# Microplastics' Detection in Honey: Development of Protocols in a Simulation
## 2 Materials and Methods
### Protocol 1: PET MPs Detection in Deionized Water through Organic Solvents  
An amount of 80 mg of pulverized PET powder was mixed with 20 mL of deionized water in a glass beaker. The suspension was filtered through metallic filters of 40, 100, and 200 mesh. After that, the suspension was equally divided into four large PP plastic Eppendorf tubes of 15 mL with a glass syringe during stirring. In each Eppendorf tube, 2.5 mL of organic solvents were placed to produce four different samples: (S1): toluene; (S2): petroleum ether; (S3): dipentene; and (S4): pentane. Each Eppendorf tube was shaken by hand for 1 min and placed on a stirrer for 5 min at 45 rpm. Afterward, the supernatant (all the organic phase) was taken with a glass pipette and placed in a metallic cup for each Eppendorf tube, respectively [36]. As sample carriers, metallic cups were cleaned and used just once for these experiments, as plastic particle carryover was observed even though cleaning was performed using acetone, ethanol, and sonication. Other materials tested as sample carriers for these experiments are presented in the discussion section. The four metallic cups were put on a heating plate to dry (Figure S1c in the Supplementary Section). Raman mapping was carried out for each sample (Figure S1d in the Supplementary Section). Later, 2.5 mL from the water phase of each sample was taken with a glass pipette and put in four metallic cups for Raman mapping, at the same conditions, after drying for one week in a fume hood. Protocol 2: Methodological Experiment: Distributions of PET MPs into Different Organic Phases according to Their Dimensions  
One scoop (80 mg) of the pulverized PET powder described above was mixed with 2.5 mL of deionized water in a glass beaker. The suspension was filtered through metallic filters of 40 mesh (twice), 60, 100, and 200 mesh, respectively. In another glass beaker, 2 g of honey was mixed with 2.5 mL of deionized water, under stirring at T = 70 \({}^{\circ}\)C for 10 min, and then the solution was left to cool down at room temperature. After that, both mixtures were put together in a 15 mL PP Eppendorf tube. The final mixture was shaken by hand for 1 min and placed on the stirrer for 5 min at 45 rpm. Then, 5 mL of pentane was added, and the solution was shaken by hand for 1 min and placed on the stirrer for 10 min at 55 rpm. After 10 min of recovering, in a fume hood, three phases were created: the supernatant (D_Y1), the colloid phase, and the remaining sample phase. The D_Y1 was taken using a glass pipette and filtered using a 0.45 \(\upmu\)m cellulose acetate (CA) filter. The colloid phase was collected and centrifuged at 1500 r/min for 10 min [37]. Subsequently, after centrifugation, the supernatant (D_YC1) was taken with a glass pipette and passed through another syringe attached to a 0.45 \(\upmu\)m CA filter. The remaining solution from the centrifugation was centrifuged again at 1500 r/min for 10 min and all the solution created except the sediment (D_C1) was taken using a clean glass pipette and passed through another syringe with a 0.45 \(\upmu\)m CA filter. The sediment was diluted in 20 \(\upmu\)L of ethanol and placed on a metallic microscopic slide. All the filtrates were stored in glass vials in a freezer at 4 \({}^{\circ}\)C for 24 h. The next day, the extraction procedure was repeated with the remaining sample (the rest of the initial sample without the supernatant and the colloid phase), by adding 5 mL of pentane. The names of the respective phases from the second extraction were \"D_Y2\" for the new supernatant, \"D_YC2\" for the new supernatant after the first centrifugation, and \"D_C2\" for the new solution after the second centrifugation without the sediment. Lastly, the new remaining sample from the second extraction (D_Final), was taken with a glass pipette and passed through another syringe attached to a 0.45 \(\upmu\)m CA filter. After 48 h, all filters were opened, and Raman mapping was carried out for each sample.