1 NOVEL PROTOCOL FOR IDENTIFICATION AND QUANTIFICATION OF

2 MICROPLASTICS IN BIOLOGICAL SAMPLES

3
4 Guilherme Malafaiaa,c,d,e#; Thiarlem Marinho da Luza; Amanda Pereira da Costa Araújoa,b, Mohamed
5 Ahmed Ibrahim Ahmed f, Teresa Rocha-Santosg, Damià Barcelóh,i,
6
a
7 Laboratório de Pesquisas Biológicas, Instituto Federal Goiano, Urutaí, GO, Brazil.
b
8 Programa de Pós-Graduação em Ciências Ambientais, Universidade Federal de Goiás, Goiânia,
9 GO, Brazil.
c
10 Programa de Pós-Graduação em Biotecnologia e Biodiversidade, Universidade Federal de Goiás,
11 Goiânia, GO, Brazil.
d
12 Programa de Pós-Graduação em Ecologia e Conservação de Recursos Naturais, Universidade
13 Federal de Uberlândia, Uberlândia, MG, Brazil.
e
14 Programa de Pós-Graduação em Conservação de Recursos Naturais do Cerrado, Instituto Federal
15 Goiano, Urutaí, GO, Brazil.
f
16 Plant Protection Department, Faculty of Agriculture, Assiut University, Assiut 71526, Egypt.
g
17 Centre for Environmental and Marine Studies (CESAM), Department of Chemistry, University of
18 Aveiro, 3810-193 Aveiro, Portugal.
h
19 Catalan Institute for Water Research (ICRA-CERCA), H2O Building, Scientific and Technological
20 Park of the University of Girona, Emili Grahit 101, 17003, Girona, Spain.
i
21 Water and Soil Quality Research Group, Department of Environmental Chemistry, Institute of
22 Environmental Assessment and Water Research (IDAEA-CSIC), JordiGirona1826, 08034,
23 Barcelona, Spain.
24
25 #Corresponding Author: Biological Research Laboratory, Goiano Federal Institution – Urutaí
26 Campus. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO, Brazil. CEP: 75790-
27 000. Phone: +55 64 3465 1995. E-mail: guilhermeifgoiano@gmail.com
28

29

30 SUPPLEMENTARY MATERIAL

1 1
2
 Figure S1. Representative images of the beakers containing the solutions obtained after washing the membranes with (AC) dimethyl
sulfoxide (DMSO) (100%, 50% and 25%, respectively), (DF) chloroform (100%, 50% and 25%, respectively), (GI) acetone (100%,
50% and 25%, respectively), (JN) ethyl alcohol (100%, 50% and 25%) and (O) purified water (via reverse osmosis).
31

3 2
4
32

5 3
6
 Figure S2. Representative photomicrographs of the solutions obtained after washing the
membranes with (A) purified water (via reverse osmosis); (B-D) dimethyl sulfoxide (DMSO)
(100%, 50% and 25%, respectively); (E-H) chloroform (100%, 50% and 25%); (I-L) acetone
(100%, 50% and 25%) and (M-T) ethyl alcohol (100%, 50% and 25%). The solutions were
analyzed in beakers under stereoscopic microscopy. The images were presented in gray scale to
facilitate the visualization of the microplastics.
33
34

7 4
8
35

9 5
10
 Figure S3. Micro-Raman spectrum recorded from the sample containing PE MPs and
acetonitrile (blue line), compared to standard spectrum for polyethylene (orange line). The
Raman spectrum was collected and identified by correlation with a spectral library
(PublicSpectra, https://publicspectra.com/).
36
37

11 6
12
 Figure S4. (A) Corrected total fluorescence of polyethylene microplastics (CTMPF) subjected or
not to the alkaline digestion process (via KOH to 10%) and (B-C) correlation analysis between
the size of plastic particles and CTMPF calculated through the ImageJ software. In “A”, the bars
represent the mean ± SEM. Distinct lowercase letters indicate significant difference between
groups of particles exposed or not to the digestion solution (i.e.: KOH to 10%) (n=30/each).
38

13 7
14