Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

This protocol describes microplastic sampling and sample analysis on the sea surface. Sea Surface Sampling. Deploy the manta net from the side of the vessel using a spinnaker boom, or A-Frame, using lines and karabiners.

Deploy the manta net out of the wake-zone in order to prevent collecting water affected by turbulence inside the wake-zone. Write down the initial GPS coordinates and initial time in the data sheet provided. Start to move in one straight direction with a speed of approximately 2-3 knots for thirty minutes and begin the time measurement.

After thirty minutes, stop the boat and write down the final GPS coordinates, the length of the route, and the average boat speed into the data sheet provided. Pick up the manta net from the water. Rinse the manta net thoroughly from the outside of the net with sea water using a submersible pump or water from the boat water reservoir.

Rinse in the direction from the manta mouth to the cod end in order to concentrate all particles adhered to the net into the cod end. Note:Never rinse the sample through the opening of the net to prevent contamination. Safely remove the cod end and sieve the sample in the cod end through a 300 micrometer mesh size sieve or less.

Rinse the cod end thoroughly from the outside and pour the rest of the sample through the sieve. Repeat this step until there are no longer any particles inside the cod end. Concentrate all material on the sieve in one part of the sieve.

With the use of a funnel, rinse the sieve into a glass jar or plastic bottle by using 70%ethanol. Close the bottle, wipe it with paper towels and label the lid and outside of the jar with the sample name and date. Microplastic separation from the sea surface samples.

If the sample does not contain any items larger than 25mm and appears to be clean, continue directly with step 3. Otherwise, pour the sample through the sieve and remove all natural or artificial litter objects of a size greater than 5mm from the sample using visual identification and tweezers. Be careful to rinse each removed object carefully with distilled water in order to remove any microplastic litter adhered to it.

Store all natural and artificial litter objects in separate containers. Dry all natural and artificial litter objects in a desiccator, or in the open air but in a closed dish, and weigh them. Identify all litter objects greater than 25mm according to the Master List of Categories of Litter Items.

After removing all larger objects, concentrate all remaining pieces in one part of the sieve using squirt bottles or tap water. Pour the sample into a glass container using a minimum amount of 70%ethanol with the help of a funnel. Take a small amount of the sample and pour it into a glass Petri dish.

Analyze the sample with the use of a stereomicroscope and search for the microplastic particles. When finding each microplastic particle, categorize it into one of the categories according to the categories found in Table 1 and put it in the Petri dish or other glass vials marked with the category name. The Petri dish needs to be closed at all times.

Put the Petri dish under a microscope with measuring equipment and measure the size of each particle. Measure the longest diagonal, except filaments, and note its color. Weigh the microplastic particles of each category separately.

Microplastic particles need to be previously dried. How To Identify Microplastic? No cell structure.

Uneven Uniform thickness. Distinctive colors When separating microplastics from your sample, be conservative and remove more than less. We can still determine the real chemical structure of particles later on.

Chemical characterization. Attenuated Reflection Fourier Transform Infrared Spectroscopy, or ATR FT-IR, is a commonly used technique for the characterization of plastics. Material composition and in some cases, also the extent of degradation, may be determined.

Prior to the analysis the detection system must be cleaned with alcohol and a lint-free cloth, and a special plate for small particles is placed on the sample holder. A background spectrum is then recorded. The sample is then placed on the sample holder and tightened.

When the collection of the sample spectrum is initiated, the detector sends a beam of infrared light through the ATR crystal in such a way that it reflects off the sample in contact with the sample. This reflection penetrates slightly into the sample which allows for the recording of a spectrum. The beam is then collected by a detector as it exits the crystal and a spectrum is finally obtained.

Spectra are characteristic for each material, so it is identified by automated comparison of the obtained spectrum with spectra in a database. ATR FT-IR microscope. An FT-IR microscope combines the function of a microscope and an infrared spectrometer.

This allows recording a spectrum on a very small area suitable for the analysis of microplastics smaller than 1mm. The microscope is most often used in ATR mode, although Transmission and Reflectance modes are possible. The analysis starts by placing the sample on a glass filter.

Other filters can be used, but their polymer nature can interfere with the characterization. The filter with the sample is placed on the automatic scanning table and the joystick is used to locate the sample and record an optical image. On this image, we mark a 20x20 micron area where the sample will be characterized.

A background measurement is then made, followed by the collection of the spectrum on the defined location. An ATR FT-IR spectrum is obtained which is compared with the spectra in the database to identify the composition of the sample.Results. The described protocol provides basic results with microplastic particles categorized in 6 categories according to their visual features.

The first category, and usually the most abundant one, is Fragments. These are rigid, thick, with sharp crooked edges and irregular shapes. They appear in a variety of different colors.

The second category is Films. These also appear in irregular shapes, but in comparison with fragments, they are thin and flexible and usually transparent. The third category is Pellets that usually originate from the Plastic Industry.

Their shapes are irregular and round and are normally larger in size, around 5mm in diameter. They are usually flat on one side and can be in different colors. The fourth category is Granules.

In comparison with Pellets, they appear in a regular round shape and usually in smaller sizes, around 1mm in diameter. They appear in natural colors. The fifth category is Filaments.

They are, next to fragments, the most abundant type of microplastic particles. They can be short to long, with different thicknessess and colors. The last category is Foams.

Often they originate from large particles of styrofoam. They are soft and irregularly shaped, and white to yellow colored. The main acquired result is the number of microplastic particles per sample.

This data can be further normalized per square kilometer. The formula used for normalization is microplastic particles per sample, divided by sampling area, wherein area is calculated with multiplication of sampling distance with manta width. Here you can see one example of normalized data presented in Table 3 and Figure 1.

In addition, particles can be analyzed with image analysis software. The results include maximum length and area of each particle. At the end, the chemical analysis of total, or highest possible number, of particles per sample is recommended.

With the use of Forier Transform Infrared Spectroscopy, a spectrum from selected particles is acquired as seen on this graph. This spectrum is then compared with the spectrums from the software library. The final results show if one particle is plastic or not, and the type of plastic regarding their chemical structure.Conclusion.

By the use of this protocol, accurate and reliable results of microplastic abundance on the sea surface are obtained and can be compared with other existing studies.