## 6 Methodology for MP Analysis
### Sample Pre-Treatment and MP Extraction for Analysis  
During laboratory work and handling with samples, it is necessary to follow the general guidelines for preventing contamination. The recommendation is that all laboratory utensils and equipment are made of glass, metal or ceramic as well as cotton clothing and latex gloves are worn during work [52; 65; 84; 99; 102]. It is necessary to use blanks or negative controls to check for plastic contamination during the preparation process [58] and to better understand the origin of potential bias [103]. To avoid contamination with MP particles from the air [104], the sample manipulation should be kept to a minimum [99]; work surfaces should be regularly cleaned with cotton cloths and ethanol [103], acetone or methanol [102]; sample preparation should be performed in a laminar [52; 65] or fume hood [84] and washed laboratory equipment should be kept covered with aluminium foil [101]. Prior to identifying and quantifying MP particles using analytical techniques, they must be separated from the rest of the sample without damage [58]. When choosing an appropriate extraction technique, it is necessary to take into account the purpose and goals of the analysis. The pre-treatment procedure depends on the type of sample being analyzed (sea water, freshwater, sediment, soil, biota, air, food, cosmetic products, etc.). Water samples can be directly filtered through a net, sieve [41] or glass or metal fiber filter [99; 101]. For clean solid samples, rinsing with distilled water may be sufficient [76]. For more complex samples (e.g., soil, sediment and biota), multiple filtering, sieving, flotation, density separation and chemical or biological digestion of organic matter are used [14; 53; 76; 95; 105]. Soil is a mixture of solids (clay and minerals), liquids and significant amounts of dead organic material [14]. Techniques for MP extraction from soil that have given good results are air flotation, density separation, heating at 130 \({}^{\circ}\)C [36] and oil extraction [14]. MPs can be separated from the larger sediment using an MP separator based on particle size or an electrostatic separator considering non-electrostatic properties [58; 99]. For smaller sediment (<1 mm), density separation (e.g., ZnCL\({}_{2}\) solution in a flotation chamber) and subsequent filtration can be used [99]. The density of MPs is relatively low and mostly varies from 0.9 to 2.3 g cm\({}^{-3}\)[41]. Density separation pre-treatment is based on adding the sample to a prepared solution of a high density, where the MPs float on the surface, while the mineral parts of the soil or sediment remain at the bottom of the solution. MPs of higher density need a denser solution for efficient separation [103]. Different salts (e.g., NaCl, CaCl\({}_{2}\), ZnCl\({}_{2}\), ZnBr\({}_{2}\), NaI, NaBr, potassium formate, sodium tungstate dihydrate and sodium polytungstate) are used to prepare solutions of known density [14; 36; 53; 65; 76; 99; 101] that have different separation efficiency on individual MP types. Separation can last up to several days (72 h), so various options for shortening this time are being investigated [53; 54]. Common laboratory glassware and accessories are most often used for density separation, but some scientists have developed specialized equipment, such as a sediment separator, an overflow column and an elutriation column for sediment samples or a small volume glass separator for ground water samples [103]. An additional step in the MP extraction procedure is the digestion of samples to remove organic matter. Chemical procedures and reagents that can be used for digestion are shown in Table 2. Digestion is more effective at a higher temperature (50-70 \({}^{\circ}\)C) for a longer time (12-96 h), but it is necessary to consider that certain plastic types melt and lose at higher temperatures [76].