Effects of aqueous suspensions of titanium dioxide nanoparticles on Artemia salina: assessment of nanoparticle aggregation, accumulation, and toxicity
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- Ates, M., Daniels, J., Arslan, Z. et al. Environ Monit Assess (2013) 185: 3339. doi:10.1007/s10661-012-2794-7
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Aquatic stability and impact of titanium dioxide nanoparticles (TiO2 NPs, 10–30 nm) were investigated using Artemia salina. Acute exposure was conducted on nauplii (larvae) and adults in seawater in a concentration range from 10 to 100 mg/L TiO2 NPs for 24 and 96 h. Rapid aggregation occurred in all suspensions of TiO2 NPs to form micrometer size particles. Yet, both nauplii and adults accumulated the aggregates significantly. Average TiO2 content in nauplii ranged from 0.47 to 3.19 and from 1.29 to 4.43 mg/g in 24 and 96 h, respectively. Accumulation in adults was higher ranging from 2.30 to 4.19 and from 4.38 to 6.20 mg/g in 24 and 96 h, respectively. Phase contrast microscopy images revealed that Artemia were unable to excrete the particles. Thus, the TiO2 aggregates filled inside the guts. No significant mortality or toxicity occurred within 24 h at any dose. Lipid peroxidation levels characterized with malondialdehyde concentrations were not statistically different from those of the controls (p > 0.05). These results suggested that suspensions of the TiO2 NPs were nontoxic to Artemia, most likely due to the formation of benign TiO2 aggregates in water. In contrast, both mortality and lipid peroxidation increased in extended exposure to 96 h. Highest mortality occurred in 100 mg/L TiO2 NP suspensions; 18 % for nauplii and 14 % for adults (LC50 > 100 mg/L). These effects were attributed to the particle loading inside the guts leading to oxidative stress as a result of impaired food uptake for a long period of time.
KeywordsTiO2 nanoparticleArtemia salinaAggregationAccumulationToxicity
Nanotechnology is the new frontier worldwide and is predicted to become a trillion US dollar industry in the near future (Schmidt 2009). As new nanomaterials and products containing nanoscale particles are manufactured, as many will inevitably reach environmental repositories. The release of nanomaterials from commercial products into the aquatic environment has already been reported (Benn and Westerhoff 2008; Geranio et al. 2009). Nevertheless, the effects on human and environmental health are poorly understood because of the complexity of factors that affect chemical and toxicological properties of nanomaterials (Chatterjee 2008; Choi et al. 2009).
Titanium dioxide nanoparticles (TiO2 NPs) exhibit photocatalytic and antibacterial properties and thus have been used in various consumer products and environmental applications, including paint, sunscreens, surface coatings, and water disinfection (Bahnemann et al. 2002; Schulz et al. 2002; Mills et al. 2004; Zeynalov and Allen 2006; Choi et al. 2006). The release of products containing TiO2 NPs into fresh and coastal waters (e.g., estuaries) is concerning as it may impact the aquatic species and marine food chain, particularly algae and zooplankton (Moore 2006; Farre et al. 2009). A number of groups have evaluated the ecotoxicity of TiO2 NPs on freshwater models, such as Daphnia magna (Hund-Rinke and Simon 2006; Lovern and Klaper 2006; Warheit et al. 2007; Handy et al. 2008; Farkas et al. 2010; Kim et al. 2010; Zhu et al. 2010). Nevertheless, our understanding about their fate and toxic effects is still in its infancy because of the controversies among the findings associated with the differences in test models, experimental conditions, and surface properties of TiO2 NPs. For instance, Wiench et al. (2009) found little acute toxicity from nanoscale and microscale TiO2 on D. magna using different test media and several NP formulations (EC50 > 100 mg/L). A similar result was reported for zebrafish (Danio rerio) embryos for which no significant toxicity was observed from TiO2 NPs at concentrations as high as 500 mg/L (Zhu et al. 2008). Conversely, TiO2 NPs induced lethal effects in a long-term exposure study in that 40 % of D. magna died when exposed to 20 mg/L levels (Adams et al. 2006). Relatively high toxicity was reported from Zhu et al. (2010) for uncoated TiO2 NPs that induced 13 % mortality on D. magna within 72 h at 0.1 mg/L level.
Artemia salina (brine shrimp) are zooplankton that are used to feed larval fish in cultures like copepods and daphnids (Sorgeloos 1980). They play an important role in the energy flow of the food chain in marine environment. In addition, they are used as a laboratory bioassay organism to develop standard toxicology assays (Vanhaecke et al. 1981; Sanchez et al. 1997; Nunes et al. 2006; Kanwar 2007). Artemia are hypo/hyper-osmotic regulators that are able to maintain hemolymph ion concentrations within narrow limits over an external salinity range from 0.26 % NaCl to supersaturated brines. With this capability, Artemia appear to be suitable model species to investigate the fate and ecotoxicity of nanomaterials in marine ecosystems through laboratory experiments.
In this study, we conducted exposure studies on Artemia, both nauplii (larvae) and adults, in aqueous suspensions of uncoated TiO2 NPs to elucidate the effects of TiO2 NPs on the marine ecosystems. Total TiO2 content (accumulation) and toxic effects (mortality and lipid peroxidation) were determined under acute exposure for 24 and 96 h. Colloidal stability of the NPs in water was also examined to understand the influences on NP accumulation and toxicity.
Materials and methods
Reagents and chemicals
Titanium dioxide nanoparticles (TiO2 NPs, 99.5 % rutile polymorph) were purchased, as uncoated nanomaterials, from Skyspring Nanomaterials Inc. in Houston, TX, USA. The NPs were spherical with an average particle size (D50) between 10 and 30 nm and approximate surface area of 50 m2/g. The morphology of the NPs was rutile with pale yellow color, which is most widely found polymorph of TiO2 in nature.
Artemia cysts (The Great Salt Lake, Utah harvest) were purchased from Artemia International LLC, Houston, TX, USA and were kept at 4 °C temperature moisture-free container in a refrigerator. Deionized water produced by Barnstead E-pure system with resistivity of 18 MΩ cm was used to prepare the exposure medium and experimental solutions. Trace metal grade nitric acid (HNO3, Fisher Scientific) and hydrofluoric acid (HF, Sigma Aldrich) were used for digestion of the Artemia collected at the end of the exposure to determine the total TiO2 contents. The use of HF was necessary for effective solubilization of TiO2 to Ti ions in solution for inductively coupled plasma mass spectrometry (ICP-MS) measurements. Stock titanium standard solution (1,000 μg/mL) was purchased from SCP Science (Champlain, NY, USA) and used for preparation of ICP-MS standards in 5 % HNO3. Carbon-coated Cu transmission electron microscopy (TEM) grids (300 mesh) were used to measure the size of NPs. The grids were purchased from Electron Microscopy Sciences, Hatfield, PA, USA.
Preparation of test organism
Artemia cysts were hatched in seawater (30 ‰ m/v). The seawater was prepared by dissolving appropriate amount of Instant Ocean® salt in deionized water, stirred for 24 h under aeration and then filtered through 30-μm Millipore cellulose filters. Artemia were hatched by using the procedure described by Persoone et al. (1989). Briefly, encysted Artemia were first hydrated in distilled water at 4 °C for 12 h and then washed to separate the floating cysts from those that sink. The sinking cysts were collected on a Buchner funnel and washed with cold deionized water. Approximately 3 g of the precleaned cysts were incubated in 1.5 L seawater in a conical plastic contained with graduations at 30 ± 1 °C. A 1,500 lx daylight was provided continuously by a fluorescent lamp. Aeration was maintained by a small line extending to the bottom of the hatching device from an aquarium air pump. Under these conditions, Artemia hatched within 24 h.
Counting hatched Artemia
The rate of hatching was variable; therefore, it was important to determine the number of Artemia nauplii and adults as accurately as possible prior to the start of exposure. The counting was performed according to the procedure described by Sorgeloos (1980). Briefly, 100 mL solution containing the hatched Artemia nauplii was taken into a clean beaker. Under continuous stirring, 1 mL of this stock was diluted to 100 mL with seawater (100-fold dilution). Next, 0.1 mL of the diluted solution was taken under stirring and placed in a petri dish. The number of nauplii was determined by counting visually in this volume (0.1 mL).
Preparation of aqueous suspensions of TiO2 NPs
Preliminary exposure studies were conducted with up to 5 mg/L TiO2 NPs on Artemia nauplii to estimate the exposure concentration. No significant immobilization or mortality occurred within 24 h; therefore, the experimental NP suspensions were prepared between 10 and 100 mg/L to achieve measurable effects.
A stock suspension of 20 % (m/v) was prepared by dispersing appropriate amount of TiO2 NP powder in deionized water. This solution was vortexed for 20 s, and then sonicated in an ultrasonic bath for about 10 min for maximum dispersion. Appropriate volumes of the stock suspension were then immediately transferred into the exposure tanks containing Artemia nauplii or adults in the seawater.
Size distribution of TiO2 NP suspensions
Morphology and size distribution of the TiO2 NPs were characterized by TEM and dynamic light scattering (DLS). For stock TiO2 NP suspensions in deionized, measurements were made immediately after the preparation of the suspension. A drop of the colloidal solution of TiO2 NPs was placed onto 50 Å thick carbon-coated copper grids and allowed to dry for TEM measurements. The images were recorded by JEOL-1011 TEM instrument providing a resolution of JEM-1011 is 0.2 nm lattice with magnification of 50–1 × 106 under the accelerating voltage of 40–100 kV. TEM images were then analyzed by using ImageJ software package. Particle size distribution was collocated for a group of 100 particles in random fields of view. DLS measurements were conducted by DynaPro DLS instrument (wavelength, 826 nm; power, 58 mW, at 100 % usage). A portion of the stock suspension solution from stock TiO2 suspension was placed in a clean cuvette and measured five times successively. Particle size measurements from exposure medium (e.g., salt water) were made similarly by both TEM and DLS. In this case, measurements were conducted 12 h after the start of the exposure to verify possible aggregation and changes in particle size.
Experimental conditions for acute exposure of Artemia to uncoated TiO2 NPs (D50 = 10–30 nm)
TiO2 NP concentration (mg/L)
Total number of Artemia
At the end of the exposure, Artemia were sampled and thoroughly washed with deionized water through 40-μm plankton net. The cleaned samples were then filtered by 0.45-mm Whatman filter paper. For instrumental analysis, about 0.1 g of wet Artemia (nauplii and adult) was weighed and digested in teflon vessels in 2 mL concentrated HNO3 and 0.5 mL HF for 2 h using digestion block (DigiPrep MS, SCP Science) at 160 °C according to protocols described elsewhere (Arslan et al. 2000, 2011). Once completely digested, the contents were diluted to 10 mL with deionized water. The sample solutions were further diluted tenfold for analysis. For quality control, pure TiO2 NP samples (ca. 10 mg, n = 5) were digested in 3 mL HNO3 and 1.0 mL HF similarly and diluted to 10 mL with water. These samples were diluted 1,000-fold before analysis. All samples were analyzed for titanium (Ti) concentration by ICP-MS using a Varian 820MS ICP-MS instrument (Varian, Australia). Titanium standard solutions in the range of 0.2–5.0 μg/mL were used for instrument calibration. These standard solutions were prepared in 5 % HNO3 and contained trace HF (e.g., <0.1 %). Titanium concentration was converted to TiO2 content to determine the total accumulation across different doses of exposure.
Thiobarbituric acid-reactive substances were measured to determine the lipid peroxidation products as a measure of oxidative stress. The values were expressed as total malondialdehyde (MDA) concentration per gram of Artemia. MDA concentration was measured as described by Van Ye et al. (1993). For MDA measurement, 0.1 g Artemia was washed with cold water and then assayed using the MDA kit (Northwest Life Science Specialties, LLC, Vancouver, WA, USA). Samples were homogenized in 2 mL phosphate buffer (pH 7.2) by ultrasonic homogenizer and then centrifuged at 6,000 rpm for 10 min. The resulting sample supernatant was immediately processed for biochemical assay, where 10 μL butylated hydroxytoluene reagents, 0.25 mL of sample supernatant, 0.25 mL of phosphoric acid reagent, and 0.25 mL of thiobarbituric acid reagent were added to a vial, respectively. A set of stock tetramethoxypropane standards in the range of 0–8 μM was prepared freshly in methanol. To prepare calibration standards, 0.25 mL of the appropriate standard solution was processed similar to the sample supernatants as described above. All samples and standards were incubated at 90 °C for 1 h and centrifuged after cooling at 13,000 rpm for 10 min to precipitate suspending tissue. The reaction mixtures were then transferred to UV-visible spectrophotometer cuvettes and the absorbances were measured at 532 nm. Measurements were performed in triplicate for controls and experimental groups.
All experiments were repeated three times independently, and data were recorded as the mean with standard deviation. One-way analysis of variance with Tukey’s multiple comparisons was used to detect significant differences in mortality and accumulation rates among the controls and treatments. In all data analyses, a p value of 0.05 was considered statistically significant.
Results and discussion
Stability of TiO2 NPs in water
Size distributions of aqueous suspensions of TiO2 NPs
TiO2 NP concentration (mg/L)
Fresh stock suspension
Dry size (TEM, nm)
Hydrodynamic size (DLS, nm)
Dry size (TEM, nm)
Hydrodynamic size (DLS, nm)
The TEM image recorded 12 h later from exposure medium containing 10 mg/L TiO2 NPs is illustrated in Fig. 1b. Considerably larger aggregates and strips of TiO2 were observed that were more stable (e.g., remained as large particles) compared with the fine NPs shown in Fig. 1a. Size of dry particles ranged from several hundred nanometers to microns in diameter. Hydrodynamic diameter of the particles also increased ranging from 280 to 2,334 nm (see Table 2). This kind of temporal increase in the size of the aggregates of TiO2 NPs was also reported by Zhu et al. (2010) for 10 mg/mL suspension of uncoated TiO2 NPs (21 nm). Moreover, they renewed the suspensions daily to maintain NP stability and concentration, but the effects were not very different from that of continuous mixing used in this study. The median size of the aggregates increased from 580.5 to 2,349 and then to 3,526 nm within 1, 12, and 24 h, respectively.
Accumulation of TiO2 NPs
Elimination of ingested TiO2 NPs
At the conclusion of the exposure, Artemia were placed into freshly prepared seawater and allowed to clean up the guts from particles for 24 h. Then they were washed and digested similarly in acid to determine the change or loss in the TiO2 content. The results are illustrated in Fig. 3. For nauplii, the concentration of TiO2 decreased by 0.015–0.42 and 0.030–0.53 mg/g following 24- and 96-h exposures, respectively. The adults showed similar elimination pattern; 0.11–0.52 mg/g for 24-h exposure and 0.22–0.74 mg/g for 96-h exposure. These concentrations correspond to about 3–12 % reduction in TiO2 content. It is evident that Artemia were unable to eliminate the ingested particles. Likewise, D. magna had difficulty in getting rid of the particles from the guts after acute exposure to 1.0 mg/L suspensions of TiO2 NPs in static water (Zhu et al. 2010). Only a fraction of ingested TiO2 were excreted from the body in 24 h, though the efficiency improved up to 50 % within 72 h. Presence of food in the medium improved the elimination efficiency, but a significant portion (ca. 20 %) still remained in the guts (Zhu et al. 2010).
The TEM and DLS data (Fig. 1b and Table 2) clearly show that TiO2 NPs were no longer nanometer size particles but aggregates in the exposure medium. Nevertheless, Artemia, even nauplii, accumulated the aggregates from water readily within 24 h (Fig. 3). The large discrepancy between the accumulation and elimination rates could be due to the continuous aggregation of ingested particles inside the guts to yield massive TiO2 particles (see Fig. 2) that could not be excreted from the guts.
Effect of exposure time on mortality
Percent mortality rates for Artemia measured for 24- and 96-h exposure to different suspensions of uncoated TiO2 NPs
TiO2 NP concentration (mg/L)
Effect of concentration of TiO2 NP suspension on mortality
Average mortality measured across tenfold concentration gradient was largely dependent on the duration of the exposure rather than the TiO2 NP concentration of the suspension (Table 3). Compared with the controls, the suspensions had no toxic effects at any concentration within 24 h, but caused mortality during 96 h. The differences among 24-h mortalities were not significant for nauplii nor for adults (p > 0.05), ranging between 3 and 6 % when the concentration of the suspension increased from 10 to 100 mg/L (Table 3). This effect was thought to be due to the reduced surface area and catalytic activity as the NPs aggregated or agglomerated to microscale particles in solution and inside the guts. The effects on D. magna were also consistent with these results (Warheit et al. 2007; Heinlaan et al. 2008). TiO2 NP suspensions as high as 20 g/L (2 orders of magnitude more concentrated than those tested here) were reported to be nontoxic to D. magna (Heinlaan et al. 2008).
Exposure for 96 h resulted in elevated mortality in all suspensions relative to the controls (p < 0.05). However, the differences among the treatments were marginal that ranged from 13 to 18 % for nauplii (p = 0.043) and 10 to 14 % for adults (p = 0.045) when NP concentration increased from 10 to 100 mg/L. Tukey’s multiple comparisons revealed that 96-h mortality rates between the adjacent treatments (e.g., 10 and 50, and 50 and 100 mg/L) were not statistically different (p > 0.05) indicating that the concentration of the NP suspensions had marginal toxic effects on Artemia. Still though, these results imply that prolonged exposure (e.g., 96 h) increases the risk of mortality on Artemia, regardless of its state of maturity and concentration of the NP suspension. The lethal effects observed could be attributed to the failure in eliminating the aggregates of TiO2 NPs from the guts and consequently depletion of food uptake from water.
Oxidative stress induced by suspensions of TiO2 NPs
Oxidative stress levels associated with exposure to the suspensions of TiO2 NPs
TiO2 NP concentration (mg/L)
22.0 ± 0.61
25.4 ± 1.7
27.9 ± 1.2
30.6 ± 2.1
22.9 ± 0.33
41.6 ± 1.7
29.0 ± 1.4
56.2 ± 1.9
23.9 ± 0.38
44.1 ± 1.6
32.4 ± 2.8
60.7 ± 2.2
24.4 ± 1.05
44.1 ± 1.4
31.2 ± 2.5
61.2 ± 3.2
Lipid peroxidation levels increase with food deprivation (Pascual et al. 2003). The relationship is attributed to the increasing generation of oxygen-free radicals as the antioxidant levels deplete as a result of starvation. Eventually, the symptoms exhibited by Artemia along with experimental data indicate that the suspensions of TiO2 NPs are nontoxic despite substantial accumulation. This result points to the fact that that the oxidative stress induced during prolonged exposure is associated with the impaired food uptake as a result of accumulation and deposition of TiO2 aggregates inside the guts. The lethal effects occurred during 96-h exposure could therefore be attributed to the oxidative stress caused by the deprivation from food or starvation rather than the chemical toxicity of the suspensions of TiO2 NPs.
In this study, we used Artemia, crustacean filter feeder, as a test model to investigate the effects of exposure to aqueous suspensions of TiO2 NPs in marine ecosystems. The results demonstrate that TiO2 NPs rapidly aggregate in saltwater to form microscale particles. However, the formation of large particles had no effect on the accumulation; both nauplii and adults readily accumulated the microscale aggregates to elevated levels such that the guts were filled with particles within 24 h. Yet, neither nauplii nor adults showed any significant mortality or oxidative stress within 24-h exposure. Thus, it was concluded that the suspensions of TiO2 NPs were nontoxic to Artemia. Extended exposure to 96 h did induce oxidative stress manifested with marginal mortality. However, these effects were most likely due to the lack of food uptake since the guts were completely filled with the aggregates of TiO2 NPs.
This project is funded in part by grants from the National Institutes of Health (NIH) through Research Centers in Minority Institutions (RCMI) Program (Grant No: G12RR013459) and the US Department of Defense (DOD) through the Engineer, Research and Development Center (Vicksburg, MS, USA); (contract #W912HZ-10-2-0045). The views expressed herein are those of the authors and do not necessarily represent the official views of the funding agencies, and any of their sub-agencies.