# Simply Applicable Method for Microplastics Determination in Environmental Samples
## 3 Materials and Methods
### Applicability of Method to Environmental Samples  
The applicability of the method was tested on four different complex mixtures. The first mixture was a clean polymer blend (PET, PS, PVC, PE, and PP), the second a fine fraction of each polymer, the third was a polymer blend in a complex organic matrix made from microalgae and organic compost, and the fourth was a mixture composed solely of PE and PP MPs in alluvial soil with mineral oil addition. The polymer blend was selected to determine the applicability of method for identification of individual polymer type in the case of method application in real environmental samples. Furthermore, to examine the possible matrix interference during MPs identification with this method, complex organic matrix, containing microalgae and organic compost was spiked with polymer blend. Microalgae and organic compost were selected to illustrate samples with high organic load. Prior to the analysis, mixture was digested to eliminate organic matter, processed as described in Section 3.2. and Section 3.3. and MPs were identified. Besides, the identification of PP and PE polymer type could be also subjected to interferences due to the presence of mineral oil in soil samples, due to their common natural hydrocarbon load. In order to confirm the decision of selecting fragment ions (85 and 142) for PP and PE identification mineral oil was added to alluvial soil and spiked with PE and PP particles. The particle sizes' range chosen to test the method applicability was from 1 to 5 mm. A detailed description of the experiments is in the following two paragraphs. To simulate the process of MP extraction and their subsequent identification from the realistic environmental matrix, domestic compost (1 g) was added to a microalgae culture of 250 mL of _Chloella vulgaris_. This mixture was spiked with an MP polymer blend (PS, PET, PVC, PP, and PE) with total mass of 89.1 mg. The culture of green microalgae _C. vulgaris_ was cultivated beforehand in BBM medium (Phyto technology Laboratories, Rancho Santa Margarita, CA, USA) (prepared according to the manufacturer's instructions) for 14 days at a room temperature (26 \({}^{\circ}\)C), under tubular fluorescent lamps (FLUORA L36W/77, OSRAM, Munich, Germany) with a photoperiod of 16 h/8 h light/dark and was continually stirred at 150 rpm on an orbital shaker (RS-OS 20, Phoenix Instruments, Garbsen, Germany). Domestic compost was dried at 70 \({}^{\circ}\)C for 48 h, before its addition to the microalgae culture and the formation of the complex organic matrix. To separate MPs from the complex microalgae and organic compost matrix, a hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) digestion and a sieving procedure were applied. The samples were digested using 30% H\({}_{2}\)O\({}_{2}\) (1:1, _v/v_; Merck, Kenilworth, NJ, USA) [46], covered with aluminum foil, and continuously stirred for three days on magnetic stirrers (IKA -Werke, GmbH, Staufen, Germany) at 200 rpm. Afterwards, the digested mixture was sieved through a 500 mm stainless steel sieve (Retsch GmbH, Haan, Germany) and dried in an oven at 65 \({}^{\circ}\)C for 24 h. Differences in the mass of undried and dried MPs polymer blend at 65 \({}^{\circ}\)C for 24 h were found to be negligible (\(\Delta=1.6\) mg). Obtained particles after filtration were analyzed and MPs were identified according to the method procedure (see Sections 3.2 and 3.3). The applicability of PE and PP identification, with selected characteristic compounds and fragment ions, was additionally tested in spiked mixtures of alluvial soil, containing 1% of mineral oil, that is on real samples with a high hydrocarbon load. The characteristics of the alluvial soil, which was used have been previously described in [PERSON] et al. [53]. In the alluvial soil (1 g) and mineral oil (11 mg) mixture, a portion of 28 mg of PP and 7 mg of PE were added and analyzed according to the method procedure (see Sections 3.2 and 3.3).