Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Toxic potential of iron oxide, CdS/Ag2 S composite, CdS and Ag2 S NPs on a fresh water alga Mougeotia sp E. Jagadeesh, Behlol Khan, Preethy Chandran, S. Sudheer Khan ∗ Centre for Nanotechnology and Advanced Biomaterials, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613401, Tamil Nadu, India a r t i c l e i n f o Article history: Received 14 May 2014 Received in revised form 27 October 2014 Accepted 6 November 2014 Available online 13 November 2014 Keywords: Iron oxide nanoparticles Cadmium sulfide nanoparticles Silver sulfide nanoparticles Nanocomposite Toxicity Mougeotia sp. a b s t r a c t Nanoparticles (NPs) are being used in many industries ranging from medical, textile, automobile, consumer products, etc. This may increase the probability of their (NPs) release into the environment and fresh water ecosystems. The present study focuses on testing the potential effect of iron oxide, nanocomposite of cadmium sulfide and silver sulfide, cadmium sulfide and silver sulfide nanoparticles (NPs) on a fresh water alga Mougeotia sp. as the model organism. The alga was treated with different concentrations of NPs (0.1–25 mg/L). The NPs exposure caused lipid peroxidation and ROS production, and suppressed the antioxidant defense system such as catalase, glutathione reductase, and superoxide dismutase. Adsorption of NPs on algal surface and membrane damage were confirmed through microscopic evaluation and increase in protein content in extracellular medium. The present investigation pointed out the ecological implications of NPs. The study warrants the need for regulatory agencies to monitor and regulate the use of NPs. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Metal oxide NPs are being manufactured in recent years at industrial level and are used in treatment of water, medicines and engineering works [1,2]. Due to the increased use of NPs in recent years it was predicted that these NPs can find their way to the environment where their behavior can pose severe threat to the various components of ecosystem [3,4]. Magnetite (Fe3 O4 ) NPs are thermodynamically unstable in the presence of oxygen and are mainly used as information carriers, drug delivery systems, etc [5,6]. CdS and Ag2 S are some of the important transition metal sulfide semiconductor nanomaterials because of their direct as well as relatively lesser band gap, chemical stability, effective high absorption coefficient and even extremely good optical properties [7,8]. For CdS NPs the energy band gap increases with the decrease of the size [9,10]. Ag2 S is being used in various nano sized optical devices, and electronic applications including IR detectors, manufacturing fuel cells and battery-based super ionic conductors, photoconductors, photovoltaic cells resistive switching and ∗ Corresponding author. Tel.: +91 9047286362. E-mail addresses: [email protected], [email protected] (S.S. Khan). http://dx.doi.org/10.1016/j.colsurfb.2014.11.008 0927-7765/© 2014 Elsevier B.V. All rights reserved. solar selective coatings [11–13]. Due to their chemical nature and size these NPs remain in the environment for a longer time. The indiscriminate use of these NPs may lead to its release into the environment. The present study evaluated the potential toxic effect of these NPs once it is released into the environment. Here we choose Mougeotia sp. as the model system to evaluate the toxicological effect of NPs to represent the toxic potential of these NPs to the aquatic organisms, since it has been shown to be tolerant to high level of heavy metal concentration [14]. Mougeotia sp. is fast growing fresh water green algae found generously in the water bodies. This alga was found to be interesting due to its vigorous and luxuriant growth in its habitat throughout the year. The present study used low concentrations of NPs, from 0.1 to 25 mg/L in comparison with previously reported studies. Most of the previous studies were used at very high concentration of NPs to test the toxic potential on algal cells and it varies from mg/l to g/L [15–18]. The main objective of our report was to characterize the toxic effects of NP in Mougeotia sp. To evaluate these effects, we investigated the growth of algae by evaluating the chlorophyll content after being exposed to NP. Furthermore, we also investigated several biomarkers related to oxidative stress as well, such as reactive oxygen species (ROS) formation, lipid peroxidation and enzymatic antioxidant activities of catalase, glutathione reductase (GR) and Superoxide dismutase (SOD). E. Jagadeesh et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 Fig. 1. Scanning electron microscopic images of (a) iron oxide, (b) CdS/Ag2 S composite, (c) CdS and (d) Ag2 S NPs. Fig. 2. Size distribution of (a) iron oxide, (b) CdS/Ag2 S composite, (c) CdS and (d) Ag2 S NPs. 285 286 E. Jagadeesh et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 Fig. 3. X-ray powder diffraction analysis of (a) iron oxide, (b) CdS/Ag2 S composite, (c) CdS and (d) Ag2 S NPs. 2. Materials and methods 2.1. Chemicals FeCl3 ·6H2 O, Trisodium citrate, polyvinyl pyrrolidone (PVP), AgNO3 , CdCl2 and Na2 S were purchased from Merck, Germany. FeCl2 and dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich, USA. Glutathione, nitro-blue Tetrazolium (NBT), 5,5 -dithiobis (2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA) were obtained from Himedia, Mumbai, India. The present study used previously characterized particles for toxicological evaluation. 2.2. Collection and culture of algae Fresh water algae that were collected from a natural pond and identified as Mougeotia sp. were filtered to remove debris, and cultured in Bold’s 3 N modified medium. Equal amount of samples were weighed (1 g) and transferred into the flasks containing medium. The flasks were incubated on shaker at room temperature, under fluorescent light at 8 h and 16 h day/night rhythm. 2.3. Synthesis of NPs The nanoparticles were synthesized as described earlier [19–21]. Briefly, 0.1 M of FeCl2 and FeCl3 were taken in a 1:2 ratio in deionized water and heated up to 80 ◦ C for 1 h on magnetic stirrer. Ammonia (acting as a base to form iron oxide NPs) was added drop wise until the solution turned dark black in color indicating the formation of iron oxide NPs. After 1 h of heating, PVP was Fig. 4. Effect of NPs on algal growth. (a) Toxicity after 5th day and (b) toxicity after 10th day. Values are the mean of n = 3 (mean ± standard error). E. Jagadeesh et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 287 Fig. 5. Microscopic image of Mougeotia sp. After interaction with NPs: (a) control image of Mougeotia sp. (without interacting with NPs), (b) iron oxide NPs, (d) cadmium sulfide NPs, (f) silver sulfide NPs and (h) nanocomposite after interaction with algae. Adsorption of NPs on algal cells can be clearly seen in the figure. Membrane damage of algal cells were observed after the interaction of (c) iron oxide NPs, (e) cadmium sulfide NPs, (g) silver sulfide NPs and (i) nanocomposite. added. The solution was centrifuged at 12,000 × g for 30 min, pellet was lyophilized and the smooth powder obtained was taken for characterization. CdS and Ag2 S NPs were prepared by adding Na2 S to CdCl2 and AgNO3 solution separately. The nanocomposite (CdS/Ag2 S) was prepared by adding Na2 S to the mixture of CdCl2 and AgNO3 solution (1:1 ratio). The pellet obtained was lyophilized and characterized. 2.4. Characterization of nanoparticles The synthesized particles were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), particle size analyzer and zeta sizer. The surface state, morphology and structure of NPs were recorded using a field emission scanning electron microscopy (JEOL JSM-6701F, Japan) at a magnification level of 6K with an acceleration voltage of 3–35 kV. The lyophilized NPs were coated in XRD grid and the spectra was recorded using Bruker AXS (Diffractometer D8, Germany) operated at voltage of 40 KV using Cu K␣ radiation used to determine lattice parameter, crystallite size and phase identification. The particle size was measured using a particle size analyzer (Microtrac Blue Ware, Nikkiso, Japan). For measuring the particle size, 10 mg of NPs were dispersed in 100 mL of de-ionized water. The particle size was calculated based on the volume% (3 mL was used for the analysis). The zeta potential measurement was done using a Malvern (UK) zeta analyzer. 2.5. Toxicity evaluation Freshly grown algae (1 g) was suspended in Bold’s 3 N modified medium along with NPs and incubated for 10 days under fluorescent light with a day/night rhythm of 8 h/16 h. Chlorophyll pigment from the samples was extracted by adding 10 mL of 80% acetone and leaving over night at 4 ◦ C followed by centrifugation at 10,000 × g for 10 min. Supernatant was collected and the absorbance was measured at 652 nm using EQUIP-TRONICS double beam spectrophotometer (EQ-824., India). Microscopic evaluation was done to understand the adsorption of particles on cell surface and to evaluate the membrane damage by the particles (Zeiss AX-10, ProgRes C5, Germany). 2.6. Lipid peroxidation assessment Lipid peroxidation was assessed by measuring the amount of thiobarbituric acid reactive species (TBARS). 0.5 g of algae was suspended into flasks containing solutions of culture medium, 1 mM NaCl and NPs, and incubated for 24 h, then centrifuged at 10,000 × g for 5 min. The pellet was collected from each sample and 288 E. Jagadeesh et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 2.8. Protein estimation The supernatant was collected after the interaction of algal species with NPs. The amount of protein left in the supernatant was calculated by Lowery method. A control experiment was run without NPs. 2.9. Enzyme assay Reduced glutathione (GSH) content was obtained by treating the algae culture with varying concentrations of different NPs which was then centrifuged and lysates were prepared by ultrasonicating the pellets using ultra sonic vibration (Sonics Vibra-Cell Ultrasonicator) suspended in 0.1 M phosphate buffer solution. GSH content was measured through the oxidation of it by 5,5 -dithiobis(2nitrobenzoic acid) (DTNB) forming 5-thio(2-nitrobenzoic acid) (TNB) [22], absorbance was measured at 405 nm using a double beam Lambda 25 UV–visible spectrophotometer (Perkin Elmer, USA). Catalase activity was calculated by measuring the decreasing amount of H2 O2 [23]. 0.5 mL of cell lysates was taken and 0.1 M H2 O2 in 0.1 M phosphate buffer was added to them. Absorbance was measured at 240 nm for 5 min with 1 min time interval. Superoxide dismutase (SOD) activity was done by adding 1.5 mM nitro-blue Tetrazolium (NBT) along with 0.1 M 2-mercaptoethanol and 10 mM MgSO4 to the lysate [22]. Absorbance was measured at 350 nm for 5 min with 1 min time interval. Glutathione reductase (GR) activity was measured by adding phosphate buffer (0.1 M) containing 1 mM EDTA, 400 M DTNB and 300 M NADPH to the lysate [23], and checking the absorbance at 405 nm for 5 min with 1 min time interval. 3. Results and discussion 3.1. Characterization of NPs Fig. 6. Effect of NPs on (a) Lipid peroxidation of algae assessed after the interaction with NPs, (b) ROS generation in algal cells due to NPs effect and (c) Extracellular protein content after interaction of algal cells with NPs. Values are the mean of n = 3 (mean ± standard error). homogenized using 5% trichloroacetic acid (TCA). The homogenates were centrifuged at 10,000 × g for 5 min. Equal volumes of supernatant and 0.5% of thiobarbituric acid (TBA) in 20% TCA were taken and incubated in water bath at 96 ◦ C for 30 min. The solutions are then centrifuged at 10,000 × g for 5 min and the absorbance of the supernatant collected was measured at 532 nm using Tecan Multi Mode Scanner in a 96 well plate. 2.7. ROS estimation ROS determination was done by measuring formation of dichlorofluorescein by the oxidation of dichlorofluorescein diacetate (DCFH-DA). Algae culture incubated with NPs was centrifuged at 10,000 × g for 10 min, suspended in phosphate buffer saline (pH 7.8) containing 30 g/mL DCFH-DA and incubated for 30 min in a shaker. The fluorescence values were measured at an excitation wavelength of 485 nm and emission wavelength of 520 nm. The synthesized NPs were subjected to SEM for the morphological evaluation. Fig. 1 shows the morphology and surface structure of iron oxide, composite of CdS/Ag2 S, CdS and Ag2 S NPs. Spherical to oval shaped particles were observed in all the particles. Fig. 2 shows the particle size distribution of the synthesized particles where the mean diameter of iron oxide, composite of CdS/Ag2 S, CdS and Ag2 S NPs were determined to be 60 ± 3, 50 ± 3, 35 ± 2 and 45 ± 3 nm respectively. Zeta potential values are a pre requisite to study the stability of the particles. The higher the zeta potential indicates the particle with greater stability. The zeta potential of iron oxide, composite of CdS/Ag2 S, CdS and Ag2 S NPs were determined to be −33.26 ± 1.57, −29.78 ± 1.37, −31.93 ± 1.76 and −29.69 ± 1.26 respectively. The XRD pattern of synthesized Fe3 O4 NPs showed their polycrystalline nature (Fig. 3a). The peak position at 2Â values of 30.3, 34.4, 43.1, 53.6, 56.2 and 62.9 are indexed to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) respectively. The results obtained agree with standard magnetite (Fe3 O4 ) XRD patterns and identified that the Fe3 O4 NPs were in a cubic spinel structure. Similarly, the XRD pattern of CdS/Ag2 S nanocomposite is shown in Fig. 3b. XRD patterns at 2Â values were matching perfectly with (1 0 1), (1 0 2), (1 1 0) and (1 1 2) crystalline planes of CdS and the diffraction peaks can be indexed to the hexagonal mesoporous structure of CdS. The 2Â values of Ag2 S matching with (1 1 1), (−1 1 2), (−1 2 1), (1 2 1), (1 0 3), (0 3 1), (2 0 0), (−1 2 3), (0 1 4) and (−2 2 3) crystalline planes and these diffraction peaks can be indexed to monoclinic ␣-Ag2 S. XRD patterns at 2Â values of 28.9, 37.3, 44.1, 47.9 and 51.7 matching perfectly with the (1 0 1), (1 0 2), (1 1 0), and (1 1 2) crystalline planes of CdS (Fig. 3c). All of the diffraction peaks can be indexed to the hexagonal CdS. No cubic phase or impurity peaks can be detected from the XRD measurement. XRD pattern of Ag2 S NPs is E. Jagadeesh et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 289 Fig. 7. Effect of NPs on different antioxidant enzyme activities (a) Catalase, (b) Superoxide dismutase, (c) Glutathione reductase and (d) Glutathione content in algal cells. Values are the mean of n = 3 (mean ± standard error). displayed in Fig. 3d. Here the main sharp peaks were assigned to (1 1 1), (1 1 2), (1 2 1), (1 0 3), (0 3 1), (2 0 0), (2 1 3) and (1 3 4) originate from Ag2 S NPs. No impurity peaks can be observed in the XRD measurement. Sharp facets that were observed in all the graphs indicates the crystalline nature of the particles. algal membrane. Adsorption of NPs on cell surface may cause the damage of cell membrane. Fig. 5c, e, g and i shows the membrane damage of Mougeotia sp. cells after NPs treatment. 3.2. Toxicity evaluation The membrane damage of algal cell membrane was supported by lipid peroxidation analysis. The lipid peroxidation of algae by NPs was performed by measuring the formation of thiobarbituric acid reactive species (TBARS). The progressive increase in the formation of TBARS was observed up on treatment with increase in concentration of NPs (Fig. 6a). The lipid peroxidation analysis was supported by ROS estimation. Excessive generation of ROS have the ability to induce algal membrane damage, lead to cell lysis and eventually cell death [26]. Fig. 6b shows the ROS generation due to NPs. The cell membrane damage was further confirmed by cellular protein estimation after the NPs-algal treatment. The damage of membrane could lead to the release of intracellular protein content to the interaction medium, thereby increasing the extra cellular protein content compared to control. The results show that the progressive increase in the concentration of extracellular protein content was observed upon treatment with increase in concentration of NPs (Fig. 6c). The results indicate the release of cellular protein content upon membrane damage. The amount of ROS formed/generated is related to the range of biological responses and oxidative stress [27]. According to Melegari et al. [15] the concentration of CuO NPs induced for ROS generation and lipid peroxidation in Chlamydomonas reinhardtii is 1000 g/mL. The present study found ROS formation and lipid peroxidation in Mougeotia sp. observed at the NPs exposure of 0.1 mg/L. It has been reported that the induction of intracellular oxidative stress by suppressing the cellular antioxidant defence system seems to be a key event of the toxicity mechanisms of many nanomaterials [28]. Once the NPs reached inside a cell, the NPs induced the intracellular oxidative Toxicity of the NPs was evaluated by the chlorophyll extraction method in which the percentage of the chlorophyll present in the acetone is measured using a visible spectrophotometer. The amount of chlorophyll present in the supernatant is directly proportional to the number of live cells remained after the interaction. On day five all the NPs showed toxicity (Fig. 4a) except iron oxide NPs which enhanced the growth in lower concentrations (0.1 and 1 mg/L), but it induced the toxicity in higher concentration of iron oxide NPs (in 5, 10 and 25 mg/L NPs). The CdS/Ag2 S nanocomposite showed relatively higher toxicity when compared to other NPs. On day 10 all the NPs including iron oxide NPs showed increase in the percentage of toxicity (Fig. 4b). Nanocomposite shows the highest toxicity compared to other NPs. Jing et al. [24] and Lee and An [25] studied the potential toxic effect of oxide NPs on Chlorella sp. and Pseudokirchneriella subcapitata respectively. They found that the metal oxide NPs were capable to induce the toxicity. More over the toxicity was induced by the dissolved metal ions. The present study used iron oxide NPs, CdS NPs, Ag2 S NPs and CdS/Ag2 S nanocomposite for the toxicological evaluation in green algae due to the lack of adequate information about the potent toxic effect of these NPs once it is released into the environment. The results show that these NPs possess toxicity and the toxicity may also be induced due to the dissolution of metal ions [24,25]. Microscopic evaluation of the samples was done in order to support the toxicity studies. The control of the algae (Fig. 5a) shows clear filament of the Mougeotia sp. whereas Fig. 5b, d, f and h shows adsorption of NPs on to the 3.3. Lipid peroxidation, ROS formation and oxidative stress 290 E. Jagadeesh et al. / Colloids and Surfaces B: Biointerfaces 125 (2015) 284–290 stress by disturbing the balance between oxidant and anti-oxidant processes. The oxidative stress on algal cells by NPs was evaluated by measuring the activity of antioxidant enzyme such as catalase, SOD and glutathione reductase. Fig. 7 shows the activity of antioxidant defence enzyme against NPs exposure. A significant decrease in the level of antioxidant enzymes activity and Glutathione content was observed. The suppression of antioxidant enzymes may be due to the excess production of ROS. The NPs may induce the cell’s apoptosis through a ROS-mediated mechanism [29]. In addition, excessive oxidative stress may also modify the cellular protein, lipid and nucleic acid components, eventually leading to the death of the cells [30]. 4. Conclusions The present study highlighted the impact of iron oxide, composite of CdS and Ag2 S, CdS and Ag2 S NPs once it is released into the environment. A fresh water algae Mougeotia sp. was used as a model organism to test the potential toxic effect. Compared to the other NPs in this study, the iron oxide NPs showed the least toxic effect. Lipid peroxidation and ROS generation were increased upon NPs treatment. NPs exposures suppressed the antioxidant defence system, thereby increasing the oxidative stress, leading to the death of the cells. The literature shows the divergent results of the nanoparticles toxicity which further suggest the need for conducting nanotoxicity with a systematic approach. Acknowledgments The authors sincerely thank Science and Engineering Research Board (SB/FT/LS-281/2012), Department of Science and Technology, Government of India and SASTRA University (TR Rajagopalan grant) for providing support to carry out the research work. The authors also thank Dr. Aswathy Ravindran, Assistant Professor, SASTRA University for proof reading our manuscript. 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