MATERIALS AND METHODS
Absolute ethanol (EtOH, ≥99.5%) was obtained from EMD Millipore Cooperation, MA, USA (CAS No: 64-17-5). Methylene Blue (CAS:7220-79-3) and Nile Red (CAS: 7385-67-3) were obtained from ACROS Organics, NJ, USA. A stereo microscope (Meiji Techno, EMZ-8TR, Japan) was used to select a subset of the isolated plastic particles. The selected subset of plastic debris was characterized after quantification using Fourier-transform infrared spectrometry (Thermo Scientific Nicolet iS10 FT-IR). Spectra were matched with polymers with an in-house library and verified using Open Specy.25,26 Scanning electron microscope (NeoScope, JCM-5000) was used to examine the surface morphology (Figure S8).
Method Development. Identification and isolation of smaller MPs (30 µm - 1 mm) within complex environmental matrices like POM, which are similar in size and density to buoyant MP, is difficult and time-consuming. The proposed method that overcomes POM hydrophobicity using a binary solvent mixture to separate MP from environmental samples is shown in Figure S1.
Sample collection and preparation. Beach wrack line debris, which included POM and MP and surface water containing microplastics (quadruplicate, n=24) were collected from three shorelines of Lavaca Bay, Texas on December 15 and 16, 2021. These sites are named Peninsula Park (PL; 28 38’30”N 96 19’23”W), Lighthouse beach (LH; 28 38’21”N 96 36’39”W) and Six Mile (SM; 28 41’37”N 96 39’45”W). The wrack line samples were placed in a Ziplock bag, while water samples were stored in amber glass bottles.
Sample Preparation and Initial Testing.The wrack line samples were air dried in the laboratory for two days before sieving with a 1 mm mesh (Figure S2). Next, the sieved sample was briefly (<10 min) soaked in deionized water to remove sand particles. Within the materials that precipitated, no suspected plastic particles were observed. This was anticipated because most MP in the wrack line is deposited during tidal fluctuations due to their buoyancy. After sand removal, the remaining materials were sieved using 30 µm mesh and air-dried for two days before 0.5 g aliquots were randomly selected and placed in test tubes. The sand and miscellaneous materials that precipitated were examined for MP under a stereo microscope. Next, 10 ml of a binary mixture of varying EtOH:water ratios (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) was added, gently shaken and allowed to soak for two hours. The behavior of POM and MPs varied across the EtOH:water ratios (Figure S4). From EtOH:water (0:10 to 2:8), nearly all material floated. For 3:7 and 4:6, some MP precipitated, while nearly all POM and most MP floated. From 5:5 to 9:1, increasing amounts of MP and POM precipitated into two distinct layers as the ratio approached 9:1 (Figure S4). All ratios from EtOH:water (10:0 to 6:4) resulted in near complete POM buoyancy loss, which is the objective of this step. Additionally, at ratio 5:5, POM also lost buoyancy, but the time required for soaking was greater than the 2 hours needed with higher ratios of EtOH.
The ratio used to validate this method was EtOH:water (8:2), but future studies could use any ratio from 10:0 to 5:5 depending on the desire to minimize EtOH use and sample throughput. Additionally, the chemical composition of POM may vary based on the vegetation source, influencing the EtOH:water ratio or soak time required. Therefore, this crucial binary solvent step must be optimized to meet the needs of each study.
After soaking the sample in the binary solvent mixture, the solution was removed by pipette, carefully avoiding the POM and MP (Figure S4A). Next, 10 mL of DI water was added, and the tubes gently shaken. Over 5 minutes, the MP previously at the bottom of the tube floated to the surface while the POM was initially re-suspended before precipitating to the bottom (Figure S6B-G). This step was repeated twice to maximize MPs separation from the POM. The isolated microplastics were transferred via pipette from the test tube and placed on a filter membrane.
To assess the source of wrack line MP, water samples (100 ml) collected adjacent to each beach site were extracted for MPs using vacuum filtration using membrane filter. The recovered MPs were cleaned up under the microscope and sub-sample characterized using FT-IR. To assess the thoroughness and extraction efficiency of this method, visual screening under a stereo microscope (40x magnification) of both the MP isolated as well as the extracted POM were carried out. Negligible amounts of POM were found within the separated MP material (Figure 1), while equally low amounts of MP were found in the extracted POM. The maximum MP particles found in any of the 12 samples of the POM extracts were 6. When these materials were analyzed using FT-IR, they matched for polyethylene (PE), indicating that an additional washing step would likely account for this minor amount of MP loss, if deemed necessary based on research objectives.
Microplastics Characterization. FT-IR analysis (Figure S7) was performed on 23-25 randomly selected items from the isolated MP (Figure S5). Also, the morphological features of some of the microplastics were captured (Figure S8) using Scanning Electron Microscopy (SEM, NeoScope, JCM-5000).