Microplastic Impacts on Microalgae Growth: Effects of Size andHumic AcidGe Liu,† Ruifen Jiang,*,† Jing You,† Derek C. G. Muir,†,‡ and Eddy Y. Zeng†

†Guangdong Key Laboratory of Environmental Pollution and Health, School of Environment, Jinan University, Guangzhou 511443,China‡Environment and Climate Change Canada, 867 Lakeshore Road, Burlington, Ontario L7S 1A1, Canada

*S Supporting Information

ABSTRACT: Research has already demonstrated the toxiceffects of microplastics (MPs) on different biota. However, theunderlying toxic mechanism of MPs remains to be elucidated,especially the effect of particle size and the presence ofdissolved organic matter in water. This study investigated theimpact on Scenedesmus obliquus exposed in five types ofpolystyrene particle suspensions with different sizes andsurface charges, in the presence and absence of humic acid(HA). Results indicated that the 50% growth inhibition rate ofS. obliquus showed no significant difference between the fivetypes of MPs, but the toxic mechanism varied with particlesize. Larger size MPs caused adverse effects by blocking thelight transport and affecting photosynthesis, while smaller onesdestroyed the cell wall by adsorbing onto the algae surface. Also, the addition of HA significantly alleviated the toxicity ofsmaller size MPs, but not of the larger ones. Scanning electron microscopy images and the reactive oxygen species assaydemonstrated that the HA could form a corona on the surface of MPs, reduce the affinity to microalgae, and minimize theadverse effect. Together, these findings identified important factors in determining the toxicity of MPs, providing valuable datafor risk assessment of MPs.

■ INTRODUCTION

Plastic debris can fragment into a wide array of particles ofvarious sizes by physical and chemical degradation processes.1

Microplastics (MPs) are commonly defined as plastic particleswith aerodynamic diameters less than 5 mm.2 Aquatic biota,including plankton,3,4 benthos,5,6 vertebrates,7,8 and evenmammals,9 can accumulate MPs and result in adverse effects.Fish may ingest MPs as they may confuse MP particles withreal prey.7,8 Zooplankton and bivalves can easily uptake MPsby filter-feeding,3,10 while microalgae mainly do so byadsorption.11 Top predators including humans may accumu-late MPs through food-chain transfer.12−14 Although the directrisk of MPs to aquatic biota in the environment remains largelyunclear, laboratory studies have shown adverse effects onfish,15,16 Daphnia magna,16 brine shrimp,17 etc. as a result ofexposure to MPs.Microalgae are responsible for primary production in aquatic

ecosystems. They convert inorganic carbon into organiccarbon through photosynthesis and are a source of food formany aquatic organisms. The growth inhibition rate,16

photosynthesis,18,19 oxidative stress,11 and gene expression17

of microalgae may be affected by MPs, perhaps in relation tothe size, surface properties, concentrations, and even exposuretime of MPs. However, algal responses to MPs remain to befully understood. Zhang et al.20 indicated that the adverse

effects were mainly caused by mechanical damage. Contactwith MPs could damage cell walls, destroy the integrity of cellstructures, and eventually lead to cell death. On the otherhand, Bhattacharya et al.11 argued that the shading effect ofMP particles was the main reason for algal toxicity becausephotosynthesis was substantially depressed when particles werepresent. Both Zhang et al.20 and Sjollema et al.18 however,claimed no significant variability in photosynthesis parametersof algae when exposed to poly(vinyl chloride) (PVC; 1 mm)and polystyrene (PS; 0.05, 0.5, and 6 μm), respectively.Overall, the toxicity mechanism of plastics of different sizes,especially the ones ranging from nano- to microscale, has notbeen fully elucidated.Aside from unclear toxicity mechanisms, the effects of

environmental factors have rarely been considered in previousstudies. Dissolved organic matter (DOM) has been demon-strated to be an important factor affecting the bioavailability ofpollutants. Particles including MPs in the environment tend tointeract with DOM such as humic acid (HA) and proteins toform a so-called eco-corona,21 which plays an important role in

Received: October 14, 2019Revised: December 1, 2019Accepted: December 6, 2019Published: December 6, 2019

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pubs.acs.org/estCite This: Environ. Sci. Technol. 2020, 54, 1782−1789

© 2019 American Chemical Society 1782 DOI: 10.1021/acs.est.9b06187Environ. Sci. Technol. 2020, 54, 1782−1789

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dictating the behavior and toxicity of particles to aquaticorganisms. A similar effect was also found on engineerednanoparticles such as TiO2,

22 AgNP,23,24 and graphene oxide25

with DOM present. Our previous study26 found that theaddition of HA significantly alleviated the toxicity of nano-polystyrene (PS) to zooplankton. Similar results were alsoreported by Fadare et al.27 and Saavedra et al.21 who reasonedthat detoxification of HA was induced as a corona was formedon the surface of nanoplastics. In contrast, Liu et al.28 foundthat nano-PS modified by natural acidic organic polymersaltered the oxidative stress and disturbed the membranefunction of Scenedesmus obliquus cells to a certain extent.Overall, the presence of DOM in water is critical to the fateand toxicity of nanoplastics. So far, no effort has been directedtoward understanding the effects of DOM on the toxicity ofplastics, especially microsized plastics.The present study attempted to clarify the underlying

mechanisms for the toxicity of MPs to microalgae. S. obliquuswas selected as the model organism, and five types ofpolystyrene (PS) particles, including negatively charged PSwith diameters of 0.1, 0.5, 1, and 2 μm and a positively chargedPS with an amino group, were chosen as representative MPs.The toxicity endpoints of S. obliquus such as growth inhibitionrate, photosynthesis parameters, and reactive oxygen species(ROS) were tested to reveal the effects of particle sizes andsurface charge. Finally, the effects of HA in the exposuresuspensions were investigated to obtain toxicity data underscenarios more similar to the real environment, so as touncover the toxicity mechanisms.

■ METHODS AND MATERIALSTarget Compounds and Materials. Polystyrene beads

with a negative charge and four diameters (n-plain-0.1 μm, n-plain-0.5 μm, n-plain-1 μm, and n-plain-2 μm) were obtainedfrom Aladdin (Shanghai, China), with 2.5% (w/v) of PS in asolution of ethanol/water (50:50 in volume). Polystyrenebeads with positively charged amino groups (p-NH2-0.1 μm)and fluorescence labeling (f-PS-0.1 μm) were purchased fromThermo Fisher Scientific (Shanghai, China), with 2% (w/v) PSin deionized water. Humic acid (fulvic acid ≥ 90%) was alsoobtained from Aladdin. All PS stock suspensions were stored at4 °C in a dark refrigerator. Lowercase letters “n”, “p”, and “f”represent negatively charged, positively charged, and fluo-rescence labeling, respectively. The properties of all target PSparticles, including hydrodynamic diameters, ζ-potential(Table S1), morphology (Figure S1), and infrared spectrum(Figure S2), are provided.Test Organisms and Culture Conditions. The fresh-

water microalgae, S. obliquus, were obtained from the School ofLife Science, Sun Yat-sen University (Guangzhou, China). Themicroalgae were cultured according to the Organization forEconomic Cooperation and Development Guidelines 201. Theglassware was soaked with 10% hydrochloric acid for 24 h,cleaned with milli-Q water, and sterilized by autoclaving beforeuse. A general culture medium, BG-11 medium, was selectedfor S. obliquus. The microalgae were cultured at 24 ± 1 °C inan incubator with a light/dark cycle of 16/8 h and used after10−14 days when a stable logarithmic growth phase wasreached. The microalgae cell density was measured with ahemocytometer and a biological microscope at 200-foldmagnification.Growth Inhibition of S. obliquus under Different

Microplastic Suspensions. Before the experiment, the PS

stock suspension was sonicated in an ultrasonic bath for 15min to disperse the particles. An HA stock solution at 1000 mgL−1 was prepared by weighing 0.1 g of HA, dissolving it in 100mL of milli-Q water, and sonicating for 20 min before use.Bioassay procedures are as follows. A certain amount ofprecultured S. obliquus suspension was diluted with BG-11medium to a desired concentration. An appropriate volume ofPS stock suspension and HA stock solution was added to a 20mL transparent glass vial and mixed well. Then, 15 mL of thediluted microalgae suspension was transferred to another 20mL vial and manually shaken to thoroughly mix the microalgaeand PS. Finally, the prepared exposure suspensions were placedin an incubator before bioassay was conducted.The dose−response curves of PS to S. obliquus were

obtained with PS concentrations ranging from 0 to 100 mgL−1. Growth inhibition experiments were conducted insuspensions with PS only (75 mg L−1), HA only (5 mgL−1), and both PS (75 mg L−1) and HA (5 mg L−1). Allexperiments were conducted at an algae concentration of 2 ×105 cells mL−1, which was selected after optimization. Theconcentration of PS was chosen based on the dose−responsecurve, in which 75 mg L−1 PS inhibited the growth of S.obliquus up to 65% depending on different sizes and surfacecharges. The concentration of HA was also selected based onthe concentration range of HA in most freshwaters (1−15 mgL−1).27

Measurement of Chlorophyll Fluorescence Parame-ters of S. obliquus. The fluorescence of the microalgae wasmeasured using a hand-held chlorophyll fluorescence meterAquaPen-C AP-C 100 (Photo Subsystem Instrument, CzechRepublic). The OJIP curves of the algae were determined, andthe biophysical parameters derived from the OJIP curves werecalculated. The following parameters were used: (1) Fv/Fm, themaximum quantum yield of primary photochemistry; (2)specific energy flux per reaction center (RC), includingabsorption energy (ABS/RC), electron transport energy(ETo/RC), trapping energy (TRo/RC), and dissipationenergy (DIo/RC); and (3) performance index (Pi_Abs) oflight energy absorption. Before measurement, the microalgaesolution was dark-adapted for 8 min. The chlorophyllfluorescence parameters were determined after 48 h exposurein different treatments, including BG-11 medium (control),with 250 mg L−1 PS alone, HA alone (15 mg L−1), and 250 mgL−1 PS + 15 mg L−1 HA. The algal concentration used in thisexperiment was 2 × 106 cells mL−1, which was 10 times higherthan the one used in growth inhibition testing, as low algalconcentrations did not provide sufficient fluorescence signal.The morphology of PS and algae after exposure with

different treatments was obtained by scanning electronmicroscopy (SEM; Hitachi SU8010, Japan). Detailedprocedures for the preparation of SEM samples are describedin the Supporting Information, Text S1. Levels of intracellularreactive oxygen species (ROS) produced by S. obliquusexposed to five PS suspensions at a concentration of 50 mgL−1 were also measured after 24 h exposure. More detailedinformation on sample preparation can be found in theSupporting Information, Text S2.

Data Analysis and QA/QC Measures. The mean andstandard deviation were determined based on four replicatesfor each experimental treatment. Statistical difference betweenthe means was assessed by the one-way analysis of variance(ANOVA) and the Fischer least significant difference (LSD)test using SPSS Version 18 (IBM, Armonk, NY). Differences

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were considered to be significant at p < 0.05. The growthinhibition rate (IR) was calculated as IR (%) = (1 − T/C) ×100%, where T and C are the cell densities of algae in theexperimental and control groups, respectively. The 50%effective concentration of inhibition rate (IR50) value wascalculated with GraphPad Prism 7 (GraphPad Software, SanDiego, CA) with a confidence interval of 95%.

■ RESULTS AND DISCUSSIONToxicity of Polystyrene Beads to S. obliquus. The

dose−response curves for five types of PS beads were obtained

at concentrations between 0 and 100 mg L−1 (Figure 1). Themean values of IR50 indicated that n-plain-0.5 μm and n-plain-

0.1 μm posed the highest and lowest toxicity to S. obliquus,respectively (Table S1). However, when the 95% confidencelimits were considered, no significant difference was observedfor different types of PS beads, except for n-plain-0.5 μm, asthe IR50 was highly variable. This was contrary to the previousfindings that the toxicity of PS was enhanced with decreasingparticle size.18 Different findings may result from the selectionof PS concentrations. When the concentration of PS washigher than 10 mg L−1, the growth inhibition of n-plain-2 μmwas lower than most other particles, while it was higher whenthe PS concentration was lower than 10 mg L−1 (Figure 1).Another reason may be the varying toxic mechanisms of PSwith different sizes. The selected MP sizes in the present study

Figure 1. Dose−response curves of algae when exposed to five typesof PS. Error bars represent standard deviations of four replicates.

Figure 2. Effects of different PS beads on the chlorophyll fluorescence parameters of S. obliquus after a 48 h exposure. The concentration of PS was250 mg L−1, and the algae concentration was 2 × 106 cells mL−1.

Figure 3. Growth ratio of S. obliquus in three types of PS suspensionswith and without HA after a 48 h exposure. Error bars representstandard deviations of four replicates. *p < 0.05; **p < 0.01.

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ranged from nano- to microsizes, which may induce differenttoxic mechanisms in S. obliquus. The shapes of the dose−response curves also implied diversified toxic mechanisms.Besides, the dose−response curves indicated that the growthinhibition effect of p-NH2-0.1 μm was higher than that of n-plain-0.1 μm for the entire selected concentration range,consistent with previously reported results.17,19 Nolte et al.19

found that the adsorption of positively charged nanoplastics tothe cell wall of Raphidocelis subcapitata was stronger than thatof negatively charged ones. Bergami et al.17 reported that thenegatively charged PS (PS-COOH; 40 nm) formed microscaleaggregations while the positively charged PS (PS-NH2; 50 nm)formed nanoscale aggregates and was more toxic to microalgaeand brine shrimps. The present study also found that p-NH2-0.1 μm showed higher growth inhibition on S. obliquus than n-plain-0.1 μm, likely resulting from the stronger adsorptionproperty of p-NH2-0.1 μm.The chlorophyll fluorescence can reveal a lot of information

on the algae photosynthesis under stress. The effects of five PStypes on six biophysical parameters were correlated to algalphotosynthesis (Figure 2). The values of Fv/Fm and Pi_Absgenerally decreased with increasing particle size, while those ofABS/RC, TRo/RC, and DIo/RC were positively correlated toparticle size. These results indicate that large particle sizessubstantially decreased the light conversion efficiency in thephotochemical reaction center (Fv/Fm) and the energyconservation parameter, Pi_Abs. The Fv/Fm values of algaein suspensions with PS sizes of 100 nm, 500 nm, 1 μm, and 2μm were 97, 92, 69, and 43% to the control group, respectively,

while the Pi_Abs values were 56, 34, 6, and 2% of the controlgroup, respectively. The absorption (ABS/RC) and trapping(TRo/RC) energy per active reaction center (RC) increaseddue to the inactivation of some RCs, while the ratio of totaldissipation to the amount of active RCs (DIo/RC) increasedbecause of high dissipation of the inactive RCs. It is worthwhileto mention that both n-plain-0.1 μm and p-NH2-0.1 μm had ansmaller impact on most of the parameters except for DIo/RCand Pi_Abs compared to the ones with larger diameters. Also,the effects on DIo/RC and Pi_Abs parameters for n-plain-0.1μm and p-NH2-0.1 μm PS were inverse, which was probablyrelated to the surface charge of the particles. The n-plain-0.1μm PS increased the value of DIo/RC and decreased Pi_Abswhen compared to the control group. On the other hand, p-NH2-0.1 μm PS decreased DIo/RC and increased its Pi_Abs.The reason behind this inverse effect has remained unknown.The effects of MPs on the photosynthetic efficiency of algae

vary in different studies. Sjollema et al.29 found a negligibleeffect (<10% inhibition compared to control) on thephotosynthetic efficiency of microalgae upon exposure to PSbeads with sizes of 0.05, 0.5, and 6 μm. In contrast, Zhang etal.20 and Mao et al.30 reported a negative impact of micro-PVC(1 μm) and micro-PS (approximately 1 μm), respectively, onthe photosynthetic efficiency of microalgae. The discrepancywas probably caused by microalgal species, plastic-type, andMP sizes, as also found in the present study. Different sizes andstructures of the algal cell wall may influence the adsorption ofplastic and sunlight. Also, different plastic types may result indifferent adsorption capacities to microalgae and then affect

Figure 4. Growth curves of S. obliquus in suspensions with (A) n-plain-0.1 μm and (B) n-plain-2 μm. The normalized growth ratio of S. obliquus indifferent treatments with (C) n-plain-0.1 μm and (D) n-plain-2 μm. Error bars represent standard deviations of Three replicates.

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the toxicity. The sizes of the MPs, as demonstrated by ourresult, could significantly affect the photosynthesis of micro-algae.Growth Rate Inhibition in Polystyrene- and Humic

Acid-Treated S. obliquus. The presence of DOM cansignificantly affect the toxicity of pollutants to aquatic biota.We already demonstrated the effect of PS with different sizeson the S. obliquues. We further investigated the effects of HAon the toxicity of PS with different sizes. Figure 3 shows theindividual and joint effects of three PS beads on the growthrate of S. obliquus after a 48 h exposure. Compared to exposureto PS only, the addition of 5 mg L−1 HA significantly decreasedthe toxicity of n-plain-0.1 μm and p-NH2-0.1 μm, but not then-plain-2 μm PS beads. Separate exposures to n-plain-0.1 μm,n-plain-2 μm, and p-NH2-0.1 μm (75 mg L−1) reduced thegrowth rate to approximately 20, 30, and 20% of the control,respectively. However, when 5 mg L−1 HA was added to theexposure suspensions, the growth rate increased to approx-imately 60% in n-plain-0.1 μm and 90% in p-NH2-0.1 μmsuspensions. No significant improvement was observed in then-plain-2 μm suspension with HA. The addition of HA

appeared to detoxify small-sized particles but not large-sizedones. The toxic mechanism of PS seemed to vary with particlesize, as also noted by Machado et al.31

The effects of HA and MP size on long-term toxicity werefurther examined through monitoring the growth curve of S.obliquus in four types of media including BG-11 (controlgroup), 5 mg L−1 HA, 75 mg L−1 PS, and the medium withboth 5 mg L−1 HA and 75 mg L−1 PS. The growth curves ofalgae in the media with HA alone and with both n-plain-0.1μm PS and HA showed no significant difference from thosewith the control group and exhibited exponential growthpatterns (Figure 4A). However, in the medium with 75 mg L−1

n-plain-0.1 μm alone, the concentration of S. obliquusdecreased during the first 48 h (20% of the control) andthen gradually increased as time went on. Similar curves wereobserved in the medium with p-NH2-0.1 μm PS (Figure S3).On the other hand, the S. obliquus seemed to be completelyinhibited by the n-plain-2 μm PS up to 144 h exposure (Figure4B), even in the medium with HA. By normalizing theconcentrations of S. obliquus in the treatment groups to thosein the control group, the growth ratio of S. obliquus at each

Figure 5. OJIP parameters of microalgae exposure in different treatments after 48 h. Each value was defined as the ratio of control. Theconcentration of HA, PS, and S. obliquus was 15 mg L−1, 250 mg L−1 and 2 × 106 cells mL−1, respectively.

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exposure time can be plotted. The reproductive capacity wasfully recovered in n-plain-0.1 μm PS medium after a 199 hexposure (Figure 4C) but had not recovered in n-plain-2 μmPS medium (Figure 4D).Apparently, all PS particles can inhibit algal growth upon

short-term exposure with specific concentrations, but the effectof small-sized particles such as n-plain-0.1 μm and p-NH2-0.1μm PS was not permanent. The algae recovered after long-term exposure but did not recover upon exposure to large-sizedPS. The recovery effect of MPs was reported by Mao et al.30

who found that the Chlorella pyrenoidosa could reduce theadverse effects of MPs by cell wall thickening, algae homo-aggregation, and algae−MP hetero-aggregation and thentriggering the algae activity, returning the cell structure tonormal. Zhang et al.20 also reported that the inhibition effect ofMPs on Skeletonema costatum photosynthesis was weakenedover time. In contrast to our findings, both Mao et al.30 and

Zhang et al.20 reported the recovery of microalgae followingexposure to microsized MPs of approximately 1 μm, while ourresults showed that the microalgae cannot be recovered in n-plain-2 μm PS medium. The discrepancy may have beencaused by the type of microalgae used, which have differentsizes and structures from S. obliquus. C. pyrenoidosa and S.costatum seem to have a larger size than S. obliquus, andmcirosized plastics may not fully affect their photosynthesis.

Photosynthesis Inhibition in Polystyrene- and HumicAcid-Treated S. obliquus. To understand the toxicmechanism of different sizes of PS, we further examined thechlorophyll fluorescence parameters (OJIP parameters) ofmicroalgae in different PS suspensions with and without HAafter a 4 h exposure. The results showed that the addition ofHA could reduce the adverse effect of n-plain-0.1 μm, n-plain-0.5 μm (Figure 5), and p-NH2-0.1 μm (Figure S4) on S.obliquus and restore the OJIP parameters to normal levels. In

Figure 6. SEM images of the interaction of PS beads and HA with microalgae after a 24 h exposure. (A) S. oblique in control group; (B) S. obliquusin medium with HA; (C) in medium with n-plain-0.1 μm; (D) C + HA; (E) in medium with n-plain-2 μm; and (F) B + HA.

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contrast, the addition of HA did not change the OJIPparameters of microalgae in n-plain-1 μm and n-plain-2 μmsuspensions.The microalgae exposed in n-plain-2 μm beads observed

greater variation on the OJIP parameters than that observed inn-plain-0.1 μm PS suspension, and this effect could not bealleviated by the addition of HA. This finding suggested thatthe toxic effect of large-sized particles was mainly caused by alight blockage, which weakened the photosynthesis of algae.Small-sized particles had little impact on algal photosynthesis.Such difference may have resulted from the comparable sizes ofn-plain-2 μm PS beads and microalgae cells, so that microalgaewere blocked from absorbing light energy and the PSII RCswere inactivated. The damage of photosynthesis RCs is anonreversible process and results in nonrecoverable growthinhibition.32,33 On the other hand, the n-plain-0.1 μm and p-NH2-0.1 μm PS beads did not significantly damage thephotosynthesis RCs and led to fully recovered reproductivecapacity.Toxicity Mechanism of Different-Sized Polystyrene

on Microalgae. The alleviation effect of HA on n-plain-0.1μm PS was also observed for D. magna.21,27 The authorsinferred that the addition of HA might form a corona aroundthe PS, which reduced the adhesion of nanosize PS to D.magna. To explore the interaction between the HA and PSbeads, we first monitored the fluorescence intensity offluorescent-labeled PS (f-PS-0.1 μm) beads in suspensionswith and without HA. The addition of HA caused an evidentdecrease in the fluorescence intensity from 3296 to 305.6 AUin f-PS-0.1 μm suspension (Figure S5). This findingdemonstrated that HA can be absorbed on the surface of f-PS, forming an “encapsulation layer” to wrap the entire PSbeads and cause fluorescence quenching of f-PS.The nanosize PS beads can be adsorbed on the surface of

microalgae and either encapsulate microalgae cells or enter thecell wall. When HA is present, the surface of PS is modified bya layer of HA, and the adhesive force between the nanoplasticbeads and the algal surface is altered. This hypothesis wasdemonstrated by the SEM images of microalgae exposed in n-plain-0.1 μm PS suspension with and without HA. The n-plain-0.1 μm PS particles were thickly dotted on the surface ofmicroalgae cells (Figure 6C), while the ones exposed insuspension with HA were covered by much fewer particles(Figure 6D). The adsorption of PS beads on microalgaesurface can affect the flow of substances and energy betweenmicroalgae cells and the environment, influence the metabo-lism of microalgae, reduce the fluidity of algal cell membranes,and thus cause the growth inhibition of algae.11,34 On the otherhand, since the wavelength of visible light (from 390 to 760nm) is on the same order of magnitude as the size of n-plain-0.1 μm PS, visible light can penetrate the PS beads and scatterin the suspension. Therefore, n-plain-0.1 μm PS showed noeffect on the photosynthesis of S. obliquus. In contrast, the sizesof n-plain-2 μm PS and S. obliquus cells are comparable to eachother and much longer than the wavelength of light; n-plain-2μm PS can easily block the light and thereby affect thephotosynthesis of algae.We also examined ROS produced by S. obliquus from

different types of MPs at 50 mg L−1. Only S. obliquus in p-NH2-0.1 μm, n-plain-0.1 μm, and n-plain-0.5 μm suspensionsgenerated a significantly higher amount of ROS compared tothe control group (Figure S6). Positively charged MPsproduced more ROS than negatively charged ones. A

subsequent increase of ROS levels suggests that S. obliquuswas under oxidative stress. This is also supported by a previousstudy that revealed that the adsorption of plastics couldpromote ROS production, and positively charged PS beadsresulted in a higher rate of ROS than the negatively chargedbeads.11 On the other hand, n-plain-1 μm and n-plain-2 μmMPs did not produce significant amounts of ROS at the testedconcentration.Apparently, the toxicity effects of MPs were size-dependent

and affected by the presence of HA. The light blockage waslikely the main mechanism responsible for the effect of thelarge-sized particles, while the adsorption of plastics tomicroalgae was the reason for the effects of the small-sizedones. Our findings also demonstrated that HA could alleviatethe effects of small-sized MPs but not the large-sized ones.Together, the present study has identified the importantfactors influencing the toxicity of MPs to algae. However,further investigation should be conducted to elucidateadditional toxic mechanisms, including the effects of larger(>2 μm) and smaller (<0.1 μm) MPs than the ones chosen inthe present study and other types of DOM present in theenvironment such as proteins, lignin, or even the extracellularpolymeric substances released by algae.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.est.9b06187.

Experimental methods of SEM (Text S1) and infraredspectrophotometry (Text S2); the SEM images andinfrared spectrophotometry of five types of MPs(Figures S1, S2); growth curves and OJIP parametersof S. obliquus exposure in p-NH2-PS suspensions withand without HA (Figures S3, 4); fluorescence intensityof f-PS-0.1 μm in the presence or absence of HA (FigureS5); and the ROS produced in different treatments(Figure S6) (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: jiangrf5@jnu.edu.cn.ORCIDRuifen Jiang: 0000-0002-0506-2775Jing You: 0000-0002-4006-8339Derek C. G. Muir: 0000-0001-6631-9776Eddy Y. Zeng: 0000-0002-0859-7572NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was financially supported by the NationalNatural Science Foundation of China (No. 21777058).

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DOI: 10.1021/acs.est.9b06187Environ. Sci. Technol. 2020, 54, 1782−1789

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