A Temporal Study on the Effects of TiO2 Nanoparticles in a Fresh Water Microcosm
Deepak Kumar, A. Rajeshwari, Rajdeep Roy, Sunandan Pakrashi, V. Iswarya, Ida Evangeline Paul, Ankita Mathur, N. Chandrasekaran, et al. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences ISSN 0369-8211 Volume 86 Number 2 Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. (2016) 86:415-420 DOI 10.1007/s40011-014-0462-0
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Author's personal copy Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. (Apr–June 2016) 86(2):415–420 DOI 10.1007/s40011-014-0462-0
RESEARCH ARTICLE
A Temporal Study on the Effects of TiO2 Nanoparticles in a Fresh Water Microcosm Deepak Kumar • A. Rajeshwari • Rajdeep Roy • Sunandan Pakrashi • V. Iswarya • Ida Evangeline Paul • Ankita Mathur • N. Chandrasekaran Amitava Mukherjee
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Received: 9 June 2014 / Revised: 5 November 2014 / Accepted: 13 November 2014 / Published online: 19 December 2014 Ó The National Academy of Sciences, India 2014
Abstract This study assesses the temporal changes in the physico-chemical behaviour of titanium dioxide nanoparticles (anatase and rutile phase) for a period of 120 h at environmentally relevant concentration of 1,000 lg/L, and the consequent impact on the microalgae population in a fresh water microcosm. The mean hydrodynamic size analysis in the medium revealed the differences in the aggregation behaviour of the two crystalline types of particles within first 12 h exposure before they had reached the micron size range. While the short term exposure (120 h) showed an immediate effect on the resident microalgae in the microcosm with respect to control, there were no significant differences in ecotoxicity effects of rutile and anatase phases of titania. The long term (90 days) exposure demonstrated a gradual recovery of the resident algal population. Summarizing the observations, the nanosized particles at low concentration may not retain the toxic potential for longer exposure time in a microcosm presumably owing to the complexity prevalent in the natural systems. Keywords Titania nanoparticles Crystallinity Microcosm Ecotoxicity
Electronic supplementary material The online version of this article (doi:10.1007/s40011-014-0462-0) contains supplementary material, which is available to authorized users. D. Kumar A. Rajeshwari R. Roy S. Pakrashi V. Iswarya I. E. Paul A. Mathur N. Chandrasekaran A. Mukherjee (&) Centre for Nanobiotechnology, VIT University, Vellore 632014, India e-mail:
[email protected];
[email protected]
Introduction Continuing increase in the usage of the engineered titanium dioxide nanoparticles (TiO2 NPs) in the consumer applications poses elevated environmental risk associated with the exposure of freshwater ecosystem to these particles thus affecting the microalgae (primary producers) [1]. These nanoparticles are one of the commonly found ingredients in the personal care products like cosmetics and sunscreen lotions. Owing to their increased presence in the daily usage products the discharge of these nanoparticles into the wastewater system has recently raised considerable public concern [2, 3]. As per recent environmental modelling data, the predicted environmental concentrations of the titanium oxide nanoparticles would be below 1,000 lg/ L concentration [4]. The three polymorphs of titania nanoparticles are rutile, anatase and brookite, each having distinctly different crystalline structure [5]. Among them, rutile and anatase are the most prevalent forms crystalizing in tetragonal shape. They acquire distinct photocatalytic properties, with anatase having good combination of photo-activity and photo-stability, in comparison to rutile. There have been only a handful of previous studies on the behaviour of the engineered nanomaterials in microcosm or mesocosm setup simulating environmental conditions over a fixed time interval. The distribution and behaviour of PVP coated silver nanoparticles during long time exposure in a mesocosm was monitored for a period of 18 months [6]. In another earlier study, the endobenthic species were exposed to CuO nanoparticles (10 lg/L) for a period of 21 days in a mesocosm [7]. In authors’ previous microcosm study [8] on behaviour and fate of the aluminium oxide nanoparticles at environmentally relevant concentrations (1,000 lg/L and below) for a period of
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210 days, definite ecotoxic response only at 1,000 lg/L concentration was observed. The short term exposure (5 days) showed an immediate effect on the resident algal population (*25 % decreased viability) and the long term (210 days) exposure showed a gradual recovery. To the best of the knowledge there is no previous report studying the toxic effects of two different crystalline phases of titania nanoparticles at low concentration in a microcosm setup replicating the freshwater ecosystem. Keeping in mind the above facts, the main aim of the current study was to provide an overview of the ecotoxic impact of the two different crystalline phases of titanium oxide nanoparticles (anatase and rutile) at low concentration (1,000 lg/L). The study was performed on the resident microalgae population in a freshwater microcosm over 90-day exposure duration.
Material and Methods Location Details
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Characterization of the TiO2 NPs TiO2 nanoparticles were procured from Sigma Aldrich (St. Louis, Missouri, USA). The details, as provided by the supplier, are as follows: Dry titanium dioxide nanopowder, 99.7 % Anatase, particle size:\25 nm, CAS No.: 1317-700, titanium dioxide nanopowder, 99.5 % rutile, particle size: \100 nm, CAS No. 1317-80-2, All other chemicals used were of analytical grade. X-ray Diffraction Analysis To characterize the crystal structure of the TiO2 nanoparticles (anatase and rutile), 250 mg of TiO2 powder was used. Powdered X-ray diffraction analysis (D8 Advanced X-ray Diffractometer, Burker, Germany) was done following the scan with 2.2 kW Cu anode radiations at ˚ produced by a Ceramic X-ray tube and wavelength 1.54 A the scanning range was set to 10–100°. Particle Size Analysis
The setup was established with the lake water collected from VIT Lake, VIT University, Vellore, India. The location of the Vellore is at 12–15° and 13–15°N and 78–20° and 79–50°E with an average height of 216 m asl. Vellore has a typical Mediterranean climate where December– January is the coldest period and April–June, experiences, dry summer.
Powdered TiO2 nanoparticles (rutile and anatase) were dispersed in Millipore water by sonicating, using an ultrasonic processor (Sonics, USA) to prepare a stock dispersion of 10,000 lg/L. Lake water was filtered, and was used to prepare the working concentration (1,000 lg/ L) from the stock solution. Particle Size Analyzer (90 Plus Particle Size Analyzer, Brookhaven Instruments Corp, USA) was used to analyse the hydrodynamic size of TiO2.
Setting Up of the Microcosm System
Aggregation of TiO2 Nanoparticles in Lake Water
The study was carried out using 20 L of water tanks imitating lake water ecosystem (Fig. 1) [8]. Normal weathering conditions were maintained throughout the experiment, and the tanks were positioned in a garden close to the laboratory with no control over rainfall and sunlight exposure (Fig. 1). There was a continuous replenishment of lake water to recompense evaporation on alternate days. The collected soil and water were reconciled. Continuous monitoring at every seventh day was done for various parameters like pH, salinity, temperature and conductivity to confirm the lake water ecosystem environment using multiparameter PCSTestr-35 Eutech instrument, (Singapore).The microcosm was found to be comparable to lake water ecosystem after 6 weeks. Stock solution of 10,000 lg/L for both titania anatase and rutile in MilliporeTM water were prepared by sonication for 20 min with a frequency of 20 kHz and output energy of 130 W [ultrasonic processor, Sonics Corp., USA]. TiO2 nanoparticles with a final exposure concentration of 1,000 lg/L were introduced in the system.
The appropriate aliquots from the stock concentration (10,000 lg/L) were added to the experimental system to attain 1,000 lg/L concentration. Filters of coarse and finesize (20 mm mesh–Whatman no. 1 filter paper–Whatman no. 42 filter paper) were used sequentially to filter 20 mL of the sample obtained from the upper and lower region of the microcosm. Hydrodynamic size distribution analysis of the filtrate was carried out for every 6 h in an 18 h time period.
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Effect of Nanoparticles on Algal Community The possible effects on the algal community in the microcosm were analysed for a span of 150 days to study the effect of TiO2 nanoparticles. The nanoparticle-stimulated toxic response was regularly examined by estimating the cell count at definite time intervals and the findings are explained in two different sections—short term analysis for a span of 120 h and long term analysis for a span of 2,160 h with regular sampling intervals. The normalization
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Fig. 1 Photograph showing microcosm setup in the garden containing the experimental conditions under natural conditions
of the experimental data was carried out, and the relative percentage was expressed by taking the control (samples devoid of nanoparticles) to be 100 %. Viability of the cells was estimated by direct counting of the cells using a haemocytometer. Water sample (10 ml) was collected as per the standard protocol. After loading of the sample on a Neubauer chamber, a cover glass was placed over the grid and the total number of whole cells was determined under bright-field microscope at 4009 magnification to compute the relative cell count in comparison to the control. Analysis of Statistical Data Each set of experiment was repeated thrice. The mean and standard error (SE) were calculated to express the data points for all the experiments. One way ANOVA followed by Dunnett’s and Neuman Keul’s post hoc test (as applicable) was further carried out for each set of experiment to show the significant variations (at p [ 0.05) at various time points.
Results and Discussion Preliminary Characterization of the Nanoparticles From the X-ray diffraction analysis of TiO2 rutile nanoparticle, six diffraction peaks (at 69.6°, 62.9°, 54.1°, 41.1°, 36.1°, and 27.2°), with highly intense peaks at 27.2°, 36.1° and 54.1° (Fig. S1, supplementary information) were noted. The XRD results were matched with a database of Joint Committee on Powder Diffraction Standards (JCPDS) card file no. 84-1286, 01-1292, which confirmed the presence of the rutile phase. The SEM image of TiO2 rutile nanoparticles showed that the particles were approximately spherical with a diameter range from 25 to 100 nm (Fig. S2, supplementary information). The X-ray diffraction patterns of TiO2 anatase nanoparticles
confirming the anatase phase and their nearly spherical morphology as seen from the TEM micrograph (Fig. S3, supplementary information) and also confirmed in a previous report [9]. The mean hydrodynamic size of both TiO2 anatase and rutile nanoparticle in Millipore water was estimated to be around 285 ± 10 and 327 ± 15 nm respectively.
Aggregation of Particles in Fresh Water Microcosm The toxicity of nanoparticles depends considerably on their size and surface chemistry in the aqueous medium. The aggregation behaviour of the particles in the test medium always plays a pivotal role in determining their toxic effect. The mean hydrodynamic size (MHD) of the lake water medium devoid of the particles was in the range between 237 ± 18 and 279 ± 12 nm in the upper part and the bottom part of the fresh water microcosm respectively. This was noted presumably owing to the presence of the nanosized natural colloids [8]. The MHD of TiO2 anatase nanoparticles was found to be 285 ± 6 and 306 ± 2 nm in the upper and lower parts of the fresh water microcosm at 0 h. The aggregation profile of anatase nanoparticles in the fresh water microcosm was recorded till 18 h and MHD was found to be 299 ± 6, 1,192 ± 102, and 1,958 ± 87 nm at 6, 12, and 18 h respectively in the upper part of the microcosm (Fig. 2a, b; Fig. S4, supplementary information). Similarly, in the bottom part of the microcosm, MHD was seen to be 352 ± 47, 1,781 ± 151, and 3,183 ± 237 nm at 6, 12, and 18 h respectively. On the other hand, the MHD of rutile nanoparticles at 0, 6, 12, and 18 h was found to be 289 ± 7, 356 ± 8, 1,442 ± 90, and 2,623 ± 167 nm in the upper part of the microcosm and 316 ± 8, 1,174 ± 29, 2,087 ± 116, and 4,482 ± 145 nm in the bottom part (Fig. 2a, b; Fig. S5, supplementary information). These results suggested that there was no significant increase in the MHD of TiO2 nanoparticles (anatase and rutile) till 6 h from the initial
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Fig. 2 Detailed particle size analysis in lake water matrix with depiction of dose associated impact of two different TiO2 NPs (anatase and rutile) from 0 to 18 h showing total aggregation profile.
a: Showing total aggregation profile of TiO2 (anatase and rutile) in upper part of microcosm. b: Showing total aggregation profile of TiO2 (anatase and rutile) in bottom part of microcosm
size in both the upper and bottom parts of the microcosm, whereas a considerable increase in size was noted only after 12 h of exposure. In the current study, it was noted that the rutile nanoparticles aggregated more rapidly as compared to anatase in both top and bottom layers of microcosm tanks. It has been reported previously by Dalai et al. [9] that the TiO2 anatase nanoparticles under laboratory conditions (pH 7 and at temperature 37 °C) were stable for over a period of 24 h in the sterilized VIT lake water. The presence of the natural organics in the freshwater may have influenced the stability of the TiO2 nanoparticles in the microcosm albeit for a short exposure time. The influence of natural organic matter on the fate and behaviour of metal oxide nanoparticles has been well emphasized in the previous reports [10, 11]. A rapid aggregation trend has been reported for ZnO and Al2O3 nanoparticles, where the initial aggregates were found to be loosely bound and subsequently formed the compact agglomerates [8, 12]. As the particle size increases, the surface charge of both anatase and rutile TiO2 nanoparticles shifts toward a more negative value. This effectively leads to a high energy barrier resulting in a higher critical coagulation concentration [11], facilitating their deposition and removal, thus decreasing their bioavailability in the system. With the continuing increase in the aggregate size of the nanoparticles in the test medium, they ultimately tend to behave like a bulk material in the system and the toxicity response would be expectedly different from that of the nanosized material [13, 14]. Therefore, the particle size analysis in the current study was discontinued after 18 h as the average size range increased beyond 1,000 nm in both the upper and bottom compartments of the microcosm.
Effect of Aggregation of Nanoparticles on Freshwater Resident Algal Population
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It is well known that the microalgae as a primary producer play a major role in the maintenance of an aquatic ecosystem. Any external perturbations to the aquatic system can be estimated by analysing the changes on algal population in the ecosystem. In the present study, the damage to the algal population owing to the intervention of nanoparticles, if any, was analysed with the help of the cell enumeration. The bioaccumulation for 1 lg/mL of TiO2 NPs in sterile lake water in the lab conditions (pH 7 and at temperature 37 °C) was found to be 78.36 L/kg by ICP–OES analysis as stated earlier [15]. The cell viability (Fig. 3) was found to decrease during the short term study (120 h) and increased during the long term study (360 h). Though both the types of particles retained the sub-micron size range only till 12 h (Fig. 2a, b), considerable toxicity (about 73.9 and 79.9 % viability for anatase and rutile nanoparticles respectively) was noted even at 120 h in the microcosm. During the first 6 h, no significant toxicity was noticed for both types of the nanoparticles. After 6 h the toxicity could be noticed in the cells. The authors also mentioned that both the types of nanoparticles could retain their sub-micron size only up to 12 h in the lake water under the given set of conditions. It means that after the first 12 h, the TiO2 nanoparticles were no longer retaining their nanometer size and were being transformed in much bigger size by aggregation. This might have naturally resulted in a decreased availability to the cells, thereby reducing the toxicity to the cells. At that point of time, the cells are being exposed not to nanoparticles but micron sized TiO2 particles. The interaction of
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Fig. 3 Depiction of dose associated impact of two different TiO2 NPs (anatase and rutile) onto fresh water algae with response to nanoparticles exposure up to 2,160 h (90 days)
NPs with algae leads to the aggregation of NPs by the exopolysaccharides (EPS) produced by algae due to the stress triggered by NP. The produced EPS may cover the surface of NPs, which may cause their gradual aggregation possibly altering their bioavailability and toxicity [9, 16, 17]. As the size of the NPs enters the micron range, there is less availability of NPs in water and thereby the toxicity of NPs on algae is significantly reduced. In a related study, it has shown that, on nTiO2 exposure, the growth rates of A. variabilis was reduced up to 90 % with concentrations ranging from 0.5 to 500 mg/L, and for various exposure periods, ranging from 24 to 144 h [1]. The anatase nanoparticles in the current study had a greater impact on the resident algal population than the rutile nanoparticles during short term exposure (120 h) possibly owing to their differential rate of aggregation as discussed in the preceding section. The aggregate size mostly controls the bioavailability and thus reactivity of the particles in an aqueous medium. Several prior reports suggest that the size is one of the major determining factors for metal oxide nanotoxicity [18–20]. Stolle et al. [21] reported that both the size and crystal structure of the nanoparticles contributed to cytotoxicity. For both anatase and rutile nanoparticle-treated systems, a significant increase in the algal population was noted after 360 h (15 days) during the long term exposure. Interestingly the algal growth reached its maximum viability of about 88 and 97 % for the anatase and the rutile treated samples respectively, on the 90th day (2,160 h). The reduction in biological load during a short exposure was replenished with time, and a restorative effect of algal population could be noted after 360 h (15 days), possibly owing to the decreased availability of TiO2 nanoparticles in
the aquatic system. The previous reports on freshwater microalgae (Scenedesmus obliquus) and the mammalian cell lines (HaCaT cell) mentioned about the adaptive nature of the cells upon prolonged exposure at low concentration of titanium dioxide nanoparticles possibly due to reduced bioavailability [22, 23]. Possible correlations between the aggregation of the nanoparticles in an aqueous medium and their crystalline structure can be drawn from the current study, which may control the bioavailability and thus the toxicity potential within a limited exposure period.
Conclusion The bioavailability of the engineered nanoparticles in a complex environmental system is one of the major criteria to be considered for studying their ecotoxicity implications. The nanoparticle availability is a function of their aggregation behaviour in the aquatic system, which in turn is strongly dependent on the environmental parameters like pH, temperature, total dissolved solids and presence of the other pollutants [the changes in parameters over the 3 month exposure duration are detailed in Fig. S6–S8, supplementary information]. This conventional interdependence makes it quite difficult to predict the nanoparticles flow pathway and toxicity potential in a realistic environment. Owing to the complexity of environmental parameters and lack of control in a microcosm setup, it is difficult to derive definite conclusions from studies of this kind. In short exposure duration, typical differences were noticed in the aggregation behaviour largely based on the crystalline phases of the particles, which in turn influenced their toxic
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impact to the microalgae in the system. However, the observations from long term tests point towards natural attenuation of possible detrimental impacts of the tiny particles irrespective of their crystallinity, when they are present at low concentration in the natural systems. Acknowledgments The authors acknowledge Sophisticated Analytical Instrumentation Facility (SAIF), Department of Science and Technology (DST) at Indian Institute of Technology, Madras for SEM analysis and Life Science Research Board-DRDO, Govt of India, for financial support. The authors declare that they have no conflict of interest.
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