# Ecotoxicological Assessment of Microplastics in Freshwater Sources--A Review
## 4 Characterization Methods for Microplastics in the Environment
### Microscopy Based Methods  
Visual identification by binocular (polarized) microscope (BM) is used to detect the physical characterization of MPs differing in color, shape, surface texture, or size. MPs come in eight common shapes [74]: fibers, spheres, pellets, lines, sheets, flakes, foam, and fragments--the latter is the most abundant shape. A visual approach is also useful to distinguish MPs from other particles of similar size and shape (e.g., clay or algae), and for particle counting [76]. Larger particles (size in a range from 1 to 5 mm) can be analyzed even by the naked eye, but there is a possibility of missing small and transparent MP particles in the samples. Analysis of smaller particles requires the application of a microscope [35; 77]; [PERSON] et al. [78] reported 1 mm in size as a detection threshold without a microscope. [PERSON] et al. [79] identified different MPs from wastewater by polarized light microscopy (PLM) and provided further confirmation by non-focal Raman microscopy. Polarized light enables the classification of materials with a certain type of anisotropy; under cross polars, they look bright. There is also strong evidence that visual sorting is not a very reliable method. Namely, [PERSON] et al. [80] reported that 70% of visually sorted particles were not identified as plastic when additional FTIR analysis was performed. Method using PLM is limited in MPs identification from non-birefringent polymers such as PVC. Nevertheless, the visual approach is simple, economical, and can be implemented in situ in a short time. However, it does not provide information about chemical composition. In summary, the visual approach can provide information about MPs abundance and even the size, which might be quite helpful when assessing adverse effects caused by MPs presence. However, in practice, more reliable techniques for MPs identification are used (spectroscopy or chromatography) to obtain more relevant information due to the high possibility of misjudgment [15; 76].  
Scanning Electron Microscopy (SEM) is used for the determination of MPs size, shape, surface changes, and roughness, as well as to confirm bacterial biofilm formation on MPs surface. Samples for SEM analysis (i) must be in solid state, non-radioactive, and non-magnetic, (ii) cannot contain moisture, (iii) have to possess stable composition, and (iv) need to be coated at high vacuum [81]. The surfaces of MPs are scanned by an electron beam to obtain clear images, high magnification, and high resolution of MP surfaces [81]. Accordingly, great insights of MP surfaces are obtained, allowing to achieve a more precise identification of MPs. SEM can be coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), producing high-resolution images of particles and providing an elemental analysis of measured objects. Thus, it is possible to distinguish MPs from inorganic particles [82]. Trace elements (like Al, Na, Ca, Mg, or Si) can be identified as well, indicating the presence of additives or particles adsorbed on MPs surface [12; 82]. Accordingly, SEM-EDS may also provide the evidence for adsorbed heavy metals (e.g., Cd, Cr, Pb, Zn, Ni) onto MPs. However, it should be also pointed out that in the case of nonconductive samples such MPs, the covering of a sample surface with a very thin layer of carbon by high vacuum evaporation coating or gold or gold/palladium alloy or platinum thin film using a plasma sputter coater is highly necessary [83]. Hence, the carbon covering may provide interference during elemental analysis. More relevant results on MPs identification can be achieved by combining SEM-EDS with spectroscopic methods such as Raman or Fourier transform-infrared spectroscopy (FTIR). [PERSON] et al. [84] combined three observation techniques to identify MPs: (i) color, size, and morphology were determined by PLM, (ii) SEM-EDS was used for specific structure, morphological properties, and chemical composition of individual MPs, and (iii) Raman spectroscopy has been applied to determine polymer types.