# Microplastic Pollution: Chemical Characterization and Impact on Wildlife
## 2 The Chemical and Physical Characterization of Microplastics
### Optical Microscopy  
After separating MPs from the sample of interest, the next step is physical and chemical characterization. Visual inspection is the quickest and most popular way to identify suspected microplastic particles. With the aid of optical microscopes, microplastic particles' size, shape, and color can be characterized. This method has the advantage of being the simplest, lowest cost, and allowing for the largest diversity of microplastic to be detected in terms of size (primary diameter or length), color, and form (as fiber, film, fragment, and spherule). As an illustration, Figure 2A presents visual features of microplastic particles observed through a Carl-Zeiss binocular microscope [38]. This is the very first stage of plastic screening for all samples. Optical microscopy is utilized as a prescreening method to reduce the number of particles that are required to be examined by SEM or alternate methods. By using optical microscopy, it is facile to identify non-plastic particles, such as organic plant or animal remnants and even some shells, and these were therefore omitted from the follow-up investigation [45]. However, a significant caveat of visual sorting is demonstrated in the work of [PERSON] et al., as results show that visual sorting may cause the concentration of microplastics to be underestimated [47]. These authors suggest that a need exists for a second, follow-up analysis to confirm the polymer nature of the particles. Visual identification of microplastics can indeed be very challenging, since the microscopist has little if any basis to optically discern particle composition. One advance which aids the microscopist has been borrowed from biology--staining microplastic particles with dyes. Plastic particles can be dyed to improve their ability to be detected because dyes adsorb plastic particles (see Figure 2B). Dyes produce different colors which allow us to visually detect the type, shape, and size of microplastic. In the study of [PERSON] et al., [48] the authors investigated staining microplastics with several dyes which adhere to plastic surfaces and turn the particles colored, or in some cases fluorescent. Multiple dyes used include Oil Red EGN, Eosin B, Rose Bengal, Hostasol Yellow 3G, and NR (Nile Red). Investigators soaked particles in the dyes for different durations between 5 min and 66 h. Nile Red was chosen as the optimal stain since it has the highest levels of adsorption and fluorescence intensity. When exposed to blue light, the dye will fluoresce and simple photography with an orange filter is used to find fluorescence emission. Fluorescent particles can be recognized and counted using image analysis. Particles that were as small as a few micrometers can be detected using magnified images that can be recorded and tiled/arranged to cover the entire filter area. Interestingly, Nile Red's solvatochromic properties provide the opportunity for plastic categorization based on the surface polarity traits of identified particles. It was established that an incubation period of between 5 min and 66 h and a dye concentration of between 1 and 1000 g mL\({}^{-1}\) were ideal for visibility. A working concentration of 10 g mL\({}^{-1}\) produced a nice balance between background signal, visibility, and speed [46]. In the work of [PERSON] et al., microplastics present within the digestive tract of commercial Kutum fish sampled from the Caspian Sea were considered [49]. To validate the visual assessment, a subsample of 24 microplastic objects were randomly chosen and stained with Nile Red (NR) after being observed and counted under a stereomicroscope. All samples that were seen were fluorescent. Both blue and green fluorescence made NR-stained microfilaments, fragments, and microbeads visible, with the first one having a more pronounced appearance. All MP with a darker background could be distinguished by their green fluorescence. Bright yellow to red fluorescence (under the blue and green filters, respectively,) was sufficiently produced for MP by staining with NR/methanol solution.