# Microplastic Pollution: Chemical Characterization and Impact on Wildlife
## 2 The Chemical and Physical Characterization of Microplastics
### Sample Preparation  
The analysis of microplastics present in animal tissues, water samples, or air presents substantial analytical (measurement) challenges since the micron-sized polymeric particles are present at ultratrace mass loadings. The analysis presents the proverbial needle within a haystack problem. In virtually all experiments, sample preparation steps are crucial to the analysis workflow. The general workflow is illustrated in Figure 1 below. Microplastics are usually collected from the samples by sieving or dissection from animal tissue of interest. Next, most samples will be treated with reagents to chemically digest or dissolve the matrix such as KOH, HNO\({}_{3}\), or H\({}_{2}\)O\({}_{2}\) before trying to separate the microplastics by filtration, or gravimetrically by utilizing differences in density [36; 37] with or without centrifugation [38]. Fortunately, the chemical stability of the microplastic particles often allows analysts to dissolve and digest various matrices while leaving the polymeric particle intact [39]. However, care must be taken--especially when using nitric acid. One of the common methods used to digest samples is the use of a solution of concentrated (30-35%) hydrogen peroxide to remove organic matter [38]. In one such work by [PERSON] et al., digested residues of water samples were mixed with 20 mL of 30% hydrogen peroxide at 60\({}^{\circ}\) Celsius for 72 h to digest organic material, then the polymer particles in the digested samples were separated by density overnight using saturated sodium chloride solution (D = 1.2 g/mL) [36]. In another study, microplastics present in air samples were collected and the sample filters were washed before digestion in 35 mL of 30% H\({}_{2}\)O\({}_{2}\) at room temperature for 10 days to eliminate organic materials. The remaining H\({}_{2}\)O\({}_{2}\) solution was then vacuum-filtered through filter paper with a 2-micron pore size to remove any remaining particle matter. Next, 50 mL of a saturated ZnCl\({}_{2}\) solution with a density of 1.6 to 1.8 g/cm\({}^{3}\) was added to each filter, and the mixture was agitated for five minutes at 350 rpm. Then, the samples were allowed to remain still for one full hour before being centrifuged for 3 min at 4000 rpm to collect all microplastics [38]. Sample preparation workflows for animal tissues are quite similar. For instance, when detecting microplastic in mussels, [PERSON] et al. first collected the mussel samples in  
Figure 1: **Top—**General workflow for microplastics analysis. Samples are initially digested to dissolve and remove inorganic and organic materials before gravimetric or filtration-based separation. Finally, chemical analysis of microplastics can be achieved. **Bottom—**Overview of microplastics and nanoplastics separation and analysis methods in simple and complex matrices. Figure reproduced from [35] with permission of the publisher. conical flasks [40]. Each conical flask then received 200 mL of 30% H\({}_{2}\)O\({}_{2}\), the bottles were covered (with foil), and the bottles were then oscillated at 65\({}^{\circ}\) Celsius and 80 rpm for 24 h, followed by 24-48 h at room temperature, depending on the soft tissue's state of digestion. Once the digestion fluid seemed clear and no visible particles were present, digestion was stopped [40]. [PERSON] et al. have carefully studied digestion environments for microplastic analysis and found that H\({}_{2}\)O\({}_{2}\) promoted by the Fenton mechanism are superior choices for organic matter removal from microplastic samples while not affecting the tested polymers [41]. One study employing this approach is from [PERSON] [42]. In this work, sampled water was filtered to remove the solids. To create the solid mass, the substance was left to dry at room temperature for the whole night to break down organic material within a solid bulk. The sample was then mixed with 20 mL of 30% H\({}_{2}\)O\({}_{2}\) and 20 mL of an Fe catalyst solution. The sample was then heated at 75\({}^{\circ}\) Celsius until the bubbles stopped expanding.