Acute Toxicity of Suspension of Nanosized Silicon Dioxide Particles to Daphnia Magna Han Bing, Wei Chen-xi, Yang Di, Hu Chuan-lu, Yu Xiao-wei, Yang Xu* Laboratory of Environmental Science College of Life Science, Huazhong Normal University Wuhan, China * Corresponding author email:
[email protected] Abstract—There is a broad application area for engineering nanoparticles (ENPs) in virtue of its special physical and chemical properties. ENPs bring a huge advance to development of human society, as well as the questions about whether the manufactured nanomaterials (MNMs) can cause disadvantages to organisms and environment. This paper analyzed the 24-h acute toxicity of water suspensions of the nanosized silicon dioxide (Nano-SiO2) and the normal sized silicon dioxide (Nor-SiO2) as its bulk counterpart to Daphnia magna with different concentration. The experiment considered EC50 value for immobilization and LC50 value for mortality as the toxicological test targets. The results show that the toxicity of Nano-SiO2 is obviously does-dependent but the Nor-SiO2 is not, evidently. The optical microscope and digital camera also been used to observe and record the biological form of Daphnia magna during the exposure and in this study we found that D. magna can uptake and adsorb Nano-SiO2 but only apparently show uptaking to its bulk counterpart. The conclusions of results suggest that there is potential harm to aquatic environment in using Nano-SiO2, and it should deserve special concern. Keywords- Nano-SiO2; Daphnia magna; Acute toxicity; Aquatic environment
I.
INTRODUCTION
As a non-metal oxide, silica (SiO2) nanoparticles have found extensive applications in chemical mechanical polishing and as additives to drugs, cosmetics, printer toners, varnishes and food. In recent years, the use of Nano-SiO2 has been extended to biomedical and biotechnological fields [1]. Engineering nanomaterials and especially nanoparticles in the free non-fixed form are currently or will soon be emitted into the environment. The most likely pathway of free nanoparticles from various sources like consumer products (cosmetics) is via the aquatic environment [2]. Therefore, the research about the ecotoxicity of Nano-SiO2 to aquatic ecological environment is very significant for human health and natural environment. The previous studies show that there is a pronounced lack of data in this field (less than 20 peer-reviewed papers are published so far), and the most frequently tested engineering nanoparticles in invertebrate tests are C60, carbon nanotubes, and titanium dioxide [3]. For example, study about the toxicity of titanium dioxide, Nano-C60 and C60HxC70Hx (a fullerene derivative) indicate that Nano-C60 was the only suspension to cause a significant change in heart rate. Exposure to both Nano-C60 and C60HxC70Hx suspensions caused hopping frequency and appendage movement to increase [4]. Daphnia 21-day
exposures resulted in a significant delay in molting and significantly reduced offspring production at 2.5 and 5 ppm nC60, which could possibly produce impacts at the population-level [5]. Just like A. Baun et al [3] said, almost all the current studies concern about the research of carbon nanomaterials and rarely touch on numerous inorganic nanomaterials. In the experiments of M. Heinlaan et al [6], which used bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus as the test organisms, show that Suspensions of nano and bulk TiO2 were less toxic (at least, they were not toxic even at 20 g/L), All Zn formulations (bulk ZnO, nanoZnO and ZnSO4·7H2O) were very toxic and Cu compounds had different toxicities: L(E)C50 (mg/L) for bulk CuO, nano CuO and CuSO4 are 3811, 79, 1.6 (V. fischeri), 165, 3.2, 0,17 (D. magna) and 95, 2.1, 0.11 (T. platyurus), respectively. Recently, the comprehensive study with regard to aquatic eco-environment of nanoparticles is the research of X. S. Zhu et al [7], which showed that the acute toxicities of all MNMs tested are dose dependent. The EC50 values ranged from 0.622 mg/L (ZnO NPs) to 114.357 mg/L (Al2O3 NPs), while the LC50 values ranged from 1.511 mg/L (ZnO NPs) to 162.392 mg/L (Al2O3 NPs). And they also reported that the D. magna can ingest nanomaterials from the test suspensions through feeding behaviors. There is a lack of studies in aquatic ecotoxicity of Nano-SiO2, although there are more and more information about MNMs are available. As a result of that, this experiment aims to research the potential influence of Nano-SiO2 to aquatic ecological environment by using aquatic invertebrate, Daphnia magna, as the test organism, and try to find out the dose–response characteristic of Nano-SiO2. The absorption and uptake of Daphnia magna in the test suspensions are simply analyzed also. II.
MATERIALS AND METHODS
A. Reagents The Nano-SiO2 was purchased from Sigma–Aldrich (St. Louis, MO, USA) with the particles size of 10- 20 nm and the purity of 99.5%. The normal sized SiO2 were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), which the particles size range from 5 to 10 μm and the degree of purity is 99.5%. Figure. 1 shows the SEM images of both two test materials. The laboratory Daphnia magna were
978-1-4244-2902-8/09/$25.00 ©2009 IEEE
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Figure. 1 SEM images of tested particles. (Left: Nor-SiO2, Right: Nano-SiO2)
presented by Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (RCEES-CAS). The other chemicals (e.g. NaHCO3, KCl, MgSO4·7H2O and CaCl2·2H2O) which were used in this study as the solutes for preparing the dilution water are all analytical reagents. B. Materials Daphnia magna is cladoceran (crustacean) which is common zooplankton found in freshwater lakes and ponds [5]. Young daphnids, aged less than 24 hours, are very sensitive which can be used as a test organism in testing of eco-toxicity of substance. The culture of Daphnia magna accorded with the National Standard of the People's Republic of China (GB/T 13266-91) [8], and Scenedesmus obliquus were used to feed Daphnia magna. C. Methods In this study, 24 hours acute toxicity tests were conducted following the Guideline 202 of Organization for Economic Co-operation and Development (OECD) [9] with modifications according to Water quality-Determination of the acute toxicity of substance to Daphnia (Daphnia magna Straus) of national standards (China, GB/T 13266-91) and the condition of our laboratory. The dilution water were prepared by adding specific amounts of solutes to distilled water, in witch the concentration of NaHCO3, KCl, MgSO4·7H2O and CaCl2·2H2O were 64.75 mg/L, 5.75 mg/L, 123.25 mg/L and 294 mg/L, respectively. Test suspensions were prepared immediately before been used by adding Nano-SiO2 particles with various amounts to the dilution water, and then they were treated by 15 min ultrasonication to make sure that the NPs were enough dispersed in test solutions. The initial concentration is 25 mg/L and five test points were arranged in a geometric series with a separation factor of 2, i.e. 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L, respectively. In order to make sure whether the particle size can influence the toxicity of Nano-SiO2, we also put the bulk counterpart of Nano-SiO2 (Nor-SiO2) to the tests, and the test points are same with the Nano-SiO2’s. Ten neonates with similar size were randomly selected and placed in a 100 mL glass conical flask containing 30 mL of test suspension. In control group, we just put 10 random neonates into a 100 mL glass conical flask containing 30 mL of dilution water. Three parallel tests were set for each concentration and control group. To avoid evaporation of the water and entry of dusts into suspensions, all of the exposure containers were covered by transparent plastic membrane with some pinholes. The glass conical flask were shook constantly at 130-140 rpm throughout the 24-h exposure time to avoidprecipitation of test particles and farthest simulate the real natural aquatic environment. All tests were carried out in the room with natural light-duck cycle and temperature ranged from 24 to 25 °C. The daphnids were not fed during the test. The immobilization and the mortality were considered as the toxicological test endpoints. Use optical microscope and digital
camera to observe and record the biological form of Daphnia magna during the exposure. Those daphnids that are not able to swim within 15 seconds after gentle agitation of the test container would be considered to be immobilized even if they can still move their antennae, and animals whose heartbeat have stopped would be considered to be dead. D. Data analysis Data were summarized in tabular form to show the numbers of immobility and death in each group, and the percentages of immobilization and death at 24 hours were plotted against test concentrations. The EPA computer probit analysis program (Version 1.5) were used to calculated the 24-h LC50 and EC50 with 95% confidence limits (p = 0.95). The statistic analysis was done by software Origin 6.0. III.
RESULTS
A. Morphological Examionation The studies about 24-h acute toxicity of Nano-SiO2 toward Daphnia magna were not found in previous reports yet. The observation of exposure duration showed a dose-response feature. When the concentrate was 25 mg/L, the movement of D. Magna was fast and smart, just like the daphnids in control groups. However, as the increased concentration, the condition of daphnids was getting worse and worse even the break of bodies, and the heartbeat of survivals were weak. There also more and more accumulation of Nano-SiO2 particles were found in the organisms as the increasing of the exposure concentration, so the color of the daphnids was getting dark (Figure. 2). We found that, in Nor-SiO2 exposure, only the guts of the test organisms were covered by the Nor-SiO2 but the other place of their bodies were not, although the exposure concentration were very high (Figure. 3). However, in the Nano-SiO2 exposure tests, the nanoparticles were evenly distributed in the most organs in the body of D. Magna (especially the gut tract and gill) (Figure. 2). Except the guts, the survivals of the conventional groups show clear bodies just like the control groups' (Figure. 3), and their heartbeats were fast. All these observed results seem to suggest that Daphnia magna can uptake both the nanosized and normal sized SiO2, but the different is that only the Nano-SiO2 can be absorbed obviously by D. Magna. These can be clearly seen by using the optical microscope (Figure. 2, Figure. 3). B. 24-h EC50 and LC50 The results of this experiment show that there are some degrees of toxicity when Nano-SiO2 contact with Daphnia magna in the aquatic environment and demonstrate overall trend of dose-dependence (Figure. 4). According to our results, the program calculated that the 24-h EC50 and LC50 are 148.871 mg/L and 660.943 mg/L, respectively (the LC50 is not accurate because the mortality of highest concentration groups did not reach or pass 50%). The 25 mg/L and 50 mg/L Nano-SiO2 had similar influence to Daphnia magna, however, the immobilization and death increased as the increasing of the following exposure concentrations, e.g. no immobilized D. Magna occurred in control groups, the percentages of
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Figure. 5
Figure. 2 Microscope images of nanosized SiO2 exposure. (A: 25mg/L, B: 50 mg/L, C: 100 mg/L, D: 200 mg/L, E: 400 mg/L and control group)
Figure. 3 Microscope images of Nor-SiO2 exposure.
immobilization and death in 25mg/L were 30% and 17%, respectively. But when it came to 400 mg/L, there were 77% daphnids were immobilized and the rate of death was 43% (Figure. 4). In the experiments of normal Nano-SiO2, just as the anticipation, the mortality and immobility were very low (immobilization rate was only 10% when the exposure concentration was 400mg/L), and the dose-dependent toxicity of Nor-SiO2 was not very obvious (Figure. 4). There were only few increasing of immobility and mortality when the concentration increased. For example, the immobility and mortality of 25 mg/L and 400mg/L were 0%, 0% and 10%, 7%, respectively (Figure. 4). IV.
DISCUSSIONS
The study shows that there is does-dependent toxicity when Nano-SiO2 contact with Daphnia magna, and the rate of immobilization and mortality would increase if the concentration of exposure tests are increased. This result is similar with previous reports which about the acute toxicity tests of inorganic nanomaterials [3, 6, 7], although there are not much available informations concerning the impact of Nano-SiO2 particles toward D. Magna. There are some methods to prepare the suspension of MNMs, including ultrasonication, stirring, using organic solvents (e.g. tetrahydrofuran) and encapsulating NPs by Hydrophilic molecules (e.g. polyvinylpyrrolidone). However, Since PVP/NP is not composed solely of NPs, the method is not applied [10]. Many researches found that using organic solvents can influence the toxicity of NPs due to the residues of the organic solvents (e.g. THE) [3, 11, 12]. Stirring does not add any extra chemicals, but the time-frames for solution preparation can be long and there is little firm evidence that nanoparticles will stay in suspension after stirring has stopped 100
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Figure. 4 Effects of suspensions of Nano-SiO2 and its bulk counterpart on the immobilization and mortality of D. magna. Error bars represent one standard deviation of the mean.
The rupture of guts and the cracking bodies in nSiO2 exposure groups
[13]. As a result of that, we chose ultrasonication to prepare these test suspensions, although someone consider the ultrasonication may increase the toxicity of nanomaterials. Filtration was not applied in this experiment, considering the total concentration of the test suspensions will change after filtration and there is no guarantee that aggregates will not reform in the filtered solution [13]. In order to simulate the real natural aquatic environment and avoid the precipitation of nanoparticles, the shaker was used to create flows in test containers. But it still has limitation because flocculation can be found at the end of the exposure, especially in the high test concentration. Theoretically, considerations suggest that the smaller the average diameter of particles is, the more toxic they are, this is because the larger specific surface area cause a higher bioactivity and bioavailability of particles, as well as their own capability of generating reactive radicals [14, 15]. From the data of Nor-SiO2 groups (Figure. 4), we could not find an evident does-dependent feature, so we did not calculate the EC50 values and the LC50 values. But there were still few immobilized and dead bodies. Considering the sharp shape of the Nor-SiO2 particles (Figure. 1), we presume that the immobilization and mortality may because of the shearing force caused by Nor-SiO2 particles in shaking water. Comparing the Nano-SiO2 test groups and the Nor-SiO2 test groups, there are some relationships between the particle size and the acute toxicity of Nano-SiO2, i.e. the smaller size cause a higher acute toxicity. These results were also found in some researches about acute toxicity of nanosized metal oxide (except ZnO) such as those studies of M. Heinlaan et al and X. S. Zhu et al [6, 7]. The small toxicity of normal particles may also due to sedimentation of the normal materials with resulting separation of organisms and the test particles [16]. Previous research shows that the rainbow trout could uptake SWCNTs form the test solution [17]. In this study, the test groups with different concentrations demonstrated that the Nano-SiO2 particles could be uptaken and absorbed by Daphnia magna (Figure. 3). This may because of the hydrophobic property of the test NPs. Hydrophobic substances have been reported to adhere easily to negatively charged biological materials; they partitioned into or onto test organism biomass and onto food or other organic detritus in the test system [7, 18]. But only uptake could be found when D. Magna touch with the normal SiO2, and the absorption was not very visible (Figure 2), although few absorbed substances were found in 400 mg/L (Figure 4). The similar result was also reported in the experiment of X. S. Zhu et al [7]. Nano-SiO2 only covered several specific places in the gut of D. Magna, while its bulk counterpart was evenly distributed in all gut tract (Figure. 2, Figure. 3), which suggest that the further digestion of Nano-SiO2, but not Nor-SiO2, may happen in D. Magna's gut. The cytotoxicity of MNMs toward human and some aquatic organisms were also reported in previous studies [1, 15,
3
17], so there is a speculation that some pathological changes of intestinal cells may occur when D. Magna exposure to Nano-SiO2 suspensions. The rupture of guts and the broken bodies (Figure. 5) found in high concentration groups also support this speculation. The absorption was found not only on the outer surface of the body (the microvillus of the body surface and the fine setae located on the thoracic legs) but also on some organs in the body (gill) (Figure. 2). It is generally assumed that particles with a suitable size are ingested without any selective mechanism. Small particles of less than 50 microns in diameter are filtered out of the water by fine setae located on the thoracic legs and are moved to the mouth [16]. As a result of that, Nano-SiO2 may be bioaccumulated and bioconcentrated in filter-feeding aquatic organisms such as Daphnia magna and transfer to the higher levels of food chain. In the nanosized SiO2 exposure, the EC50 value for immobilization and the LC50 value for mortality are 148.871 mg/L and 660.943 mg/L, respectively. Which indicate the toxicity of Nano-SiO2 is relatively small by contrast with some other inorganic nanoparticles, e.g. in the study of X. S. Zhu et al [7], the 48-h EC50 and LC50 of Al2O3 with least toxicity are 114.357 mg/L and 162.392 mg/L, respectively. Although the small toxicity, we could still find immobilization and mortality after Nano-SiO2 exposure. There may be some points: (1) the uptake of NPs result some physiochemical changes in gut tract and eventually lead to the immobilization and death. (2) Nanoparticles cause the blocking of the gills and restrict respiration. V.
CONCLUSION
This paper researched the acute toxicity of nanosized SiO2 particle to Daphnia magna. Based on the experimental results, the following conclusion can be obtained. (1) Nano-SiO2 has acute toxicity to Daphnia magna and the influence is does-dependent. (2) There may some relationships between the toxicity of Nano-SiO2 and its particle size; the smaller particles are likely to be more toxic. But the toxicity of normal sized SiO2 is not very obvious. (3) Daphnia magna can uptake and absorb Nano-SiO2 particles, while only uptake can be found in Nor-SiO2 exposure tests. Nano-SiO2, but not Nor-SiO2, can be further digested and cause physiochemical changes in the gut of D. Magna. (4) Filter-feeding aquatic organisms such as Daphnia magna can bioaccumulate and bioconcentrate Nano-SiO2 and transfer it to the other trophic levels. The cytotoxicity of nanosized SiO2 can cause the decline of the population level of Daphnia magna and other filter-feeding aquatic organisms. All these may impact the aquatic eco-environment even the entire ecological system and deserve extra attention.
This work was supported by the National Key Technologies R&D Programs of China (2006BAJ02A10-1; 2006BAI19B05). REFERENCES [1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
ACKNOWLEDGMENT The authors want to thank Prof. Wang Zijian of Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences for his kind help and his providing of laboratory Daphnia magna for this study.
[18]
W. Lin, Y. W. Huang, X. D. Zhou, and Y. Ma, “In vitro toxicity of silica nanoparticles in human lung cancer cells,” Toxicol. Appl. Pharmacol., vol. 217, pp. 252-259, December 2006. S. Ottofuelling, F. V. D. Kammer, T. Hofmann, “Nanoparticles in the aquatic environment–aggregation behavior of TiO2 nanoparticles studied in a simplified aqueous test matrix (SAM),” Geophys. Res. Abs., vol. 9, SRef-ID: 1607-7962/ gra/ EGU2007-A-08876, 2007. A. Baun, N. B. Hartmann, K. Grieger, and K. O. Kusk, “Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing,” Ecotoxicology, vol. 17, pp. 387-395, April 2008. S. B. Lovern, J. R. Strickler, and R. Klaper, “Behavioral and physiological changes in Daphnia magna when exposed to nanoparticle suspensions (titanium dioxide, nano-C60, and C60HxC70Hx),” Environ. Sci. Technol., vol. 41, pp. 4465-4470, June 2007. E. Oberdörstera, S. Q. Zhu, T. M. Blickley, P. McClellan-Green, and M. L. Haasch, “Ecotoxicology of carbon-based engineered nanoparticles: Effects of fullerene (C60) on aquatic organisms,” Carbon N Y, vol. 44, pp. 1112-1120, 2006. M. Heinlaan, A. Ivask, I. Blinova, H. C. Dubourguier, and A. Kahru, “Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus,” Chemosphere, vol. 71, pp. 1308-1316, April 2008. X. S. Zhu, L. Zhu, Y. S. Chen, and S. Y. Tian, “Acute toxicities of six manufactured nanomaterial suspensions to Daphnia magna,” J Nanopart Res, in press. “Water quality-Determination of the acute toxicity of substance to Daphnia (Daphnia magna Straus),” China National Standard, GB/T 13266-91, August 1992. “OECD guideline for the testing of chemicals. ‘Daphnia sp., acute immobilisation test’,” OECD, No. 202, November 2004. D. Y. Lyon, L. K. Adams, J. C. Falkner, and P. J. Alvarezt, “Antibacterial Activity of Fullerene Water Suspensions: Effects of Preparation Method and Particle Size,” Environ. Sci. Technol., vol. 15, pp. 4360-4366, July 2006. S. Zhu, E. Oberdörster, and M. L. Haasch, “Toxicity of an engineered nanoparticle (fullerene, C60) in two aquatic species, Daphnia and fathead minnow,” Mar. Environ. Res., vol. 62, pp. s5-s9, July 2006. J. Brant, H. Lecoanet, M. Hotze, and M. Wiesner, “Comparison of electrokinetic properties of colloidal fullerenes (n-C60) formed using two procedures,” Environ. Sci. Technol., vol. 39, pp. 6343-6351, September 2005. M. Crane, R. D. Handy, J. Garrod, and R. Owen, “Ecotoxicity test methods and environmental hazard assessment for engineered nanoparticles,” Ecotoxicology, vol. 17, pp. 421-437, April 2008. L. K. Adams, D. Y. Lyon, and P. J. Alvarez, “Comparative eco-toxicity of nanoscale TiO2, SiO2, and ZnO water suspensions,” Water Res., vol. 40, pp. 3527-3532, September 2006. M. N. Moore, “Do nanoparticles present ecotoxicological risks for the health of the aquatic environment,” Environ Int, vol. 32, pp. 967-976, July 2006. K. Hund-Rinke, and M. Simon, “Ecotoxic effect of photocatalytic active nanoparticles (TiO2) on algae and daphnids,” Environ Sci Pollut Res Int, vol. 13, pp. 225-232, July 2006. C. J. Smith, B. J. Shaw, and R. D. Handy, “Toxicity of single walled carbon nanotubes to rainbow trout, (Oncorhynchus mykiss): respiratory toxicity, organ pathologies, and other physiological effects,” Aquat. Toxicol., vol. 82, pp. 94-109, February 2007. “OECD Series on Testing and Assessment: guidance document on aquatic toxicity testing of difficult substances and mixtures,” OECD, No. 23, June 2006.
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