Articleshttps://doi.org/10.1038/s41893-020-0567-9

Effective uptake of submicrometre plastics by crop plants via a crack-entry modeLianzhen Li1,2, Yongming Luo   1,2,3 ✉, Ruijie Li1, Qian Zhou1, Willie J. G. M. Peijnenburg4,5, Na Yin1, Jie Yang3, Chen Tu1,2 and Yunchao Zhang1

1CAS Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, China. 2Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China. 3CAS Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China. 4Center for Safety of Substances and Products, National Institute of Public Health and the Environment, Bilthoven, The Netherlands. 5Institute of Environmental Sciences (CML), Leiden University, Leiden, The Netherlands. ✉e-mail: ymluo@issas.ac.cn

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NaTuRE SuSTaiNabiLiTY | www.nature.com/natsustain

1

Supplementary Information for

Effective uptake of submicrometer-plastics by crop plants via a crack-

entry mode

Lianzhen Li1,3, Yongming Luo1,2,3*, Ruijie Li1 , Qian Zhou1, Willie J.G.M. Peijnenburg4, 5, Na

Yin1, Jie Yang2, Chen Tu1,3, Yunchao Zhang1

1 CAS Key Laboratory of Coastal Zone Environmental Processes and Ecological Remediation, Yantai Institute

of Coastal Zone Research, Chinese Academy of Sciences;

2 CAS Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese

Academy of Sciences;

3 Center for Ocean Mega-Science, Chinese Academy of Sciences;

4 National Institute of Public Health and the Environment, Center for Safety of Substances and Products, P.O.

Box 1, 3720 BA Bilthoven, The Netherlands;

5 Institute of Environmental Sciences (CML), Leiden University, Leiden, The Netherlands

* Corresponding author, E-mail address: ymluo@issas.ac.cn; ymluo@yic.ac.cn

Tel.: 86-25-86881101; fax: 86-25-86881128.

This file includes the following:

Supplementary Fig. 1 to 37

2

Supplementary Fig. 1 Typical confocal microscopy images of 2.0 μm red (Nile blue labeled) and green (4-

chloro-7-nitro-1,2,3-benzoxadiazole labeled) fluorescently labeled polystyrene (a, b) and polymethyl

methacrylate (c, d) beads. The beads were viewed using a confocal laser scanning microscope (FluoView

FV1000; Olympus, Japan) at excitation/emission wavelengths of 635/680 nm or 488/515 nm for red and green fluorescence respectively. Scale bar, 50 μm.

a b

c d

3

Supplementary Fig. 2 Confocal images of section of lettuce (upper) and wheat (lower) roots, stem and leaf

with various excitation wavelengths.

root

leaf

stem

405 nm 488 nm 559 nm 635 nm

4

a b c

d e f

g h i

j k l

5

Supplementary Fig. 3 Bright-field and confocal images of sections from different segments of wheat (upper)

and lettuce (lower) that were not treated with fluorescently labeled polystyrene (PS) beads. Images a, d, g and j

are the corresponding merged images of images b and c, e and f, h and i, and k and l, respectively. Sections b, e,

h and k were viewed using a confocal laser scanning microscope (FluoView FV1000; Olympus, Japan) at

excitation/emission wavelengths of 635/680 nm or 488/515 nm for roots or stems and leaves, respectively. (a-c)

Transverse section of the mature zone of a root 40 mm from the apex; (d-f) longitudinal section of the mature

zone of a root 40 mm from the apex; (g-i) transverse section of a stem; (j-l) transverse section of a leaf. Scale

bar, 100 μm.

a b c

d e f

g h i

j k l

6

Supplementary Fig. 4 Longitudinal sections of the mature zone of roots of wheat grown in Hoagland solution

with 0.2 μm polystyrene (PS) beads at concentrations of 0, 0.5, 5.0 or 50 mg L-1 for 10 d. PS beads were

labeled with Nile blue. The accumulation of PS beads was analyzed under bright-field conditions and in the

red channel using CLSM. Scale bar, 100 μm.

0 mg L-1

635 nm excitation Bright field Overlay

0.5 mg L-1

5.0 mg L-1

50 mg L-1

7

Supplementary Fig. 5 Accumulation of 2.0 μm fluorescently labeled polystyrene (PS) microbeads.

Longitudinal sections of different primary root segments from wheat (a-d) and lettuce (e-h) treated for 10 d with

a 50 mg L−1 solution of Nile blue-labeled 2.0 μm polystyrene microbeads. The uptake of PS beads was analyzed

in the red channel using CLSM. (a, e) Root tip (up to 2 mm from the apex), (b, e) mature zone (10 mm from the

apex), (c, g) lateral root zone (50 mm from the apex), (d, h) lateral root zone (more than 70 mm from the apex).

These are merged bright-field and confocal images. Scale bar, 100 μm.

a b c d

e f g h

8

Supplementary Fig. 6 Confocal images of transverse and longitudinal sections in the root zone 70 mm from the

root apex of wheat (left) and lettuce (right) treated for 10 d with a 50 mg L−1 solution of 5 μm (a, b), 7 μm (c, d)

or 10 μm (e, f) polystyrene (PS) microbeads labeled with Nile blue. The accumulation of PS beads was analyzed

in the red channel using CLSM. These are merged bright-field and confocal images. Scale bar, 100 μm.

Supplementary Fig. 7 Optical microscopy image (a) and scanning electron microscopy image (b) of 0.2 μm

polystyrene beads trapped by the root cap of a wheat plant.

a b

c d

e f

a b

c d

e f

ba

9

Supplementary Fig. 8 Root tips of lettuce (a) and wheat (b) plants treated with a 50 mg L−1 solution of

fluorescently labeled 0.2 μm polystyrene beads. The dark green tips of the lettuce plants can be clearly observed

(red arrow).

a b

10

Supplementary Fig. 9 Localization of fluorescently labeled polystyrene (PS) beads in a wheat root tip after

exposure to a 50 mg L-1 solution of 0.2 μm Nile blue-labeled PS beads as a function of time and analyzed under

bright-field conditions and in the red channel using CLSM. (a-c) A two-week-old control root tip showing no

signal (i.e., no red fluorescence). (d-f) At 0.5 h, PS beads attached to the root surface in the root cap. (g-i) At 2

h, fluorescently labeled PS beads were found in the internal layers of the columella and root cap and in the apical

meristem. (j-l) At 12 h, areas of bright red fluorescence (i.e., columella and root cap) indicated bead accumulation

in the root cap, lateral root cap, and epidermis. Images c, f, i and l are the corresponding merged images of

images a and b, d and e, g and h, and j and k, respectively.

0.5 h

2 h

0 h

12 h

a b c

d e f

g h i

j k l

11

Supplementary Fig. 10 Localization of fluorescent 0.2 μm polystyrene (PS) beads in a transverse section of a

wheat root after exposure to a 50 mg L-1 solution of PS beads labeled with Nile blue as a function of time and

analyzed under bright-field conditions and in the red channel using CLSM. Thus, any red fluorescence represents

beads, which were found in different tissues of the wheat root after different treatment durations. (a-c) At 0 h

(control plant), the transverse section from the root of a two-week-old control plant exhibited no signal (i.e., no

red fluorescence). (d-f) At 0.5 h, PS beads were found in xylem vessels and in the epidermis. (g-i) At 2 h,

additional fluorescent PS beads were found in the vascular cylinder, and beads had diffused to the endodermis.

(j-l) At 12 h, areas of bright red fluorescence indicated additional bead accumulation in the xylem, epidermis,

and cortical tissues. Images c, f, i and l are the corresponding merged images of images a and b, d and e, g and

h, and j and k, respectively.

0.5 h

2h

0 h

12h

a

d

g

j

b

e

h

k

c

f

i

l

12

Supplementary Fig. 11 Transverse and longitudinal sections of different segments of wheat (upper) and lettuce

(lower) primary roots treated for 10 d with a 50 mg L−1 solution of 0.2 μm polystyrene (PS) beads labeled with

Nile blue. The accumulation of PS beads was analyzed in the red channel using CLSM. (a and b) Root tip (up to

2 mm from the apex), (c and d) mature zone (10 mm from the apex), (e and f) lateral root zone (50 mm from the

apex). These are merged bright-field and confocal images. Scale bar, 100 μm.

a

f

e

d

c

b

a ec

b d f

13

Supplementary Fig. 12 Accumulation of 2.0 μm fluorescently labeled polystyrene (PS) microbeads.

Longitudinal sections of different segments of wheat roots lacking secondaries treated for 5 d with a 50 mg L−1

solution of Nile blue-labeled 2.0 μm polystyrene microbeads. The uptake of PS beads was analyzed under bright-

field conditions and in the red channel using CLSM. (a-c) Root tip (up to 2 mm from the apex), (d-f) mature

zone (20 mm from the apex). Images a and d are the corresponding merged images of images b and c and images

e and f, respectively. Bar = 100 μm.

a

fed

cb

14

Supplementary Fig. 13. Environmental scanning electron microscopy (FEI Quanta 250 FEG ESEM, Eindhoven,

The Netherlands) of the lateral root formation of a 7-day-old wheat seedling (upper) and one-month-old lettuce

plants (lower). (a) Tightly packed epidermal cells. (b, c) Separation of outer tissues as the primordium emerges.

(d) Large gaps between the epidermal cells at the site of lateral root emergence.

a

dc

b

a

dc

b

15

Supplementary Fig. 14 Impact of lateral root emergence on the primary root structure of wheat. As lateral root

initiation occurs deep within the primary root, it is critical that overlying tissues undergo cell separation to allow

primordium emergence. (a) Three different tissues must be traversed: the endodermis, the cortex, and the

epidermis, resulting in a dramatic impact on the primary root structure. (b) The secondary xylem of the lateral

root is connected to the vascular cylinder of the primary root at the point where there is a break in the Casparian

strip, thus providing an opportunity for plastic beads to traverse the membrane to enter the stele.

a b

16

High transpiration rate Low transpiration rate

0 h

0.5 h

12h

24 h

48 h

17

Supplementary Fig. 15 Localization of 0.2 μm (upper) and 2.0 μm (lower) fluorescent polystyrene (PS) beads

in a transverse section of a wheat root at the junction of the primary root and secondary root as a function of time under high transpiration conditions compared to that under low transpiration conditions. The plants were exposed

to a 50 mg L-1 solution of Nile blue-labeled PS beads. The uptake of PS beads was analyzed under bright-field

conditions and in the red channel using CLSM. Bar = 100 μm.

High transpiration rate Low transpiration rate

0 h

0.5 h

12 h

24 h

48 h

18

Supplementary Fig. 16 Presence of 2.0 μm (a, b) and 0.2 μm (d, e) polystyrene (PS) beads in the xylem sap of

lettuce (a, d) and wheat (b, e) after exposure to a 50 mg L-1 solution of PS beads for 10 d. To verify whether PS

beads can move from the roots to the shoots, the xylem sap of a plant exposed to a 50 mg L-1 solution of PS

beads was analyzed using an Olympus-CX31 microscope (Olympus, Tokyo, Japan) at a magnification of 400×

(for 2.0 μm beads) or 1000× (for 0.2 μm beads), and beads were identified based on size, morphology and color.

The size and morphology of the beads observed in the xylem sap was similar to that of pristine PS beads in the

Hoagland culture solution (c, f). Beads were not observed in control plants grown in Hoagland solution without

a PS bead suspension.

a

b

c

d

e

f

19

Supplementary Fig. 17 Stomatal conductance (mol m−2 s−1) and transpiration rate (mmol m−2 s−1) of wheat (a)

and lettuce (b) plants under two different transpiration rate condition. They were measured using an open

photosynthetic system (LI-6400XTR, Li-Cor, Lincoln, NE, USA) at a photon flux density of 1000 μmol m−2 s−1

and ambient CO2 concentration of 400 μmol mol−1. Shown are the value ± SD, n=5.

0.0

0.1

0.2

0.3

0.4

High transpiration

Stomatal conductance

Low transpiration0

2

4

6

Sto

ma

tal

Co

nd

uct

an

ce(m

ol

H2

O m

-2 s

-1)

Transpiration rate

Tra

nsp

ira

tio

n r

ate

(m

mo

l H

2O

m-2

s-1)

a

Low transpiration High transpiration0.00

0.05

0.10

0.15

Sto

ma

tal

con

du

cta

nce

(m

ol

H2O

m-2 s

-1)

Stomatal conductance

0

1

2

3

4

Tra

nsp

ira

tio

n r

ate

(m

mo

l H

2O

m-2

s-1)

Transpiration rate

b

20

a b c

d e f

g h i

j k l

21

a b c

d e f

g h I

j k l

i

22

Supplementary Fig. 18 Accumulation of fluorescently labeled polystyrene (PS) beads in roots of wheat grown

for 20 days with 150 mg kg-1 of 0.2 μm Nile blue-labeled PS beads in sand (upper) or 500 mg kg-1 of 0.2 μm

Nile blue-labeled PS beads in a sandy soil (middle) and 150 mg kg-1 of 2.0 μm Nile blue-labeled PS beads in

sand (lower). The uptake of PS beads was analyzed under bright-field conditions and in the red channel using

CLSM. (a-c) Longitudinal section of a root tip (up to 2 mm from the apex), (d-f) longitudinal section of a mature

zone (10 mm from the apex), (g-i) longitudinal section of a lateral root zone (50 mm from the apex), j-l,

transverse section of a lateral root zone (50 mm from apex). Images c, f, i and l are the corresponding merged

images of images a and b, d and e, g and h, and j andk, respectively. Bar = 100 μm.

a b c

d e f

g h i

j k l

23

a b c

fd e

g h

j

i

k l

24

Supplementary Fig. 19 Accumulation of fluorescently labeled polystyrene (PS) beads in lettuce grown in a

sand matrix for 20 days with 150 mg kg-1 of 0.2 μm (upper) or 2 μm (lower) Nile blue-labeled PS beads. The

uptake of PS beads was analyzed under bright-field conditions and in the red channel using CLSM. (a-c)

Longitudinal section of a root tip (up to 2 mm from the apex), (d-f) longitudinal section of a mature zone (10

mm from the apex), (g-i) longitudinal section of a lateral root zone (50 mm from the apex), j-l, transverse section

of a lateral root zone (50 mm from apex). Images c, f, i and l are the corresponding merged images of images a

and b, d and e, g and h, and j and k, respectively. Bar = 100 μm.

a

d

g

j

b c

e f

h i

k l

25

Supplementary Fig. 20 Scanning electron microscopy (SEM) images of 0.2 μm and 2.0 μm polystyrene bead

localization in the root of a lettuce plant. One-month-old plants were grown in a sand matrix exposed to 150 mg

kg-1 of PS beads for 20 d. (a, b) 0.2 μm beads in the xylem vessels and cortical tissue at the sites of lateral root

emergence. (c, d) 0.2 μm PS beads in the lateral root of the lettuce plant. (e, f) 2.0 μm beads at the junction of

the primary root and lateral root of the lettuce plant. The b, d and f images are enlargements of the area indicated

by the red square in images a, c and e.

a b

c d

e f

26

Supplementary Fig. 21 Presence of 2.0 μm (a, b) and 0.2 μm (d, e) polystyrene (PS) beads in the xylem sap of

lettuce (a, d) and wheat (b, e) after exposure to a 150 mg kg-1 PS beads for 20 d in sand culture. To verify whether

PS beads can move from the roots to the shoots, the xylem sap of a plant was analyzed using an Olympus-CX31

microscope (Olympus, Tokyo, Japan) at a magnification of 400× (for 2.0 μm beads) or 1000× (for 0.2 μm beads),

and beads were identified based on size, morphology and color. The size and morphology of the beads observed

in the xylem sap was similar to that of pristine PS beads in the Hoagland culture solution (c, f). Beads were not

observed in control plants grown in Hoagland solution without a PS bead suspension.

a

b

c

d

e

f

27

Fig. S22 Localization of 0.2 μm fluorescent polymethyl methacrylate (PMMA) beads in transverse and

longitudinal sections of a lettuce root at root apex (A-F; a-f) and at the junction of the primary root and secondary

root (G-L; g-l) after exposure in treated wastewater spiked with 50 mg L-1 PMMA beads under high transpiration

conditions compared to that under low transpiration conditions. The uptake of PMMA beads was analyzed in the

red channel using CLSM. These are merged bright-field and confocal images. Bar = 100 μm.

A B C

D E F

G IH

LKJ

a b c

d e f

g ih

lkj

High transpiration rate Low transpiration rate

28

Supplementary Fig. 23 Localization of 2.0 μm fluorescent polymethyl methacrylate (PMMA) beads in

transverse and longitudinal sections of a lettuce root at root apex (A-F; a-f) and at the junction of the primary

root and secondary root (G-L; g-l) after exposure in treated wastewater spiked with 50 mg L-1 PMMA beads

under high transpiration conditions compared to that under low transpiration conditions. The uptake of PMMA

beads was analyzed in the red channel using CLSM. These are merged bright-field and confocal images. Bar =

100 μm.

Supplementary Fig. 24 Localization of 0.2 μm (A-C; a-c) and 2.0 μm (D-F; d-f) fluorescent polymethyl

methacrylate (PMMA) beads in transverse sections of a lettuce leaf vein after exposure in treated wastewater

spiked with 50 mg L-1 PMMA beads under high transpiration conditions compared to that under low transpiration

conditions. The uptake of PMMA beads was analyzed in the red channel using CLSM. These are merged bright-field and confocal images. Bar = 100 μm.

A B C

D E F

G IH

LKJ

a b c

d e f

g ih

lkj

High transpiration rate Low transpiration rate

A CB

D E F

a b

d

c

e f

High transpiration rate Low transpiration rate

29

Supplementary Fig. 25 Scanning electron microscopy (SEM) images of 0.2 μm polymethyl methacrylate

(PMMA) bead localization in the root and leaf of a lettuce plant. One-month-old plant were exposed treated

wastewater spiked with 50 mg L–1

PMMA beads for 10 d under high transpiration regimes. (a-b) PMMA beads

in the root xylem vessels. (c-d) PMMA beads in the root epidermal tissue. (e-f) PMMA beads in the leaf vein.

(b, d and f) are enlargements of the area marked by the red square in (a, c and e), respectively. The insets show

an enlargement of the area indicated by the red square

a b

c d

e f

30

Supplementary Fig. 26 Scanning electron microscopy (SEM) images of 2.0 μm polymethyl methacrylate

(PMMA) bead localization in the root and leaf of a lettuce plant. One-month-old plant were exposed treated

wastewater spiked with 50 mg L–1 PMMA beads for 10 d under high transpiration regimes. (a-b) PMMA beads

in the root xylem vessels. (c-d) PMMA beads in the root epidermal tissue. (e-f) PMMA beads in the crack

between the epidermal tissue of the main root and the secondary root. (g-h) PMMA beads in the leaf vein. (b, d,

f and h) are enlargements of the area marked by the red square in (a, c, e and g), respectively. The insets show

an enlargement of the area indicated by the red square.

a b

dc

e f

g h

31

Supplementary Fig. 27 Accumulation of 0.2 μm fluorescently labeled polystyrene (PS) beads in a wheat root.

The plants were grown in a sand matrix irrigating with PS bead spiked wastewater at a concentration of 150 mg

kg-1 beads for 20 d. The uptake of PS beads was analyzed under bright-field conditions and in the red channel

using CLSM. (a-c) Longitudinal section of a root tip (up to 2 mm from the apex), (d-f) transverse section of a

mature zone (10 mm from the apex), (g-i) transverse section of a lateral root zone (50 mm from apex). Images

a, d, and g are the corresponding merged images of images b and c, e and f, and h and i, respectively. Bar = 100

μm.

a b c

d fe

g h i

32

Supplementary Fig. 28 Accumulation of 2 μm fluorescently labeled polystyrene (PS) microbeads in a wheat

root. The plants were grown in a sand matrix irrigating with PS bead spiked wastewater at a concentration of

150 mg kg-1 microbeads for 20 d. The uptake of PS beads was analyzed under bright-field conditions and in the

red channel using CLSM. (a-c) Longitudinal section of a root tip (up to 2 mm from the apex), (d-f) transverse

section of a mature zone (10 mm from the apex), (g-i) transverse section of a lateral root zone (50 mm from

apex). Images a, d, and g are the corresponding merged images of images b and c, e and f, and h and i,

respectively. Bar = 100 μm.

a cb

fd e

ihg

33

A B

DC

FE

G H

34

Supplementary Fig. 29 Scanning electron microscopy (SEM) images of 0.2μm and 2.0 μm polystyrene (PS)

bead localization in the root of a wheat plant. The wheat plants were grown in a sand matrix irrigating with

spiked wastewater with a concentration of 150 mg kg-1 of PS beads for 20 d. (a-b) 0.2μm PS beads in the root

xylem vessels. (c-d) 0.2 μm PS beads in the crack between the epidermal tissue of the main root and the

secondary root. (e-f) 2.0 μm PS beads in the root xylem vessels. (g-h) 2.0 μm PS beads in the crack between the

epidermal tissue of the main root and the secondary root. (b, d, f and h) are enlargements of the area marked by

the red square in a, c, e and g, respectively. The insets show an enlargement of the area indicated by the red

square.

a b

dc

fe

g h

35

Supplementary Fig. 30 Presence of 2.0 μm (c) and 0.2 μm (d) polystyrene (PS) beads in the xylem sap of wheat

after grown in a sand matrix irrigating with spiked wastewater with a concentration of 150 mg kg-1 of PS beads

for 20 d. To verify whether PS beads can move from the roots to the shoots, the xylem sap of a plant was analyzed

using an Olympus-CX31 microscope (Olympus, Tokyo, Japan) at a magnification of 400× (for 2.0 μm beads) or

1000× (for 0.2 μm beads), and beads were identified based on size, morphology and color. The size and

morphology of the beads observed in the xylem sap was similar to that of PS beads in the treated wastewater (a,

b). Beads were not observed in control plants grown in sand matrix irrigating with treated wastewater without a

PS bead suspension.

a

b

c

d

36

Supplementary Fig. 31 Transverse and longitudinal sections of different segments of wheat treated for 10 d

with a 50 mg L−1 solution of 0.2 μm fluorescent silica nanoparticles covalently labeled with a kind aminocyanine

dye. The section was analyzed in the red channel using CLSM. (a -c) Root tip, (d and f) lateral root zone (20 mm

from the apex). Images c and f are the corresponding merged images of images a and b, d and e, respectively.

Bar = 100 μm.

a

fe

cb

d

37

Supplementary Fig. 32 Typical scanning electron microscopy (SEM) images showing 0.2 μm (a) and 2.0 μm

(b, c) PS beads deformed in the intercellular space of the vascular tissue of the wheat root.

a

b

c

38

Supplementary Fig. 33 Mechanical properties of 0.2 μm and 2.0 μm polystyrene (PS) and SiO2 beads.

Young’s modulus images of 0.2 μm (a) and 2.0 μm (b) SiO2 beads, in comparison with the modulus of 0.2 μm

(c) and 2.0 μm (d) PS beads obtained at 100 nN using a Dimension Icon AFM system (Bruker, Santa Barbara,

CA, USA) in PeakForce QNM mode.

a b

c d

39

Supplementary Fig. 34 Leakage of the fluorescent dye 4-chloro-7-nitro-1,2,3-benzoxadiazole from PS bead in

lettuce exposure medium measured using centrifugal ultrafiltration technique during the exposure duration of

10 days. The centrifugal filter units (Amiconultra, 3kDamolarmass cutoff) were first pre-equilibrated with the

experimental medium used for algal exposures. Subsequently, 4 mL of each sample were centrifuged at 3700g

for 20 min. To ensure that adsorptive losses of dyes were negligible, both the supernatant and the filtrate were

discarded and the protocol was repeated three times. Following fourth centrifugation cycle, potential leaked

dyes were determined in the filtrate. Shown are the free fluorescence of the PS particles as percentage of total

fluorescence intensity ± SD, n=4.

0 50 100 150 200 250

0

10

20

30

40

50

Time (hours)

Fre

e f

luo

res

cen

ce

dy

e (

%)

0.2 m PS beads in lettuce treated wastewater exposure

2.0 m PS beads in lettuce treated wastewater exposure

40

Supplementary Fig. 35 Typical scanning electron microscopy (SEM) images of 0.2 μm (a) and 2.0 μm (b)

polystyrene (PS) as well as 0.2 μm (c) and 2.0 μm (d) polymethyl methacrylate (PMMA) polymer beads used

in this study.

a

d

b

c

41

Supplementary Fig. 36 Raman spectra of the 0.2 μm (a) and 2.0 μm (b) polystyrene (PS) beads used for

exposure in experiments in comparison with a reference PS (red). The beads were confirmed to be polystyrene

by Raman spectroscopy using a DXR Raman microscope (Thermo Scientific, USA). A 780 nm laser was focused

by a 10×microscope objective for the sample dried on a glass slide.

Supplementary Fig. 37 FTIR spectra of 0.2 μm (a) and 2.0 μm (b) polymethyl methacrylate (PMMA) beads

used for exposure in experiments. The beads were verified by using a Fourier Transform Infrared Spectrometer

(FTIR, Nicolet iS5, Thermo Company, USA) equipped with an iD7 ATR accessory and diamond crystal on a

single reflection plate. The FTIR was operated with a resolution of 4cm and a mid-IR range of 650-4000 cm−1

at a rate of 32 scans per analysis. All spectra were compared with a database (Hummel Polymers and Additives,

Putuzu, Thermo-Fisher) to verify the composition of the plastic particles.

4500 4000 3500 3000 2500 2000 1500 1000 500

0

500

1000

1500

2000

2500

Ram

an

cou

nts

PS beads

wavenumber (cm-1)

reference PS

4500 4000 3500 3000 2500 2000 1500 1000 500

0

200

400

600

800

1000

Ram

an

cou

nts

referencePS

wavenumber (cm-1)

PS beads

a b

500 1000 1500 2000 2500 3000 3500 4000 4500

0.0

0.2

0.4

Ab

so

rban

ce

Wavenumber (cm-1)

500 1000 1500 2000 2500 3000 3500 4000 4500

0.0

0.2

0.4

Ab

so

rban

ce

Wavenumber (cm-1)

a b