Appendix A

Supporting Figures and Tables

Figure S1. Process for filtering data to calculate the proportion of accurate chemical IDs using each spectroscopic
method.
Figure S2. Comparison of particle types identified using microscopy, and identified using FTIR
microspectroscopy, used to investigate subsampling bias. Here, only results from laboratories that used both
methods in the study are included.
Figure S3. Comparison of particle types identified using microscopy, and identified using Raman
microspectroscopy, used to investigate subsampling bias. Here, only results from laboratories that used both
methods in the study are included.
Table S1. Correlation results between metadata variables and overall accuracy among laboratories using FTIR
and Raman microspectroscopy for the analysis of microplastics.

FTIR microspectroscopy
Variables Correlation SD + SD -
Polymer type 0.52 0.08 0.07
Material type 0.29 0.09 0.09
Particle Morphology 0.27 0.06 0.07
Spectral Collection Mode 0.21 0.05 0.04
Particle Color 0.21 0.07 0.05
Size Fraction 0.2 0.07 0.05
Matching Threshold 0.19 0.05 0.05
Time for Measurement 0.19 0.07 0.03
Accessories Used 0.15 0.06 0.04
Number of Scans 0.15 0.05 0.04
Raman microspectroscopy
Variables Correlation SD + SD -
Material Type 0.63 0.04 0.05
Polymer type 0.51 0.03 0.02
Particle Color 0.47 0.03 0.03
Particle Morphology 0.42 0.04 0.03
Spectral Range 0.3 0.02 0.02
Spectrometer Grating 0.27 0.03 0.04
Spectral Resolution 0.27 0.03 0.02
Time for Measurement 0.24 0.02 0.02
Laser Wavelength 0.23 0.03 0.02
Matching Procedure 0.22 0.02 0.03
Figure S4. Comparison of accuracy of chemical identification using FTIR microspectroscopy among color
categories of spiked particles. Numbers next to each horizontal bar state the number of particles of each
category that were analyzed among laboratories using this method.
Figure S5. Comparison of accuracy of chemical identification using FTIR microspectroscopy among morphology
categories of spiked particles. Numbers next to each horizontal bar state the number of particles of each
category that were analyzed among laboratories using this method.
Figure S6. Comparison of accuracy of chemical identification using FTIR microspectroscopy among spectral
collection modes used by participating laboratories. Numbers next to each horizontal bar state the number of
particles of each category that were analyzed among laboratories using this method.
Figure S7. Analysis time spent per particle (minutes) for each laboratory using FTIR microspectroscopy for
chemical identification. Among the 7 laboratories that achieved greater than 95% accuracy, 71% spent less than
10 minutes analyzing each particle.
Figure S8. Comparison of accuracy of chemical identification using FTIR microspectroscopy among all spiked
polymer types. Categories with one or more chemical ID e.g., PS or PVC or PET are particles within the 1-212 µm
size fractions that visually appeared the same. Numbers next to each horizontal bar state the number of
particles of each category that were analyzed among laboratories using this method.
Figure S9. Comparison of accuracy using FTIR microspectroscopy, compared across the reported minimum
spectral matching threshold (HQI) used by participants. Numbers next to each horizontal bar state the number
of particles of each category that were analyzed among laboratories using this method.
Figure S10. Comparison of accuracy using Raman microspectroscopy, compared across the color categories of
spiked particles. Numbers next to each horizontal bar state the number of particles of each category that were
analyzed among laboratories using this method.
Figure S11. Comparison of accuracy using Raman microspectroscopy, compared across the morphology
categories of spiked particles. Numbers next to each horizontal bar state the number of particles of each
category that were analyzed among laboratories using this method.
Figure S12. Comparison of methods for spectral baseline correction used by participants using Raman
microspectroscopy to analyze particles within the study. Numbers next to each horizontal bar state the number
of particles of each category that were analyzed among laboratories using this method.
Figure S13. Comparison of accuracy of chemical identification using Raman microspectroscopy among spiked
natural material types. Categories with one or more chemical ID e.g., PS or PVC or PET are particles within the 1-
212 µm size fractions that visually appeared the same. Numbers next to each horizontal bar state the number of
particles of each category that were analyzed among laboratories using this method.
Table S1. Method parameters used by laboratories in the analysis of particles using Raman microspectroscopy.

Lab ID Accuracy (%) Excitation laser Spectral Spectral Comment

wavelength resolution Range
C 47 785 nm 1 cm-1 400-4000
CC 86 532 nm 1.4 cm-1 400-4000
H 65 785 nm 1 cm-1 100-3200
Q 93 532 nm 6.7 cm-1 200-5200
QQ 98 532 nm 1 cm-1 250-3500
RR 99 532 nm 3.3 cm-1 59-3500
S 100 633 nm 5.5 cm-1 800-3100
U 100 532 nm 3.5 cm-1 800-3100 > 500 m
only
785 nm 1.7 cm-1
Z 100 473 nm 2.7 cm-1 185-3400
WW 100 785 nm 2 cm-1 746-1835

Standard Operating Procedures

SOP for subsampling particles for chemical ID
 Pick (subsample) the first 30 particles identified from each color/morphology category (e.g. blue fiber,
black fragment) within each size fraction. Store the subsampled particles on a substrate relevant to the
method of chemical identification you will be using, e.g., double-sided tape on projector paper or a glass
slide for particles that will be analyzed via Raman or benchtop ATR-FTIR, a reflective surface for
reflectance FTIR spectroscopy. If less than 30 particles are identified for a certain particle category, pick
as many as you find. After 30 particles have been picked, no longer pick from that category, but continue
to count and characterize all other plastic particles you find.

SOP for chemical identification

1. SOPs for conducting chemical analysis can be found in Appendix H for FTIR spectroscopy and Appendix I
for Raman spectroscopy.
2. All subsampled particles (≤30 of each category per size fraction) must be chemically identified.
3. Particles too small for manual picking and subsampling may be analyzed directly from a filter. The choice
of method for this is yours to make.
 4. Whichever method is used for analysis, submit all details and references used.
5. Record all results in data sheets provided and back up the data electronically.

Metadata collection

Table S3. Metadata required upon results submission for labs using FTIR Spectroscopy

FIELD NAME TYPE REQUIRED DESCRIPTION

LabID Text Yes Anonymized and coded by letter

SampleID Text Yes The ID assigned to the sample
“LabID_Matrix_Number”
SizeFraction Text Yes e.g. 1-20 um, 20-212 um, 212-500 um, >500
um. (see Size Fraction lookup list)
SpectraCollectionMode Text Yes The method used to acquire spectra e.g. ATR,
reflectance or transmission.
Accessories Text No Describe any extra accessories, used, besides
a filter, for sample presentation and analysis
with the instrument.
CrystalType Text Yes Type of crystal used on the FTIR (e.g.
diamond, germanium).
Background Yes Measurement of background spectra e.g.
before spectrum, after spectrum, every 300
minutes.
SpectralRange Integer Yes Wavenumber range of the spectra, reported
in cm-1
SpectralResolution Decimal Yes The maximum number of spectral peaks that
the spectrometer can resolve, reported in cm -
1
/pixel
NumberScans Integer Yes Number of scans performed.
Smoothing Text Yes, if used Pre-processing step to minimize background
noise and interference.
BaselineCorrection Text Yes, if used Pre-processing step to flatten baseline and
minimize signal interference
DataTransformation Text Yes, if used Spectral data processing technique, often
used to normalize signal intensity values.
MatchThreshold Integer No If used, minimum hit quality index (HQI) value
for ‘acceptable’ matches, reported as a
percentage.
SubsamplingMethod Text No Method used for subsampling, e.g. novel or
from literature. Cite all relevant literature.
(Relevant for smallest size fractions where
alternative subsampling methods may be
 used).
Time Integer Yes Time taken to complete FTIR analysis on all
subsampled particles, for each size fraction, in
hours.
Comments Text No Any comments related to the analysis of the
microplastics via FTIR.

Table S4. Metadata required upon results submission for labs using Raman Spectroscopy

FIELD NAME TYPE REQUIRED DESCRIPTION

LabID Text Yes Anonymized and coded by letter

SampleID Text Yes The ID assigned to the sample
“LabID_Matrix_Number”
SizeFraction Text Yes e.g. 1-20 um, 20-212 um, 212-500 um, >500
um. (see Size Fraction lookup list)
LaserPower Integer Yes The laser power of the instrument should be
reported (in mW).
LaserWavelength Integer Yes e.g. 785nm, 532nm. (must be in nanometers)
SpectrometerGrating Integer Yes e.g. 1200nm, 600nm. (must be in
nanometers)
SpectralResolution Decimal Yes The maximum number of spectral peaks that
the instrument can resolve, reported in nm
or cm-1/pixel
SpikeFilter Text Yes, if used Correction of cosmic spikes within the
spectra, ON or OFF.
ICSCorrection Text Yes, if used Relative intensity correction of spectra
setting, ON or OFF.
Smoothing Text Yes, if used Pre-processing step to minimize background
noise and interference
BaselineCorrection Text Yes, if used Pre-processing step to flatten baseline and
minimize signal interference (e.g. line,
polynomial, manual).
DataTransformation Text Yes, if used Spectral data processing technique, often
used to normalize signal intensity values.
SpectralRange Integer Yes Wavenumber range of the spectra, reported
in cm-1.
MatchThreshold Integer Yes, if used Minimum hit quality index (HQI) value for
‘acceptable’ matches, reported as a
percentage.
MatchingProcedure Text Yes Software matching procedure (e.g. ‘ID Expert’
or ‘Search It’ when using Bio Rad ‘KnowItAll’
software). Where the top match was not
 chosen, what procedures were used to
identify the most accurate match.
SubsamplingMethod Text No Method used for subsampling, e.g. novel or
from literature. Cite all relevant literature.
(Relevant for smallest size fractions where
alternative subsampling methods may be
used).
Time Integer Yes Time taken to complete Raman analysis on all
subsampled particles, for each size fraction,
in hours.
Comments Text No Any comments related to the analysis of the
microplastics by the Raman used.

Table S5. Proportion of accurate chemical identification results reported using FTIR spectroscopy for spiked
plastic particles, separated by color, morphology and size fraction. Results were pooled among all samples and
labs and are reported as the proportion of particles for which the chemical identification result matched that of
the spiked particle type in question.
Proportion correct ID’s No. particles identified
Color
Clear 1 292
Blue 1 138
White 0.9 68
Green 0.92 934
Red 0.88 73
Orange 0.66 58
Morphology
Fragment 0.97 825
Sphere 0.93 531
Fiber 0.76 207
Size Fraction (μm)
>500 0.98 688
212-500 0.91 429
20-212 0.87 309
1-20 0.33 3

Table S6. Proportion of accurate chemical identification results reported using Raman spectroscopy for spiked
plastic particles, separated by color, morphology and size fraction. Results were pooled among all samples and
labs and are reported as the proportion of particles for which the chemical identification result matched that of
the spiked particle type in question.
Proportion correct ID’s No. particles identified
Color
Clear 0.99 67
 Blue 0.98 117
White 0.91 1096
Green 0.87 240
Red 0.05 168
Orange 0.8 94
Morphology
Fragment 0.96 803
Sphere 0.89 700
Fiber 0.3 279
Size Fraction (μm)
>500 0.86 614
212-500 0.8 698
20-212 0.8 446
1-20 1 24

Statistical software and packages

R version 4.0.3 (2020-10-10)

Platform: x86_64-w64-mingw32/x64 (64-bit)

Running under: Windows 10 x64 (build 22000)

Matrix products: default

locale:

[1] LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252

[3] LC_MONETARY=English_United States.1252 LC_NUMERIC=C

[5] LC_TIME=English_United States.1252

attached base packages:

[1] stats graphics grDevices datasets utils methods base

other attached packages:

[1] safejoin_0.1.0 readxl_1.3.1 data.table_1.13.6 boot_1.3-25

[5] htmlwidgets_1.5.3 rcompanion_2.4.0 skimr_2.1.3 plotly_4.9.3

[9] renv_0.12.5 Factoshiny_2.3 FactoInvestigate_1.7 shiny_1.5.0

[13] FactoMineR_2.4 ggpubr_0.4.0 ggrepel_0.9.0 forcats_0.5.0

[17] stringr_1.4.0 dplyr_1.0.2 purrr_0.3.4 readr_1.4.0

[21] tidyr_1.1.2 tibble_3.0.4 ggplot2_3.3.3 tidyverse_1.3.0