To see the version in Japanese (日本語), please click here
Monitoring methods are published in Michida, Y. et al., “Guidelines for harmonizing ocean surface microplastic monitoring methods”, Ministry of the Environment, Japan, 71 pp, 2019
Microplastic damage assessment by digestion methods are published in Alfonso, M. B., K. Takashima, S. Yamaguchi, M. Tanaka, and A. Isobe "Microplastics on plankton samples: multiple digestion techniques assesment based on weight, size, and FTIR spectoroscopy analyses" Marine Pollution Bulletin, 173, 113027, 2021.
To see the image gallery click here
Disclaimer: This method is based on our experience and recommendation. Please, consider that any changes in the steps and/or equipment would lead to different results.
This protocol is to collect all visible plastic pieces between 0.3 mm to 5 cm
Those items that are lost/damaged during lab work cannot be measured
Necessary equipment:
Lab coat, mask, nitrile gloves, paper towels, PTFE rinsing bottle, purified water, 0.3 mm mesh, 2 L plastic jar, funnel, metal clips, and bottle for sample storage.
Safety measures
Because 2% of formalin was added to prevent sample organic matter decomposition, be careful of inhalation and adhesion to the skin, etc. Wear a lab coat, mask, and gloves. When handling the sample stock solution, open it under the fume hood. Store the formalin waste liquid in a 20 L tank and dispose of it properly.
Procedure:
(1) Adjust the 0.3 mm mesh net to a funnel using metal clips, then pour the seawater sample into the 2 L plastic jar. Wash the inner wall of the sample container with a rinsing bottle and purified water several times, and pour it in the same way as the sample
Note: If the sample contains too much natural debris, plankton, fish, etc., work in several steps to avoid sample overflow in the funnel.
(2) Reserve the filtered water in the sample container and keep it refrigerated until further inspection.
(3) With a rinsing bottle, wash the sample content in the mesh to remove any formalin residual, and then pass it into a plastic beaker with purified water. Wash both net sides, so there is nothing left behind. If you do not proceed with plastic sorting immediately, transfer it to a bottle for sample storage and store it in a refrigerator. Wash the inner wall of the plastic beaker again so that there is nothing left.
Note: If you process multiple samples simultaneously, use sample numbers/ID tags for each sample bottle during work and any temporary storage flask to avoid errors.
If organic matter digestion is needed, proceed to step 2 (Organic matter digestion), if not, proceed to step 3 (Plastic sorting).
If the sample contains a large amount of organic matter (algae, plankton, small fish, etc.), you can perform two-step digestion using KOH and Fenton reactions. This method does not affect the size or polymer infrared spectrum in different plastics (see the paper here ).
Necessary equipment and reagents:
Lab coat, mask, nitrile gloves, protective glasses, potassium hydroxide (special grade), purified water, rising bottle, electronic scale, graduated cylinder, medicine metal spoon, 500 ml glass beaker, stirrer bar, stirrer, magnet (for fixing stirrer), stork, reagent bottle, 500 ml glass jar, thermostat or dryer (that can be set at 40 °C), 0.3 mm mesh net, plastic beaker (1 L or 2 L), fixing clips, tweezers, 20 L waste liquid tank (For alkaline liquid waste).
Safety measures:
Work will be done under the fume hood. Wear a lab coat, mask, gloves, and protective goggles.
Because potassium hydroxide is a deleterious substance, refer to the Safety Data information and be careful when handling/storing it.
How to prepare KOH:
*Be sure to perform in this order!
1- Put 450 ml of purified water in a 500 ml glass beaker.
2- Add 50 g of potassium hydroxide to (1) in 2 to 3 portions, and stir with a stirrer bar to dissolve.
3- Transfer to a reagent bottle using a glass funnel and store it.
Procedure:
(1) As in step 1 (Sample opening), filter the seawater sample with a 0.3 mm mesh net. The filtered seawater will be filtered later as described in step 4 (Filtration) below, so please, store it for later. Remove with forceps any natural debris and plastics larger than ~ 2-3 cm with tweezers, washing them with purified water to not lose any microplastic. Discard the natural debris. If there is a plastic of 5 cm or less, measure it, take a picture, and store it for reference.
(2) Drain all the water from the sample on the net, and then transfer all the content into a 500 ml glass jar using a spatula. Wash the sample adhered to the net by spraying a 10% potassium hydroxide solution from a rising bottle into a 1 L plastic beaker and transferring it into the 500 ml glass jar. Rinse into a glass jar with 10% potassium hydroxide solution so that nothing remains in the 1 L plastic beaker.
(3) Add 10% potassium hydroxide solution until the sample (natural debris+plastics) is completely immersed in the solution. As a guideline, use ~100 ml per 10 g of the sample weight.
(4) Close the lid of the glass jar, and put it in an incubator or dryer, setting a temperature of 40 °C for 72 hours.
(5) Continue with step 2.2 (Fenton reaction).
Necessary equipment and reagents:
Lab coat, mask, nitrile gloves, protective glasses, 30% hydrogen peroxide (special grade), iron sulfate heptahydrate (special grade), concentrated sulfuric acid (98%, special grade), purified water, rising bottle, electronic scale, graduated cylinder, medicine spoon, 500 ml glass beaker, thick hole graduated pipette, funnel, reagent bottle, fume hood with harmful gas removal device (← required when using concentrated sulfuric acid), 1 L glass tall beaker, stirrer bar, hot stirrer, magnet (for fixing stirrer), 50 ml graduated cylinder (for hydrogen peroxide, 2 for Fe (II) solution), thermometer (1 for each beaker to process chemicals), aluminum foil, bath container (sample receiver in step 2.1, etc.), 0.2 mm mesh net, plastic beaker (1 L or 2 L), fixing clips, 20 L waste liquid tank (for inorganic waste liquid w/heavy metal), funnel and coffee filter for waste liquid.
Safety measures:
Work will be done in the fume hood. Wear a lab coat, mask, nitrile gloves, and protective goggles.
Because 30% hydrogen peroxide and concentrated sulfuric acid are deleterious substances, refer to the ‘Safety Data information’ and be careful when handling/storing it.
Iron sulfate heptahydrate becomes unstable at temperatures above 30 °C, so be careful in the summer when storing.
After the reaction, if the iron-derived components precipitate, it is advisable to filter the waste liquid with a coffee filter before disposing it in the waste tank.
**The reaction between hydrogen peroxide and ferrous iron is exothermic and will raise the sample temperature. If the temperature is too high, the shape/size of the plastic may change, deteriorate, or even be destroyed, so always control the temperature of the sample liquid and adjust the temperature by using a water bath or adding purified water so that it does not exceed 60 °C.**
How to prepare Fe (2+) solution:
*Be sure to perform in this order!
1- Weigh 7.5 g of iron sulfate heptahydrate and put it into a 500 ml glass beaker.
2- Add 500 ml of purified water to (1) and stir gently with a stirrer to dissolve.
3- Measure 3 ml of concentrated sulfuric acid with a thick-hole measuring pipette and stir it.
4- Transfer the final reagent solution to a reagent bottle using a funnel and store appropriately.
Procedure:
(1) Filter the sample that has completed step 2.1 (Potassium hydroxide (KOH) reaction) with a 0.2 mm mesh net, and spray purified water to thoroughly wash away the potassium hydroxide solution from the sample.
(2) Carefully drain the water from the sample contents on the net, and put ~2 spatula (10 to 20 g) in a 1 L tall beaker.
(3) Spray purified water onto the net from which the sample has been filtered using a rinsing bottle to wash the front and back, receive the liquid in a plastic beaker, and store it for use in a later procedure. ☆). Cover with a watch glass or wrap.
(4) Measure with a separate 50 ml graduated cylinder 40 ml of 30% hydrogen peroxide solution and 40 ml of Fe (II) solution and put in the tall beaker containing the sample of step (2).
(5) Set the thermometer in step (4) and cover it with aluminum foil.
(6) After gently stirring so that the reagents are mixed, heat on a hot plate until the liquid temperature reaches about 50 °C. After adding the reagent, continue the procedure for 20 minutes.
(7) When the liquid temperature reaches about 50 °C, remove it from the hot plate and watch the temperature. The liquid temperature continuously rises with the reaction, so when it reaches about 55 °C, cool the sample in a water bath. If the temperature still rises, add purified water to adjust the temperature to 60 °C or less. If the reaction temperature drops below 60 °C, remove it from the water bath.
(8) Add 40 ml of 30% hydrogen peroxide solution again and repeat steps (5) to (7).
(9) If any organic matter remains after 20 min reaction (see image A below), repeat step (8) up to 3 times (1 hour is recommended to avoid plastic damage).
(*) As shown in image B, the state of the liquid after the reaction differs depending on whether concentrated sulfuric acid is added to the Fe (II) solution. When adding concentrated sulfuric acid, it is possible to filter the transparent reaction solution and take out the plastic on the filter paper if there is almost no undissolved residue of the organic substance.
If there is undissolved residue even after adding sulfuric acid, it is difficult to check the plastic on the filter paper, so continue with the following step (10).
(10) Fix a 0.2 mm mesh net (*) in a plastic beaker (1 L), and fix the stirrer with a magnet to pour the sample into it after chemical treatment. Wash the inner wall of the glass beaker several times and pour it in the same way.
(*) Since the pore diameter of the 0.2 mm mesh is less than 0.3 mm, it is considered that all the plastic pieces of 0.3 mm or more will remain on the net unless they have a very elongated shape. Therefore, the liquid passed through the net will be discarded in the inorganic waste liquid tank.
(11) Use a washing bottle to spray purified water on the sample contents remaining on the net to wash off the sample surface, and then wash the sample contents remaining on the net in a plastic beaker with tap water. Wash the front and back of the net to have nothing left.
(12) The sample obtained in step (11) will be used for step 3 (Plastic sorting). If you do not proceed immediately, transfer it to a sample storage bottle and store it in a refrigerator. For this, wash the inner wall of the plastic beaker thoroughly so that there is nothing left.
Note: When concentrated sulfuric acid is not added to the Fe (II) solution, an iron-derived precipitate is formed in the reaction solution. To remove the precipitate, when discarding the liquid that has passed through the net with a 0.2 mm mesh in step (10) set the coffee filter in the funnel for waste liquid as shown in the image and pour it little by little.
Image A
Image B
Sample A before chemical treatment
Sample A after 2-step digestion (most natural debris was removed)
Necessary equipment:
Stainless steel receiver, sticky notes, 2 tweezers (use one with a thin tip and curved tip), petri dishes.
Procedure:
(1) Put ~ 50 ml of each sample in the sample stainless steel receiver, add purified water up to ~1/2 the height of the receiver, and let the plastic float to the surface. Check both, the surface and the bottom of the recipient.
(2) Use tweezers to obtain plastic particles, wash the surface one by one with water (because another plastic may adhere to the surface of the plastic), and arrange them in a large petri dish.
Notes: Please note that items that are lost or damaged during work cannot be measured. If natural debris still remains and is difficult to look at plastics in step (1), work with only 10 ml each time, or use tweezers to remove any natural debris, but always look for any adhered plastic.
(3) Classify into 3 categories’: pl (plastic), es (Styrofoam), and fb (fishing nets and fishing lines) in different petri dishes. Also, separate them according to similar size ranges.
(4) After collecting all plastic pieces and natural debris that can be visually identified and removed, put the remaining liquid into a receiver container and store it in a refrigerator until the filtration procedure.
Note: paper towels with formalin should be discarded as industrial waste.
Sample receivers (~15 cm diameter) Stainless Steel: AS ONE Part No. 4-5640-03 Plastic: Sanoya Sangyo Co., Ltd. 150-1
Sample petri dishes: pl (left), es (center), and fb (right) (Items > 8 mm are sorted separately)
Polystyrene petri dishes :9 cm and 5.5 cm diameter (AS ONE Part No.: 1-8549-02)
Seawater that passed through the neuston net with a 0.3 mm mesh when the sample was opened, and the liquid that remained in the receiver after the removal work was suction-filtered to check and remove fine plastic pieces
Necessary equipment:
Lab coat, mask, nitrile gloves, rinsing bottle, tap water, 2 L (or 1 L) plastic beaker (with hand), spatula, filter tweezers, small petri dish (for drying filter paper), lupe (15x), small petri dish (new), large petri dish, fine tweezers, 20 L waste liquid tank (inorganic/organic waste liquid tank depending on the presence or absence of chemical treatment). Filter paper (Whatman, Grade 4: Particle retention capacity 20-25 μm, CAT No. 1004-047), Black filter paper (ADVANTEC, No.2, particle retention capacity 5 μm, product name: JIS P 3801), Membrane filter (ADVANTEC, pore diameter 0.8 μm, material: mixed cellulose ester, diameter 47 mm, ITEM No. A080P047A, black),
Filter equipment: Filter holder manifold 5 series (AS ONE), 47mm holder (ADVANTEC, model: KP-47U), air pump (15L / min), suction filtration bottle (500 ml), silicon tube, tube joint, rubber stopper
Safety measures:
Work will be done in the fume hood. Wear a lab coat, mask, and gloves.
Waste liquid after filtration is classified as inorganic waste liquid just in case.
Procedure:
(1) Set the filter paper or membrane filter in the filter equipment.
The filter to be used is selected according to the condition of the sample liquid to be filtered.
(Example 1) If the sample liquid is turbid or has a high viscosity and is expected to be easily clogged, select filter paper (Whatman, Grade 4: particle retention capacity 20-25 μm).
(Example 2) If a large number of microplastics (about 1000 or more in a 2 L sample bottle) are confirmed at the time of removal, and if there are few foreign substances (plankton, algae, etc.) contained and the sample liquid is transparent, black filter paper (example 2) Select ADVANTEC, No. 2, particle retention capacity 5 μm) or membrane filter (ADVANTEC, pore size 0.8 μm). However, the membrane filter is very easy to clog, so starting with about 20 to 30 ml is recommended.
(2) Transfer all the sample liquid to a 2 L (or 1 L) plastic beaker. After stirring with a spatula, dispense an appropriate amount (*) of the sample solution, turn on the pump connected to the suction filter, and perform suction filtration.
(*) The amount of liquid to be filtered for each filter paper is about 30 ml to 100 ml. The amount of liquid to be filtered for each membrane filter is about 20 ml to 50 ml. (It is assumed that it will not clog, but as mentioned above, first try from about 20 to 30 ml each so that the amount of organic matter adhering to one filter paper is not too large and it is not difficult to check the filter paper.
(3) After filtering, if waste liquid accumulates, it is discharged to the waste liquid tank each time. Dispose of the waste liquid properly.
(No chemical treatment → Organic waste liquid tank, Chemical treatment → Inorganic waste liquid tank)
(4) After filtering, arrange the filter papers one by one in a small petri dish using filter tweezers, dry overnight at room temperature, and then search for the plastic piece on the filter paper with a lupe (one in a large petri dish so that the entire filter paper can be seen easily). Put them on each and check them, and take them out with tweezers on a new small petri dish.
(5) Use step (4) filter and take photographs and proceed to FTIR particle analysis.
Filter equipment
Paper filters on small petri dishes for drying overnight.
Visual check with lupe looking for microplastics
Different paper filters used
When an appropriate amount of sample is filtered, the filter paper sample spreads thinly and evenly, making it easy to find the plastic
When an excessive amount of sample is filtered, plankton overlaps, and it is difficult to find the plastic
Necessary equipment:
Stereomicroscope (OLYMPUS SZX7, eyepiece WHSZ10, objective lens DFPL 0.75 × -4), USB camera (AS ONE HDCE-X3N), microscope software (scope image), micrometer, PC, Fourier Transform Infrared Spectrophotometer (hereinafter FTIR), Size measurement software (Image J), ethanol (special grade), paper towel, fine-tipped tweezers, petri dish, parafilm, label (vinyl tape).
Important notes:
First, check and set the magnification of the microscope. After focusing on the micrometer and taking a picture with a microscope, check the number of pixels per 1 mm (required when converting to the actual size) with ImageJ. See section 5 below for how to use ImageJ.
When using multiple microscopes in the same laboratory, adjust the magnification with the zoom handle so that the number of pixels per 1 mm is the same for the second and subsequent microscopes (so that there is no error for each microscope).
After fixing the zoom handle with tape so that it does not move, be sure to reconfirm that the magnification does not change even after fixing.
Using a metal frame (wire width 0.3 mm, spacing between squares 5 mm) is recommended when shooting.
(1) Start scope image.
(2) Align the metal frame with the shooting screen, and place the microplastic samples in the center of the square. Click the "Capture" button, enter the image file name, and save it in the image folder for each sample. Enter pl for plastic pieces, es for styrofoam, and fb for fishing nets and fishing lines. Number them as 001,002,003 ..., respectively.
(Example) Enter "pl001" for the first piece of plastic, "pl002" for the second piece, and similarly, enter "es001" for the first piece of styrofoam and "fb001" for the first piece of fishing net/fishing line.
(3) Perform FTIR analysis every time one shot is taken (see <FTIR analysis> below for FTIR analysis) to distinguish between plastic and other suspended matter.
After the analysis, store the sample in a small petri dish (classified into 3 types, pl, es, and fb. Label the petri dish and fill in so that the contents can be understood). Plastic samples whose shape has changed by FTIR analysis are stored together in a separate petri dish.
(4) Take a picture of the plastic piece taken out from the filter paper in the same way.
(5) Do not shoot a sample too big to fit on the PC screen; store it in a separate petri dish, and measure the size later with a caliper. If it is not possible to visually determine whether it is plastic, check the material with FTIR. Plastic samples whose size has been measured with a caliper are also given a sample name and stored in a petri dish like other samples.
Microscope OLYMPUS SZX7
USB Digital Camera AS ONE Model: HDCE-X3N
Micrometer Co., Ltd. Fuji Kogaku OSM1
Fix the zoom handle if you want to set the number of pixels per 1 mm
Metal frame: put the plastic particle in the center
Always label every petri dish
(1) Start FTIR.
(2) Before placing the sample on the sample holder, clean it using tissue (or cotton swab) with ethanol. When is dry, proceed to make a background measurement. (background measurement is performed about once an hour during analysis. It is also performed each time a negative peak appears or the baseline is disturbed)
(3) After placing the sample on the holder, lower the bar and press the particle. Fine-tune the height of the bar to make the spectrum clearer.
(4) Press the measurement start button. Perform a spectral search and compare with the library data (it is also effective to check the peak position) to determine the type of plastic. Most of the plastics contained in the samples collected on the sea surface are polyethylene, polypropylene, and polystyrene, but other plastics occasionally appear.
Note: If the piece is thick or if any organic matter has adhered to the surface, it will be difficult to determine the polymer, so perform the analysis by cutting with a cutter or wiping the surface.
(5) If the piece is broken during analysis, add x at the end of the image file name, such as "pl001x". If the piece is too small or lost after analysis and cannot be recovered, add xx at the end, such as "pl001xx".
(6) Before analyzing the following sample, clean the sample holder with ethanol again. After that, repeat steps (3) to (6).
Bruker FTIR ALPHA
SHIMADZU FTIR IR Affinity-1S
Notes:
Before measuring the size, check if there are any duplicates or missing numbers in the image file name.
If the number of plastic pieces exceeds 1000, the work will be smoother when measuring the size with imageJ if the image folders are separated from the 1001st.
Procedure:
1) After shooting, measure the size with ImageJ. Start ImageJ, open the image file you want to measure with File → open, and click the fifth straight line tool from the left (Image C). Click and drag the mouse to follow the longest part of the plastic piece (Image D), and measure the length with the M key on the keyboard. In the table displayed by pressing the M key (Image E), the actual size can be calculated based on the number of pixels at the right end (value in the Length column).
(2) Open the following photo with Shift + O and measure the length of all the samples in the same way. Size measurement is performed by pl, fb, and es, respectively.
(3) After measuring the size, select all ImageJ tables (Ctrl + A) and copy (Ctrl + C) to obtain the Excel summary table (☆ Excel summary table is available here * Caution: per 1 mm The number of pixels is 250. It cannot be used with other conversion factors, so change it and use it) Paste it in the specified position (columns O to U). The Excel schedule has tabs corresponding to pl, fb, and es, so be careful where you paste them.
(4) If the sample size is too large to fit on the PC screen, measure the size with a caliper and manually enter it in the Excel summary table (column D).
(5) The number and the total number of pl, fb, and es, by size are displayed in the "Number" tab of the Excel summary table.
Image C: Select the straight line tool indicated by the cursor.
Image D: Select the maximum diameter of the plastic piece. Click one end, drag it to the other end, and click again.
Image E: Measurement results (Results) are displayed. Dividing the length value by the conversion factor (number of pixels per 1 mm) obtained with a micrometer, gives the actual size (unit: mm).