Supporting Information Jian Zhao,1,2 Xuesong Cao,1 Xiaoyu Liu,3 Zhenyu Wang,*,1,2 Chenchen Zhang,1 Jason

C. White,4 and Baoshan Xing*,5

1 Institute of Costal Environmental Pollution Control, and Ministry of Education Key

Laboratory of Marine Environment and Ecology, Ocean University of China, Qingdao

266100, China

2 Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory

for Marine Science and Technology, Qingdao 266071, China

3 Institute of Materials Science and Engineering, Ocean University of China, Qingdao

266100, China

4 Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New

Haven, CT 06504, United States

5 Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts

01003 United States

*Corresponding authors

Tel.: +1 413 545 5212

E-mail address: bx@umass.edu (Dr. Baoshan Xing); wang0628@ouc.edu.cn (Dr. Zhenyu

Wang)

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Table S1. The components of SE and modified SE mediums. Component SE medium Content

(g/L)Modified SE medium

(g/L)NaNO3 0.25 0.125K2HPO4· 3H2O 0.075 0.0375MgSO4· 7H2O 0.075 0.0375CaCl2· 2H2O 0.025 0.0125KH2PO4

NaClFeCl3· 6H2OFe-EDTA a

0.1750.0250.0051 mL

0.08750.01250.00250.1 mL

A5 (Trace mental solution) a 1 mL 0.5 mLa The detailed components of Fe-EDTA and A5 are shown in Table S2.

Table S2. The components of stock solutions of Fe-EDTA and A5 (trace metal solution) in SE mediums.

Components Concentration

Fe-EDTANa2EDTA 10 g/LFeCl3· 6H2O 0.81 g/LHCl 0.05 M

A5 (Trace metal solution)

H3BO3 2.86 g/LMnCl2· 4H2O 1.86 g/LZnSO4· 7H2O 0.22 g/L(NH4)6Mo7O24. 4H2O 0.039 g/L

Table S3. Selected physicochemical properties of CuO NPs.Parameters ValuesSize from manufacturer (nm) <50Size from TEM (nm) 40±10Purity (%) >99.9Surface area (m2/g) a 13.3Hydrodynamic diameter b (nm) 503±35Zeta potential (mV) b −30.7Released Cu2+ (mg/L) c 0.57a: Calculated from N2 adsorption-desorption isotherms; b: Measured in algal medium; c: Measured in modified SE medium after dissolving for 5 days.

S2

0 24 48 72 96 1200

0.1

0.2

0.3

0.4

0.5

0.6

1500 g

35000 g

UF 10KDa

Time (h)

Dis

sove

d C

u2+

(mg/

L)

Figure S1. Dissolution of CuO NPs (10 mg/L) in the modified SE medium as a function of exposure times. CuO NPs were separated using low-speed centrifugation (1500×g, 30 min) and filtration (0.22 μm, twice), high-speed centrifugation (35,000×g, 30 min) and filtration (0.22 μm, twice), and ultrafiltration (UF, 10 kDa) respectively.

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Figure S2. Optical microscope images of algae before (A) and after CuO NPs (10 mg/L) (B) exposure (24 h).

S4

A

B

sEPS bEPS0

0.4

0.8

1.2

1.6

a

a

b

b

ControlCuO NPs treatment

EPS

conc

entra

tion

of a

lga

(pg/

cell)

Figure S3. The effect of CuO NPs (10 mg/L) on the exudation of algal soluble EPS (s-EPS) and algal bound EPS (b-EPS) after exposure. For a given EPS type, different letters represent significant differences between CuO NPs treatment and Control group (i.e., without the exposure of CuO NPs) (p < 0.05, LSD), n = 3.

EPS content determination. For the quantitative analysis of EPS, algal cells were exposed to CuO NPs (10 mg/L). The algal suspension was separated to two fractions including the supernatant and the algal pellet through centrifugation (2,000×g, 15 min). Soluble EPS (s-EPS) in the supernatant was determined using a Total organic carbon (TOC) analyzer (liquid TOC II, Elementar, Germany). The obtained algal pellet was used to extract the bound EPS (B-EPS) by following the procedure of Zhang et al. (2008). The obtained B-EPS was quantified with TOC analyzer.

References:Zhang S, Xu C, Santschi PH. 2008. Chemical composition and 234 Th (IV) binding of extracellular polymeric substances (EPS) produced by the marine diatom Amphora sp. Mar Chem 112: 81-92.

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Figure S4. TEM images of CuO NPs-treated algal cells. This image showed that the thickness of the EPS layer was increased in comparison with the untreated cell in Figure 3A, and the CuO NPs aggregates (red arrow) were present in the layer of EPS.

Figure S5. TEM image of CuO NPs that unattached to the algal cells (A), and the electron diffraction pattern of CuO NPs (indicated with red arrow) (B). In panel A, the distance between alga and CuO NPs is around 1 µm.

Figure S6. TEM image of CuO NPs that is around 100 nm from the algal cell (A), and the electron diffraction pattern of CuO NPs (indicated with red arrow) (B).

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CuO (110)

CuO (002)

CuO (110)

CuO (002)

CuO NPs

CuO NPs

A B

A B

CuO NPs

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 50.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

ck NPs

Time (day)

Alg

al c

ells

(cel

ls/m

L)

Figure S7. The effect of shading by CuO NPs (10 mg/L) on the growth of algae.

Figure S8. Flow cytometry images of PI-dyed algal cells after 0-120 h exposure. The algal cells were analyzed by side scatter and FL2 detectors (564-606 nm) (green region: intact cells; red region: membrane-damaged cells).

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CuO NPs

Cu2+

Control

0 h 2 h 24 h 48 h 72 h 96 h 120 h

Figure S9. Ultrastructure change of algal cells after exposure to CuO NPs. (A) Untreated algal cell; (B) Algal cell treated with Cu2+ (0.6 mg/L); (C-F) Algal cell treated with CuO NPs (10 mg/L). CW: cell wall; N: cell nucleus; Chl: chloroplast; P: pyrenoid; V: vacuole. The place pointed by red arrow on the images indicated possible damage.

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NV

CW

CW

CW

ChlNP

P

P

0.5 µm 0.5 µm0.2 µm

CBA

D E F

0.5 µm0.2 µm 0.2 µm

PN

ChlChl

CW CW CW

Figure S10. HRTEM of Cu2+-treated algal cells. Panel B was enlarged from the selected area of Panel A. Panel C was the HRTEM image of the area that enlarged from Panel B. Panel D was the FFT of the selected area in Panel C. No crystal lattice or diffraction ring was observed from Panels C and D, indicating that CuO NPs were not formed in algal cells.

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A B

CD

Figure S11. HRTEM of extracellular particles in Cu2+-treated algal cells. Panel B was enlarged from the selected area of Panel A. Panel C was the HRTEM image of the area that enlarged from Panel B. Panel D was the FFT of the selected area in Panel C. No crystal lattice or diffraction ring was observed from Panels C and D, indicating that CuO NPs were not extracellularly formed.

0

2

4

6

8

10

12

14

16

a

ab

c

b

b

Control

CuO NPs

Cu2+

Cu

cont

ent (

mg

Cu/

108

Cel

ls)

Figure S12. Contents of Cu bound on cell walls and internalized by algal cells after exposure to CuO NPs for 72 h. Concentrations of CuO NPs and Cu2+ were 10 and 0.6 mg/L, respectively. Different letters represent significant differences among the three treatments (control, CuO NPs, and Cu2+) (p < 0.05, LSD), n = 3.

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Cell wall-bound Cu Internalized Cu

A B

CD

CuO NaN3 Chlor Amilo Cyclo0

1

2

3

4

5

6

7 a

b

b

a aIn

tern

aliz

aed

Cu

(a. u

.)

Figure S13. Effect of NaN3 (3 mM, energy-dependent endocytosis inhibitor), chlorpromazine (Chlor, 3 μg/mL, clathrin-mediated endocytosis inhibitor), amiloride (Amilo, 50 μM, macropinocytosis endocytosis inhibitor), methyl-β-cyclodextrin (Cyclo, 50 μM, lipid-raft-mediated endocytosis inhibitor) on uptake of CuO NPs (10 mg/L) by algal cells after 24 h exposure. Different letters represent significant difference among the five treatments (p < 0.05, LSD), n = 3. The results showed that NaN3 and Chlor significantly reduced the internalized Cu content after CuO NPs exposure, indicating that the uptake of CuO NPs was energy-dependent and clathrin-mediated endocytosis.

Endocytic Inhibition AssayThe algae were cultured in modified SE medium and the initial concentration of algal cells was 1×106 cells/mL. Different endocytosis inhibitors including NaN3 (3 mM, energy-dependent endocytosis inhibitor, ALEXIS Biochemicals, Switzerland), chlorpromazine (3 μg/mL, clathrin-mediated endocytosis inhibitor, Sigma, USA), methyl-β-cyclodextrin (50 μM, lipid-raft-mediated endocytosis inhibitor, ALEXIS Biochemicals, Switzerland), and amiloride (50 μM, macropinocytosis endocytosis inhibitor, ALEXIS Biochemicals, Switzerland) were used for endocytic inhibition tests (Linares et al., 2014; Chen et al., 2015; Pooja et al., 2015; Wang et al., 2011). After exposure to CuO NPs and the above inhibitors for 24 h, the algal cells were collected and washed with 0.02 M EDTA three times to remove the Cu bound onto the cell wall. The algal cells were acid-digested for 2 h, and the intracellular Cu was measured using an inductively coupled plasma mass spectrometry (iCAP Qc, Thermo fisher, USA).

ReferenceLinares J, Matesanz MC, Vila M, Feito MJ, Goncalves G, Vallet-Regí M, Marques

PAAP, Portoles MT. 2014. Endocytic mechanisms of graphene oxide nanosheets in osteoblasts, hepatocytes and macrophages. ACS Appl Mater Interfaces 6:13697-13706.

Chen G, Liu D, He C, Gannett TR, Lin W, Weizmann Y. 2015. Enzymatic synthesis of

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periodic DNA nanoribbons for intracellular pH sensing and gene silencing. J Am Chem Soc 137: 3844-3851.

Pooja S, Pushpanathan M, Gunasekaran P, Rajendhran J. 2015. Endocytosis‒mediated invasion and pathogenicity of streptococcus agalactiae in rat cardiomyocyte (H9C2). PloS one 10: e0139733.

Wang Z, Li J, Zhao J, Xing B. 2011. Toxicity and internalization of CuO nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter. Environ Sci Technol 45: 6032-6040.

20 30 40 50 60 70 802θ (0)

Inte

nsity

(Cou

nts)

(110)

(11-1)

(002)

(111)

(200)

(020)

(20-2)

(11-2)(202)

(11-3) (31-1) (220)(311)(222)

(110)

(200)

(111) (211)

(220) (311)

(222)

Figure S14. X-ray diffraction patterns of CuO NPs. The peaks in the XRD spectrum indicated with red lines belong to CuO NPs. The locations indicated with blue lines are the planes of Cu2O NPs. Clearly, there is no peak on the location of Cu2O planes, showing that no Cu2O was present in original CuO NPs.

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