Grantee Research Project Results
2008 Progress Report: Methodology Development for Manufactured Nanomaterial Bioaccumulation Test
EPA Grant Number: R833327Title: Methodology Development for Manufactured Nanomaterial Bioaccumulation Test
Investigators: Chen, Yongsheng , Crittenden, John C. , Huang, C. P. , Sommerfeld, Milton , Hu, Qiang , Chang, Yung
Institution: Arizona State University , University of Delaware
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2006 through August 31, 2009
Project Period Covered by this Report: September 1, 2007 through August 31,2008
Project Amount: $399,768
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: a Joint Research Solicitation-EPA, NSF, NIOSH, NIEHS (2006) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
Nanomaterials, also known as manufactured or engineered nanomaterials, have one or more dimensions in the range of 1 to 100 nanometers and are manufactured in a controllable or manipulatable fashion (National Research Council, 2002). Most manufactured nanomaterials are made of carbon, silicon, transition metals, or metal oxides; others are made from nanocrystals composed of multiple compounds, such as silicon and metals (i.e., quantum dots) (Dreher, 2004). Due to their wide application, the discharge of nanomaterials into the environment could be significant in the near future. However, their potential adverse health and environmental effects have received adequate attention. Especially, no data are available on whether manufactured nanomaterials are toxic within months or years. Thus, these nanomaterials could constitute a completely new class of non-biodegradable pollutants that can accumulate in food chains. Due to the lack of information about bioaccumulation, biotoxicity, and mutagenic effects, the risks related to the transfer and persistence of nanomaterials in the environment and food chain must be evaluated.
The objectives of this project are: 1) to develop suitable manufactured nanomaterial bioaccumulation testing procedures to assure data accuracy and precision, test replicability, and comparability test results; 2) to evaluate how the forms of manufactured nanomaterials affect their potential bioavailability and bioconcentration factor (BCF) in phytoplankton; 3) to determine the potential biomagnification of manufactured nanomaterials in zooplankton; and 4) to determine the potential biomagnification of manufactured nanomaterials in fish. This report summarizes the progress made over the first year of the project.
Progress Summary:
I. Determine the Bioavailability of Nanomaterials in Water
Materials and Methods
Source of Nanomaterials
This research examined two classes of manufactured nanomaterials: organic carbon-based nanoparticles (NPs) such as fullerenes (C60), single wall carbon nanotubes (SWCNTs), and multiple wall carbon nanotubes (MWCNTs); and inorganic metal oxide NPs such as titanium oxide (nTiO2), zinc oxide (nZnO), and alumina (nAl2O3). All of these NPs are commercially available. C60 powder was obtained from SES (Houston, TX, USA). Both SWCNTs and MWCNTs were obtained from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China). Carbon black particles, which served as the bulk counterparts of the carbon-based NPs in this study, were purchased from Tianjin Jinqiushi Chemical Port Co., Ltd. (Tianjin, China). Nanoscale TiO2 (nTiO2, anatase), nanoscale ZnO (nZnO), and nanoscale Al2O3 (nAl2O3) were purchased from Nanjing High Technology NANO CO., LTD. (Nanjing, China); the bulk counterparts of these NPs, ZnO/bulk, TiO2/bulk (anatase) and Al2O3/bulk, were purchased from The Third Chemical Regent Factory of Tianjin (Tianjin, China). Table 1 summarizes the basic physicochemical properties of these compounds using information provided by the manufacturers, including particle size and purity. None of the NPs or bulk counterparts had functional groups.
Table 1 Nanomaterials and their bulk counterparts used in this research
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Particles
|
particle size
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Purity (%)
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Source
|
|
C60
|
< 200 nm
|
99.5
|
SES, Houston, USA
|
|
SWCNTs
|
D < 2 nm
L = 5-15 μm
|
CNTs>90
SWCNTs>60
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Shenzhen Nanotech Port Co., Ltd.
|
|
MWCNTs
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D = 10-20 nm
L = 5-15 μm
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> 98.0
|
Shenzhen Nanotech Port Co., Ltd.
|
|
Carbon Black
|
20,000 nm
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> 95.0
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Tianjin Jinqiushi Chemical Port Co., Ltd.
|
|
nZnO
|
20 nm
|
> 99.6
|
Nanjing High Technology NANO Co., Ltd.
|
|
ZnO/Bulk
|
1,000 nm
|
> 99.0
|
The Third Chemical Regent Factory of Tianjin
|
|
nTiO2
|
≤ 20 nm
|
> 99.5
|
Nanjing High Technology NANO Co., Ltd.
|
|
TiO2/Bulk
|
10,000 nm
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> 99.0
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The Third Chemical Regent Factory of Tianjin
|
|
nAl2O3
|
80 nm
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> 99.9
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Nanjing High Technology NANO Co., Ltd.
|
|
Al2O3/Bulk
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90,000 nm
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> 99.0
|
The Third Chemical Regent Factory of Tianjin
|
Note: “D” is diameter; “L” is length.
Preparation and Storage of Nanoparticle Suspensions
Nanoparticle stock suspensions of 1 g/L concentration were prepared by suspending 1 g of dry nanopowder in 1 L of nanopure water (Nanopure Diamond, Barnstead Thermolyne Corp., IA) and sonicating for 15 minutes at 20 KHz and an intensity of 200 W/L (Model 2000U, Ultrasonic Power Corp., IL). Nanopure water has a conductivity of less than 5×10-6 Ω-1cm-1 and a pH of 5.6+0.2. Stock suspensions were stored at room temperature (25 oC) for no longer than 2 days before preparation of the test suspensions.
Nanomaterial Toxicity to Green Algae
Algal strain and culture conditions
Green algae (e.g., Scenedesmus obliquus) are the primary producers that form the base of the food chain in aquatic ecoregions. Thus, the algae growth inhibition test can be used to evaluate the potential effects of a substance on an aquatic ecosystem. In these experiments, Scenedesmus obliquus was obtained from the Freshwater Algae Culture Collection, Institute of Hydrobiology, Chinese Academy of Sciences, and cultivated in 100 mL of Organisation for Economic Co-operation and Development (OECD) test medium (OECD, 1984) in 250-mL sterile glass flasks, and illuminated with cool-white fluorescent lights (70 μE/m2/s) on a 14:10 light-dark (LD) cycle. Temperature was maintained at 25 ◦C in an air-conditioned growth chamber. Figure 1 provides a representative growth curve for Scenedesmus oblignus cultured under these conditions. Cells in the exponential growth phase (days 3-6) were collected from stock cultures and diluted to a concentration of 2×104 cells/mL using the sterile OECD test medium. The resulting solution was used as the inocula for the following experiments.
Exposure procedure
NP test solutions were prepared immediately prior to use by diluting the stocks mentioned above with OECD test medium. NP test solutions (30 mL) and the algae inocula (30 mL) were mixed such that the initial concentration of Scenedesmus oblignus was 1×104 cells/mL; and Table 2 lists the concentrations of NPs used. Growth medium containing a high NP concentration (>1 mg/L) appeared turbid. Thus, revised standard culture conditions for these experiments were adapted as follows: 1) 250 mL glass flasks contained 60 mL (rather than the 100 mL in the OECD guidelines) of test mixture; 2) temperature of 25 ± 2◦C, light intensity of 180 μE/m2/s (>60-120 μE/m2/s requested in OECD guideline) with 14:10 LD cycle; and 3) agitation on a shaker table at a speed of 120±5 rpm (to maintain the suspension at as stable a concentration as possible). By increasing the light intensity, reducing the culture volume, and shaking appropriately, this method minimizes the shading effects of the particles and provides a more direct measure of the potential inherent chemical toxicity of these NPs to algae. Additionally, (Cleuvers and Ratteb, 2002) reported that high light intensities and lower culture volumes result in lower coefficients of variation, thus improving the sensitivity of the test for statistical calculation of toxicity data.
Figure 1 The growth curve of Scenedesmus oblignus.
Table 2 Concentration gradients of particles in green algae toxicity tests
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Particle Suspensions
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Concentration Gradient (mg/L)
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||||||
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nTiO2
|
500
|
100
|
50
|
10
|
5
|
1
|
0.5
|
|
nAl2O3
|
1000
|
500
|
100
|
50
|
10
|
5
|
1
|
|
nZnO
|
10
|
5
|
1
|
0.5
|
0.1
|
0.05
|
0.01
|
|
C60
|
200
|
100
|
50
|
10
|
5
|
1
|
0.5
|
|
SWCNTs
|
100
|
50
|
10
|
5
|
1
|
0.5
|
0.1
|
|
MWCNTs
|
100
|
50
|
10
|
5
|
1
|
0.5
|
0.1
|
Data analysis
From day 0 to day 4, samples were taken daily from both treatment groups and controls, and cell numbers were counted with a hemocytometer under a microscope. As recommended by OECD, to determine the concentration effect relationship, the growth rate and the inhibition percentage for each NP concentration was calculated as the difference between the area under the control growth curve and the area under the growth curve at each test NP concentration (OECD, 1984). The growth inhibition percentages were compared with the concentration data in a regression analysis, and the exact value of the 96 h EC50 was determined. The dose response equation was χ2 tested with 95% confidence. Furthermore, the adhesion and/or adsorption of NPs by Scenedesmus oblignus were also observed and documented using a microscope with a digital camera.
Nanomaterial Toxicity to Daphnia
Culture conditions and exposure procedure
D. magna, a common zooplankton found in freshwater lakes and ponds, is one of the most sensitive organisms used in ecotoxicity tests (Alberdi et al., 1996). The U.S. Environmental Protection Agency (EPA), OECD, and International Organization for Standardization (ISO) standard protocols have used it as a standard test organism (OECD, 2004). In this study, acute (48 h) toxicity tests were conducted following OECD Guideline 202 with slight modifications according to the OECD draft guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD, 2000 and 2004). Test solutions were prepared immediately prior to use by diluting the nanoparticle stocks mentioned above with reconstituted water. In this procedure, the stock solution/mixture was continuously stirred with a magnetic stirrer to maintain the suspension at as stable a concentration as possible. Table 3 lists the concentration gradients of the NPs and their bulk counterparts. Toxicity tests were conducted with the bulk counterparts of the NPs (i.e., carbon black, ZnO/bulk, TiO2/bulk and Al2O3/bulk) to determine whether the sizes of NPs may affect their toxicities to aquatic organisms. These tests employed a completely random design consisting of 5-7 treatment groups and a control group per nanoparticle or bulk counterpart. Ten randomly selected neonates (< 24 h old) were placed in a 100 mL glass exposure beaker containing 30 mL of test solution. To ensure a constant concentration, all beakers were covered with transparent plastic film containing several apertures and then shifted to a shaker. The beakers shook constantly at 140 rpm throughout the 48 h exposure time. Shaking was chosen to minimize sedimentation of particles, but we also considered it less disturbing because of the constant random flows experienced by the animals in nature. Three replicate exposure beakers were employed per treatment or control group. The daphnids were not fed during the tests, and all tests were conducted indoors at a constant temperature (23±2°C) with a natural light-dark cycle. After 48 h of exposure, the immobilization and mortality of the individuals in each container were assessed using a Leica microscope equipped with a digital camera (Leica, Germany). Animals that are unable to swim within 15 seconds of gentle agitation of the test container are considered immobile while those whose heartbeats have stopped are considered dead. Furthermore, the uptake and adsorption of nanomaterials by D. magna were observed and documented using a microscope with a digital camera. During the exposure period, mortality in the control groups was less than 10%.
Table 3 Concentration gradients of particles in Daphnia acute toxicity tests
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Particle Suspensions
|
Concentration Gradient (mg/L)
|
||||||
|
nTiO2
|
500
|
100
|
50
|
10
|
5
|
1
|
0.5
|
|
TiO2/Bulk
|
500
|
100
|
50
|
10
|
5
|
1
|
0.5
|
|
nAl2O3
|
1000
|
500
|
100
|
50
|
10
|
--
|
--
|
|
Al2O3/Bulk
|
1000
|
500
|
100
|
50
|
10
|
--
|
--
|
|
nZnO
|
5
|
1
|
0.5
|
0.1
|
0.05
|
0.01
|
--
|
| Other project views: | All 17 publications | 8 publications in selected types | All 8 journal articles |
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| Type | Citation | ||
|---|---|---|---|
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Sun H, Zhang X, Niu Q, Chen Y, Crittenden JC. Enhanced accumulation of arsenate in carp in the presence of titanium dioxide nanoparticles. Water, Air, & Soil Pollution 2007;178(1-4):245-254. |
R833327 (2008) R833327 (Final) |
Exit |
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Wang J, Zhang X, Chen Y, Sommerfeld M, Hu Q. Toxicity assessment of manufactured nanomaterials using the unicellular green alga Chlamydomonas reinhardtii. Chemosphere 2008;73(7):1121-1128. |
R833327 (2008) R833327 (Final) |
Exit Exit |
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Zhang X, Sun H, Zhang Z, Niu Q, Chen Y, Crittenden JC. Enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticles. Chemosphere 2007;67(1):160-166. |
R833327 (2008) R833327 (Final) |
Exit Exit |
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Zhu X, Zhu L, Lang Y, Chen Y. Oxidative stress and growth inhibition in the freshwater fish Carassius auratus induced by chronic exposure to sublethal fullerene aggregates. Environmental Toxicology and Chemistry 2008;27(9):1979-1985. |
R833327 (2008) R833327 (Final) |
Exit |
Supplemental Keywords:
Health, Scientific Discipline, PHYSICAL ASPECTS, Health Risk Assessment, Physical Processes, Risk Assessments, bioavailability, nanomaterials, fish-borne toxicants, bioaccumulation, exposure, fate and transport, nanoparticle toxicity, nanotechnology, food chainProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.