The Phases of WS2 Nanosheets Influence Uptake, Oxidative Stress

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Ecotoxicology and Human Environmental Health 2

The Phases of WS Nanosheets Influence Uptake, Oxidative Stress, Lipid Peroxidation, Membrane Damage and Metabolism in Algae Peng Yuan, Qixing Zhou, and Xiangang Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04444 • Publication Date (Web): 24 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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Environmental Science & Technology

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The Phases of WS2 Nanosheets Influence Uptake, Oxidative Stress, Lipid

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Peroxidation, Membrane Damage and Metabolism in Algae

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Peng Yuan, Qixing Zhou, Xiangang Hu*

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Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

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Education)/Tianjin Key Laboratory of Environmental Remediation and Pollution Control,

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College of Environmental Science and Engineering, Nankai University, Tianjin 300350,

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China

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Corresponding author: Xiangang Hu, [email protected]

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Fax: 0086-022-23507800

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Tel.: 0086-022-23507800

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ABSTRACT:

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Applications of transition metal dichalcogenide (TMDC) nanosheets with different

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phases have attracted much attention in various fields. However, the effects of TMDC

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phases on environmental biology remain largely unknown. In this study, chemically

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exfoliated WS2 nanosheets (Ce-WS2, mainly the 1T phase) and annealed exfoliated WS2

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nanosheets (Ae-WS2, 2H phase) were fabricated to serve as representative TMDC

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nanomaterials. Ce-WS2 showed higher levels of cellular uptake, oxidative stress, lipid 1

ACS Paragon Plus Environment


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peroxidation, membrane damage and inhibition of photosynthesis than Ae-WS2 in

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Chlorella vulgaris. These differences were attributed to the higher electron conductivity

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and higher separation efficiency of electrons and holes in the 1T phase, a typical feature

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of Ce-WS2. Correspondingly, 2H-phase Ae-WS2 exhibited lower

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photooxidation/reduction activity and a lower ability to generate reactive oxygen species

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(mainly •OH) under visible-light irradiation. 1T-phase Ce-WS2 dissolved more readily

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than Ae-WS2 and released more W ions into aqueous environments, but the W ions

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exhibited negligible toxicity. Metabolomic analysis revealed that Ce-WS2 induced more

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obvious alterations in metabolites (e.g., amino acids and fatty acids) and metabolic

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pathways (e.g., starch and sucrose metabolism) than Ae-WS2. These alterations correlated

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with cell membrane damage, oxidative stress and photosynthesis inhibition. The present

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work provides insights into the environmentally friendly design of two-dimensional

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TMDCs.

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KEYWORDS:

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Phytotoxicity, nanotoxicology, oxidative stress, phase, Chlorella vulgaris

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INTRODUCTION

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Two-dimensional (2D) transition metal dichalcogenide (TMDC) nanosheets have shown 2


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tremendous potential for use in various fields (e.g., electronic instruments, human

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healthcare, energy storage and conversion, sensors and environmental protection).1-6

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TMDC materials usually exist in more than one phase, and phase is a critical factor in

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their fabrication and application.7 The structure of TDMCs, consisting of transition metal

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atoms sandwiched between chalcogen atoms, can be either in octahedral coordination (1T

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phase) or in trigonal prismatic coordination (2H phase), creating subtle differences in

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electronic structures and other properties.1, 8 For example, the 1T phase is metallic, in

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sharp contrast to the semiconducting 2H phase.2, 9 The phases of TMDCs affect the

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surface activity of nanomaterials, and their roles in environmental applications and

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behaviors have been studied.2, 10, 11 However, the effects of different phases of TMDC

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nanosheets on environmental biology remain largely unknown, although the relevant

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information is critical for evaluating the ecological risks and design of TMDCs.

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Nanomaterials are exposed to a variety of environments during different parts of

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their life cycles, such as fabrication, use and disposal.12, 13 Given the same elemental

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composition, nanoscale forms with high surface activity usually induce more obvious

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toxicity than bulk forms.14-16 It is well known that the composition, lateral size, thickness

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and surface functionalization of nanomaterials play key roles in nanotoxicology.17-20

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Phases determine the atomic arrangement of the nanomaterial surface and influence the

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interface interactions between nanomaterials and the surrounding microenvironment.21

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However, the effects of nanomaterial phases on organisms in the environment, especially 3



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aquatic organisms, are poorly studied. In the present study, WS2 nanosheets, a

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representative 2D TMDC,21 were chosen to study the roles of phase in biological

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responses to nanomaterials. In the present work, chemically exfoliated WS2 nanosheets

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(Ce-WS2, mainly 1T phase) and annealed exfoliated WS2 nanosheets (Ae-WS2, 2H

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phase) were fabricated and characterized prior to the biological experiments. Chlorella

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vulgaris is widely used as a model organism to test the toxic effects of heavy metals,

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organic pollutants and nanoparticles in aquatic environments.22 In the present study, the

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effects of different phases of WS2 nanosheets on the biological responses of Chlorella

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vulgaris, such as nanomaterial uptake, membrane damage, lipid peroxidation,

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photosynthesis and toxicological mechanisms, were investigated.

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In contrast to genes and proteins, metabolites are the direct output of biochemical

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activity and are easily associated with biological phenomena and cellular biochemistry.23

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However, studies examining only a few metabolites may overlook key information on

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biological responses. Metabolomics with an untargeted analysis strategy can provide

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global views of biological responses and identify the specific molecular mechanisms of

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adverse effects.23, 24 Thus, in this study, the metabolic mechanisms of biological

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responses are investigated using metabolomics. The present work provides insights into

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the roles of nanomaterial phases, in particular for TMDCs, and their environmental risks,

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which could be a useful reference for the evaluation of nanotoxicity and nanomaterial

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design. 4


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MATERIALS AND METHODS

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Preparation and Characterization of WS2 Nanosheets

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Chemically exfoliated WS2 nanosheets (Ce-WS2) prepared by lithium intercalation were

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obtained from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). Annealed

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exfoliated WS2 nanosheets (Ae-WS2) were fabricated by annealing the Ce-WS2 samples

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at 350℃ for 3 h under an argon atmosphere and then cooled to room temperature. To

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reduce nanosheet restacking, WS2 nanosheet suspension was performed in an ultrasonic

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dispersion (150 W for 10 min in an ice-water bath) prior to the characterization and

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toxicology experiments. Detailed characterizations of Ce-WS2 and Ae-WS2 are provided

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in the Supporting Information.

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Algal Culture and Growth Inhibition by WS2 Nanosheets

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Chlorella vulgaris (No. FACHB-8) was obtained from the Freshwater Algae Culture

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Collection at the Institute of Hydrobiology, Wuhan, China. The algae were cultured in an

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illumination incubator (14:10 h light:dark, 1800 lx, 24 ± 1 °C, 80% humidity). For the

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WS2 nanosheets exposure experiment, algae in the logarithmic growth phase were

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collected, washed, and then diluted to an initial concentration of 5 × 105 cells/mL in

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250-mL glass flasks containing 100 mL of blue-green (BG-11) medium. To explore the

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toxic mechanisms of nanomaterial phases and compare them with the reported results for

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other 2D nanomaterials,15, 16, 25 we tested WS2 nanosheets (Ce-WS2 and Ae-WS2) at 0.1, 5



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1, and 10 mg/L by applying them to algal cells. Algae without nanomaterial exposure

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were used as a control. To study the toxic effects of tungsten ions on Chlorella vulgaris,

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we also exposed the algae to different concentrations (up to 50 μM) of tungstate salt

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(Na2WO4). The Chlorella vulgaris was cultured from an initial concentration of 5 × 105

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cells/mL, reached the stationary phase at 96 h and then gradually died afterward.26, 27

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Therefore, the inhibitory effects of WS2 nanosheets and tungstate salt on algae growth

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were evaluated by counting the cell numbers (Nt) using flow cytometry (FCM, Accuri

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C6, Becton-Dickinson, USA) at 24 h, 48 h, 72 h and 96 h. The algal cells in the control

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group (Nc) were also counted. The growth inhibition (%) = (Nc – Nt)/Nc × 100.

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Electron Microscopic Observations

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The surface alteration and cellular ultrastructure of algal cells were observed by scanning

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electron microscopy (SEM) and transmission electron microscopy (TEM), respectively.

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The details are presented in the Supporting Information.

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Analysis of Photosynthesis

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The chlorophyll fluorescence parameters of Chlorella vulgaris were measured every 24 h

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using a pulse-amplitude-modulation fluorimeter (WATER-PAM, Heinz Walz, Effeltrich,

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Germany).28 The maximum photochemical quantum yield of the photosystem (Fv/Fm)

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was selected as an indicator of the photosynthetic capacity of Chlorella vulgaris. Algal

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cells were dark-adapted for 10 min. The minimum fluorescence (F0) was measured using

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a modulated light with low intensity to avoid the reduction of the PSII primary electron 6


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acceptor. The maximum fluorescence (Fm) was induced with a short saturating pulse of

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white light. The Fv/Fm was calculated as (Fm – F0)/Fm.

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Assessment of Oxidative Stress

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The level of intracellular reactive oxygen species (ROS) was detected using a fluorescent

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probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA). The intracellular total reduced

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glutathione (GSH) content was determined using a kit (catalog number: A006-2) from

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Nanjing Jiancheng Bioengineering Institute, China. Abiotic GSH oxidation by WS2

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nanosheets was quantified using Ellman's assay. The details are presented in the

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Supporting Information.

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Lipid Peroxidation of the Cell Membrane

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The lipid peroxidation of the cell membrane was determined by analyzing the content of

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malondialdehyde (MDA). The cell collection, disruption and centrifugation steps were

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the same as for the assessment of oxidative stress. The supernatants were used to analyze

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the MDA content using a kit (Catalog Number: A003-1) from Nanjing Jiancheng

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Bioengineering Institute, China, as directed in the manual.

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Hemolysis Assay

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Red blood cells (RBCs) have no fluid phase or receptor-mediated endocytosis and are

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widely used to study nanomaterial interactions with the cell membrane29. WS2-induced

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membrane damage was further investigated using a hemolysis assay in RBCs. Mouse

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RBCs were diluted to 1 × 108 cells/mL in phosphate-buffered saline (PBS). 7



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Subsequently, 490 μL of RBC suspension was mixed with 10 μL of WS2 nanosheets to

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achieve final concentrations of 0.1-0 mg/L. PBS and Triton X-100 were used as negative

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and positive controls, respectively. The mixtures were gently stirred and incubated for 3 h

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at 37 °C. The samples were centrifuged at 1000 g, and the absorbance of the supernatants

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was measured at 541 nm on a microplate reader (Tecan Spark 10M, Switzerland). The %

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hemolysis = (sample absorbance – negative control absorbance)/(positive control

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absorbance – negative control absorbance) × 100.

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Permeability of Cell Membranes

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Algal cells were collected by centrifugation at 1500g for 5 min after treatment with WS2

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nanosheets or tungstate salt for 96 h and then washed and resuspended in PBS. To assess

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the permeabilization of algae, we used the fluorescent dye propidium iodide (PI) at 1

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μg/L to stain cellular nucleic acids. The samples were incubated for 5 min in the dark at

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room temperature. The fluorescence emission was measured by FCM.

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Comet assay

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After exposure to WS2 nanosheets for 96 h, algal cells were collected. DNA damage was

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assessed using a comet assay kit (Catalog number, KGA240-50, Keygen Biotech, China)

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according to the manufacturer’s specifications. The details are presented in the

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Supporting Information.

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Electron Spin Resonance

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Electron spin resonance (ESR) measurements were carried out at ambient temperature 8


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using a Magnettech MiniScope 400 ESR spectrometer operated at a microwave

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frequency of 9.4 GHz and a magnetic field modulation frequency of 100 kHz. A solar

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simulator consisting of a 300 W xenon lamp filtered to provide simulated sunlight was

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used in the ESR studies. The superoxide and hydroxyl radicals were determined using the

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spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).30 2,2,6,6-Tetramethylpiperidine

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(TEMP) was used to demonstrate the generation of singlet oxygen.30 The presence of free

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electrons was detected using the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl

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(TEMPO).30

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Dissolution of WS2 Nanosheets

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WS2 nanosheets were prepared at 10 mg/L in deionized water and, separately, in BG-11

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medium. The dissolved ions from 10 mg/L WS2 nanosheets were separated by

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centrifuging 10 mL samples at 5000 g in a centrifugal ultrafilter (Amicon Ultra-15 3 kD,

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Millipore, MA) for 30 min after different experimental durations (3 h, 24 h, 48 h, 72 h

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and 96 h). The concentrations of dissolved W ions were directly determined using

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inductively coupled plasma mass spectrometry (ICP-MS, Elan drc-e, PerkinElmer, USA).

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Cellular Uptake

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After 96 h of exposure, algal cells were washed repeatedly with PBS, then collected and

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digested using HNO3/H2O2 (3:1) until no color was observed. After the cell digest was

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filtered through a 0.22-µm water membrane, the concentrations of W ions were measured

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by ICP-MS. Physical and pharmacological inhibitors were used to further investigate the 9



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pathway of nanosheet uptake by algal cells. The cells were precooled at 4 °C for 1 h to

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inhibit energy-dependent uptake, and then WS2 nanosheets were added and incubated

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with the algae for another 1 h. To investigate the specific mechanism of endocytosis, the

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cells were pretreated with methyl-beta-cyclodextrin (MβCD, 20 mM), chlorpromazine

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hydrochloride (CPZ, 100 μM), or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 μM) for 1

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h. Subsequently, WS2 nanosheets were added and coincubated with the algae for 1 h. The

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internalized WS2 nanosheets were quantified using ICP-MS.

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Metabolomic Analysis

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After exposure to WS2 nanosheets for 96 h, the algal cells were collected. Intracellular

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metabolites were extracted from cells by liquid–liquid extraction and subjected to

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nitrogen blow-off, lyophilization, derivatization, and subsequent analysis by gas

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chromatography−mass spectrometry (GC-MS, 6890N/5973, Agilent, USA).22 The details

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are presented in the Supporting Information.

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Statistical Analysis

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All treatments included three replicates, and the results are presented as the mean ±

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standard deviation. The data were analyzed using one-way analysis of variance

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(ANOVA) and compared using Tukey’s test with SPSS 22.0 software. A p value less

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than 0.05 was considered statistically significant. The metabolomic data were imported

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into SPSS 22.0 software for univariate analysis. Metabolic pathway analysis was

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performed using MetaboAnalyst version 4.0 (http://www.metaboanalyst.ca), and 10


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SIMCA-P 13.0 software was used for multivariate analysis of metabolites.

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RESULTS AND DISCUSSION

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Characterization of Ce-WS2 and Ae-WS2

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As shown in Figure S1, the lateral sizes of Ce-WS2 ranged from 77 nm to 133 nm, and

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the peak of the lateral size distribution was located at 94 nm. Similarly, the lateral sizes of

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Ae-WS2 ranged from 51 nm to 146 nm, and the peak of the lateral size distribution was

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located at 98 nm. The average thicknesses of Ce-WS2 and Ae-WS2 were 1.4 nm and 1.3

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nm, respectively, which were very close to the height of monolayer WS2 2.

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The alteration of hydrodynamic diameters was used to analyze nanomaterial

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aggregation.11, 31 As shown in Figure S2, the hydrodynamic diameters increased by

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107-128%, suggesting the aggregation of nanomaterials in the BG-11 medium. The

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above results are consistent with a previous study in which high ionic strength was shown

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to stimulate nanomaterial aggregation.32, 33 The data concerning zeta potential in Figure

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S2 also confirmed that the tested nanosheets were metastable in BG-11 at a pH of

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approximately 7.0. Increased zeta potential and aggregation led to the precipitation of

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nanomaterials, which, in turn, affected the interaction of the nanomaterials with

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organisms. In the present study, Ce-WS2 and Ae-WS2 showed no significant difference in

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zeta potential or aggregation, meaning that these factors will not interfere with the study

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of the role of phase in WS2 nanosheet toxicity.

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Figure 1a shows X-ray powder diffraction (XRD) patterns for the Ce-WS2, Ae-WS2 11



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and bulk 2H WS2 samples. Ae-WS2 showed similar peaks to bulk 2H WS2. In contrast to

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the standard 14.3° (002) peak observed in the Ae-WS2 and bulk 2H-WS2 samples, the

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XRD patterns of Ce-WS2 samples showed a new (002)new peak at 9.4°, which was

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consistent with 1T-WS2.9 The Raman spectra of the Ce-WS2 and Ae-WS2 nanosheets are

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provided in Figure 1b. Two prominent peaks corresponding to the in-plane E12g and

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out-of-plane A1g modes of 2H WS2 were observed for all samples. However, the

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spectrum of Ce-WS2 exhibited much richer Raman features than Ae-WS2, with several

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new peaks in the low frequency region, which corresponded to the distorted 1T-phase

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Raman active modes and were not possible in the 2H phase. The intensities of Raman

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modes in the low frequency region (J1, J2, and J3) for Ae-WS2 almost disappeared after

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annealing treatment. The Raman spectrum of Ae-WS2 was very similar to that of the 2H

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phase,2, 9 indicating that Ce-WS2 was transformed into 2H-WS2 by the annealing

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treatment. The above results were also supported by the results of high-resolution

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transmission electron microscopy (HRTEM). As shown in Figure 1c and 1d, the trigonal

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lattice and honeycomb lattice facets for the 1T phase and 2H phase can be clearly

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visualized in Ce-WS2 and Ae-WS2 nanosheets, respectively. XPS is an efficient

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technique to evaluate the composition of 2D TMDC materials in the 1T and 2H phases.10,

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34

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the W4+ 4f7/2 and W4+ 4f5/2 in the 2H-WS2 (1T-WS2) phase, respectively. Ce-WS2

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contained the signals of both the 1T and 2H forms, indicating that Ce-WS2 was a mixture

The signals at ~ 33 (32) eV and ~ 35 (34) eV, shown in Figure 1e, are attributable to

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of the 1T and 2H phases. 1T was the main phase of Ce-WS2, accounting for ~ 61%.

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When the nanosheets annealed into Ae-WS2, they transformed to 100% 2H phase and

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closely matched the bulk 2H-phase WS2.

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Growth Inhibition

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As shown in Figure S3a, dose-dependent algal growth inhibition was evident for all

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durations of exposure. After 24 h of exposure, there was no significant inhibitory effect at

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0.1 or 1 mg/L, but algal growth was significantly inhibited at 10 mg/L. The inhibition

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rates were 24.0%, 11.3%, and 7.2% for Ce-WS2, Ae-WS2 and bulk WS2, respectively.

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The inhibition effect was lower than those of other 2D materials, such as graphene oxide

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and MoS2.27, 35 For all the materials, the growth inhibition increased as exposure time

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increase (24-72 h). After 72 h, the inhibition rate decreased with increasing exposure

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time. The above phenomenon may be due to the increase in algal cell density from 5.0 ×

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105 to 6.2 × 106 cells/mL. In addition, the nanomaterials exhibited obvious aggregation

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after 72 h (Figure S2), which may reduce their toxicity. Aggregated graphene exhibited

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lower toxicity than the dispersed form.36 The inhibition rate induced by Ce-WS2 was

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significantly higher than that induced by Ae-WS2 during the entire exposure experiment.

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The differences in specific toxicity are discussed below, along with the mechanisms.

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Morphological and Ultrastructural Changes in Algal Cells

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The SEM images of the control group showed intact, smooth and well-shaped surfaces

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(Figure 2a). After exposure to WS2 nanosheets, the cells shrunk, and some wrinkles 13



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(denoted by black arrows) and holes (denoted by red arrows) appeared on the surface

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(Figure 2b and 2c). TEM images in Figure 2d-2i show the ultrastructural differences in

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algal cells between the control and exposed groups. In the control group, an intact cell

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wall was observed, and the cytoplasm was closely attached to the cell membrane (Figure

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2d). In contrast, the cell walls exposed to WS2 nanosheets were damaged (as denoted by

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the red arrows), and serious plasmolysis was also observed (as denoted by black arrows)

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in Figure 2e and 2f. Similar phenomena were also observed in graphene-, graphene

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oxide- and MoS2-exposed Chlorella vulgaris in our previous study27, 35, demonstrating

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that these two-dimensional nanomaterials caused similar physical damage to algal cells.

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In particular, the adsorption of WS2 nanomaterials on cell walls was observed (as denoted

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by the purple arrows in Figure 2h and 2i). Cell wrapping by the aggregated sheets is an

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important mechanism that may be involved in 2D sheet toxicity. Wrapping by nanosheets

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hinders matter exchange (e.g., nutrient substances) between cells and the surrounding

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environment, resulting in damage to the cell wall and membrane37, 38 . Moreover,

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nanoparticles could also be found inside the cells, especially in chloroplasts, as denoted

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by the green arrows in Figure 2h and 2i. The specific uptake pathways of nanomaterials

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are discussed below.

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Cellular Uptake

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The cellular uptake of nanoparticles and the associated pathway can have direct

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consequences for intracellular localization and cytotoxicity39, 40. ICP-MS showed 14


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quantitatively that the cellular Ce-WS2 and Ae-WS2 content reached 0.335 and 0.155

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μg/106 cells, respectively, after 10 mg/L exposure (Figure 2j). The cellular internalization

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of WS2 was significantly lower than the internalization of MoS2 by Chlorella vulgaris27,

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which was consistent with the fact that the toxicity of WS2 nanosheets was lower than

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that of MoS2 nanosheets. Moreover, the cellular content of Ce-WS2 was 1.79-fold higher

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than that of Ae-WS2 at 1 mg/L. The correlation coefficient between cellular uptake and

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growth inhibition rate was determined to be 0.93 (Figure S4a). To further evaluate the

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effect of phase on the uptake mechanism, we used ICP-MS to quantify the uptake of

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nanosheets under physical and pharmacological inhibition. Energy-dependent

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endocytosis can be inhibited by exposing algal cells to low temperatures40. The uptake of

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Ce-WS2 and Ae-WS2 was reduced by 37.09% and 38.23%, respectively, at 4 °C

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compared to control conditions, indicating the energy dependence of the internalization

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process (Figure 2k). However, the internalization of WS2 was still detected at 4 °C,

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indicating the existence of energy-independent pathways as well, for example, passive

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diffusion.39 To further identify the energy-dependent pathways, we treated algae with

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MβCD to block caveolae-mediated endocytosis5, 40, CPZ to prevent clathrin-mediated

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endocytosis5, and EIPA to prevent uptake by micropinocytosis.41 MβCD and EIPA

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reduced the uptake of Ce-WS2 by 19.03% and 34.68%, respectively (Figure 2k). MβCD,

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CPZ, and EIPA reduced the uptake of Ae-WS2 by 41.50%, 31.30% and 52.77%,

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respectively (Figure 2k), suggesting a difference between the uptake pathways of Ce-WS2 15



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and Ae-WS2. The above data suggest that macropinocytosis played a dominant role in the

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uptake of both phases of WS2 nanosheets. In contrast, clathrin-mediated endocytosis was

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not very important in the uptake process, especially for Ce-WS2. Moreover, Ae-WS2 was

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more sensitive than Ce-WS2 to these pharmacological inhibitors.

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Photosynthesis and Oxidative Stress

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As shown in Figure S5, after the algae were exposed to WS2 nanosheets for 24 h, the

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Fv/Fm ratios (the maximum photochemical quantum yields) were slightly higher than

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those of control cells. As the exposure time increased, the Fv/Fm ratios decreased. The

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Fv/Fm values in the treated group at 72 h were significantly decreased compared with

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those in the control, implying the inhibition of photosystem II (PSII).42 Then, the Fv/Fm

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values increased slightly at 96 h, which was consistent with the results showing growth

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inhibition (Figure S3). In addition, the Fv/Fm ratios in the Ce-WS2 groups were lower

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than those in the Ae-WS2 groups, which was also consistent with the internalization of

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WS2 nanosheets by algae. To explore the mechanisms underlying the decrease in

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photosynthesis, we investigated oxidative stress. Both Ce-WS2 and Ae-WS2 triggered an

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increase in intracellular ROS levels in algal cells, and the former induced significantly

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higher intracellular ROS levels than the latter (Figure S3b). Figure S3c shows that WS2

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nanosheets at 1 mg/L and 10 mg/L inhibited reduced glutathione (GSH) by 16.1-68.6%,

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with especially strong inhibition by Ce-WS2. The correlation coefficients of ROS

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generation with cellular GSH and growth inhibition ratio were determined to be 0.90 and 16


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0.88, respectively (Figure S4b), suggesting that oxidative stress played an important role

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in nanotoxicity. Furthermore, abiotic GSH oxidation was used as a model system to

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examine the possibility of oxidative stress mediated by Ce-WS2 and Ae-WS2 using

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Ellman's assay. In Figure S6, Ce-WS2 had stronger GSH oxidation potential than

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Ae-WS2, which was consistent with the results of biotic experiments in algal cells.

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Lipid Peroxidation and Membrane Damage

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Cell membrane damage is an important mechanism of nanomaterial cytotoxicity. In

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addition to the physical damage to the cell membrane inflicted by their sharp edges or

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rough surfaces, nanomaterials can cause membrane damage and cytotoxicity through

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ROS generation and lipid peroxidation.43, 44 As shown in Figure 3a, both Ce-WS2 and

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Ae-WS2, but especially Ce-WS2, caused significant lipid peroxidation of the membrane,

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which was consistent with ROS generation and the failure of membrane integrity in TEM

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images. RBCs have no fluid phase or receptor-mediated endocytosis and are widely used

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to study nanomaterial interactions with the cell membrane.29 WS2 nanosheet-induced

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membrane damage was investigated using a hemolysis assay in RBCs. As shown in

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Figure 3b, WS2 nanosheets exhibited a slight hemolytic effect, and the hemolytic effect

337

of Ce-WS2 was stronger than that of Ae-WS2. Furthermore, the cellular permeability and

338

integrity of algal cells were examined by PI uptake. The green (P2) and red (P1) regions

339

in the flow cytometry images indicate intact cells and membrane-damaged cells

340

(membrane permeabilization), respectively (Figure 3c). Ce-WS2 induced a much higher 17



341

level of membrane permeabilization than Ae-WS2, which was consistent with the changes

342

in membrane peroxidation.

343

Genotoxicity

344

Shape, size and surface chemicals are important parameters that can affect the potential

345

cytotoxicity of nanomaterials20,29,36. The main difference between the two tested WS2

346

nanosheets was the phases between 1T and 2H, and the roles of the phases of WS2

347

nanosheets in nanotoxicity are largely unknown. Previous reports showed that

348

graphene-based 2D nanomaterials induce genetic damage45, 46. In the present work, the

349

genotoxicity induced by WS2 nanosheets was measured by monitoring DNA damage to

350

algal cells using a comet assay. As shown in Figure S7, there was a slight increase in tail

351

DNA in the WS2 nanosheet-treated group at 10 mg/L, and there was no significant

352

difference (p > 0.05) between Ce-WS2 and Ae-WS2. This indicates that 10 mg/L WS2 has

353

low genotoxicity, which is consistent with the observation that WS2 is less toxic than

354

graphene materials.25

355

ESR Detection of ROS and Photogenerated Electrons

356

Generation of oxidative stress in cells is considered a major factor in

357

nanoparticle-induced cytotoxicity.47 The algal exposure experiment was performed under

358

visible-light irradiation. Through semiconductor photocatalysis, WS2 nanosheets may

359

produce photogenerated ROS, causing damage to the exposed organism.48, 49 To test this

360

hypothesis, we measured the generation of hydroxyl radical (•OH) and superoxide (O2•−) 18


Page 18 of 44

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361

from irradiated Ce-WS2 and Ae-WS2. No ESR signals were observed for Ce-WS2 or

362

Ae-WS2 without irradiation (Figure 4a and 4b). After 30 min of irradiation in the

363

presence of Ce-WS2 and Ae-WS2 nanosheets, four lines with relative intensities of

364

1:2:2:1 were observed – the characteristic spectrum of the spin adduct DMPO/•OH.30

365

Furthermore, the addition of DMSO (a specific scavenger for hydroxyl radicals) almost

366

completely suppressed the signal intensity of irradiated Ce-WS2 and Ae-WS2,

367

demonstrating that hydroxyl radicals were generated from irradiated Ce-WS2 and

368

Ae-WS2, whereas no superoxide radicals were produced. With the same amount and

369

recording time, the ESR signal intensity generated from photoexcited Ce-WS2 was

370

approximately 1.33-fold higher than that from Ae-WS2, indicating that Ce-WS2 has a

371

stronger ability to photogenerate hydroxyl radicals. TEMP itself is ESR silent but can

372

react with singlet oxygen to produce a nitroxide radical with a distinctive three-line ESR

373

spectrum.30 Figure S8 shows that no singlet oxygen was generated from irradiated

374

Ce-WS2 or Ae-WS2.

375

Previous research has shown that both visible and near-infrared (NIR) light excitation

376

can cause electron-hole pairs in WS2 nanosheets50. To explore the differential ability of

377

light to generate electrons and holes on the surface of Ce-WS2 and Ae-WS2 nanosheets,

378

we used ESR spectroscopy to investigate the light-induced formation of electrons. The

379

spin label TEMPO was shown not to react with oxidative intermediates formed in

380

photoexcitation of photocatalysis, such as •OH, O2•−, 1O2 and holes.51 In the present 19



381

study, TEMPO was used to verify electrons photogenerated by nanomaterials under

382

simulated sunlight. TEMPO exhibited a stable triplet ESR spectrum, with a relative

383

intensity of 1:1:1. As shown in Figure 4c and 4d, the signal intensity was unchanged

384

when TEMPO was mixed with WS2 nanosheets before irradiation or irradiated without

385

WS2 nanosheets (control). In contrast, an obvious reduction in the ESR signal intensity

386

was observed for the reaction of TEMPO with WS2 nanosheets under irradiation for 15

387

min. The reduction of TEMPO signal intensity caused by Ce-WS2 and Ae-WS2 was

388

calculated to be 68.23% and 59.68%, respectively. After 30 min of irradiation, the signal

389

intensity of TEMPO was reduced by 96.44% and 90.34% for Ce-WS2 and Ae-WS2,

390

respectively. The above results suggested that Ce-WS2 produced more electrons than

391

Ae-WS2 under simulated sunlight.

392

Photogenerated electrons are related to the band gaps of semiconductors. Low band

393

gaps are facile for the photogeneration of electrons.30, 52, 53 As shown in Figure S9, the

394

band gap of Ce-WS2 (1.67 eV) was lower than that of Ae-WS2 (2.04 eV), indicating that

395

Ce-WS2 was more likely to generate electrons and holes. In addition, due to the presence

396

of the metallic 1T phase, Ce-WS2 presented higher electron conductivity than that of

397

Ae-WS2 54, 55, which substantially improved the charge transfer efficiency and separation

398

efficiency of electrons and holes, leading to an increase in ROS (mainly •OH) levels and

399

nanotoxicity.3, 55

400

Release of Dissolved Ions 20


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401

Toxic ions released from nanomaterials may contribute to nanotoxicity.56 Figure S10

402

shows that the amount of dissolved W from WS2 nanosheets was greater in BG-11

403

culture medium than in deionized water. In addition, ions were released much more

404

rapidly from Ce-WS2 than from Ae-WS2 in both deionized water and BG-11 culture

405

medium. Similar results were reported by Wang et al.,11 who demonstrated that the

406

oxidation dissolution of chemically exfoliated MoS2 nanosheets was faster than that of

407

ultrasonically exfoliated nanosheets due to the presence of the 1T phase in the former, as

408

opposed to the pure 2H phase in the latter. Shang et al.57 demonstrated that the

409

enhancement of WS2 nanoparticle (NP) dissolution by UV light was due to the

410

photocorrosion of NPs by the holes: WS2 + 6h+

411

4d, Ce-WS2 produced more electrons and holes than Ae-WS2, which explains why

412

Ce-WS2 released ions much more quickly than Ae-WS2 under irradiation. The dissolution

413

products of WS2 nanosheets usually contain tungstate, W(VI).11, 27, 57 Tungstate salt

414

(Na2WO4) was chosen as the control to study the toxic effects of the ions on algae. As

415

shown in Figure S11a, tungstate salt showed no statistically significant inhibitory effect

416

on cell growth at a concentration of 50 μM (corresponding to WS2 nanosheets at

417

approximately 12.4 mg/L). Intracellular ROS and cell membrane integrity were also not

418

affected by tungstate salt (Figure S11b and S11c), suggesting that the differences in

419

toxicity between Ce-WS2 and Ae-WS2 were derived from nanosheets rather than the

420

released ions.

ℎ𝑣

W6+ + 2S. As shown in Figure 4c and

21



421

Metabolic Mechanisms of Biological Responses

422

The identified metabolites included amino acids, fatty acids, small-molecule acids,

423

carbohydrates, and other biomolecules. The relative levels of the metabolites are

424

presented using heat maps in Figure S12. Using hierarchical clustering (HCL) analysis,

425

Ce-WS2 induced more obvious metabolite alteration than Ae-WS2 relative to the control

426

treatment. The two-dimensional score plots of the principal component analysis (PCA)

427

and partial least squares-discriminant analysis (PLS-DA) models both demonstrated that

428

the Ce-WS2 and Ae-WS2 groups were separated from each other and from the control

429

group (Figure 5a and 5b), indicating that WS2 nanosheets affected cellular metabolism.

430

As shown in Figure S13, WS2 nanosheet exposure significantly upregulated the levels of

431

amino acids and fatty acids. Previous reports suggested that MoS2 nanosheets exposed to

432

human dermal fibroblasts upregulate intracellular amino acids via the degradation of

433

abnormal proteins.58 The high levels of free fatty acids are related to β-oxidation

434

processes, and the upregulation of fatty acids acts as an organismal adaptation to

435

membrane oxidative stress.59, 60 Herein, Ce-WS2 induced more obvious perturbation of

436

the algal metabolic profile of amino acids and fatty acids than Ae-WS2 (Figure S13),

437

which was consistent with the higher toxicity of Ce-WS2. Palmitic acid and stearic acid

438

were significantly upregulated. Previous reports showed that the upregulation of palmitic

439

acid and stearic acid results in cell membrane damage,61 and damage to cell plasma

440

membranes is observed in Figure 3c. Moreover, increases in palmitic acid and stearic 22


Page 22 of 44

Page 23 of 44


441

acid levels inhibit electron transport in photosynthesis and disintegrate phycobilin from

442

the thylakoid membrane,61, 62 which is consistent with the decrease in maximum

443

photosynthesis induced by WS2 nanosheets (Figure S5). The differentially regulated

444

metabolites were further defined according to a p value < 0.05 and false discovery rate

445

(FDR) < 0.05. There were 28 and 16 specific metabolites that distinguished Ce-WS2 and

446

Ae-WS2, respectively, from the control group (Figure S14). Next, the differentially

447

regulated metabolites were mapped onto their corresponding metabolic pathways. As

448

shown in Figure 5c and 5d, both Ce-WS2 and Ae-WS2 upregulated the pathways of

449

alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism,

450

arginine and proline metabolism and glutathione metabolism. Metabolic pathways such

451

as glycine, serine, threonine metabolism and glutamate metabolism are involved in GSH

452

biosynthesis and metabolism.63,64 However, the results in Figure S3c show a decrease in

453

intracellular GSH levels. ROS generated by WS2 nanosheets depleted GSH and then

454

stimulated cells to promote GSH synthesis by compensation. In addition, Ce-WS2

455

downregulated starch and sucrose metabolism and glycerolipid metabolism, while

456

Ae-WS2 downregulated only glycerolipid metabolism (Figure 5c and 5d). Starch and

457

sucrose are important sources of energy storage in algae. The downregulation of starch

458

and sucrose metabolism may be associated with the reduced photosynthetic activities

459

under WS2 nanosheet stress.65 The above results showed that metabolic analysis can

460

provide new insights into the differences in toxicity between Ce-WS2 and Ae-WS2. 23



461

The phase of a nanomaterial is a critical determinant of its applications and

462

environmental fate, such as pollutant adsorption, catalysis, antibacterial activity and

463

degradation.2, 9, 66 However, the biological responses to different phases of WS2

464

nanosheets remain largely unknown, which limits their environmental risk evaluation and

465

their suitability for environmentally friendly design. The results of the present study

466

demonstrated that the phases of WS2 nanosheets affected oxidative stress, membrane

467

damage, lipid peroxidation, uptake and cytotoxicity in algae. The metallic 1T phase

468

showed higher toxicity than the semiconducting 2H phase. Noticeably, WS2 nanosheets

469

at 0.1 mg/L did not trigger remarkable toxicity to algae and seemed safe at predicted

470

environmental concentrations (e.g., μg/L).67, 68 However, new nanomaterials, especially

471

2D nanomaterials, are being developed quickly, and investigations into these novel

472

nanomaterials might reveal materials of higher environmental concern.69 Compared with

473

the well-studied effects of size, shape and surface modifications of nanomaterials on

474

nanotoxicology, little is known about the effects of phase. The results of the present study

475

highlight the importance of considering the phase of a nanomaterial when evaluating its

476

environmental risk.

477 478

ASSOCIATED CONTENT

479

Supporting Information Available

480

Methods are provided for WS2 nanosheet characterization, electron microscopic 24


Page 24 of 44

Page 25 of 44


481

observations, oxidative stress, the comet assay and metabolomic analysis. Figures S1-S14

482

show the results of WS2 nanosheet characterization, growth inhibition, ROS levels, GSH

483

content, correlation analysis, photosynthetic efficiency, abiotic oxidation, the comet

484

assay, ESR spectra, band gap, cytotoxicity and metabolomic analysis.

485 486

AUTHOR INFORMATION

487

Corresponding author:

488

*Email: [email protected] (X.H.).

489

Tel.: 86-22-23507800; fax: 86-22-23507800.

490 491

NOTES

492

The authors declare no competing financial interests.

493 494

ACKNOWLEDGMENTS

495

This work was financially supported by the National Natural Science Foundation of

496

China (grant nos. 21577070 and 21307061).

497 498

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Figure Captions

719

Figure 1. Characterizations of Ce-WS2 and Ae-WS2. a, XRD patterns of Ce-WS2 and

720

Ae-WS2z compared with the peak line of bulk 2H-WS2 from the Joint Committee on

721

Powder Diffraction Standards (JCPDS) card; b, Raman spectra of Ce-WS2, Ae-WS2 and

722

bulk WS2; c, high-resolution TEM images of Ce-WS2; d, high-resolution TEM images of

723

Ae-WS2. To show the crystalline structure clearly, enlarged views of the basal planes are

724

inserted in c and d. e, XPS spectra and deconvolution analysis of Ce-WS2, Ae-WS2 and

725

bulk WS2.

726 727

Figure 2. Uptake of Ce-WS2 and Ae-WS2 and damage to the cellular structure. a-c, SEM

728

images; d-I, TEM images of algae exposed to WS2 nanosheets of different phases. j and

729

k, Cellular uptake rate and pathway of WS2 nanosheets. a, d and g, Control without

730

nanomaterial exposure; b, e, and h, Ce-WS2 at 10 mg/L; c, f and i, Ae-WS2 at 10 mg/L; j,

731

cellular nanomaterial content; k, effect of physical inhibition and pharmacological

732

inhibitors on the uptake of WS2 nanosheets in algal cells. “∗”, represents statistical

733

significance at p < 0.05.

734 735

Figure 3. Lipid peroxidation and membrane damage induced by WS2 nanosheets of

736

different phases in Chlorella vulgaris. A, MDA content; b, red blood cell hemolysis; c,

737

flow cytometry images of PI-dyed algal cells after 96 h of exposure. “∗” represents 37



738

statistical significance at p < 0.05.

739 740

Figure 4. ESR spectra of ROS and photogenerated electrons. ESR spectra were obtained

741

from samples containing DMPO with Ce-WS2 (a) and DMPO with Ae-WS2 (b) with and

742

without the addition of DMSO. ESR spectra were obtained from Ce-WS2 with TEMPO

743

(c) and Ae-WS2 with TEMPO (d).

744 745

Figure 5. Metabolic analysis of Chlorella vulgaris exposed to WS2 nanosheets. PCA

746

score plot (a) and PLS-DA score plot (b) of metabolites in algal cells exposed to WS2

747

nanosheets. Significantly disturbed metabolic pathways in algal cells influenced by

748

Ce-WS2 (c) and Ae-WS2 (d) treatment. The red and green arrows indicate the up- and

749

downregulated metabolic pathways, respectively.

750 751 752

38


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753 754

Figure 1.

39



755 756

Figure 2.

40


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757 758

Figure 3.

759 760

41



761 762

Figure 4.

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Figure 5.

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The Phases of WS2 Nanosheets Influence Uptake, Oxidative Stress

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