2008 Progress Report: Methodology Development for Manufactured Nanomaterial Bioaccumulation Test

EPA Grant Number: R833327
Title: 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 - Main Campus , 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: Health Effects , Nanotechnology , Health , 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

Particles
particle size
Purity (%)
Source
C60
< 200 nm
99.5
SES, Houston, USA
SWCNTs
D < 2 nm
L = 5-15 μm
CNTs>90
SWCNTs>60
Shenzhen Nanotech Port Co., Ltd.
MWCNTs
D = 10-20 nm
L = 5-15 μm
> 98.0
Shenzhen Nanotech Port Co., Ltd.
Carbon Black
20,000 nm
> 95.0
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
> 99.0
The Third Chemical Regent Factory of Tianjin
nAl2O3
80 nm
> 99.9
Nanjing High Technology NANO Co., Ltd.
Al2O3/Bulk
90,000 nm
> 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 ± 2C, 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

Particle Suspensions
Concentration Gradient (mg/L)
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
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
--
ZnO/Bulk
5
1
0.5
0.1
0.05
0.01
--
C60
100
50
25
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
Carbon black
100
75
50
25
10
5
1
Note: “--” means no test was done.
 
Data analysis
The 48 h EC50 and LC50, and their associated 95% confidence intervals (95% CIs), were calculated using the EPA computer probit analysis program (Version 1.5). Statistical analyses were carried out using standard ANOVA techniques followed by Tukey’s honestly significant difference test. Differences were statistically significant when p < 0.05.
 

Results and Discussion

 

Toxicity of NPs to Scenedesmus oblignus
Figure 2 shows the dose-effect relationships between NPs and the growth of Scenedesmus oblignus while Figure 3 presents the growth inhibition due to exposure to different NPs. Algal growth in all NP treatments was significantly inhibited when the total concentration of NPs in the culture was increased. The correlation coefficients between the inhibition percentage and total NP concentrations were 0.954, 0.928, 0.547, 0.899, 0.843, and 0.996 for nZnO, nTiO2, nAl2O3, C60, SWCNTs and MWCNTs suspensions, respectively (Table 4). As calculated by regression analysis, the 96 h EC50 of nZnO, nTiO2, nAl2O3, C60, SWCNTs and MWCNTs were 1.049, 15.262, >1000, 13.122, 22.633, 15.488 mg/L, respectively. Obviously, the nZnO suspension is the most toxic to Scenedesmus oblignus, while nAl2O3 is the least toxic NP tested. Based on an ANOVA analysis of the 96 h EC50, the rank order of NPs for the algae growth inhibition is: nZnO > C60, nTiO2, MWCNTs, SWCNTs > nAl2O3. These results demonstrate that NPs with different compositions may exert widely varying effects on aquatic organisms.
 
Figure 2 Dose-effect relationships between various NPs and the growth of Scenedesmus oblignus.  A: The area under the growth curve.
Figure 3 Percentage of growth inhibition of Scenedesmus oblignus due to exposure to NP suspensions.
 
Table 4 The 96 h EC50 of several NP suspensions on the growth of Scenedesmus oblignus.
NPs
Regression Equation
Correlation Coefficient
EC50(mg/L)
nZnO Suspension
y = 38.862x + 49.194
R2 = 0.9542
1.049±0.565
C60 Suspension
y = 26.42x + 20.456
R2 = 0.8988
13.122±4.182
nTiO2 Suspension
y = 39.902x + 2.7719
R2 = 0.9275
15.262±6.968
MWCNTs Suspension
y = 38.468x + 4.3117
R2 = 0.9964
15.488±7.108
SWCNTs Suspension
y = 27.978x + 12.097
R2 = 0.8434
22.633±9.605
nAl2O3 Suspension
y = 14.204x - 10.044
R2 = 0.5471
>1000
 
Adhesion and/or adsorption of NPs by Scenedesmus oblignus
 
Figure 4 shows the adhesion and/or adsorption of NPs by Scenedesmus oblignus after 96 h of incubation. All of the NPs tested were found on the cell walls of the algae. However, the NPs exhibited different adhesion and/or adsorption behaviors. Among the metal oxide NPs, the weakest adhesion and/or adsorption was found in nAl2O3 aggregates, even in the highest treatment concentration of 1000 mg/L (Figure 4D). This may be because nAl2O3 aggregates are larger than those of the other metal oxide NPs. The adhesion and/or adsorption of nZnO look also unobvious in Figure 4C, possibly because the NP concentration was never more than 10 mg/L. Among the carbon-based NPs, the aggregates appear to be larger than the algal cells, thus some NPs aggregates may not be adhered or adsorbed on the algae surface but enwrap wholly or partially around the algal cells (Figure 4E,F,G). Nevertheless, when observed under the microscope, smaller-sized C60 or carbon nanotubes are attached to the cell wall (inset pictures in Figure 4). The observed adhesion and/or adsorption of NPs by Scenedesmus oblignus indicates that the NPs have the potential to transfer through the food chain, which may pose a threat to algal predators, such as the zooplankton Daphnia magna.
 
Moreover, as demonstrated in the inset pictures in Figure 4, morphologic changes and damage to Scenedesmus oblignus is apparent after 96 h of exposure to the tested NPs except for nAl2O3. More research is needed to determine the exact mechanisms for these morphologic changes or damage.
 
Figure 4 The adhesion / adsorption and/or enwrapping of NPs to Scenedesmus oblignus as well as the resulting morphologic changes or damage after 96 h incubation (20×10).
A: Control; B: 100 mg/L nTiO2; C: 10 mg/L nZnO; D: 1000 mg/L nAl2O3; E: 100 mg/L C60; F: 100 mg/L SWCNTs; G: 100 mg/L MWCNTs.
Toxicity of NPs to Daphnia magna
 
In these experiments, the acute toxicities of all NPs and their bulk counterparts to D. magna increased with increasing particle concentration (Figure 5), demonstrating dose-dependency. nZnO at concentrations of 0.01 mg/L produced a slight immobilization (6.67% of the control) and 0% mortality in D. magna; this was not significantly different from the control (p > 0.05). A concentration of 5 mg/L, however, caused 100% immobilization and mortality. The EC50 of immobilization and LC50 of mortality for nZnO were calculated as 0.622 mg/L and 1.511 mg/L, respectively.
 
The toxicity of nAl2O3 to D. magna has not been previously reported in the literature. In the present study, 10 mg/L nAl2O3 did not cause immobilization or mortality, 1,000 mg/L resulted in 100% immobilization and mortality (Figure 5). Nanoscale TiO2 also exhibited a gradual increase in toxicity toward D. magna, with 100% immobilization and 90% mortality occurring in the 500 mg/L exposure group. However, at 10 mg/L it caused only 20% immobilization and 0% mortality (Figure 5).
 
Similarly, increasing concentrations of the carbon-based NPs (i.e., C60, SWCNTs and MWCNTs) caused increases in toxicity to daphnids (Figure 5). The concentration range of such NPs was 0.1 to 100 mg/L because 100% mortality occurred at 100 mg/L. The EC50 of immobilization and LC50 of mortality for C60 NPs were calculated as 9.344 mg/L and 10.515 mg/L, respectively. Similarly, the EC50 and LC50 for MWCNT NPs were 8.723 mg/L and 22.751 mg/L, respectively. SWCNT NPs, however, demonstrated lower EC50 and LC50 values of 1.306 mg/L and 2.425 mg/L, respectively (Table 5).
 
Table 5 tabulates these 48 h EC50 and LC50 values. to The EC50 values for immobilization ranged from 0.622 mg/L (nZnO) to 114.357 mg/L (nAl2O3), whereas the LC50 values for mortality ranged from 1.511 mg/L (nZnO) to 162.392 mg/L (nAl2O3). In these experiments, the nZnO suspension was the most toxic to D. magna, while nAl2O3 was the least toxic. Based on the ANOVA results, the NPs affected the immobilization of D. magna in the order nZnO > SWCNTs > C60, MWCNTs > nTiO2 > nAl2O3; for mortality, the order was nZnO, SWCNTs > C60 > MWCNTs > nTiO2, nAl2O3.
 
The bulk counterparts of the NPs tested in this work (i.e., ZnO/bulk, TiO2/bulk, Al2O3/bulk, and carbon black) also exhibited a gradual increase in toxicity toward D. magna with increasing concentration (Figure 5). Table 5 lists their 48 h EC50 and LC50 values. For Al2O3/bulk, although some immobilization and mortality occurred, the 48 h EC50 and LC50 were both calculated to be more than 500 mg/L. However, the values of the 48 h and 96 h LC50 for ZnO/bulk were only 0.481 mg/L and 1.250 mg/L, respectively, similar to those of the ZnO NPs.
 
Figure 5 Effects of suspensions of six NPs and their bulk counterparts on the immobilization and mortality of D. magna.
 
Table 5 The 48 h EC50 and LC50 to D. magna of water suspensions of the tested materials
Material suspensions
EC50 (mg/L)
95% CI
LC50 (mg/L)
95% CI
nAl2O3
114.357
111.232-191.100
162.392
124.325-214.803
Al2O3/Bulk
>500
n.d.
>500
n.d.
nTiO2
35.306
25.627-48.928
143.387
106.466-202.818
TiO2/Bulk
275.277
170.661-570.045
>500
n.d.
nZnO
0.622
0.411-0.805
1.511
1.120-2.108
ZnO/Bulk
0.481
0.301-0.667
1.250
0.985-1.848
SWCNTs
1.306
0.821-1.994
2.425
1.639-3.550
MWCNTs
8.723
6.284-12.128
22.751
15.678-34.388
C60
9.344
7.757-11.262
10.515
8.658-12.757
Carbon black
37.563
33.076-41.968
61.547
54.546-68.232
Note: n.d. = not determined
 
Uptake and adsorption of NPs by D. magna
Large amounts of dark material were found in the gut tract of D. magna after NP exposure (Figures 6 and 7) but not in the control. D. magna can clearly ingest all NPs tested in this study, and therefore, accumulation could occur in the gut (Figures 2 and 3). In addition, at high concentrations some NPs (MWCNTs, etc.) were observed not only sticking to the glass beakers containing the test solution but also adhering to the external surface of the daphnids. This occurred within the first 24 h and extended to the end of the 48 h exposure period. In some cases, the ingestion and adsorption of NPs were significant enough to prevent ambulation of the daphnids through the water column and caused them to sink to the bottom of the beakers. However, in the low concentration exposure groups, only ingestion was observed, as shown in Figure 6. Other studies have also indicated that D. magna and rainbow trout can take up SWCNTs from test solutions (Roberts et al., 2007; Smith et al., 2007). These results thus suggest that exposure of aquatic organisms to such NPs could pose a risk of bioaccumulation, especially for filter-feeding copepods such as D. magna.
 
Figure 6 The uptake and adsorption of MWCNTs by D. magna after 48 h exposure. (2×10)
   

H: 48 h Control

 
 
    

 

Figure 7 Dead D. magna after 48 h exposure to different nanomaterials. (2×10)
 
Comparative toxicities of NPs to Scenedesmus oblignus and Daphnia magna
Table 6 compares the ecotoxicities of NPs to Scenedesmus oblignus and Daphnia magna. Most of the NPs tested, except for nTiO2, were more dangerous to daphnia based on EC50 values, indicating that daphnia may be more sensitive to NP exposure. However, Scenedesmus oblignus was more sensitive to nTiO2 exposure. These contrary results imply that the evaluation of the ecotoxicity of NPs that may be released into aquatic environments is not straightforward. Multiple organisms, especially those at different trophic levels, should be used to explore the real ecotoxicity of NPs.
 
Table 6 Comparison of NP ecotoxicities to Scenedesmus oblignus and D. magna.
Note: LOEC is the Lowest Observed Effective Concentration.
 

Conclusion

 

This study showed that:
1) These six nanomaterials with different chemical compositions and/or nanostructures were all able to inhibit the growth of Scenedesmus obliquus and the behavior of Daphnia magna to varying degrees, displaying obvious, dose-dependent ecotoxicities. The 96 h EC50 values for nZnO, nTiO2, nAl2O3, C60, SWCNTs and MWCNTs on the growth of Scenedesmus obliquus were 1.049, 15.262, >1000, 13.122, 22.633, and 15.488 mg/L, respectively. Based on the 96 h EC50, the toxicity of these six manufactured nanomaterials has the order nZnO > C60, nTiO2, MWCNTs, SWCNTs > nAl2O3. The 48 h immobilization EC50 values for nZnO, nTiO2, nAl2O3, C60, SWCNTs and MWCNTs on Daphnia magna were 0.622, 35.306, 114.357, 9.344, 1.306 and 8.273 mg/L, respectively. Based on these results, the toxicity of these manufactured nanomaterials had the order nZnO > SWCNTs > C60, MWCNTs > nTiO2 > nAl2O3. The nZnO suspension was the most toxic to both organisms, and Daphnia magna was more susceptible to its effects. The toxic effects of the three carbon nanomaterials (C60, SWCNTs and MWCNTs) to both algae and daphnia were generally similar, with their EC50 values varying only within one order of magnitude.
 
2) In the present tests, NPs were more toxic than “traditional” chemicals (i.e., the bulk counterparts of the NPs), which suggests that current regulations that focus on such “traditional” chemicals should be evaluated with respect to their applicability to nanomaterials. New regulations should be based not only on chemical components but also on size, as chemical properties change at the nanoscale.
 
3) Different organisms have different sensitivities to NP exposure. Multiple organisms, especially those at different trophic levels, should be used to explore the real ecotoxicity of NPs.
 
4) These experimental results confirm that, with some revision, the current standard ecological effects testing schemes and test protocols could meet the particular requirements of examining the ecological effects of nanoparticles as well as be adapted for ecotoxicity comparison. Poorly water-soluble manufactured nanomaterials and/or their aqueous dispersions may absorb photosynthetically active light and hence limit the growth of algal cultures in tests, leading to an overestimation of toxicity. In practice, it is better than method in OECD standard guideline to change the incubation system by shortening the light path, increasing the light intensity, and maintaining turbulence to compensate for internal light absorption and thus to minimize the dispersion effect. Observing Daphnia magna is also difficult in test media with a high concentration of nanoparticles. Placing test vessels on a light box or transferring the contents of the test vessels to shallow containers for scoring may help.
 

II. Determine the Potential Bioconcentration of Manufactured Nanomaterials in Zooplankton

 

Materials and Methods

 

Experimental design
As described above, aquatic exposures of aquatic organisms to manufactured NPs could pose a risk of bioaccumulation, especially for filter-feeding copepods such as D. magna. Therefore, bioconcentration experiments, including a 24 h uptake period followed a 72 h depuration period (Total experimental time is 96 h), were conducted 1) to investigate if daphnia can ingest nanoparticles from test solutions, and, if so, 2) to identify the accumulation profile for daphnia exposed to nanoparticles.
 
In this experiment, nTiO2 test solution was prepared immediately prior to use by diluting the stock solution (1 g nTiO2 suspended in 1 L nanopure water) with reconstituted water. Adult D. magna (8-10 d old), which had not been fed for 24 h, were exposed to 0.1 mg/L nTiO2 test solution for 24 h (uptake period) followed by 72 h of depuration. This concentration was selected based on the results from the above toxicity experiments. (The concentration of a substance in a bioaccumulation experiments should be below that which elicits toxicity or leads to changes in the behavior of the organisms). One hundred D. magna were sampled and pooled for particle content analyses (see below) after 0 (as control), 3, 6, 12 and 24 h of exposure (uptake period). No water change occurred during this period. And then, the rest of the D. magna were transferred to fresh reconstituted water for depuration. After 6, 12, 24, 48 and 72 of depuration, 100 daphnia were removed and pooled for particle content analyses. Water renewal was performed at a 24 h interval in depuration period. Mortality and immobilization as well as behavior changes were also observed during the experiment. Furthermore, this experiment was also repeated at a higher nTiO2 concentration of 1.0 mg/L in order to identify the concentration effects on accumulation profile in D. magna, but we also considered that higher concentration of NPs may occur at where close to a NPs manufacture plant or the source of an unintended release of NPs. All the above experiments were carried out in triplicate. 
 
All the experiments were performed under the conditions required by OECD Guideline 202 (22 ± 2°C, 48 h; diffuse light, 16:8 light-dark cycle; no food was provided). Samples of the test solution were also collected at the 0 h (control) and 24 h of the exposure period as well as the end of the 72 h depuration period. These samples were tested for pH, dissolved oxygen, ammonia, alkalinity and hardness. nTiO2 concentration in 0 h and 24 h water samples was also measured.
 
To determine the amount of nTiO2 bioaccumulated by the daphnids, the sampled organisms were thoroughly washed with nanopure water, collected, and blotted dry on filter paper. The wet weight was measured on an electronic microbalance. The samples were then dried to constant weight at 70°C in an oven overnight to determine dry weight. Dried samples were digested in 70% HNO3 (0.5 ml for 1 mg of dried sample) at 150 °C for 60 min. The digestions were then evaporated to dryness. TiO2 nanoparticles released by digestion were decomposed into titanium (IV) ion by heating with 1 ml of sulfuric acid – ammonium sulfate solution. After cooling, the solution was transferred quantitatively to a volumetric flask and diluted to 10 ml with nanopure water. The TiO2 concentrations of the digested samples were then determined by Inductively Coupled Plasma Optical Emmission Spectrometer (ICP-OES). The instrumental parameters were: RF power 1150 W; nebulizer pressure 15.2 MPa; carrier gas (Ar) flow rate 0.5 l/min; peristaltic pump rate 130 rpm; integration time High WL Range 5 s, Low WL Range 30 s; and wavelength 336.12 nm.
 
Data analysis
Michaelis–Menten kinetics was used to analyze nTiO2 uptake data:
Where C is whole body nTiO2 concentration at time t; Csat is the whole body nTiO2 concentration at saturated state (maximal concentration); and KM is the Michaelis–Menten constant, which is the exposure time needed to reach half of whole body nTiO2 concentration at saturated state. Using Michaelis–Menten kinetics, the exposure time needed for accumulated nTiO2 to reach 50% (tu0.5 or KM) and 90% (tu0.9) of saturation was determined. Time needed to depurate 50% (td0.5) and 90% (td0.9) of the whole body nTiO2 was also determined using first order kinetics. The nTiO2 bioconcentration factor (BCF) for D. magna was calculated as the ratio of the whole body nTiO2 concentration (based on the dry weight) to the nTiO2 concentration in the water at steady state.
 

Results and Discussion

 

During the study, the average temperature, pH, dissolved oxygen (DO), and ammonia were 23 ± 2°C, 7.9 ± 0.1, 7.7 ± 0.4 mg/L and 0.25 ± 0.09 mg/L. The average hardness and alkalinity were 204 ± 13 and 188 ± 11 mg/L as CaCO3, respectively. In the uptake portion of the experiment, the nTiO2 concentration in the water at steady state was measured to be 0.08 and 0.52 mg/L for 0.1 and 1.0 mg/L nTiO2 treatment respectively.
This study found that daphnia accumulated nTiO2 during the exposure phase and eliminated nTiO2 during the depuration phase. Whole body nTiO2 concentrations increased with exposure time and reached a plateau (saturation) after 12 h (Fig. 8). The whole body nTiO2 concentration at saturation was measured to be 4.52 g/kg (average values of 12 and 24 h) in 0.1 mg/L nTiO2 treatment group, and 61.09 g/kg (values of 24 h only) in 1.0 mg/L nTiO2 treatment group (Table 7). The exposure times needed for the accumulation of nTiO2 to reach 50% (tu0.5) and 90% (tu0.9) of the saturation level, based on the Michaelis–Menten kinetics model, were calculated to be 3.87 hours and 34.84 hours, respectively, in 0.1 mg/L nTiO2 treatment group. These values were not significantly to those measured in 1.0 mg/L nTiO2 treatment group (Table 7). However, the values of td0.5 (29.60 h) and td0.9 (69.45 h) (times to depurate 50% and 90% of the accumulated nTiO2) were found significantly less than those in 1.0 mg/L nTiO2 treatment group (Table 7). Thus, it can be speculated that Daphnia may be more difficult to depurate nTiO2 after exposure to high concentration of nTiO2 (e.g., 1.0 mg/L).
 
The BCFs for nTiO2 in D.magna were calculated at steady state. A high value of BCF was found to be 56562.50 even in the low concentration (0.1 mg/L) treatment groups. And also, the BCF value in 1.0 mg/L nTiO2 treatment group was high to 118062.84. Interestingly, the time needed for daphnia to accumulate nTiO2 to half of the saturated nTiO2 concentration (KM) was shorter than that needed to depurate half of the saturated nTiO2 concentration (td0.5) (Table 7). Therefore, the very high BCF may be due to the fact that the uptake process was faster than the depuration process. This phenomena also suggests that exposure of daphnia to low nTiO2 concentrations could result in high nTiO2 accumulation.
 
Figure 8 Uptake and depuration of nTiO2 in D. magna.
Table 7 Uptake and depuration rate constants and bioconcentration factor (BCF) for nTiO2 in D. magna.
Dose (mg/L)
Exact dosea (mg/L)
Whole body concentration(dw) (g/kg)
BCFs      (l/kg)
KM=tu0.5 (h)
tu0.9 (h)
td0.5 (h)
td0.9 (h)
0.10
0.08
4.52b
56562.50
3.87
34.84
26.76
88.90
1.0
0.52
61.09
118062.84
3.72
33.51
74.52
247.59
a Exact concentration of nTiO2 in culture medium after exposure for 24 hours.
b Average concentrations at times of 12 and 24 hour.
 

III. Determine the Potential Bioconcentration of Manufactured Nanomaterials in Fish

 

Materials and Methods

 

Experimental design
Although the amount of research on the toxicology of nanomaterials is increasing, little is currently known about their transfer and fate in aquatic environments, or about their potential influence on the transfer and fate of co-pollutants. This study examined the adsorption of cadmium (Cd) and arsenic (As) onto nTiO2 and the potential of nTiO2 to facilitate the transport of these elements into carp (Cyprinus carpio).
 
Reagents and solutions
All reagents used were analytical-reagent grade except for acids, which were trace metal analysis grade. Nanopure water was used for preparation of the stock solution. Laboratory equipment and containers were dipped in 25% (v/v) HNO3 solution for at least 12 h prior to use. The Cd stock solution (1000 mg/L) was prepared by dissolving 1.0 g Cd metal in 5 mL HNO3 (1 + 1) and then diluted to 1 L.  Na3AsO4•12H2O was used for preparation of As (V) stock solutions (100 mg/L). The standard stock solutions were stored in glass bottles kept at 4°C in the dark. Working solutions were freshly prepared from the stock solution for each experimental run. A reductant solution of 0.4% (w/v) NaBH4 dissolved in 0.5% (w/v) NaOH was used for hydride generation; this was prepared immediately prior to use.
 
Adsorption of Cd and As onto TiO2 nanoparticles
Batch experiments were used to study adsorption kinetics and isotherms. Dechlorinated tap water was used to maintain water circumstances similar to those in the bioaccumulation tests. In each experiment, a series of 250 mL Pyrex glass Erlenmeyer flasks were used, 100 mL of 10 mg/L nTiO2 suspension was added to each flask. The required amount of Cd or As standard solution was added to initiate the adsorption. The flasks were shaken at 150 rpm in a reciprocating shaker and kept in the dark at 25 ± 1 °C. At 0, 10, 20, 30, 60, 120, 180, and 360 min, a 10 mL sample of each suspension was removed and centrifuged twice for 10 min at 12,000 rpm using a high speed centrifuger (Hermle Z323, Germany). Residual aqueous Cd or As concentrations were analyzed using an atomic fluorescence spectrometer equipped with hydride generation (HG-AFS 2201, Haiguang Co., Beijing, China). In adsorption isotherm experiments, the initial Cd or As (V) concentration was varied while the nTiO2 suspension concentration of 10 mg/L was held constant. Isotherm tests were conducted for 2 h and then the residual aqueous Cd or As concentration was analyzed. The amount of Cd or As adsorbed was calculated by mass balance between the initial and final solution concentrations. To correct for any loss of Cd or As due to adsorption to the containers, control experiments were carried out without the adsorbent; negligible adsorption by the container walls occurred (<5%).
 
Accumulation experiment
Carp (Cyprinus carpio) were purchased from a local pet shop. The initial body weight and length of the fish were 6.1 ± 1.2 g and 4.0 ± 0.7 cm, respectively. All fish were acclimatized in dechlorinated tap water with a natural light-dark cycle for ten days before experiment.
 
In the accumulation tests, seven glass tanks containing 16 l dechlorinated tap water were divided to seven groups: water control, 3 mg/L nTiO2 , 10 mg/L nTiO2, As, As+10 mg/L nTiO2, Cd and Cd+10 mg/L nTiO2. The initial concentration of Cd was 97.3 ± 6.9 μg/L. (The initial concentration of As was 200.0 ± 10.2 μg/L.). 32 Carp were placed in each tank after 2 h. The fish were fed a commercial food twice a day during the experiment. To maintain a relatively stable concentration of nTiO2 in solution, the tanks were aerated slightly throughout the tests. Fish were transferred to freshly made solution every day. During the tests, the water temperature was maintained at 23 ± 2 °C for each exposure. The pH of the exposure water was 7.8. Three fish were removed and sacrificed on days 2, 5, 10, 15, 20, and 25; on day 20, 6 carp from the water control, 3 mg/L nTiO2 and 10 mg/L nTiO2 treatments were dissected into skin and scales, muscle, gills, and viscera. After pretreatment, Cd, As and nTiO2 concentrations in the whole body or different parts of carp were analyzed.
 
Analytical procedures
Cd and As concentrations in the water were measured by Hydride Generation Atomic Fluorescence Spectrometry (HG-AFS). Before these measurements, samples containing Cd were pretreated with 1.0 mL concentrated HCl, 5.0 mL thiourea solution (5%) and 0.5 mL CoCl2 solution (100 mg/L). A thiourea (5%)–ascorbic acid (5%) mixing reagent was used to pre-reduce arsenate, and hydrochloric acid (5%) media was used for hydride generation.
 
To determine the Cd, As and nTiO2 concentrations in the whole body or parts of the fish, the fish were rinsed with dechlorinated tap water, dried at 105°C, and ground into powder. Approximately 0.20 g dried sample and 4.0 mL concentrated HNO3 were added into each of 6 PTFE (Polytetrafluoroethylene) digestion tubes. After 10 min, the vessels were sealed and put in the microwave. The samples were digested using a three-stage protocol (5 min at 150 °C, 5 min at 180 °C and 5 min at 190 °C). Afterward, the vessel was cooled. Sample filtration was not required because dissolution was complete. For Cd and As analysis, the excess acid was removed from the digested solutions by heating them to near dryness at 90 °C using an electric furnace. Cd and As concentrations were then determined following the procedure for water samples. For nTiO2 analysis, the digests were transferred to triangular flasks and evaporated to dryness. The nTiO2 released by digestion was decomposed into titanium (IV) ion by heating with 5 ml of sulfuric acid - ammonium sulfate solution. After cooling, the solution was transferred quantitatively to a 25 ml volumetric flask. The nTiO2 concentrations in the digested samples were determined as described in the daphnia bioconcentration experiments.
 
Data analysis
Mean Cd, As and nTiO2 concentrations were calculated from the three replicates and expressed with standard deviation (n = 3). The homogeneity of variance was checked and a one-way analysis of variance (ANOVA) was performed to assess the significance of the differences observed between Cd concentrations in fish exposed to Cd versus Cd + nTiO2 (between As concentrations in fish exposed to As versus As + nTiO2). All statistical analyses were conducted at a significance level of 0.05.
Experimental data for As(V) and Cd adsorption onto nTiO2 were described by the Freundlich isotherm:
                                                                                                     (1)
Where q (mg/g) is the amount of adsorbed As(V) or Cd, Ce is the equilibrium As(V) or Cd concentration in solution (mg/l), and KF and 1/n are the Freundlich constants. The Freundlich parameters were obtained by nonlinear least-squares regression analysis. The accumulation of Cd, As and nTiO2 was described using the standard exponential equation (2) (Pendleton et al., 1995):
                                                                                                             (2)
Where Ct is the substance concentration in whole fish (lg/g dry weight), A is the substance concentration at equilibrium (μg/g dry weight), B is the first-order rate constant (d-1), which gives insight into how rapidly the element is accumulated, and t is the exposure time (d). The BCF of different treatment groups was calculated from the following equation (3):
                                                (3)
 

Results and Discussion

 

Adsorption of Cd and As onto TiO2 nanoparticles
Sorption kinetics was observed for 6 h. The adsorption processes of Cd and As onto nTiO2 were fast, reaching equilibrium within 30 min. At equilibrium, the amounts of Cd and As adsorbed by nTiO2 were approximately 65% and 25%, respectively, indicating that nTiO2 has a stronger adsorption capability for Cd than As. The adsorption isotherm was determined using 2 h of equilibrium time. Experimental data for Cd and As adsorption onto nTiO2 fit the Freundlich isotherm well; the correlation coefficients were 0.959 and 0.946, respectively. Figure 9 presents the plots. The Freundlich parameters were obtained by nonlinear least-squares regression analysis. The constants KF and 1/n were found to be 250 mg/g and 0.962 for Cd, and 20.71 mg/g and 0.58 for As(V).
 

 

Figure 9  Freundlich plots for the adsorption of Cd (A) and As(V) (B) onto TiO2 nanoparticles.
Bioconcentration and distribution of nTiO2 in carp
 
nTiO2 concentrations in water and fish samples were determined using ICP-OES after treatment with a solution of sulfuric acid and ammonium sulfate. The bioconcentration of nTiO2 in carp was found to be significant (Figure 10); the nTiO2 concentrations in carp exposed 3 mg/L and 10 mg/L nTiO2 suspensions for 25 days were calculated to be 211 mg/g and 518 mg/g, respectively. The BCF values for different concentrations at equilibrium are 675.15 and 595.14, respectively. Figure 11 shows the nTiO2 concentrations in different parts of the carp. Significant  nTiO2 accumulation occurs in viscera and gills of fish, while the bioconcentration of nTiOin muscle is relatively small (Table 8).
 
Figure 10 Accumulation kinetics of nTiO2 in carp. 10mg/L exposure, 3 mg/L exposure,  control.
 
 
Figure 11 nTiO2 concentrations in different parts of carp. control, 3 mg/L exposure, 10mg/L exposure.
 
Table 8 BCFs for nTiO2 in different parts and the whole body of carp.
nTiO2 concentrations
Skin and scales
Muscle
Gill
Stomach
Whole body
3 mg/L
103.3
52.5
348.5
2 096.7
675.5
10 mg/L
61.0
43.2
222.3
1 426.0
595.4
 
Accumulation of Cd and As by carp in the presence of nTiO2
Figures 12 and 13 present the accumulations of Cd (or As) in carp exposed to Cd (or As) versus Cd (or As) + nTiO2 as a function of exposure time. The Cd concentration in carp exposed to Cd contaminated water increased gradually, reaching 9.07 μg/g after 25 d of exposure. When exposed to Cd-contaminated water in the presence of nTiO2, however, the carp accumulated considerably more Cd. As can be seen from Figure 12, the Cd concentration in the carp increased sharply, reaching 22.3 μg/g at the 25th day. This is a 146% increase over that without nTiO2.The presence of nTiO2 also greatly enhanced the accumulation of As in carp, as shown in Figure 13. The arsenic concentration in carp exposed to As-contaminated water increased gradually, reaching 2.96 μg/g after 25 days exposure. The As concentration in the carp increased sharply in the presence of nTiO2, however, reaching 6.86 μg/g after 25 days exposure, an increase of 132% over the levels without nTiO2.
 
Table 9 presents the results of the regression analysis of the experimental data using Equation (2) as well as the BCFs calculated according to Equation (3) using the equilibrium concentrations. Cd (or As) concentration in carp at equilibrium (A value) in the presence of nTiO2 is much higher than that in carp exposed to Cd (or As) only. Also, the BCFs for Cd and As at equilibrium in the presence of nTiO2 are almost fivefold and twofold higher, respectively, than those in the absence of nTiO2. Thus, nTiO2 appears to facilitate the transportation of heavy metals (e.g., Cd and As) into fish.
 
 
Figure 12 The accumulation of Cd in carp exposed to Cd () or Cd + nTiO2 (). The curves represent the exponential regression of the mean values.
Figure 13 The accumulation of As in carp exposed to As () or As + nTiO2 (). The curves represent the exponential regression of the mean values.
 
Table 9 The exponential accumulation parameters and BCFs.
Exposure media
A (μg/g)
B (d−1)
R2
BCF
Cd
6.98
0.143
0.631
64.4
Cd + nTiO2
29.3
0.063
0.942
325.0
As
3.40
0.109
0.983
22.67
As + nTiO2
8.34
0.074
0.991
55.60
 

Conclusion

 

Due to their small particle size, large specific surface area, and strong electrostatic attraction, nTiO2 nanoparticles have a high adsorption capacity for heavy metals, such as Cd and As. Moreover, this study found significant nTiO2 bioconcentration by the carp, with BCF values at equilibrium of more than 500. Significant nTiO2 accumulation occurs in the viscera and gills of fish, while bioconcentration of nTiO2 in muscle is relatively small. Interestingly, the presence of nTiO2 was found to greatly enhance the accumulation of Cd and As in carp. After 25 d of exposure, Cd and As concentrations in carp increased by 146% and 132%, respectively.

Future Activities:

 


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 17 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article 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)
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  • Journal Article 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)
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  • Journal Article 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)
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  • Journal Article 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)
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  • Supplemental Keywords:

    Health, Scientific Discipline, PHYSICAL ASPECTS, Health Risk Assessment, Risk Assessments, Physical Processes, fate and transport, food chain, bioavailability, exposure, nanotechnology, nanomaterials, nanoparticle toxicity, bioaccumulation, fish-borne toxicants

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    Original Abstract
  • 2007
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