Microplastic Characterization and Quantification

The physical analysis of all microplastic samples was performed in Portugal. Samples were first processed to remove organic material. A chemical digestion using potassium hydroxide (KOH) was used to extract the microplastics from biological matrices, according to (Pagter et al., 2018 (link); Supplementary information, protocol, SI-P1) and analyzed in entirety except for the Yarkon samples from summer which due to the high amount of microplastics present were subsampled six times using a Folsom Plankton Splitter (Wildco® 1831-F10). All plastic particles were observed using a stereoscopic microscope (Leica DFC480) equipped with a camera (Leica Flexacam). The particles were then counted, measured, and separated according to their typology (fragment, filament, pellet, film, microbead, and sponge/foam) and color (black, blue, white, transparent, red, green, multicolor, and others). Examples for typologies and colors are presented in Supplementary Figure S1.
ImageJ software (v1.48, National Institutes of Health) and Leica LAS-X measurement software (Leica Application Suite X-LAS X) were used to measure the microplastics. The plastic particles that were removed for DNA extraction were also included in the total plastic particles. A blank was used (filter exposed to air) throughout the procedure and microplastics found in the blanks that were similar to those found in the samples were removed from the analyzes.
IBM® SPSS® Statistics 25.0 software was used for the statistical analysis of the microplastics. Kolmogorov–Smirnov and Shapiro–Wilk tests were used to test the normality of variables. Nonparametric tests were selected since the data did not follow a normal distribution. To compare the abundance of plastics, between seasons (summer and winter) and locations (Yarkon and Sado) the non-parametric Kruskal-Wallis H (KW) test was used. Mann Whitney U-test was used to perform Pairwise comparisons between two independent groups. A significance level of 0.05 was considered for all statistical analyzes.
Fourier transformed infrared spectroscopy (FTIR Spectrum Two™ ATR Universal spectrometer, Perkin Elmer, United States) in attenuated total reflectance (ATR) was used to collect spectra in the absorbance mode in the region of 4,000 cm-1 to 450 cm-1 with a data range of cm-1. The resolution was fixed at 4 cm-1 (4 scans). Plastic samples were pressed against the diamond crystal with a force of 80–120 N. Spectra were obtained by absorbance (A) and analyzed using PerkinElmer Spectrum IR software (version 10.7.2). The results were compared with several reference spectra from different Perkin Elmer databases and pre-existing spectral libraries (Hummel, 2002 ; Coates, 2006 ; Jung et al., 2018 (link)) to identify the chemical nature of the plastics. The acceptance level was established at >90% similarity match, between the sample spectrum and the reference spectra database. Statistical analyzes were performed using IBM® SPSS® Statistics 25.0 software (Chicago, IL, United States).