当前位置:首页 >> 冶金/矿山/地质 >>

Cytotoxicity of ZnO NPs towards fresh water algae Scenedesmus obliquus at low exposure concentration


Aquatic Toxicology 162 (2015) 29–38

Contents lists available at ScienceDirect

Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Cytotoxicity of ZnO NPs towards fresh water algae Scenedesmus obliquus at low exposure concentrations in UV-C, visible and dark conditions
M. Bhuvaneshwari a , V. Iswarya a , S. Archanaa b , G.M. Madhu c , G.K. Suraish Kumar b , R. Nagarajan d , N. Chandrasekaran a , Amitava Mukherjee a,?
a

Centre for Nanobiotechnology, VIT University, Vellore 632014, India Department of Biotechnology, IIT Madras, India c Department of Chemical Engineering, M.S. Ramaiah Institute of Technology, Bangalore, India d Department of Chemical Engineering, IIT Madras, India
b

a r t i c l e

i n f o

a b s t r a c t
Continuous increase in the usage of ZnO nanoparticles in commercial products has exacerbated the risk of release of these particles into the aquatic environment with possible harmful effects on the biota. In the current study, cytotoxic effects of two types of ZnO nanoparticles, having different initial effective diameters in ?ltered and sterilized lake water medium [487.5 ± 2.55 nm for ZnO-1 NPs and 616.2 ± 38.5 nm for ZnO-2 NPs] were evaluated towards a dominant freshwater algal isolate Scenedesmus obliquus in UV-C, visible and dark conditions at three exposure concentrations: 0.25, 0.5 and 1 mg/L. The toxic effects were found to be strongly dependent on the initial hydrodynamic particle size in the medium, the exposure concentrations and the irradiation conditions. The loss in viability, LDH release and ROS generation were signi?cantly enhanced in the case of the smaller sized ZnO-1 NPs than in the case of ZnO-2 NPs under comparable test conditions. The toxicity of both types of ZnO NPs was considerably elevated under UV-C irradiation in comparison to that in dark and visible light conditions, the effects being more enhanced in case of ZnO-1 NPs. The size dependent dissolution of the ZnO NPs in the test medium and possible toxicity due to the released Zn2+ ions was also noted. The surface adsorption of the nanoparticles was substantiated by scanning electron microscopy. The internalization/uptake of the NPs by the algal cells was con?rmed by ?uorescence microscopy, transmission electron microscopy, and elemental analyses. ? 2015 Elsevier B.V. All rights reserved.

Article history: Received 30 September 2014 Received in revised form 3 March 2015 Accepted 4 March 2015 Available online 7 March 2015 Keywords: Effective particle size ZnO NPs Exposure condition Dissolution Internalization ROS

1. Introduction The current production rate of ZnO nanoparticles worldwide is about 528 t/year (Zhang and Saebfar, 2010), and they are increasingly used in paint formulations, sun-screen creams, hair care products, food additives (as an essential nutrient) and tooth pastes (Heng et al., 2010; Osmond and Mccall, 2010; Serpone et al., 2007). The ZnO nanoparticles from these products may be released into the aquatic environment, and thus, cause considerable harm to the aquatic organisms. The effect of the metal oxide nanoparticles needs to be assessed on the various aquatic organisms as an integral part of environmental risk assessment (Blinova et al., 2010).

? Corresponding author. Tel.: +91 416 2202620; fax: +91 416 2243092. E-mail addresses: amit.mookerjea@gmail.com, amitav@vit.ac.in (A. Mukherjee). http://dx.doi.org/10.1016/j.aquatox.2015.03.004 0166-445X/? 2015 Elsevier B.V. All rights reserved.

As a model organism, various species of freshwater algae have been subjected to toxicological studies as they play an important role in the aquatic ecosystem, including biomass production and as primary producers in the aquatic food chain. The microalgae constitute the basis of aquatic food webs and also assist in the puri?cation of water (Adams et al., 2006; Wang et al., 2009; Ji et al., 2011). Microalgae have been used as the bio-indicators of fresh water pollutants owing to their high bioaccumulation abilities (Barhoumi and Dewez, 2013). The correlation between water quality parameters, algal growth and the amount of photosynthetic pigment produced was highlighted by Zhou et al. (2012). The eco-toxicological data of metal oxide nanoparticles is still in a nascent stage, even though their usage in consumer products has sharply increased recently (Kahru et al., 2008). The toxicity of ZnO nanoparticles on the aquatic organisms was observed to depend strongly on their particle size (Gurr et al., 2005) and stability in the medium (Panessa-Warren et al., 2009). Peng et al. (2011)

30

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

studied the toxic effect of ZnO spheres of various diameters – 6.3 nm (small spherical) and 15.7 nm (large spherical) and concluded that the small spherical shaped nanoparticles exhibited elevated toxicity against the diatoms. The dissolution of Zn2+ from ZnO NPs can also account for its toxic potentiality (Aruoja et al., 2009; Lee and An, 2013; Chen et al., 2012). A few researchers (Lee and An, 2013; Chen et al., 2012) have noted that the ZnO NPs caused destabilization of the cell membrane, which in turn inhibited the growth of algae in a linear proportion to the NPs concentration. Ji et al. (2011) observed that the toxicity of ZnO NPs towards green algae Chlorella sp. followed a decreasing order of Zn2+ > nano-ZnO > bulkZnO, when the NPs concentration was lower than 50 mg/L. As the concentration increased beyond 50 mg/L, the nano scale ZnO particles had a higher toxic potential than Zn2+ ions. In another case, Aruoja et al. (2009) reported that the bulk and nano ZnO particles exhibited the same level of toxicity as compared to ZnSO4 (72 h, EC50 ? 0.04 mg/L). Green microalgae Chlamydomonas reinhardtii were noted to be highly sensitive towards ZnO NPs even at low exposure concentrations (8-day EC50 ≥ 0.01 mg/L) (Gunawan et al., 2013). The toxicity of the photo-activated ZnO NPs on the green algae Pseudokirchneriella subcapitata was recently assessed by Lee and An (2013) who reported that the growth of algae was inhibited under UVA, UVB and visible light conditions without any signi?cant difference among the three. Summarizing the previous studies on algal toxicity behaviour of the ZnO nanoparticle, it can be inferred that exposure concentrations, colloidal stability and ionic dissolution in the medium, as well as irradiation conditions, contributed signi?cantly towards toxic potential. UV-C is a high energy radiation with a wavelength less than 280 nm, than the other UV irradiations, such as UV-A and UV-B (Vileno et al., 2007). ZnO nanoparticles have a higher photocatalytic capacity under UV-C irradiation compared to UV-A and UV-B conditions. Hence, ZnO nanoparticles–UV-C combination has been utilised widely in photodegradation studies (K?rans ? an et al., 2015) and for the disinfection of drinking water (Noroozi et al., 2011). According to previous studies, UV-C irradiation signi?cantly accelerated the dissolution of ZnO (Han et al., 2010) and photochemical ROS generation (Ma et al., 2014). Thus, the effects of ionic dissolution as well as ROS gneretaion would be exacerbated if ZnO–UV-C combination is employed for toxicity tests. However, only few studies are available on the effect of UV-C light on the toxicity of ZnO NPs. Yang and Ma (2014) reported that the cytotoxicity of 60–80 nm ZnO NPs to A549 cells was wavelength dependent. Very recently, the effect of UV-C irradiation on toxic effects of two phases of titania nanoparticles, anatase and rutile, and their binary combination was studied with freshwater algae (Iswarya et al., 2015). Taking into account all these factors it was worthwhile to study the impact of UV-C irradiation on algal toxicity of ZnO NPs in comparison with visible light and dark conditions to elucidate the effect of photo conditions. It may be hypothesised that the differences in the effective size of the nanoparticles in the test medium will strongly in?uence their interaction with the aquatic organisms in the medium. However, a thorough literature survey reveals an absence of any detailed report regarding the effect of different sizes of ZnO NPs on cytotoxic effects towards freshwater alga. Speci?cally the current study differs from the previous ones with differently sized ZnO NPs (Table S1, Supplementary information) in terms of the following points: (a) reaction medium used (the particles behaviour would be markedly different in nutrient supplemented medium, freshwater, and freshwater simulates the environmental conditions better), (b) irradiation conditions employed (none of studies covered UV-C, visible and dark conditions together), (c) comprehensive coverage of the mechanistic endpoints (chlorophyll content, oxidative stress analyses, and membrane damage by LDH analyses have not been covered in any of the prior studies).

Therefore, the aim of the present study was to investigate the toxic potential of two ZnO NPs with similar primary size but different initial effective diameters towards the fresh water algal isolates Scenedesmus obliquus at three exposure concentrations: 0.25, 0.5 and 1 mg/L of nanoparticles under UV-C, dark and visible light conditions. The preliminary nanoparticle characterization was performed using UV–vis spectroscopy, X- ray diffraction, microscopy and dynamic light scattering. Possible ionic dissolution from the nanoparticles in the experimental medium was studied by atomic absorption spectroscopy. The toxicity effect of both the sizes of ZnO NP towards the algae was determined by cell enumeration and photosynthetic pigment content analysis. The evaluation of oxidative stress and membrane integrity was performed by DCFHDA and LDH assays, respectively. The surface interactions of the NPs were con?rmed by scanning electron microscopy. The internalization/uptake of the NPs was quanti?ed by elemental analyses, and further con?rmed by ?uorescence and transmission electron microscopic studies. 2. Materials and methods 2.1. Preliminary characterization of the nanoparticles In the present study, two different sizes of ZnO NPs are tagged as ZnO-1 NPs and ZnO-2 NPs. ZnO-1NPs were procured from Sigma–Aldrich (particle size <100 nm: purity: 99.5% speci?c surface area: 15–25 m2 g?1 ). ZnO-2 NPs were synthesized by the process of gel combustion yielding an average particle size of about 40 nm (SEM) with speci?c surface area of 80.425 m2 g?1 (Venkatesham et al., 2013). The hydrodynamic size of the particles after being sonicated (65 kHz, 530 W, 20 min) into Millipore water with the particle concentration of 1 mg/L at 0 h was measured using dynamic light scattering method (90 Plus Particle Size Analyzer, Brookhaven instruments Corporation, USA). The primary particle size and morphology was analysed using scanning electron microscopy (SEM, Model S400, HITACHI, Japan). The crystallinity and purity of two ZnO NPs were assessed using an X-Ray diffractometer with CuK? radiation of wavelength = 1.5406 in the scan range 2? = 20–80? . The absorption spectra of both the varieties of ZnO NPs were studied using a UV–vis spectrophotometer (UV–vis spectrophotometer 2201, Systronics, India). 2.2. Particle stability and chemical dissolution in ?ltered and sterilized lake water matrix The dynamic light scattering (DLS) technique was employed to study the effective hydrodynamic size of the nanoparticles at various concentrations in the sterilized and ?ltered lake water medium at different time intervals of 0, 4, and 8 h. The amount of Zn2+ ion leached from the ZnO NPs in the sterilized and ?ltered lake water medium was quanti?ed by atomic absorption spectroscopy (AAnalyst 400, PerkinElmer, USA). The ZnO NPs of various concentrations (0.25, 0.5 and 1 mg/L) were dispersed in 5 mL of sterilized lake water (as detailed in Pakrashi et al., 2013) and exposed under UV-C, dark, and visible light at different time intervals for 24, 48 and 72 h. For UV irradiation, the test samples were placed in UV-C lamp (TUV 15W/G15 T8, Philips, India) with intensity of 0.44 mW cm?2 , wavelength < 280 nm. For dark, the samples were covered with an opaque sheet. Fluorescence lamps (TL-D super 80 linear ?uorescent tube, Philips, India) with a light intensity of 0.16 mW cm?2 ,wavelength > 400 nm was used for the visible condition. After the interaction, the suspension was centrifuged at 12,000 rpm at 4 ? C for 20 min, and the supernatant was re-centrifuged for 20 min and ?ltered through 0.1 ?m followed by a 3 kDa membrane ?lter (Dimkpa et al., 2006; Pakrashi et al., 2013).

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

31

The Zn2+ ions were measured in the ?ltrate using AAS at wavelength of 315 nm. 2.3. Toxicity assessment 2.3.1. Experimental setup Fresh water dominant algal isolate S. obliquus was used to study the toxic effect of ZnO NPs of two different sizes. All the toxicity studies are carried out using lake water matrix which was collected from VIT Lake, Vellore (Tamil Nadu, India) and ?ltered through Whatman No. 1 followed by sterilization to get rid of the biological interferences (as detailed in Pakrashi et al., 2013). Toxicity studies were carried out employing the three irradiation conditions: UVC, dark and visible light conditions. All the samples were kept in a static condition for an interaction period of 72 h. Initial studies on UV-C revealed that UV-C had a trivial effect on the growth of algal cells (94.2% growth in comparison with visible light). 2.3.2. Cytotoxicity assessment of ZnO NPs The algal cells were harvested during the exponential stage of their growth and subjected to centrifugation (7000 rpm, 4 ? C, 10 min), and the obtained pellet was washed twice with sterile lake water (as detailed in Dalai et al., 2013). Algal cells with 0.5OD were interacted with different concentrations 0.25, 0.5 and 1 mg/L (0.25 mM, 0.5 mM, 1 mM) of nanoparticles for 72 h along with a control that was devoid of nanoparticles, and exposed under UV-C, dark, visible light irradiation. The algal toxicity testing strictly adhered to OECD guidelines (OECD, 1984). The interacted algal cultures after 72 h were taken to study the possible damages, agglomeration and cell enumeration using optical microscopy (Zeiss Axiostar Optical Microscope, USA). For cell enumeration, the control and nanoparticles treated algal samples were loaded into the Neubauer chamber, and the intact cells (without any changes in size and morphology) were counted. The percentage loss in cell viability of treated cells was calculated with respect to control cells. Since ZnO NPs ionized during exposure, the probable toxic effect of released Zn2+ from the ZnO-1 and ZnO-2 NPs was analysed. The dissolved Zn2+ ions from 1 mg/L of both sizes of NPs exposed under UV-C, dark and visible light irradiation were interacted with algal culture for 72 h. Further, the toxicity was assessed by cell enumeration and estimation of photosynthetic pigment content. For the estimation of photosynthetic pigment, the control and nanoparticles treated cells were pelleted by centrifugation at 7000 rpm for 10 min, to which 1 mL of DMF was added and incubated overnight under dark condition. The samples were recentrifuged at 7000 rpm for 10 min, and supernatant was collected for chlorophyll estimation by measuring the absorbance at 470, 649 and 665 nm using UV–vis Spectrophotometer 2201, Systronics, India (Dalai et al., 2013). 2.3.3. Oxidative stress assessment (ROS) Intracellular reactive oxygen species (ROS) such as hydroxyl radical OH? and superoxide anion were detected using cell-permeable dye, 2 -7 -dichloro?uorescein-diacetate (DCFH-DA) (Wang et al., 2008; Dalai et al., 2014). For cellular ROS staining, 100 ?M DCFH-DA was added to 5 mL of control and NPs-treated culture, followed by 30 min incubation under dark condition. Dichloro?uorescence was measured by the spectro?uorometer (SL174, ELICO, India) using an excitation wavelength of 485 nm and emission at 530 nm. DCFH-DA stained cells were subjected to ?uorescence microscopy to visualize in vivoROS generation by interacting 1 mg/L ZnO NPs with the algae (Rastogi et al., 2010). 2.3.4. Determination of membrane integrity (LDH assay) The cell membrane integrity was determined by the amount of enzyme lactate dehydrogenase released during exposure of ZnO

NPs. After a 72 h interaction period, algal culture was centrifuged at 7000 rpm for 10 min and supernatant was taken for detection of LDH release. In 100 ?L of supernatant, reagents such as 100 ?L of 30 mM sodium pyruvate, 2.8 mL of 0.2 M Tris–HCl and NADH 100 ?L were added just before the analysis. The decrease in the absorbance at 340 nm was measured using UV–vis double beam spectrophotometry (UV–vis spectrophotometer 2201, Systronics, India). The amount of LDH release was compared with the control and treated samples by the rate of absorbance decline (Brown et al., 2001; Dalai et al., 2014). 2.4. Determination of total organic carbon (TOC) The possible changes in the nutritional status of ZnO NPs interacted lake water matrix in the presence and absence of algal cells under different irradiation conditions, such as UV-C, dark and visible was measured. After a 72 h interaction, the samples were centrifuged for 10 min at 7000 rpm and the supernatant was ?ltered through Whatman No. 1. The difference in TOC content with respect to control was observed using TOC analyzer (Shimadzu, Total Organic Carbon Analyser L). 2.5. Surface interaction of the Cell-NPs Scanning electron microscopy was used to examine the attachment of NPs onto the surface of algal cells and the subsequent effect of exopolysaccharides (EPS) secretion on agglomeration of algal cells. The control and treated (1 mg/L) cells were coated on a thin glass slide, air dried and observed under the scanning electron microscopy (SEM, Model S400, HITACHI, Japan) (Ji et al., 2011; Pakrashi et al., 2013). 2.6. Internalization and uptake of NPs 2.6.1. Fluorescence microscopy Nuclear matrix disruption and leakage were studied by means of ?uorescence microscopy (DM-2500, Leica, Germany). The nuclear speci?c stain Acridine orange (AO) of 10 ?L (15 ?g/mL in PBS) was added to both control and NPs (1 mg/L) treated algal cells. Observation was made using BP 450-490, LP 590 ?lter under a ?uorescence microscope (Dalai et al., 2013). 2.6.2. Transmission electron microscopy Internalization of NPs and deformity in cellular organelles was observed under transmission electron microscopy (TEM, Philips CM12, Netherlands). Ultrathin sections of control and treated (1 mg/L) algal cells were attached onto a copper grid and examined under an electron microscope (Pakrashi et al., 2013). 2.6.3. Quanti?cation of intracellular and extracellular ZnO NPs The concentration of the ZnO NPs present in the supernatant as well as on the surface of the algal cells (extracellular) and internalized into the algal cells (intracellular) was quanti?ed by elemental Zn analyses using a atomic absorption spectroscopy (AA analyst400, PerkinElmer) (as detailed in Dalai et al., 2013). Brie?y, the nanoparticle (1 mg/L of ZnO-1 and ZnO-2) treated algal culture was centrifuged at 4000 rpm for 20 min, and the supernatant was taken separately to quantify the NPs remaining in the solution. To the pellet, 5 mL of 0.02 M EDTA was added, and the tubes were inverted for 30 s remove the extracellular ZnO NPs (Zhou et al., 2012). The suspension was subjected to centrifugation at 4000 rpm for 20 min to quantify the extracellular nanoparticles. The algal pellets were used for quanti?cation of intracellular metal content. Samples in triplicate were acid digested with excess of concentrated HNO3 and estimated in AAS.

32

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

2.7. Statistical analysis All experiments were carried out in the triplicates, and the standard errors were calculated. Signi?cance difference between the groups of two nanoparticles was calculated using two-way ANOVA (Bonferroni posttests) by software, Graph Pad Prism. Statistical signi?cance was accepted at a level of p < 0.05. 3. Results 3.1. Primary characterization of ZnO NPs Scanning electron micrographs showed a uniform distribution of irregular, spherically-shaped particles with similar average particle size of about 40–44 nm for both the ZnO-1 and ZnO-2 NPs (Fig. S1, Supplementary information). SEM images indicate that the primary particle size of the two ZnO NPs does not differ by much. The effective diameters of ZnO-1 and ZnO-2 NPs (1 mg/L) were found to be 212.3 ± 10, 483 ± 13 nm in Millipore water and 487.5 ± 2.5, 616.2 ± 38.5 nm in ?ltered and sterilized lake water medium, respectively. There was a signi?cant difference (p < 0.05) between the initial effective diameter of the two particles in Millipore water and lake water medium though their primary particle size did not differ signi?cantly. The XRD pattern con?rmed that both the ZnO-1 and ZnO-2 NPs were of wurtzite phase and hexagonal in structure (Fig. S2, Supplementary information). The zeta potential for ZnO-1 and ZnO-2 NPs in the lake water medium was found to be +63.89 mV and +133.93 mV, respectively. The absorption spectra of ZnO-1 NPs at 377 nm and ZnO-2 NPs at 370 nm con?rmed their chemical structure (as shown, Fig. S3, Supplementary information). 3.2. Colloidal and chemical stability of nanoparticles in the lake water medium 3.2.1. Nanoparticle stability in lake water medium The stability of nanoparticles in the ?ltered and sterilized lake water medium was studied at different time intervals such as 0, 4 and 8 h (Fig. 1). The effective diameter of ZnO-1 NPs (1 mg/L) in lake water medium was observed to be 487.5 ± 2.5, 593.1 ± 81.1 and 1210.5 ± 9.5 nm at 0, 4 and 8 h, respectively. For suspensions with 0.25 and 0.5 mg/L concentrations also, the particle size increased signi?cantly (p < 0.01) with respect to time (0, 4 and 8 h). Similarly, the effective diameter of ZnO-2 NPs (1 mg/L) was observed to be about 616.1 ± 38.5, 885.3 ± 62.5 and 1380 ± 44.5 nm at 0, 4 and 8 h, respectively. Similar, time-dependent increase in effective diameter of ZnO-2 NPs was also observed for 0.5 mg/L. However, the effective diameter was not increased signi?cantly

(p > 0.05) at 4 and 8 h of exposure to 0.25 mg/L compared to 0 h data. From this result, it is inferred that the effective diameter of ZnO-1 was signi?cantly smaller than the ZnO-2 NPs in lake water medium for 0.25, 0.5 and 1 mg/L at all time intervals. 3.2.2. Chemical dissolution of the ZnO NPs in lake water medium ZnO nanoparticles in the colloidal suspension are known to release free Zn2+ ions (Gunawan et al., 2013). The abiotic dissolution of both ZnO-1 and ZnO-2 NPs under UV-C, dark and visible light irradiation was quanti?ed for the durations (24, 48 and 72 h). The dissolution was found to be dependent on the initial concentrations of nanoparticles used. There was no signi?cant increase in the release of Zn2+ ions with respect to time at lower exposure concentrations (0.25 and 0.5 mg/L) under all irradiation conditions, while a signi?cant difference was noticed at 1 mg/L NPs concentration. The release of Zn2+ ions was observed to be maximal at 48 h of exposure time, whereas an insigni?cant increase in dissolution was observed between 48 and 72 h. The dissolution of 1 mg/L ZnO1 NPs at 48 h was about 0.4 mg/L under UV-C, 0.3 mg/L under dark, followed by 0.2 mg/L under visible light exposure (Fig. 2A). Similarly, the release of Zn2+ ions from ZnO-2 NPs was found to be concentration dependent under UV-C, dark and visible light irradiation. The concentration of released Zn2+ ions from 1 mg/L of ZnO-2 NPs under UV-C, dark and visible light irradiation was measured to be 0.3 mg/L, 0.2 ± 0.1 mg/L and 0.2 mg/L, respectively at 48 h exposure time (Fig. 2B). Similar trend was observed for 0.25 and 0.5 mg/L of ZnO-2 NPs. The concentration of released Zn2+ ions from ZnO-1 and ZnO-2 NPs under UV-C irradiation was observed to be statistically signi?cant (p < 0.001) with respect to dark and visible light irradiation condition. An insigni?cant increase in dissolution was noticed between dark and visible light exposure. The dissolution was found to be signi?cantly (p < 0.001) higher for

Fig. 1. Stability of ZnO-1 and ZnO-2 nanoparticles in lake water medium with respect to exposure duration (0, 4 and 8 h) at three different concentrations 0.25, 0.5 and 1 mg/L. The data are presented as mean ± SE, n = 3.

Fig. 2. The dissolution of ZnO NPs in lake water medium with respect to exposure duration (24, 48 and 72 h) at concentrations 0.25, 0.5 and 1 mg/L under UV-C, dark and visible light irradiation. (A) Zn2+ ion released from ZnO-1NPs. (B) Zn2+ ion released form ZnO-2 NPs. The data are presented as mean ± SE, n = 3.

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

33

1 mg/L of ZnO-1 NPs as compared with 1 mg/L of ZnO-2 under all the irradiation conditions at 48 h. 3.3. Cytotoxicity assessment of ZnO NPs 3.3.1. The loss of cell viability and the corresponding decrease in the photosynthetic pigment The ZnO NPs treated, and untreated algal cells were examined under optical microscopy. The untreated algal cells showed intact morphological features (Fig. S4A, Supplementary information), whereas cell wall breakage, membrane damages, aggregation of algal cells and nanoparticle agglomerates encapsulated on algae were observed at 1 mg/L ZnO NPs treated condition (Fig. S4B–D, Supplementary information). For ZnO-1 and ZnO-2 NPs, a signi?cant loss in cell viability with respect to concentration was observed under all the irradiation conditions (p < 0.001) (Fig. 3). At 1 mg/L of ZnO-1 NPs exposure, the loss in cell viability was about 44.1 ± 0.8%, 30.2 ± 2.7% and 25.8 ± 1.8% under UV-C irradiation, dark and visible light, respectively. Algal cells treated with ZnO-2 at 1 mg/L, the loss of cell viability was noted to be 32.1 ± 2.0%, 23.9 ± 1.2% and 23.7 ± 1.6% under UV-C, dark and visible light conditions, respectively. Similar trend was observed for 0.25 and 0.5 mg/L of ZnO-1 and ZnO-2 NPs. The loss in cell viability was signi?cantly higher for UV-C irradiation than dark and visible light whereas, no signi?cant differences were noted between dark and visible light conditions for both ZnO-1 and ZnO-2 NPs. This study revealed that smaller sized ZnO-1 NPs were more toxic to S. obliquus than ZnO-2 NPs under all the irradiation conditions (p < 0.05). The toxicity of ZnO NPs was further con?rmed by quantifying the amount of photosynthetic pigment content (Fig. S5, Supplementary information). The concentration-dependent decrease in chlorophyll a (CA ) content was observed for both ZnO-1 and ZnO-2 NPs treated algal cells under all irradiation conditions. Upon exposure to 1 mg/L of ZnO-1 NPs, the reduction in chlorophyll a (CA ) content was estimated to be 77.7 ± 1.1%, 49.8 ± 0.7% and 2.2 ± 0.6% under UV-C irradiation, dark and visible light, respectively. In case of ZnO-2 NPs at 1 mg/L, the reduction was only about 34.5 ± 1.5%, 28.5 ± 1.2%, 1.0 ± 1.2% under UV-C irradiation, dark and visible light, respectively, which is signi?cantly lower than ZnO-1. Similar trend was observed for 0.25 and 0.5 mg/L of ZnO-1 and ZnO-2 NPs. 3.3.2. Toxicity of dissolved Zn2+ ions Since, ZnO NPs are soluble in aqueous suspension, and the probable cytotoxic effect of dissolved Zn2+ ions in lake water was studied

Fig. 4. The loss of cell viability induced by released Zn2+ ion from 1 mg/L ZnO-1 and ZnO-2 NPs under UV-C, dark and visible light irradiation. The data are presented as mean ± SE, n = 3.

on algae. The dissolved Zn2+ ions from 1 mg/L of ZnO-1 NPs caused 32.2 ± 4.4%, 26.5 ± 3.9%, and 2.7 ± 1.9% loss in cell viability under UV-C, dark and visible light irradiation, respectively, which was signi?cant with respect to control. Similarly, the ?ltrate containing Zn2+ ions from 1 mg/L of ZnO-2 NPs showed loss in cell viability of about 24.4 ± 3.6%, 24.1 ± 3.6%, and 0.8 ± 0.1% under UV-C, dark and visible light irradiation, respectively (Fig. 4). The cytotoxicity of Zn2+ ions was observed to be lower than ZnO NPs as such on algae, suggesting that Zn2+ is one of the dominant factors involved in the loss in cell viability. 3.3.3. Oxidative stress assessment The cytotoxicity of nanoparticles can be correlated with the intracellular ROS production. A concentration-dependent increase in ROS production with respect to control was observed in algal cells treated with both the ZnO NPs. For ZnO-1, the ROS generation was found to be 195.9 ± 0.1%, 140.5 ± 1.9%, and 125.5 ± 0.3% at 1 mg/L upon exposure to UV-C, dark and visible light, respectively. Similarly, for ZnO-2 NPs at 1 mg/L, the ROS production was about 174.7 ± 0.8%, 112.4 ± 4.6%, and 112.5% under UV-C, dark and visible light, respectively. Similar trend was observed for 0.25 and 0.5 mg/L of ZnO-1 and ZnO-2 nanoparticles. UV-C irradiated samples induced signi?cantly higher (p < 0.001) ROS production as compared to dark and visible light condition, whereas no signi?cant (p > 0.05) differences were observed between dark and visible light exposure. The ROS production was found to be signi?cantly higher for ZnO-1 than ZnO-2 NPs under all the conditions (Fig. 5). For further con?rmation, the in vivo ROS production was observed

Fig. 3. The loss of cell viability of algal cells upon exposure to ZnO-1 and ZnO-2 nanoparticles at different concentrations (0.25, 0.5 and 1 mg/L, 72 h) under UV-C, dark and visible light irradiation. The data are presented as mean ± SE, n = 3.

Fig. 5. Generation of ROS by ZnO-1 and ZnO-2 nanoparticles under irradiation conditions UV-C, dark and visible light. The data are presented as mean ± SE, n = 3.

34

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

3.6. Internalization and uptake of ZnO NPs 3.6.1. Fluorescence microscopy The probable genotoxicity of ZnO NPs in the algal cells was estimated by staining with nuclear speci?c stain, Acridine orange. The control cells were of red ?uorescence with intact cell membrane (Fig. S7A, Supplementary information). The interaction of algal cells with 1 mg/L of ZnO-1 and ZnO-2 NPs result in uptake of nuclear speci?c dye, Acridine orange, which clearly indicated the compromised cell membrane and nuclear matrix degradation (Fig. S7B and C, Supplementary information). 3.6.2. Transmission electron microscopy The TEM micrographs of algal cells treated with 1 mg/L of ZnO-1 under UV-C (Fig. 8A–C) show an irregular cell structure, degraded cellular organelles, internalized ZnO-1 NPs into algal cell walls, excess of phosphate granules and lipid vesicles formation. Further, the dark treated cells exhibited a deformed cell membrane, cell shrinkage and starch–pyrenoid complex formation (Fig. 8D and E). Algal cells treated with ZnO-2 NPs caused comparatively less nanoparticles uptake and phosphate granules formation under UVC (Fig. 8F and G) and dark exposure (Fig. 8H and I). Degradation of organelles such as the nucleus, chloroplast and plasmolysis of algal cells was also observed by the stress induced by the ZnO nanoparticles. The difference in effect of ZnO-1 and ZnO-2 could not be demarked clearly by transmission electron microscopy. 3.6.3. Quanti?cation of intracellular and extracellular ZnO NPs After 72 h of exposure, the intracellular and extracellular Zn content was quanti?ed. The increased cellular uptake of Zn was obtained in UV-C irradiated ZnO-1 NPs treatment (1 mg/L) of about 31.0% than dark (13.0%) and visible light (14.0%). The extracellular Zn content was quanti?ed to be about 7.2%, 22.5% and 15.25% under UV-C, dark and visible light condition, respectively. In the supernatant, the Zn content was about 60.6%, 73.1%, and 47.5% under UV-C, dark and visible light. The internalization of ZnO-1 NPs was found to be signi?cantly higher than adsorption on algal cells (extracellular NPs) under UV-C irradiation. Similar effect was observed in the case of ZnO-2 NPs. The maximum uptake of Zn from ZnO-2 treated cells (1 mg/L) was observed in UV-C (16.0%) than dark (14.0%) and visible light (0.7%). The extracellular Zn content was found to be 17.0%, 12.14% and 14.0% under UV-C, dark and visible light condition, respectively. In the supernatant, the Zn content was about 64.8%, 57.3%, and 18% under UV-C, dark and visible light. Though the adsorption and internalization were facilitated by ZnO-1 and ZnO-2 NPs, the uptake was more in smaller sized ZnO-1 interacted algal cells than ZnO-2 NPs interacted cells under UV-C irradiation condition. 4. Discussion 4.1. Stability of ZnO NPs in lakewater medium The hydrodynamic size of the nanoparticles in the test medium plays a signi?cant role in toxicity of ZnO NPs towards the test organism (Kato et al., 2009; Pauluhn, 2009). Though the primary particle size of both the particles was similar in the current study, their hydrodynamic size differed signi?cantly in lake water medium initially as well as during the exposure (Fig. 1). The difference in effective diameter between the two ZnO NPs may be due to difference surface coatings (e.g. surfactants, polymers, and polyelectrolytes) result in changes in dispersion characteristics of the nanoparticles. Adsorbed surfactants prevent aggregation of NPs by increasing surface charge and also by reducing interfacial energy between particle and solvent (Rosen, 2004). With an increase in both the exposure duration and the concentration, the NP aggregate

Fig. 6. Estimation of membrane integrity by release of LDH upon interaction with ZnO-1 and ZnO-2 nanoparticles at different concentration (0.25, 0.5 and 1 mg/L, 72 h). The data are presented as mean ± SE, n = 3.

by staining the algal cells with DCFH-DA dye (as detailed in Supplementary information, Fig. S6).

3.3.4. Determination of membrane damage The membrane integrity of algal cells treated with ZnO NPs was estimated by release of the cytosolic enzyme, lactate dehydrogenase. The algal cells exposed to ZnO-1 and ZnO-2 NPs showed a concentration-dependent increase in LDH release with respect to control (Fig. 6). The effect of particle size on LDH release was signi?cantly (p < 0.01) higher for ZnO-1 NPs than for ZnO-2 NPs under all the irradiation conditions.

3.4. Changes in the total organic carbon (TOC) in the medium due to NP interactions The change in nutritional content of sterilized lake water interacted with 1 mg/L of nanoparticles in the presence and absence of algae was studied. There was no signi?cant change in the TOC content of ?ltered, and sterilized lake water medium interacted with 1 mg/L of ZnO-1 and ZnO-2 NPs in absence of algae, irrespective of irradiation. In the presence of algae, TOC content of lake water interacted with 1 mg/L of ZnO-1 NPs was about 27.0 ± 0.5 mg/L, 8.75 ± 0.4 mg/L and 6.85 ± 0.3 mg/L under UV-C, dark and visible light irradiation, respectively, with respect to the control. Upon exposure to 1 mg/L of ZnO-2 NPs, the TOC content was found to be 15.5 ± 2.5 mg/L, 0.3 mg/L and 0.57 mg/L under UV-C irradiation, dark and visible light, respectively. TOC content was found to be signi?cantly (p < 0.01) more for ZnO-1 NPs treated condition than ZnO-2 NPs.

3.5. Surface interactions of ZnO NPs The scanning electron microscopic image of untreated algal cells shows an intact cell membrane with a smooth surface (Fig. 7A). The adsorption of nanoparticles on the algal cell surface was observed upon exposure to ZnO-1 NPs (Fig. 7B) and ZnO-2 NPs (Fig. 7C) treated cells under UV-C irradiation. The ZnO-1 and ZnO-2 NPs interaction apparently damaged the algal cell walls and excess of algal exudates like exopolysaccharides (EPS) were released, which further caused agglomeration of algal cells (Fig. 7D and E).

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

35

Fig. 7. Scanning electron micrograph of algal cells (A) control cells with intact cell membrane; (B) ZnO-1 NPs adsorption onto the cell surface and deformed cell membrane under UV irradiated condition; (C) ZnO-2 treated cells with damages in cell structure; (D and E) aggregation of algal cells by secretion of exo-polymeric substances after interacted with ZnO-1 and ZnO-2 NPs.

sizes reached the micron ranges. The formation of larger aggregates of ZnO NPs was possibly due to the interactions with the natural organic matter (e.g. proteins, polysaccharides, nucleic acids, lipids) from algae, plants and fungi present in the lake water (Navarro et al., 2008; Liu et al., 2014). Prathna et al. (2011) suggested that the aggregation of metallic nanoparticles in lakewater medium depends on the surface functionalization and preparation methods. Adams et al. (2006) also reported that the primary particle size cannot be considered as a major factor in assessing their toxicity, when aggregation of NPs occurred.

4.2. Dissolution of ZnO nanoparticles The ionic dissolution of nanoparticles depends strongly on the surface area, which again can be inversely correlated with particle size (Wong et al., 2013; Lopes et al., 2014). In this study, an enhanced chemical dissolution (in the absence of algae) was noted for the smaller-sized ZnO-1 NPs than for ZnO-2 NPs at 48 h of exposure to UV-C irradiation. Increased absorbance of the ZnO NPs in UV range enhanced the photo-decomposition of NPs under UV-C irradiation, which in turn resulted in more dissolution (Han et al.,

36

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

Fig. 8. Transmission electron micrographs of algal cells (A) ZnO-1 NPs exposed cells to UV irradiation shows internalization of NPs into algal cell wall, excess of phosphate granule deposition; (B) lipid vesicle formation due to nanoparticle stress; (C) shrinkage, NPs aggregates, degradation of nucleus and chlorophyll; (D and E) ZnO-2 NPs treated cells under UV-C irradiation with internalized nanoparticles; (F and G) ZnO-1 NPs treated cells under dark with starch–pyrenoid complex formation and damaged cell membrane; (H and I) ZnO-2 treated cells under dark with degradation of starch–pyrenoid complex and degradation of cellular organelles. Note: Yellow arrow – NPs internalization; green arrow – phosphate granule; blue arrow – lipid vesicles; red arrow – membrane damages. (For interpretation of the references to colour in this ?gure legend, the reader is referred to the web version of this article.)

2010). Similar to these ?ndings, Lee and An (2013) and Kim and An (2012) reported a substantial ionic dissolution from ZnO NPs at different exposure conditions (UV-A, UV-B and visible light). However, the differences in the dissolution were found to be insigni?cant between dark and visible light irradiation for both ZnO-1 and ZnO-2 NPs.

4.3. Toxicity of ZnO NPs: ROS production and cell membrane damage Toxicity of ZnO NPs towards S. obliquus was found to depend on particle size in the test medium, concentration and irradiation condition for both ZnO-1 and ZnO-2 NPs. The toxicity induced by smaller sized ZnO-1 was signi?cantly (p < 0.001) more than ZnO2 NPs. Smaller sized nanoparticles with greater surface area could effectively interact, easily stick to/cross the cellular membrane and get internalized into algal cells (Bystrzejewska-Piotrowska et al., 2009), whereas the formation of larger aggregates makes them less available to the cells. A previous study by Peng et al. (2011) noted that the smaller sized ZnO spheres of 6.3 nm had more adverse effect on marine algae than 15.7 nm particles. Similarly, Khare et al. (2011) reported that ZnO NPs of 25 nm were more toxic to the C. elegans than the 100 nm particles.

The particle size and photo-catalytic activity are interrelated. With a decrease in particle size, the photo-catalytic activity increases due to an increase in speci?c surface area and pore volume (Metzler et al., 2011). The toxicity of ZnO-1 and ZnO-2 NPs under UV-C irradiation was signi?cantly (p < 0.001) more compared to visible light conditions, possibly due to enhanced UV absorption ef?ciency. One of the factors contributing to this phototoxic behaviour is reactive oxygen species (ROS) generation (Zhou et al., 2014; Gunawan et al., 2013). ZnO nanoparticles are known to produce photocatalytic ROS under UV-C and visible light condition (Choi and Hu, 2008). The smaller-sized particles have an increased speci?c surface area with enhanced speci?c quantum yield, and thus generate more ROS (Tseng et al., 2006). The particle size-dependent reactive oxygen species generation in cell lines by Ag nanoparticles was already reported by Carlson et al. (2008). Similarly, it is noted in the current study that the smaller sized ZnO-1NPs produce signi?cantly more ROS than ZnO-2 NPs under both UV-C and visible light conditions. Further, the intracellular ROS production was con?rmed by green DCF ?uorescence of algal cells exposed to ZnO NPs (Fig. S5, Supplementary information). In vivo ROS production was observed in all the three different irradiation conditions; however, the UV-C irradiated algal cells showed more DCF ?uorescence than under dark and visible light conditions. Rastogi et al. (2010) and He and Hader (2002)

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38

37

also reported in vivo ROS generation in Anabaena sp. under UV-B irradiation. In the current study, signi?cant toxic effects and considerable intracellular ROS generation were noted even in dark conditions. The toxicity under dark condition is an interesting observation and was previously reported by Ji et al. (2011) in Chlorella sp. by ZnO and TiO2 NPs, with a higher exposure concentration (6-day EC30 of 30 mg/L). Further, Dalai et al. (2013) observed notable toxicity at lower concentration of TiO2 (1 mg/L) under dark condition. The considerable intracellular ROS production under dark condition may be related to nanoparticle stress as reported by Dalai et al. (2013), and Adams et al. (2006). The interactions of the nanoparticles with algae may cause damage to the cell membrane and release LDH enzyme in the test solution, which may be one of the toxicity mechanisms inducing cell death. A signi?cantly-enhanced LDH release was observed for ZnO-1 treated cells as compared to ZnO-2, and the UV-C irradiated algal cells also released signi?cantly more LDH than the cells treated in dark and visible light conditions. Increased LDH release under UV-C was owing to photo-induced toxicity for ZnO NPs, as discussed in the preceding section. Interestingly, substantial membrane damage was also noted under dark conditions for both ZnO-1 and ZnO-2 NPs. Signi?cant membrane damage under dark condition was reported previously by Simon-Deckers et al. (2009) upon exposure to TiO2 NPs in Cupriavidus metallidurans and by Dalai et al. (2013) in S. obliquus. The current observation corroborates with these ?ndings. 4.4. ZnO adsorption and uptake The surface charge analyses reveal that the positively-charged ZnO-1 and ZnO-2 NPs could electrostatically interact with negatively-charged algal cells, facilitating their adsorption. The adsorption of particles on the cell surface and the release of the extracellular substances exacerbated aggregation of the algal cells (Fig. 7). The deformed cell membrane and larger aggregates of nanoparticles entrapping the algae were evident from optical microscopy (Fig. S4, Supplementary information). A similar effect was observed by Chen et al. (2012) on Chlorella sp. upon being treated with ZnO NPs. Ji et al. (2011) also noticed entrapment of algae by ZnO NPs. The adsorption of NPs on algal cell wall may be attributed to the presence of numerous binding (polysaccharides, cellulose and glycoproteins) sites on the algal cell wall (Chen et al., 2012). The adhesion of ZnO NPs on algae may either cause mechanical damage (Lin and Xing, 2008) or, by releasing the Zn2+ ions, disrupt the cellular metabolic pathway of algae. Furthermore, an internal degradation of the cellular organelles, such as nucleus and chloroplast, and substantial damages to the cell wall were observed in the ZnO NPs treated algal cells (TEM images in Fig. 8). The degradation of the nucleus was con?rmed by uptake of nuclear speci?c stain by interacted algal cells (Fig. S7, Supplementary information). The presence of larger aggregates of ZnO NPs internalized into the algal cell membrane could be noted (Fig. 8). The adsorbed and internalized nanoparticles were quanti?ed by elemental analyses. Under UV-C irradiation, the ZnO-1 treated cells showed signi?cantly more internalization than adsorption, which reveals that the enhanced cytotoxicity under UV-C irradiation may be principally due to the internalized nanoparticles. ZnO-1 NPs treatment under UV-C irradiation facilitated the formation of phosphate granules, starch pyrenoid complex and lipid droplets (Fig. 8). ZnO NPs induced phosphate granule formation in Chlorella pyrenoidosa and S. obliquus has been reported previously by Zhou et al. (2013). Similarly, Kim et al. (2013) reported the formation of lipid droplets in C. reinhardtii and Chlorella vulgaris under the stress conditions. In the current work, the lipid content was quanti?ed by gas chromatography–mass spectroscopy (GC–MS) for

ZnO-1 NPs (since they showed higher toxicity compared to ZnO-2) treated algal cells under UV-C irradiation (Fig. S8, Supplementary information). The presence of poly unsaturated fatty acids, such as palmitic acid and linolenic acid methyl ester, could be con?rmed. 4.5. Effect of Zn2+ ions on algal viability Along with the nanoparticles, the Zn2+ ion released in the medium also contributed to the toxicity effects. However, the loss of cell viability caused solely by dissolved Zn2+ ion under UVC, dark and visible light irradiation was substantially lower than that caused by the toxicity of both ZnO-1 and ZnO-2 nanoparticles. This ?nding is similar to reports of previous study by Ji et al. (2011), where the ZnO NPs had higher cytotoxicity than Zn2+ ions on Chlorella sp. 5. Conclusion In summary, the hydrodynamic size of the nanoparticles in the test medium played a signi?cant role in the cytotoxicity of algae. In addition to Zn2+ ion release, there were some other mechanistic factors such as ROS production, LDH release (membrane damage), adsorption and internalization of nanoparticles that could have contributed towards the toxicity on algae S. obliquus. The photocatalytic activity of ZnO NPs under UV-C irradiation enhanced the cytotoxic effects. Acknowledgements We acknowledge Sophisticated Analytical Instrumentation Facility (SAIF) for SEM and Christian Medical College, Vellore, India, for the Transmission Electron Microscopy used for this study. We acknowledge the research grant provided by the LSRB-DRDO (DLS/81/48222/LSRB-262/BTB/2012), Government of India, for carrying out the present study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox. 2015.03.004. References
Adams, L.K., Lyon, D.Y., Alvarez, D.J., 2006. Comparative eco-toxicity of nano-scale TiO2 , SiO2 , and ZnO water suspensions. Water Res. 40, 3527–3532. Aruoja, V., Dubourguier, H.C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 1461–1468. Barhoumi, L., Dewez, D., 2013. Toxicity of superparamagnetic iron oxide nanoparticles on green alga Chlorella vulgaris. BioMed. Res. Int. 2013, 11. Blinova, I., Ivask, A., Heinlaan, M., Mortimer, M., Kahru, A., 2010. Ecotoxicity of nanoparticles of CuO and ZnO in natural water. Environ. Pollut. 158, 41–47. Brown, D.M., Wilson, M.R., MacNee, W., Stone, V., Donaldson, K., 2001. Size dependent proin?ammatory effects of ultra?ne polystyrenes particles: a role for surface area and oxidative stress in the enhanced activity of ultra?nes. Toxicol. Appl. Pharmacol. 175, 191–199. Bystrzejewska-Piotrowska, G., Golimowski, J., Urban, P.L., 2009. Nanoparticles: their potential toxicity, waste and environmental management. Waste Manage. 29, 2587–2595. Carlson, C., Hussain, S.M., Schrand, A.M., Braydich-Stolle, L.K., Hess, K.L., Jones, R.L., Schlager, J.J., 2008. Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. J. Phys. Chem. B 112, 13608–13619. Chen, P., Powell, B.A., Mortimer, M., Ke, P.C., 2012. Adaptive interactions between zinc oxide nanoparticles and Chlorella sp. Environ. Sci. Technol. 46, 12178–12185. Choi, O., Hu, Z., 2008. Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environ. Sci. Technol. 42, 4583–4588. Dalai, S., Pakrashi, S., Joyce Nirmala, M., Chaudhri, A., Chandrasekaran, N., Mandal, A.B., Mukherjee, A., 2013. Cytotoxicity of TiO2 nanoparticles and their detoxi?cation in a freshwater system. Aquat. Toxicol. 138, 1–11.

38

M. Bhuvaneshwari et al. / Aquatic Toxicology 162 (2015) 29–38 Noroozi, R., Mehdinezhad, M.H., zafarzadeh, A., 2011. Photocatalytic removal of Escherichia coli by ZnO activated by ultraviolet-C light from aqueous solution. mljgoums 5 (2), 52–61. Organisation for Economic Cooperation and Development (OECD), 1984. Algal Growth Inhibition Test. OECD Guidelines for Testing of Chemicals 201. OECD, Paris, France. Osmond, Mccall, M.J., 2010. Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard. Nanotoxicology 4, 15–41. Panessa-Warren, B.J., Maye, M.M., Warren, J.B., Crosson, K.M., 2009. Single walled carbon nanotube reactivity and cytotoxicity following extended aqueous exposure. Environ. Pollut. 157, 1140–1151. Pakrashi, S., Dalai, S., Prathna, T.C., Trivedi, S., Myneni, R., Raichur, A.M., Mukherjee, A., 2013. Cytotoxicity of aluminium oxide nanoparticles towards fresh water algal isolate at low exposure concentrations. Aquat. Toxicol. 132, 34–45. Pauluhn, J., 2009. Pulmonary toxicity and fate of agglomerated 10 and 40 nm aluminum oxyhydroxides following 4-week inhalation exposure of rats: toxic effects are determined by agglomerated, not primary particle size. Toxicol. Sci. 109, 152–167. Peng, X., Palma, S., Fisher, N.S., Wong, S.S., 2011. Effect of morphology of ZnO nanostructures on their toxicity to marine algae. Aquat. Toxicol. 102, 186–196. Prathna, T.C., Chandrasekaran, N., Mukherjee, A., 2011. Studies on aggregation behaviour of silver nanoparticles in aqueous matrices: effect of surface functionalization and matrix composition. Colloids Surf. A: Physicochem. Eng. Aspects 390, 216–224. Rastogi, R.P., Singh, S.P., Hader, D.P., Sinha, R.P., 2010. Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2 ,7 -dichlorodihydro?uorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937. Biochem. Biophys. Res. Commun. 397, 603–607. Rosen, M.J., 2004. Surfactants and Interfacial Phenomena, 3rd ed. Wiley-Interscience, New York. Serpone, N., Dondi, D., Albini, A., 2007. Inorganic and organic UV ?lters: their role and ef?cacy in sunscreens and suncare products. Inorg. Chim. Acta 360, 794–802. Simon-Deckers, A.L., Loo, S., L’hermite, M.M., Boime, N.H., Menguy, N., Reynaud, C.C., Gouget, B., Carriere, M., 2009. Size, composition and shape dependent toxicological impact of metal oxide nanoparticles and carbon nanotubes towards bacteria. Environ. Sci. Technol. 43, 8423–8429. Tseng, Y.H., Lin, H.Y., Kuo, C.S., Li, Y.Y., Huang, C.P., 2006. Thermostability of nano TiO2 and its photocatalytic activity. React. Kinet. Catal. Lett. 89, 63–69. Venkatesham, V., Madhu, G.M., Satyanarayana, S.V., Preetham, H.S., 2013. Adsorption of lead on gel combustion derived nano ZnO. Procedia Eng. 51, 308–313. Vileno, B., Lekka, M., Sienkiewicz, A., Jeney, S., Stoessel, G., Lekki, J., Forró, L., Stachura, Z., 2007. Stiffness alterations of single cells induced by UV in the presence of nano TiO2 . Environ. Sci. Technol. 41, 5149–5153. Wang, H., Wick, R.L., Xing, B., 2009. Toxicity of nanoparticulate and bulk ZnO, Al2 O3 and TiO2 to the nematode Caenorhabditis elegans. Environ. Pollut. 157, 1171–1177. Wang, J., Zhang, X., Chen, Y., Sommerfeld, M., Hu, Q., 2008. Toxicity assessment of manufactured nanomaterials using the unicellular green alga Chlamydomonas reinhardti. Chemosphere 73, 1121–1128. ? A.B., 2013. A comprehensive review on the Wong, S.W., Leung, K.M., Djuriˇ sic, aquatic toxicity of engineered nanomaterials. Rev. Nanosci. Nanotechnol. 2, 79–105. Yang, Q., Ma, Y., 2014. Irradiation-enhanced cytotoxicity of zinc oxide nanoparticles. Int. J. Toxicol. 33 (3), 187–203. Zhang, S., Saebfar, H., 2010. Chemical Information Call-in Candidate: Nano Zinc Oxide. California Dept. of Toxic Substances Control, pp. 1–11 http://www.dtsc.ca.gov/TechnologyDevelopment/Nanotechnology/ upload/NanoZincOxide.pdf Zhou, G.J., Peng, F.Q., Zhang, L.J., Ying, G.G., 2012. Biosorption of zinc and copper from aqueous solutions by two freshwater green microalgae Chlorella pyrenoidosa and Scenedesmus obliquus. Environ. Sci. Pollut. Res. 19, 2918–2929. Zhou, G.J., Peng, F.Q., Yang, B., Ying, G.G., 2013. Cellular responses and bioremoval of nonylphenol and octylphenol in the freshwater green microalga Scenedesmus obliquus. Ecotoxicol. Environ. Saf. 87, 10–16. Zhou, H., Wang, X., Zhou, Y., Yao, H., Ahmad, F., 2014. Evaluation of the toxicity of ZnO nanoparticles to Chlorella vulgaris by use of the chiral perturbation approach. Anal. Bioanal. Chem. 406–415, 3689–3695.

Dalai, S., Pakrashi, S., Bhuvaneshwari, M., Iswarya, V., Chandrasekaran, N., Mukherjee, A., 2014. Toxic effect of Cr(VI) in presence of nTiO2 and nAl2 O3 particles towards freshwater microalgae. Aquat. Toxicol. 146, 28–37. Dimkpa, C.O., Calder, A., Britt, D.W., McLean, J.E., Anderson, A.J., 2006. Responses of a soil bacterium, Pseudomonas chlororaphis O6 to commercial metal oxide nanoparticles compared with their metal ions. Environ. Pollut. 159, 1749–1756. Gunawan, C., Sirimanoonphan, A., Teoh, W.Y., Marquis, C.P., Amal, R., 2013. Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii. J. Hazard. Mater. 260, 984–992. Gurr, J.R., Wang, A.S., Chen, C.H., Jan, K.Y., 2005. Ultra?ne titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells. Toxicology 213, 66–73. Han, J., Qiu, W., Gao, W., 2010. Potential dissolution and photo-dissolution of ZnO thin ?lms. J. Hazard. Mater. 178, 115–122. He, Y.Y., Hader, D.P., 2002. UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp. protective effects of ascorbic acid and N-acetyl-l-cysteine. J. Photochem. Photobiol. B: Biol. 66, 115–124. Heng, B.C., Zhao, X., Xiong, S., Woei Ng, K., Yin-Chiang Boey, F., Say-Chye Loo, J., 2010. Toxicity of zinc oxide (ZnO) nanoparticles on human bronchial epithelial cells (BEAS-2B) is accentuated by oxidative stress. Food Chem. Toxicol. 48, 1762–1766. Iswarya, V., Bhuvaneshwari, M., Alex, S.A., Iyer, S., Chaudhuri, G., Chandrasekaran, P.T., Bhalerao, G.M., Chakravarty, S., Raichur, A.M., Chandrasekaran, N., Mukherjee, A., 2015. Combined toxicity of two crystalline phases (anatase and rutile) of Titania nanoparticles towards freshwater microalgae: Chlorella sp. Aquat. Toxicol. 161, 154–169. Ji, J., Long, Z., Lin, D., 2011. Toxicity of oxide nanoparticles to the green algae Chlorella sp. Chem. Eng. J. 170, 525–530. Kahru, A., Dubourguier, H.C., Blinova, I., Ivask, A., Kasemets, K., 2008. Biotests and biosensors for ecotoxicology of metal oxide nanoparticles: a minireview. Sensors 8, 5153–5170. Kato, H., Suzuki, M., Fujita, K., Horie, M., Endoh, S., Yoshida, Y., 2009. Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment. Toxicol. In Vitro 23, 927. Khare, P., Sonane, M., Pandey, R., Ali, S., Gupta, K.C., Satish, A., 2011. Adverse effects of TiO2 and ZnO nanoparticles in soil nematode, Caenorhabditis elegans. J. Biomed. Nanotechnol. 7, 116–117. Kim, S.W., An, Y.J., 2012. Effect of ZnO and TiO2 nanoparticles preilluminated with UVA and UVB light on Escherichia coli and Bacillus subtilis. Appl. Microbiol. Biotechnol. 95, 243–253. Kim, S., Kim, H., Ko, D., Yamaoka, Y., Otsuru, M., Kawai-Yamada, M., Lee, Y., 2013. Rapid induction of lipid droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by brefeldin A. PLoS One 8, e81978. K?rans ? an, M., Khataee, A., Karaca, S., Sheydaei, M., 2015. Arti?cial neural network modeling of photocatalytic removal of a disperse dye using synthesized of ZnO nanoparticles on montmorillonite. Spectrochim. Acta Part A: Mol. Biom. Spectrosc. 140, 465–473. Lee, W.M., An, Y.J., 2013. Effects of zinc oxide and titanium dioxide nanoparticles on green algae under visible, UVA, and UVB irradiations: no evidence of enhanced algal toxicity under UV pre-irradiation. Chemosphere 91, 536–544. Lin, D., Xing, B., 2008. Root uptake and phytotoxicity of ZnO nanoparticles. Environ. Sci. Technol. 42, 5580–5585. Liu, Y., Tourbin, M., Lachaize, S., Guiraud, P., 2014. Nanoparticles in wastewaters: hazards, fate and remediation. Powder Technol. 255, 149–156. Lopes, S., Ribeiro, F., Wojnarowicz, J., ?ojkowski, W., Jurkschat, K., Crossley, A., Loureiro, S., 2014. Zinc oxide nanoparticles toxicity to Daphnia magna: size-dependent effects and dissolution. Environ. Toxicol. Chem. 33, 190–198. Ma, H., Wallis, L.K., Diamond, S., Li, S., Canas-Carrell, J., Parra, A., 2014. Impact of solar UV radiation on toxicity of ZnO nanoparticles through photocatalytic reactive oxygen species (ROS) generation and photo-induced dissolution. Environ. Pollut. 193, 165–172. Metzler, D.M., Li, M., Erdem, A., Huang, C., 2011. Responses of algae to photocatalytic nano-TiO2 particles with an emphasis on the effect of particle size. Chem. Eng. J. 170, 538–546. Navarro, E., Baun, A., Behra, R., Hartmann, N.B., Filser, J., Miao, A.J., Sigg, L., 2008. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 17, 372–386.


相关文章:
更多相关标签: