sample to be tested is obtained.

[0115] S61, Filtering: Shake the collected water sample thoroughly, and pass it through a 50-mesh (0.300 mm) stainless steel screen to remove large particles;

[0116] S62, Digestion: The filtered water sample is transferred to a conical flask, and 30% H2O2 is added with a volume ratio of 1:10 to the water sample. The H2O2 is aspirated using a 0.45 um filter membrane before use to avoid introducing other microplastics into the sample. The conical flask is placed in a constant temperature shaking incubator for 1 hour;

[0117] S63, density separation: pour the digested water sample into a graduated cylinder, rinse the conical flask with ultrapure water to ensure that there is no residue on the wall of the flask, add saturated sodium chloride solution, and stand for 24 hours;

[0118] S64, suction filtration of supernatant: remove the supernatant with microplastics floating on the upper part of the graduated cylinder and filter through a dyed filter membrane with a pore size of 22 μm and a diameter of 25 mm. Rinse the beaker wall and filter cup wall with ultrapure water during the suction filtration process until there is no residual microplastic. Place the filter membrane flat on a petri dish for storage and dry for later use.

[0119] S65, take a picture of the filter membrane using an industrial CCD. Take a picture of the microplastic standard filter membrane prepared in step S64 using an industrial CCD. Obtain the microplastic standard filter membrane picture as shown in Figure 4 , the specific steps are as follows:

[0120] S651, calibration: use software to calibrate the precision parameters of the CCD lens;

[0121] S652, take a picture: adjust the magnification and height of the objective lens until the surface of the filter membrane is clear and complete. Focus on the entire area of the filter membrane. 653, set the standard: use the camera's built-in software to set the ruler in the lower right corner of the picture, and avoid the filter membrane area.

[0122] S66, image processing, use Photoshop software to remove the background of the picture, and use Image J software to perform binary processing on the picture after removing the background and measure the area of the microplastic to obtain the corresponding relationship between the concentration of the microplastic water suspension and the area. The specific steps are as follows:

[0123] S661, remove the background

[0124] S6611, form a selection area: import the picture, click the "pen tool" in the left menu bar, and draw points along the edge of the filter membrane. After closing, select the closed area and press "Ctrl+Enter" to form a selection area.

[0125] S6612, export the picture: click "Select" in the upper menu bar, then click "Invert", and finally press "Delete" to complete the cutout and export the picture in jpg format.

[0126] S662, binary processing

[0127] S6621, set the ruler: open the filter membrane picture with Image J and use the Straight line tool to trace the ruler. Click Analyze-Set scale, fill in the length 446 in the Known distance column, fill in the length unit as μm in the Unit of Length column, and check Global.

[0128] S6622, Gray picture: click Image-Type-8bit;

[0129] S6623, Binary picture: click Image-Adjust-Threshold, select B&W;

[0130] S6624, Adjust threshold: click Set, set Lower threshold level as 80, set Upper threshold level as 190, apply the Lower threshold level and Upper threshold level to each picture, as shown in Figure 6

[0131] S663, Measure area

[0132] S6631, click Analyze-Set measurements, check Area and Limit to threshold; click Analyze-Analyze Particles, set Size and Circularity, check Clear results, Exclude on edges and Summarize; click OK.

[0133] S6632, export data: the dialog box popped up is the statistical data, click File-Save As to save.

[0134] S67, calculate concentration: the total area of microplastics on the filter membrane is 6071012 μm 2 The concentration is calculated according to the standard curve, and the result shows that the concentration of microplastics in the sample is 13.769 mg.

[0135] Those skilled in the art will appreciate that the embodiments described herein are intended to facilitate the reader's understanding of the principles of the present application, and should be understood as not limiting the scope of protection of the present application to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations according to the technical inspiration disclosed in the present application without departing from the essence of the present application, and these modifications and combinations are still within the scope of protection of the present application.​

Claims

1. A method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology, characterized in that: Includes the following steps: S1, staining Microplastics or filter membranes are dyed to obtain dyed microplastics or filter membranes; S2. Prepare standard solutions Weigh the microplastics described in step S1 to prepare a standard concentration of microplastic aqueous suspension, and obtain n portions of a series of microplastic aqueous suspension standard solutions with different concentrations, wherein the concentrations of the series of microplastic aqueous suspension standard solutions are all distributed within the range of 1 to 10 mg / L. S3, Filtering The microplastics in the series of microplastic aqueous suspension standard solutions obtained in step S2 are filtered onto the filter membrane and dried to obtain n series of microplastic standard filter membranes. S4, Image Acquisition Take photographs of the microplastic standard filter membranes prepared in step S3 to obtain n images of the microplastic standard filter membranes; S5, Image Processing The microplastic standard filter membrane image obtained in step S4 is processed by image processing software to remove the background. The image after background removal is binarized, and then the area of ​​microplastics is measured using image processing software to obtain the correspondence between the concentration and area of ​​microplastic aqueous suspension. S6. Detection of sample concentration The microplastic aqueous suspension sample to be tested was pretreated to obtain the microplastics in the sample liquid. The obtained microplastics were then subjected to the same staining, filtration, image acquisition and image processing. The concentration of microplastics in the microplastic aqueous suspension sample was obtained based on the correspondence between the concentration and area of ​​the microplastic aqueous suspension.

2. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 1, characterized in that: In step S1, the microplastic dyeing step is as follows: Nile red is dissolved in an organic solvent to prepare a dyeing working solution, the microplastics are dyed in the dyeing working solution, heated to a predetermined temperature, filtered and dried for storage; the filter membrane dyeing step is as follows: methylene blue solution is prepared, the filter membrane is immersed and dyed in the methylene blue solution, taken out and dried for storage.

3. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 2, characterized in that: The concentration range of Nile Red is 5–10 mg / L; the organic solvent is selected from acetone, methanol, n-hexane or chloroform.

4. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 2, characterized in that, In the microplastic dyeing process, the product is heated in a water bath at 30–60°C for 30–60 minutes.

5. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 1, characterized in that: The filter membrane has a pore size of 0.22 μm to 0.45 μm.

6. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 1, characterized in that: In step S4, an industrial camera is used to photograph the microplastic standard filter membrane, and a scale is added to the image of the microplastic standard filter membrane.

7. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 1, characterized in that: ImageJ software was used for image binarization and area measurement. The image binarization and area measurement operations were as follows: (1) Import the background-removed image of the microplastic standard filter membrane into ImageJ software; (2) Use the Set Scale tool in the software to set the scale parameters, match the pixels of the image itself with the actual length, and obtain the actual length of the image; (3) Use the software type tool to convert the image type to an 8-bit grayscale image; (4) Use the software adjust-Threshold tool to adjust the image threshold. After application, the image is converted into a binary image. At this time, the microplastics appear black or white, and the background between the microplastics is white or black. (5) Using the Set measurements software, measure the area of ​​the colored area of ​​the microplastics in the selected area to obtain the total area S of the microplastics.

8. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 1, characterized in that: In step S5, a standard curve is plotted based on the relationship between the concentration and area of ​​the microplastic aqueous suspension. A scatter plot is then generated using Origin software, a trend line is added, and the coefficient of determination R is determined. 2 Not less than 0.

99.

9. The method for detecting microplastic concentration in water based on advanced oxidation and image recognition technology according to claim 1, characterized in that: In step S6, the pretreatment steps for the microplastic aqueous suspension sample to be tested are as follows: (1) The microplastic aqueous suspension sample to be tested was filtered with a mesh screen with a pore size of 300μm~600μm to remove large particulate impurities; (2) A 30% H2O2 solution was added to the filtered water sample as a digestion solution to form a mixed solution. The mixed solution was then placed in a constant temperature shaking box to complete the digestion and purification. (3) Add saturated sodium chloride solution to the digested water sample, let it stand for 24-48 hours, remove the supernatant containing microplastics, and filter to obtain microplastics.

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