STOTEN-17105; No of Pages 9 Science of the Total Environment xxx (2014) xxx–xxx

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Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization Bin Xia ⁎, Bijuan Chen, Xuemei Sun, Keming Qu, Feifei Ma, Meirong Du Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

H I G H L I G H T S • • • •

Inhibition of marine microalgae by TiO2 NPs and bulk particles was evaluated. Aggregation of TiO2 NPs and bulk particles was observed in marine algal test medium. TiO2 NPs induced damage to algal cell membranes as detected by flow cytometry. Increased TiO2 nanotoxicity to algal cells was caused by internalization of NPs.

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Article history: Received 21 September 2014 Received in revised form 21 November 2014 Accepted 21 November 2014 Available online xxxx Editor: D. Barcelo Keywords: TiO2 nanoparticles Marine microalga Titanium dioxide Oxidative stress Membrane integrity Internalization

a b s t r a c t The toxicity of TiO2 engineered nanoparticles (NPs) to the marine microalga Nitzschia closterium was investigated by examining growth inhibition, oxidative stress and uptake. The results indicated that the toxicity of TiO2 particles to algal cells significantly increased with decreasing nominal particle size, which was evidenced by the 96 EC50 values of 88.78, 118.80 and 179.05 mg/L for 21 nm, 60 nm and 400 nm TiO2 particles, respectively. The growth rate was significantly inhibited when the alga was exposed to 5 mg/L TiO2 NPs (21 nm). Measurements of antioxidant enzyme activities showed that superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were first induced and subsequently inhibited following exposure to 5 mg/L TiO2 NPs. The depletion of antioxidant enzymes with a concomitant increase in malondialdehyde (MDA) levels and reactive oxygen species (ROS) posed a hazard to membrane integrity. A combination of flow cytometry analysis, transmission electron microscopy and Ti content measurement indicated that TiO2 NPs were internalized in N. closterium cells. The level of extracellular ROS, which was induced by TiO2 NPs under visible light, was negligible when compared with the intracellular ROS level (accounting for less than 6.0% of the total ROS level). These findings suggest that elevated TiO2 nanotoxicity in marine environments is related to increased ROS levels caused by internalization of TiO2 NPs. © 2014 Published by Elsevier B.V.

1. Introduction The production of engineered nanomaterials has increased exponentially over the past several years. As a result of this increase, engineered nanoparticles (NPs) are inevitably released into aquatic systems from the common sources, including personal care products, urban and industrial sewage and anti-fouling components of paints— eventually reaching the ocean (Handy et al., 2008; Klaine et al., 2008; Matranga and Corsi, 2012). Consequently, coastal waters are expected to represent the ultimate sink for NPs (Canesi et al., 2012). The stability of NPs is mainly related to their physicochemical properties (e.g., size, charge, and coating) and environmental factors (e.g., ionic strength and dissolved organic materials) (Liu et al., 2014; Zhao et al., 2014). For example, multi-walled carbon nanotubes can be ⁎ Corresponding author. E-mail address: [email protected] (B. Xia).

stably suspended in water as a result of dissolved organic materials. Humic acids (HAs) greatly enhanced suspension of both P- and CMWCNTs. The suspension enhancement was attributed to HA sorption, which increased electrostatic repulsion and steric hindrance between individual MWCNTs (Zhou et al., 2012). However, the high ionic strength can result in aggregation of TiO2 NPs (Zhu et al., 2014). Thus, the dispersion of NPs in seawater is likely to be more difficult than that in freshwater. It is reasonable to assume that the mechanisms by which NPs exert toxic effects on marine organisms are more complex than the mechanisms by which NPs exert toxic effects in freshwater. Therefore, findings from NP toxicity investigations in fresh water (Li et al., 2014a, 2014b; Ma et al., 2014; Zhao et al., 2013) must be taken into account but cannot be extrapolated to marine biota that exist in seawater. Among the various types of NPs, TiO2 NPs are currently of great interest. TiO2 NPs are widely used for many applications such as sunscreens, paints, coatings, solar cells, and photocatalytic water purification (Botta

http://dx.doi.org/10.1016/j.scitotenv.2014.11.066 0048-9697/© 2014 Published by Elsevier B.V.

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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et al., 2011). For Europe and USA, the highest predicted environmental concentrations in surface water were projected for TiO2 NPs compared with other NPs such as ZnO NPs, Ag NPs, carbon nanotubes (CNTs), and fullerenes (Gottschalk et al., 2009). The concentration of TiO2 NPs (20 and 300 nm) released from painted facades was reported to be as high as 3.5 × 108 particles/L in runoff water (Kaegi et al., 2008). Much attention has been devoted to the risk of increased TiO2 NP release on various ecosystems (Cañas-Carrell et al., 2011; Li et al., 2014a, 2014b; Wallis et al, 2014). Importantly, recent eco-toxicological data showed that TiO2 NPs were toxic to marine organisms, such as invertebrates (Barmo et al., 2013; Canesi et al., 2010; Zhu et al., 2011a, 2011b), cyanobacteria (Cherchi and Gu, 2010), and polychaetes (Galloway et al., 2010). Miller et al. (2010) reported that TiO2 NPs had no measurable effect on the population growth rates of marine phytoplankton (Thalassiosira pseudonana, Skeletonema marinoi, Dunaliella tertiolecta and Isochrysis galbana). On the contrary, the same group (Miller et al., 2012) reported that TiO2 NPs can be phytotoxic to the same marine phytoplankton under natural levels of ultraviolet radiation. However, these authors did not characterize intracellular and extracellular reactive oxygen species (ROS) production. Furthermore, they did not examine the impact of TiO2 NPs on membrane permeability. As such, the mechanism of TiO2 NP toxicity on marine phytoplankton remains unclear. In this study, we focus on the cytotoxicity of TiO2 NPs on marine phytoplankton, a key primary producer of the food web within the marine ecosystem. Nitzschia closterium, a marine eukaryotic unicellular diatom, was selected because it is widely present in the marine ecosystem and is a high-quality food for the culture of bivalves. The aim of this study was to investigate (1) the growth inhibition of marine microalgae caused by TiO2 NPs compared with their bulk counterparts; (2) the mechanism by which TiO2 NPs exert cytotoxic effects on algae cells, which was determined by examining antioxidant enzyme activities and ROS generation over time; and (3) the possibility that the NPs are internalized within cells. These results will enhance our understanding of the mechanism by which TiO2 NPs exert toxic effects on marine organisms. In addition, this work will provide essential data for studying the downstream effects of NPs on the microalgae-bivalve food chain in future studies. 2. Materials and methods 2.1. Materials TiO2 particles of three different sizes were used in this study: 21 nm NPs were acquired from Sigma Aldrich Company Ltd., China, while 60 nm NPs and 400 nm particles were obtained from Aladdin Reagent Inc., China. All the experiments were carried out using a TiO2 NP stock suspension (1000 mg/L) prepared in marine algal f/2 medium. A 30 min sonication (200 W; 4 × 104 Hz) was performed to homogenize the stock suspensions immediately before further use. The size distribution and zeta potential of TiO2 NPs and bulk particles (BPs) in the algal test media were measured with a particle size analyzer (Nano ZS Malvern Instruments, USA). The crystalline structure of the powder was examined using X-ray diffraction (XRD; Bruker D8 Advance TXS, Germany). In addition, the morphology of all the particles was observed using a transmission electron microscope (TEM, JEM-2100, JEOL, Japan). 2.2. Algal growth assays The unicellular marine diatom N. closterium was purchased from the Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, China. During the growth inhibition experiments, the algal cells were cultured in f/2 medium under 3500 lx light intensity with a 12:12 light–dark cycle (temperature: 20 ± 1 °C; salinity: 33.5 ± 0.5; pH: 7.8 ± 0.1). The light source in the tests was a cool fluorescent tube emitting light in the visible spectrum with a wavelength range of 400–700 nm. Stock solutions of TiO2 NPs and BPs were added to the algal medium for treatment at different concentrations (5, 10, 20, 40,

80, and 100 mg/L for 21 nm TiO2 NPs; 10, 20, 40, 80, 160, 240, and 360 mg/L for 60 nm TiO2 NPs; and 20, 40, 80, 160, 320, and 500 mg/L for TiO2 BPs). Algae (1 × 106 cells/mL) in the exponential phase of growth were exposed to TiO2 particles of three different sizes, and the algal biomass measurements were performed from 0 to 96 h. All treatments were performed in triplicate. Cells were counted using a microscope (Olympus AX70, Japan), and in each sample, the algal cells were counted a minimum of three times. The concentration of TiO2 suspended in the medium was measured using UV spectrophotometry and calculated according to the method described by Federici et al. (2007). 2.3. SEM and TEM imaging A scanning electron microscope (SEM, HITACHI S-4800) was used to observe the morphology of algae after TiO2 exposure. Transmission electron microscopy (TEM, JEM-2100, JEOL, Japan) was employed to characterize the size and morphology of TiO2 NPs as well as the interaction between TiO2 NPs and algal cells. After exposure to NPs for 24 h, all algal cells were fixed using 2.5% glutaraldehyde. Then, the samples were post-fixed in 1% osmic acid for 1 h and washed three times with phosphate-buffed saline (PBS, pH 7.2). The samples were dehydrated in increasing concentrations of acetone (30, 50, 70, 80, 90, and 100%; 20 min each time) at room temperature. EPON812 resin was used to permeate and impregnate the samples for 5 h. Ultrathin sections were made and placed on nickel grids for TEM observation and energy dispersive spectroscopy (EDS, INCA100, Oxfordshire, UK) analysis. 2.4. Enzymatic activity, lipid peroxidation and reactive oxygen species Time-dependent determination of the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), lipid peroxidation (LPO) and ROS generation in algal cells was conducted after exposure to TiO2 NPs (5 mg/L) for 2, 6, 12, 24, and 48 h. The algal cells were collected by centrifugation (5000 rpm) for 5 min at 4 °C, and the supernatant was removed for extracellular ROS determination. Meanwhile, the ROS levels of the algal culture medium in the absence or presence of 5 mg/L TiO2 NPs were determined after 2 h of exposure. The harvested algae were suspended in 2 mL of buffer solution (PBS, pH 7.2) and immediately disrupted by sonication (Ningbo Haishu Kesheng Ultrasonic Equipments Co., Ltd, KS-500F, China) for 3 min with a 3 s pause after each 3 s pulse in an ice bath. The cell homogenate was centrifuged at 2500 rpm for 10 min at 4 °C, and the supernatant was used for the measurement of enzyme activities, malondialdehyde (MDA) level and intracellular ROS generation. The activity of SOD was measured based on its ability to inhibit the reduction of nitro blue tetrazolium (NBT) by superoxide radicals generated with xanthine/xanthine oxidase. One unit of SOD activity (U) is defined as the amount of protein that inhibits the rate of NBT reduction by 50% in 1 mL of the reaction solution (Sun et al., 1988). CAT activity was determined according to Claiborne (1985) by measuring the initial rate of decrease in absorbance at 240 nm as a consequence of H 2 O2 consumption over 1 min. POD activity was assayed using guaiacol as a hydrogen donor by measuring the change at 470 nm over 1 min as described by Chance and Maehly (1955). The enzyme activities were calculated per mg of protein. Protein concentrations in the cell extracts were determined at 595 nm using the method developed by Bradford (1976), with bovine serum albumin as the standard. Enzyme assays were conducted using a BIO-RAD iMark microplate reader (Bio-RAD, Hercules, CA, USA). The LPO level was determined in terms of MDA (a product of lipid peroxidation) content, which was measured using the thiobarbituric acid (TBA) reaction as described previously (Erdelmeier et al., 1998). ROS generation was measured according to the method described

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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Fig. 1. Microalgae cell growth inhibition after exposure to 0–100 mg/L nominal concentrations of 21 nm TiO2 NPs (0–100 mg/L) (A), 60 nm TiO2 NPs (0–360 mg/L) (B) and TiO2 BPs (0–500 mg/L) (C). Panel D is the summary of 96 h EC50 of the three TiO2 particles. Data are showed as mean value ± standard deviations, n = 6. Bars followed by different letters (a– c) demonstrate significant differences among different treatments (p b 0.05).

by Wang et al. (2011). The sample (2.0 mL) was mixed with 50 μL of 10 μM DCFH-DA (2,7-dichlorofluorescein diacetate) and incubated at 37 °C for 1 h in darkness. The formation of DCF (2,7-dichlorofluorescein) was determined at an emission wavelength of 535 nm and an excitation wavelength of 485 nm using a fluorospectrophotometer (Hitachi, F4600, Japan). The ROS level in the presence of algae was expressed as the ratio of fluorescence emission intensity to cell density (cell number/mL).

2.5. Flow cytometry analysis At the end of the exposure period (48 h), the interaction between the microalgal cells and the TiO2 NPs was investigated for all the test solutions to determine whether the particles can enter the cells. Prior to the start of the experiment, the cells in the treatment solutions were washed with PBS buffer solution to remove NPs adsorbed onto the cells. This interaction was analyzed using a flow cytometer (BD FACS Calibur, USA). The side scatter (SSC) and forward scatter (FSC) intensities, which reflect the cellular density of microalgae (granularity) and the cell size, respectively, were recorded. Membrane integrity after exposure to TiO2 NPs for 2, 6, 12, 24, 48, 72, 96, and 120 h was examined using a flow cytometer. Propidium iodide (PI) was utilized to assess cell membrane integrity (Xiao et al., 2011). PI cannot cross the membrane and intercalate with nucleic acids inside the cell if the membrane is intact. The PI stock solution (100 μg/mL) was prepared by dissolving PI in PBS buffer (pH 7.2) and stored at 4 °C until use. The samples were incubated for 20 min in the PI solution (10 μg/mL for 2 × 106 cells) and were analyzed after staining. The fluorescence of PI was detected with an FL2 detector, and at least 20,000 events were collected and analyzed for each sample. The membrane integrity was expressed as follows: the percentage of membranedamaged cells induced by TiO2 NPs (%) = (the total number of membrane-damaged cells − the membrane-damaged cells in the blank control) × 100 / the total number of cells with intact membranes.

2.6. Intracellular and cell-wall-bound Ti contents The intracellular and cell-wall-bound Ti contents were measured using the method described by Franklin et al. (2000), with slight modifications. The algae were incubated in test media containing 5 mg/L TiO2 NPs for 48 h. After centrifugation, the supernatants, including TiO2 NPs, were removed. The remaining algal pellet was rinsed with PBS buffer three times to eliminate any unbound Ti. EDTA (5 mL) was added to the collected algal cells to combine the bound Ti from the cell walls, and the cells were then centrifuged (4000 rpm) for 30 min. The supernatant was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine the cell-wall-bound Ti content. The remaining algal cells were acid-digested with HNO3 for 12 h, and diluted with deionized water. ICP-MS was then used to measure the intracellular Ti content. 2.7. Statistical analysis The results are expressed as the means ± standard deviation (SD) of three replicates. The 96 h EC50 values (TiO2 NP concentration required to cause a 50% reduction in growth) and their associated 95% confidence intervals were calculated using the standard US EPA Probit Analysis Program (version 1.5). A one-way analysis of variance (ANOVA) with Tukey's range test was performed to identify significant differences among different groups (p b 0.05). 3. Results and discussion 3.1. Characteristics of TiO2 particles The characteristics of the TiO2 particles are shown in Table S1. The surface areas of the TiO2 particles of three different sizes increased with decreasing primary particle size. Aggregation of the three particle samples was observed in algal test media. The relatively low negative zeta potentials (from −13.2 mV to −6.4 mV) may lead to disequilibrium with

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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Fig. 2. Activities of SOD (A), CAT (B), POD (C), and MDA concentrations (D), intra- (E) and extra-cellular (F) ROS level of algal cells after exposed to TiO2 NPs (5 mg/L) for 2, 6, 12, 24, and 48 h. ROS level was the DCF-fluorescence normalized to cell density (cell number/mL). An asterisk demonstrates significant differences between the treatment and controls at the same time (p b 0.05).

chemicals in the surrounding water, causing charge heterogeneity, which leads to NP aggregation. The TEM images (Fig. S1) confirmed the aggregation and agglomeration. The propensity to aggregate in seawater has been reported in several previous studies for different types of nanoparticles. Morelli et al. (2012) showed that quantum dots (QDs) tended to aggregate and precipitate in raw seawater. Similar findings were observed by García-Negrete et al. (2013) for Au-citrate nanoparticles. In contrast to the clear surface and sharp edges of the TiO2 NPs in the ultrapure water, the edges of TiO2 NPs in the culture medium were unclear, most likely due to a coating of organic components from the medium (Lin et al., 2012). Notable aggregation and agglomeration of TiO2 NPs in algal culture medium were observed when compared with their BP counterparts. However, it cannot be concluded that smaller aggregates or individual NPs were absent from the TiO2 suspension prepared in algal medium. It was reported that salinity was the most dominant factor influencing the stability and aggregation of TiO2 NPs, followed by dissolved organic matter (Wang et al., 2014). In the marine environment, there were differences in the degree of aggregation between the NPs and BPs that resulted in distinctive cellular toxicological responses (Gunawan et al., 2013; Limbach et al., 2005). Thus,

the cytotoxicity of TiO2 NPs and BPs to marine microalgal cells was examined. 3.2. Effect of TiO2 particles on the growth of algae Fig. 1 shows the growth inhibition of N. closterium caused by TiO2 NPs and BPs. For TiO2 BPs, a concentration–response relationship was observed, with a calculated 96 h EC50 of 179.05 ± 20.76 mg/L. Similar concentration–response curves were recorded for both TiO2 NP treatments. Compared with TiO2 BP treatments, TiO2 NP treatments showed a less significant concentration–response relationship, likely due to the aggregation and agglomeration of TiO2 NPs (Fig. 1, S2). The calculated 96 h EC50 values for 21 and 60 nm TiO2 NPs were 88.78 ± 6.43 and 118.80 ± 12.78 mg/L, respectively, suggesting that the 21 nm TiO2 NPs were more toxic than the 60 nm TiO2 NPs and the TiO2 BPs. Furthermore, significant differences were detected in the 96 h EC50 values of the three different particles tested, indicating the size effect of TiO2 NPs. This result supported previous findings that toxicity toward freshwater microalgae was related to the primary particle size and, consequently, to the available surface area of the particles (Aruoja et al., 2009;

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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Fig. 3. Membrane integrity of algal cells detected using PI dyes by flow cytometry (A) and percentage of membrane-damage cells calculated from the data in panel A (B). Flow cytometry images of Nitzschia closterium cells were obtained after exposure to 5 mg/L TiO2 NPs for 2–120 h. All the samples were determined by side scatter and FL2 detector (564–606 nm) (R1, red region: cells with impaired membrane; R2, blue region: intact cells). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Hartmann et al., 2010). Primary size-dependent effects have also been reported by researchers examining other types of NPs, such as CeO2 (Manier et al., 2013; Van Hoecke et al., 2009). Additionally, considering that the dissolution of TiO2 NPs was negligible in water (Dasari et al., 2013; Shi et al., 2013), the effects were mainly related to the particle size of the NPs. Based on the data reported here, the 21 nm TiO2 NPs were selected for the subsequent toxicity studies. In order to study the effect of TiO2 NPs at lower concentrations on the N. closterium, the concentration of TiO2 NPs should be confirmed.

A

Figure S3 illustrates the ability of TiO2 NPs with a primary particle size of 21 nm to inhibit the growth of marine microalgae. Growth promotion of the algal cells treated by TiO2 NPs at low concentrations (1, 5, and 10 mg/L) was detected, which may be explained by the enhanced ROS generation (Fig. 2E and F). This phenomenon was attributed to hormetic responses. Hormesis is the modest stimulation of a particular function (e.g., growth rate) at low doses and inhibition of the same function at high doses, which can be attributed to an adaptive response to toxicants (Calabrese et al., 2012). A similar result was observed by Hartmann et al.

B

Fig. 4. SEM image of algal cells after 48 h exposure (A: control, B: 5 mg/L of TiO2 NPs). Arrows indicate attachment of TiO2 NPs to microalgae cells.

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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A

C

B

D

Fig. 5. TEM image of Nitzschia closterium after exposure to TiO2 NPs for 48 h. (A) Untreated algal cell; (B) algal cells after being treated with 5 mg/L TiO2 NPs; (C) high magnification TEM image of the chosen region from (B); and (D) the corresponding EDX of the chosen region from (C).

(2010). It was reported that the enhanced ROS, induced by TiO2 NPs, can stimulate the growth of plants due to the promotion of total antioxidant capacity and the high rate of photosynthesis (Carol and Dolan, 2006; Gao et al., 2008). Subsequently, the toxicity of TiO2 NPs began to appear after exposure for 4 h. At a high concentration of TiO2 NPs (20 mg/L), inhibition of the algal cells was observed throughout the exposure time (24 h). In the subsequent 20 h, except for 1 mg/L of exposure concentration, algal cells were more significantly inhibited by exposure to TiO2 NPs at 5, 10, and 20 mg/L concentrations compared with controls, with growth inhibition rates of more than 10%. Therefore, 5 mg/L was chosen for use in the subsequent study on cellular biochemical responses. Meanwhile, 1 mg/L was selected as the dietary exposure concentration to study the downstream effect of TiO2 NPs on the microalgae–bivalve food chain in the future work. This choice was supported by Aruoja et al. (2009), who determined that the 72 h noobserved-effect concentrations (NOEC) and EC50 values of TiO2 NPs were 0.98 and 5.83 mg/L, respectively, in the presence of the microalga Pseudokirchneriella subcapitata. On the other hand, the behavior and toxicity of NPs in seawater are likely very different compared to that in freshwater (Matranga and Corsi, 2012). In marine environment, the self-aggregation and agglomeration of NPs were related to the toxicity on the marine organisms (Baker et al., 2014). 3.3. Oxidative stress, ROS generation and membrane integrity To minimize the aggregation of NPs in the algal culture medium and changes in the speciation of NPs, the toxicity of TiO2 NPs to algal cells was determined over 48 h exposure periods. We hypothesized that

the toxicity of TiO2 NPs was dependent on ROS derived from internalization and/or photoactivity; therefore, we examined the responses of the antioxidant defense system to intracellular and extracellular ROS generation in N. closterium. The antioxidant enzyme activities, MDA values and ROS generation over time (at 0, 2, 6, 12, 24 and 48 h) are depicted in Fig. 2. After 2 h of exposure to NPs, the intracellular ROS level in N. closterium was significantly higher (two-fold) than that of the control. Meanwhile, the activities of SOD, CAT, and POD in NP treated cells increased 1.1, 2.9, and 1.7 times when compared with the controls, respectively. With the exception of SOD, CAT and POD activities, the algal cells in the treatment groups exhibited significant differences with respect to the controls (p b 0.05). This observed increase in the enzyme activities of algal cells was attributed to the early stress responses induced by excessive intracellular ROS generation. SOD is an antioxidant enzyme that uses free radicals as a substrate and is responsible for catalyzing the dismutation of the superoxide radical O•− 2 to H2O2 and O2. Hence, increased SOD activities are expected to cause an increase in the level of H2O2, which must be scavenged by CAT and POD. Based on our results, the increased SOD activities were coupled with increases in CAT and POD activities. This effect was considered to be the hormesis response, which has been widely described as a stimulatory effect caused by short-term exposure to a low dose of a toxicant (Calabrese et al., 2012). Subsequently, depletion of antioxidant enzyme activities (6–48 h), including the activities of SOD, CAT and POD, was detected along with ROS accumulation, suggesting that the enhanced ROS level had exceeded the scavenging ability of the antioxidant enzymes (Fig. 2). TiO2 NPs could act as a photocatalyst to produce ROS, which are often

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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B

Percentage of more complexe cells in the algae population(%)

7.00 6.00

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5.00 4.00 3.00 2.00 1.00 0.00 Control

NPs

Fig. 6. Scatter analysis of TiO2 NP interaction with Nitzschia closterium after 48 h exposure. (A) and (B) express the side scatter (SSC) and forward scatter (FSC) for control and exposed cells with TiO2 NPs, respectively. (C) The graphic indicates the percentage of more complex cells (mean ± standard deviation, n = 3) in the algae population after being untreated and treated by 5 mg/L TiO2 NPs. Asterisk demonstrates significant differences between the treatments and controls (p b 0.05).

phototoxic to cells in vitro. For example, increased levels of ROS were detected in TiO2 anatase nanoparticles (50 nm) with visible light (Lipovsky et al., 2012). Similarly, Miller et al. (2012) reported that TiO2 NPs (81% anatase, 19% rutile, 15–30 nm in size) were toxic to marine phytoplankton under cool white fluorescent lights. As shown in Fig. S4, a significant elevation in the ROS level was observed in culture medium treated with 5 mg/L TiO2 NPs for 2 h compared with the controls, which confirmed the photoactivity of TiO2 NPs. Additionally, it

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a* Ti (10-5 μg/cell)

20

Control NPs

16 12 8

b* 4 0 Cell-wall-bound

Intracellular

Fig. 7. Intracellular and cell-wall-bound Ti contents of control cells and cells treated with 5 mg/L TiO2 NPs in algal test media. Asterisk demonstrates significant differences between the treatment and controls (p b 0.05). Different letters (a–b) indicate significant differences between the intracellular and cell wall-bound Ti contents.

has been reported that the induction of intracellular ROS by NPs was the dominant effect in nanotoxicity (Menard et al., 2011). However, studies examining the induction of both intracellular and extracellular ROS by TiO2 NPs have not been reported until now. In the present study, although the extracellular ROS level of treated algal cells was significantly higher than that of the untreated algal cells for all the exposure times (p b 0.05), the extracellular ROS generation induced by TiO2 NPs under visible light was negligible compared with the intracellular ROS level, and it accounted for less than 6.0% of the total ROS. The blue regions (R2) and red regions (R1) in each image in Fig. 3 indicate intact algal cells and membrane-damaged cells, respectively. A large number of red dots indicate that more algal cell membranes were damaged. The percentage of membrane-damaged cells induced by NP exposure is shown in Fig. 3B. After 2 h of exposure, 6.16% of the cells were membrane-damaged. Over 6 h, the highest percentage of membrane-damaged cells (up to 7.97%) was detected; subsequently, the values decreased until 24 h (0.79%). After this period, the percentage of membrane-damaged cells increased over time. Similar to our result, Wang et al. (2011) reported that CuO NPs posed a hazard to the membrane integrity of Microcystis aeruginosa. Similarly, bacterial (Escherichia coli) membranes were highly damaged by CuO NPs (18%) (Zhao et al., 2013). A sequence of membrane damage, resistance, recovery and damage was observed in microalgal cells incubated with TiO2 NPs. The time-dependent trends in the percentage of membranedamaged cells were consistent with the intracellular ROS level (Fig. 2E), which confirmed that the excessive intracellular ROS production was the dominant factor causing oxidative stress in marine algal cells exposed to TiO2 NPs.

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

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3.4. Internalization of NPs by algal cells The SEM images in Fig. 4 show that the TiO2 NPs were adsorbed onto the algal cell wall after 48 h of interaction with TiO2 NPs (5 mg/L). Additional TEM observations (Fig. 5) confirmed this finding. Algal cells were partially covered by a larger number of TiO2 NPs according to EDX analysis. As observed in the TEM images (Fig. 5), particles of 20–30 nm adhered to the algal cells, illustrating that TiO2 NPs could interact with the marine microalgae in seawater. Furthermore, it is possible that TiO2 NPs could destroy the cell wall and enter the cells, inducing plasmolysis (Fig. S5). This finding was supported by Gong et al. (2011) and Chen et al. (2012). The adsorption of TiO2 NPs on microalgae could contribute to the observed toxicity of TiO2 NPs through a direct physical effect, such as the disruption of the cell wall (Lin et al., 2012), or an indirect physical effect, such as the reduction of the available light necessary for algal growth (shading effect) (Aruoja et al., 2009; Ji et al., 2011) and/or the limitation of nutrient uptake by the algal cells (Hartmann et al., 2010). Ji et al. (2011) reported that mechanisms other than the shading effect of NPs alone regulated the nanotoxicity of NPs to algae. Similarly, Aruoja et al. (2009) showed that NPs had a negligible shading effect on P. subcapitata microalgae. Additionally, Van Hoecke et al. (2009) reported that adsorption of approximately 50% phosphate to the particle surface was observed in the 32 mg/L CeO2 NP suspensions. However, the reduction of phosphate in the algal medium had no significant effect on the algal growth rate. These findings suggest that the shading effect and nutrient depletion might not be the dominant causes of the algal toxicity of NPs. In addition, given the low level of dissolution of TiO2 NPs in water, as previously reported by Dasari et al. (2013) and Shi et al. (2013), it can be assumed that the effects of dissolved Ti can be considered to be negligible in this study. Fig. 6 presents the results of flow cytometry analysis. A significant interaction (adsorption and/or uptake) between TiO2 NPs and the microalga N. closterium was detected in N. closterium cells, as evidenced by an increase in the intensity of side scatter (SSC, Fig. 6B) compared with the control (Fig. 6A) without modulation of FSC-H (indicating intact algal cells). The percentage of more complex cells increased to 5.34% after exposure to a 5 mg/L TiO2 NP suspension compared with the controls, as indicated by the SSC intensity. The pronounced increase in SSC intensity (indicating cellular granularity) can indicate the uptake/ internalization of TiO2 NPs by the algae, as all cells were pre-rinsed with PBS to remove the TiO2 NPs on the surface of the cells. In previous studies, the intracellular presence of solid particulates has been analyzed by flow cytometry and SSC measurement in both algae and bacteria (Gunawan et al., 2013; Khatoon et al., 2011; Kumar et al., 2011; Manier et al., 2013). Additionally, the intracellular and cell-wall-bound Ti contents of N. closterium were determined after the algae were treated with 5 mg/L TiO2 NPs for 48 h (Fig. 7). The significantly higher intracellular Ti content in exposed cells when compared with untreated cells (p b 0.05) confirmed the internalization of TiO2 NPs in N. closterium. Moreover, the cell-wall-bound Ti content was significantly higher than the intracellular Ti content (p b 0.05). It is known that the cell wall is the primary site for NP interaction with algae and acts as a key barrier to the uptake of NPs. Vrieling et al. (1999) reported that the pore size of the cell wall in diatoms varied from 3 to 50 nm. If the size of NPs is smaller than that of the pore, the NPs can enter the cell by endocytosis, diffusion or the action of carrier proteins (Navarro et al., 2008). Changes in the permeability of the cell wall occur during reproduction. The newly synthesized cell wall could more readily allow the entrance of NPs (Ovecka et al., 2005). Additionally, the interaction between cells and NPs can induce the formation of new pores that are larger in size compared with general pores, which may increase the likelihood of NP internalization through the cell wall (Navarro et al., 2008). It is expected that TiO2 NPs ranging in size from 20 to 30 nm, as determined by TEM observation (Fig. 5C), pass through the cell wall of N. closterium. Similar observations were published by Cherchi et al.

(2011), who demonstrated internalization of TiO2 NPs into Anabaena variabilis cells through their membranes, and Wang et al. (2011), who reported CuO NP internalization in M. aeruginosa cells. 4. Conclusions To the best of our knowledge, this is one of the first systematic studies examining the effects of TiO2 NPs on marine microalgae and investigating the mechanisms of growth inhibition, biological responses and cytotoxicity. TiO2 NPs exhibited greater toxicity than BPs, and a significant size-dependent relationship was revealed according to the EC50 values. When exposed to TiO2 NPs, the growth of marine microalgae is inhibited due to NP-induced oxidative stress, as demonstrated by an overwhelmed antioxidant defense system, increased lipid peroxidation, and decreased membrane integrity. The enhanced internalization of particles contributed to the toxic effect of TiO2 NPs on marine algal cells. Based on our findings, the potential effects of engineered nanomaterials in the marine environment should be considered. Further research is required to study the biotransformative and genotoxic effects of engineered nanomaterials on marine microalgae and other species of marine phytoplankton as well as the cascading effect of NPs on the microalgae-bivalve food chain. Acknowledgments This work was funded by the Special Scientific Research Funds for Central Non-profit Institute, Chinese Academy of Fishery Sciences (2013A02YQ02); the National Natural Science Foundation of China (41206100); and the Special Scientific Research Funds for Central Non-profit Institutes, Yellow Sea Fisheries Research Institutes (20603022012020). We thank Qian Han, Lili Huang, Xiao Shi, and Qingting Zhou for their technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.11.066. References Aruoja V, Dubourguier H-C, Kasemets K, Kahru A. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 2009;407:1461–8. Baker TJ, Tyler CR, Galloway TS. Impacts of metal and metal oxide nanoparticles on marine organisms. Environ. Pollut. 2014;186:257–71. Barmo C, Ciacci C, Canonico B, Fabbri R, Cortese K, Balbi T, et al. In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve Mytilus galloprovincialis. Aquat. Toxicol. 2013;132:9–18. Botta C, Labille J, Auffan M, Borschneck D, Miche H, Cabie M, et al. TiO2-based nanoparticles released in water from commercialized sunscreens in a life-cycle perspective: structures and quantities. Environ. Pollut. 2011;159:1543–8. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72(1): 248–54. Calabrese EJ, Iavicoli I, Calabrese V. Hormesis: why it is important to biogerontologists. Biogerontology 2012;13:215–35. Cañas-Carrell JE, Qi B, Li S, Maul JD, Cox SB, Das S, et al. Acute and reproductive toxicity of nano-sized metal oxides (ZnO and TiO2) to earthworms (Eisenia fetida). J. Environ. Monit. 2011;13:3351–7. Canesi L, Fabbri R, Gallo G, Vallotto D, Marcomini A, Pojana G. Biomarkers in Mytilus galloprovincialis exposed to suspensions of selected nanoparticles (Nano carbon black, C60 fullerene, Nano-TiO2, Nano-SiO2). Aquat. Toxicol. 2010;100:168–77. Canesi L, Ciacci C, Fabbri R, Marcomini A, Pojana G, Gallo G. Bivalve molluscs as a unique target group for nanoparticle toxicity. Mar. Environ. Res. 2012;76:16–21. Carol RJ, Dolan L. The role of reactive oxygen species in cell growth: lessons from root hairs. J. Exp. Bot. 2006;57:1829–34. Chance B, Maehly AC. Assay of catalase and peroxide. Methods Enzymol. 1955;2:764–75. Chen L, Zhou L, Liu Y, Deng S, Wu H, Wang G. Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii. Ecotoxicol. Environ. Saf. 2012;84: 155–62. Cherchi C, Gu AZ. Impact of titanium dioxide nanomaterials on nitrogen fixation rate and intracellular nitrogen storage in Anabaena variabilis. Environ. Sci. Technol. 2010;44: 8302–7.

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

B. Xia et al. / Science of the Total Environment xxx (2014) xxx–xxx Cherchi C, Chernenko T, Diem M, Gu AZ. Impact of nano titanium dioxide exposure on cellular structure of Anabaena variabilis and evidence of internalization. Environ. Toxicol. Chem. 2011;30:861–9. Claiborne A. Catalase Activity. Boca Raton: CRC Press; 1985. Dasari TP, Pathakoti K, Hwang H-M. Determination of the mechanism of photoinduced toxicity of selected metal oxide nanoparticles (ZnO, CuO, Co3O4 and TiO2) to E. coli bacteria. J. Environ. Sci. (China) 2013;25:882–8. Erdelmeier I, Gérard-Monnier D, Yadan JC, Chaudière J. Reactions of N-methyl-2phenylindole with malondialdehyde and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric assay of lipid peroxidation. Chem. Res. Toxicol. 1998;11:1184–94. Federici G, Shaw BJ, Handy RD. Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects. Aquat. Toxicol. 2007;84:415–30. Franklin NM, Stauber JL, Markich SJ, Lim RP. pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.). Aquat. Toxicol. 2000;48:275–89. Galloway T, Lewis C, Dolciotti I, Johnston BD, Moger J, Regoli F. Sublethal toxicity of nanotitanium dioxide and carbon nanotubes in a sediment dwelling marine polychaete. Environ. Pollut. 2010;158:1748–55. Gao F, Liu C, Qu C, Zheng L, Yang F, Su M, et al. Was improvement of spinach growth by nano-TiO2 treatment related to the changes of Rubisco activase? Biometals 2008; 21:211–7. García-Negrete CA, Blasco J, Volland M, Rojas TC, Hampel M, García-Negrete A, et al. Behaviour of Au-citrate nanoparticles in seawater and accumulation in bivalves at environmentally relevant concentrations. Environ. Pollut. 2013;174:134–41. Gong N, Shao K, Feng W, Lin Z, Liang C, Sun Y. Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris. Chemosphere 2011;83:510–6. Gottschalk F, Sonderer T, Scholz RW, Nowack B. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for different regions. Environ. Sci. Technol. 2009;43:9216–22. Gunawan C, Sirimanoonphan A, Teoh WY, Marquis CP, Amal R. Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii. J. Hazard. Mater. 2013;260:984–92. Handy RD, Owen R, Valsami-Jones E. The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs. Ecotoxicology 2008;17:315–25. Hartmann NB, Von der Kammer F, Hofmann T, Baalousha M, Ottofuelling S, Baun A. Algal testing of titanium dioxide nanoparticles—testing considerations, inhibitory effects and modification of cadmium bioavailability. Toxicology 2010;269:190–7. Ji J, Long Z, Lin D. Toxicity of oxide nanoparticles to the green algae Chlorella sp. Chem. Eng. J. 2011;170:525–30. Kaegi R, Ulrich A, Sinnet B, Vonbank R, Wichser A, Zuleeg S, et al. Synthetic TiO2 nanoparticle emission from exterior facades into the aquatic environment. Environ. Pollut. 2008;156:233–9. Khatoon I, Vajpayee P, Singh G, Pandey AK, Dhawan A, Gupta KC, et al. Determination of internalization of chromium oxide nano-particles in Escherichia coli by flow cytometry. J. Biomed. Nanotechnol. 2011;7:168–9. Klaine SJ, Alvarez PJJ, Batley GE, Fernandes TF, Handy RD, Lyon DY, et al. Nanomaterials in the environment: behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 2008;27:1825–51. Kumar A, Pandey AK, Singh SS, Shanker R, Dhawan A. A flow cytometric method to assess nanoparticle uptake in bacteria. Cytometry A 2011;79A:707–12. Li S, Pan X, Wallis LK, Fan Z, Chen Z, Diamond SA. Comparison of TiO2 nanoparticle and graphene–TiO2 nanoparticle composite phototoxicity to Daphnia magna and Oryzias latipes. Chemosphere 2014a;112:62–9. Li S, Wallis LK, Ma H, Diamond SA. Phototoxicity of TiO2 nanoparticles to a freshwater benthic amphipod: are benthic systems at risk? Sci. Total Environ. 2014b;466–467: 800–8. Limbach LK, Li YC, Grass RN, Brunner TJ, Hintermann MA, Muller M, et al. Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Environ. Sci. Technol. 2005;39:9370–6. Lin D, Ji J, Long Z, Yang K, Wu F. The influence of dissolved and surface-bound humic acid on the toxicity of TiO2 nanoparticles to Chlorella sp. Water Res. 2012;46:4477–87.

9

Lipovsky A, Levitski L, Tzitrinovich Z, Gedanken A, Lubart R. The different behavior of rutile and anatase nanoparticles in forming oxy radicals upon illumination with visible light: an EPR study. Photochem. Photobiol. 2012;88:14–20. Liu Y, Li S, Chen Z, Megharaj M, Naidu R. Influence of zero-valent iron nanoparticles on nitrate removal by Paracoccus sp. Chemosphere 2014;108:426–32. Ma H, Wallis LK, Diamond S, Li S, Canas-Carrell J, Parra A. Impact of solar UV radiation on toxicity of ZnO nanoparticles through photocatalytic reactive oxygen species (ROS) generation and photo-induced dissolution. Environ. Pollut. 2014;193:165–72. Manier N, Bado-Nilles A, Delalain P, Aguerre-Chariol O, Pandard P. Ecotoxicity of nonaged and aged CeO2 nanomaterials towards freshwater microalgae. Environ. Pollut. 2013;180:63–70. Matranga V, Corsi I. Toxic effects of engineered nanoparticles in the marine environment: model organisms and molecular approaches. Mar. Environ. Res. 2012;76:32–40. Menard A, Drobne D, Jemec A. Ecotoxicity of nanosized TiO2. Review of in vivo data. Environ. Pollut. 2011;159:677–84. Miller RJ, Lenihan HS, Muller EB, Tseng N, Hanna SK, Keller AA. Impacts of metal oxide nanoparticles on marine phytoplankton. Environ. Sci. Technol. 2010;44:7329–34. Miller RJ, Bennett S, Keller AA, Pease S, Lenihan HS. TiO2 nanoparticles are phototoxic to marine phytoplankton. PLoS One 2012;7:e30321. Morelli E, Cioni P, Posarelli M, Gabellieri E. Chemical stability of CdSe quantum dots in seawater and their effects on a marine microalga. Aquat. Toxicol. 2012;122:153–62. Navarro E, Baun A, Behra R, Hartmann NB, Filser J, Miao A-J, et al. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008;17:372–86. Ovecka M, Lang I, Baluska F, Ismail A, Illes P, Lichtscheidl IK. Endocytosis and vesicle trafficking during tip growth of root hairs. Protoplasma 2005;226:39–54. Shi HB, Magaye R, Castranova V, Zhao JS. Titanium dioxide nanoparticles: a review of current toxicological data. Part. Fibre Toxicol. 2013:10. Sun YI, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 1988;34(3):497–500. Van Hoecke K, Quik JTK, Mankiewicz-Boczek J, De Schamphelaere KAC, Elsaesser A, Van der Meeren P, et al. Fate and effects of CeO2 nanoparticles in aquatic ecotoxicity tests. Environ. Sci. Technol. 2009;43:4537–46. Vrieling EG, Beelen TPM, van Santen RA, Gieskes WWC. Diatom silicon biomineralization as an inspirational source of new approaches to silica production. J. Biotechnol. 1999; 70:39–51. Wallis LK, Diamond SA, Ma H, Hoff DJ, Al-Abed SR, Li S. Chronic TiO2 nanoparticle exposure to a benthic organism, Hyalella azteca: impact of solar UV radiation and material surface coatings on toxicity. Sci. Total Environ. 2014;499:356–62. Wang ZY, Li J, Zhao J, Xing BS. Toxicity and internalization of CuO nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter. Environ. Sci. Technol. 2011;45:6032–40. Wang H, Burgess RM, Cantwell MG, Portis LM, Perron MM, Wu F, et al. Stability and aggregation of silver and titanium dioxide nanoparticles in seawater: role of salinity and dissolved organic carbon. Environ. Toxicol. Chem. 2014;33:1023–9. Xiao X, Han ZY, Chen YX, Liang XQ, Li H, Qian YC. Optimization of FDA–PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa. Phys. Chem. Earth A/B/C 2011;36:424–9. Zhao J, Wang ZY, Dai YH, Xing BS. Mitigation of CuO nanoparticle-induced bacterial membrane damage by dissolved organic matter. Water Res. 2013;47:4169–78. Zhao J, Wang ZY, White JC, Xing BS. Graphene in the aquatic environment: adsorption, dispersion, toxicity and transformation. Environ. Sci. Technol. 2014:9995–10009. Zhou XZ, Shu L, Zhao HB, Guo XY, Wang XL, Tao S, et al. Suspending multi-walled carbon nanotubes by humic acids from a peat soil. Environ. Sci. Technol. 2012;46:3891–7. Zhu XS, Zhou J, Cai ZH. TiO2 nanoparticles in the marine environment: impact on the toxicity of tributyltin to abalone (Haliotis diversicolor supertexta) embryos. Environ. Sci. Technol. 2011a;45:3753–8. Zhu XS, Zhou J, Cai ZH. The toxicity and oxidative stress of TiO2 nanoparticles in marine abalone (Haliotis diversicolor supertexta). Mar. Pollut. Bull. 2011b;63:334–8. Zhu M, Wang HT, Keller AA, Wang T, Li FT. The effect of humic acid on the aggregation of titanium dioxide nanoparticles under different pH and ionic strengths. Sci. Total Environ. 2014;487:375–80.

Please cite this article as: Xia B, et al, Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: Growth inhibition, oxidative stress and internalization..., Sci Total Environ (2014), http://dx.doi.org/10.1016/j.scitotenv.2014.11.066

Interaction of TiO2 nanoparticles with the marine microalga Nitzschia closterium: growth inhibition, oxidative stress and internalization.

The toxicity of TiO2 engineered nanoparticles (NPs) to the marine microalga Nitzschia closterium was investigated by examining growth inhibition, oxid...
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