# Microplastics' Detection in Honey: Development of Protocols in a Simulation
## 3 Results and Discussion
### Contamination Check
#### 3.4.2 Contamination Due to Materials Used  
To confirm that no signal interference was introduced from the materials used in the experiments above, each tool accessory was tested under Raman spectroscopy. To avoid any MP contamination, no plastic equipment was used with a similar Raman fingerprint as the PET powder. The identification of each material was conducted using the \"KnowItAll\" Informatics System Bio-Rad Laboratories database. All Raman signals from the plastic tools used in this project (syringes, filters, etc.) are presented in Supplementary Section in comparison to the PET powder. It was fortunate that the Raman spectrum of PET was differentiated from the Raman signals of each plastic accessory used. Methodological Experiment: Distributions of PET MPs into Different Organic Phases according to Their Dimensions  
To study the MPs' phase of preference, pentane was used as a density separator, to disperse the MPs. As mentioned in Sections 2.4 and 3.1, each of the different phases passed through the CA syringe filters to collect the MPs. PET MPs on CA filters were observed and identified with the optical Raman microscope, and random selected areas were measured with Raman mapping. Raman maps acquired were then analyzed with the ImageJ application to measure the number and dimensions of MPs present in each extraction phase. MP measurements were then compared among all phases, referring to the number of MPs found in each specific selected filter area (610 \(\times\) 500 \(\upmu\)m). It was found that the D_Final had the highest number of MPs found, 121 particles, in the selected filter area compared to the other phases, while MPs' dimensions ranged between 3 \(\upmu\)m and 75 \(\upmu\)m. The second phase with the most MPs found was the D_C1, with 111 particles of PET MPs in the selected filter area. MPs with dimensions between 5-8 \(\upmu\)m were recovered in five out of the seven phases. Smaller dimensions close to the nanometric scale (1-2 \(\upmu\)m) were recovered only from supernatants and the D_C1. However, MPs in the D_YC1 (Figure 2c) were not observed with optical microscopic observation and ImageJ in the whole filter area or with Raman mapping. Also, it was expected that the pentane isolates PET MPs with small dimensions (1-2 \(\upmu\)m) and that gravity acts more on the larger MPs. In more detail, D_Y1, D_Y2, and D_C1 contained the smallest dimensions of MPs, between 1 and 20 \(\upmu\)m. It was remarkable that in D_Y1, D_Y2, and D_C1, Raman spectroscopy was able to detect MPs  
Figure 9: (**a**) PET powder; (**b**) honey sample; and (**c**) Raman spectra for pure honey (black line) and PET powder (red line), used for the experiments. with dimensions up to 1 \(\upmu\)m. The quantity of MPs was not so large, but their dispersion was sufficient to give satisfactory discrimination of the smallest dimensions (1-3 \(\upmu\)m). The D_YC2 had larger dimensions of MPs ranging between 18 and 50 \(\upmu\)m, which coexisted with MPs having smaller dimensions of 2-3 \(\upmu\)m. An interesting result was observed between the two colloid phases. In the D_C1, the MPs' dimensions were the smallest, from 1 \(\upmu\)m up to 30 \(\upmu\)m, which was in contrast with the D_C2, where the MPs' dimensions were between medium and large (18-25 \(\upmu\)m), including aggregates of 100 \(\upmu\)m. Interestingly, these aggregates were larger than what the metallic filter mesh allowed (200 mesh/74 \(\upmu\)m), assuming that they were created during the centrifugation of the colloidal phase. As a result, those aggregates did not permit us to calculate the MPs' dimensions. Sediments and the D_Final included mostly of MPs of large dimensions, which was expected, since gravity pulls down the heavy MPs together with the rest of the solid honey particles such as pollen, pollen grains, wax, and propolis. These particles presented high photoluminescence that covered the Raman signals.