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).