Grantee Research Project Results
Final Report: Short-term Chronic Toxicity of Photocatalytic Nanoparticles to Bacteria, Algae, and Zooplankton
EPA Grant Number: R831721Title: Short-term Chronic Toxicity of Photocatalytic Nanoparticles to Bacteria, Algae, and Zooplankton
Investigators: Huang, C. P. , Cha, Daniel K. , Ismat, Shah S.
Institution: University of Delaware
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2005 through September 30, 2007
Project Amount: $334,881
RFA: Exploratory Research to Anticipate Future Environmental Issues: Impacts of Manufactured Nanomaterials on Human Health and the Environment (2003) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Human Health , Nanotechnology
Objective:
The goal of this research project was to study the short-term toxicity of photocatalytic TiO2 nanoparticles toward three aquatic organisms: bacteria, algae, and zooplankton. The specific objectives were to determine (1) the short-term chronic toxicity of photocatalytic nanoparticles to pure bacterial culture including Gram-negative bacteria (e.g., E. coli TB1 and K12), and Gram-positive bacteria (B. subtilis), (2) the short-term chronic toxicity of photocatalytic nanoparticles to daphniad, Ceriodaphnia dubia, (3) the short-term chronic toxicity of photocatalytic nanoparticles to algae, Selenastrum capricornutum (new name: Pseduokirchneriella subcapatitata), and (4) the short-term chronic toxicity of copper(II) to Selenastrum capricornutum in the presence of photocatalytic nanoparticles.
Summary/Accomplishments (Outputs/Outcomes):
The effect of primary particle size was the focus of this research project. In order to test the effect of particle size on the responses of selected organisms, a total of 29 TiO2 nanoparticles of different particle size were selected and studied. Focus was on comparing the response of the testing organisms to photocatalytic nano-TiO2 particles under various growth conditions. The end points for expressing the response of bacteria were bacterial density, the rate of the production of Malondialdehyde (MDA) and TTC. MDA is a measurement of the degree of oxidation of membrane lipids and TTC is a measurement of the extent of respiration. The toxic effect was expressed in LC50 or EC50. For algae, two different experiments were conducted: bio-uptake of and exposure to nano-TiO2 particles. Experiments were conducted to determine the uptake of nano-TiO2 particles onto algal cell surfaces. The series of experiments was conducted to assess the effect of nano-TiO2 particles on the biological activities of algae, including cell counts, chlorophyll, and MDA. The effect of nano-TiO2 on the toxicity of copper to algae was also studied. For Daphnia, experiments were conducted to assess the effect of primary and secondary particle size of nano-TiO2 on the survival of daphnia. Since the major effort was to assess the effect of particle size on the responses of selected organisms to nano-TiO2 photocatalyst, reliable particle size information is essential. We have decided to establish the particle size using same method. Table 1 lists the surface areas of all TiO2 particles studied.
Exposure and responses of bacteria to photocatalytic TiO2 nanoparticles
Two different species of bacteria, i.e., E. coli representing Gram(-) and B. subtilis representing Gram(+), were employed to detect the effect of bacterial types on the survival and physiological responses which were measured in terms of malondialdehyde (MDA) production and TTC. E. coli were grown at 37°C and B. subtilis were cultured at 30°C in 100 mL of 171 mM Luria-Bertani (LB) broth on a rotary shaker (200 rpm) for 18 h. One mL of cell aliquots was added to the 100 mL of 71 mM Min salt media for further reactions. Nano-TiO2 taken from stock solutions was included in the cell-media mixture prior to the reactions. The bacterial cell membrane was composed of layers of lipids (20 to 30%) and proteins (80 to 70%). The composition is different in different species of bacteria. For example, gram-negative bacteria such as E. coli, have an additional outer membrane that is also composed of lipids and proteins. The differences between the outer and inner membranes are the enzymes that do the electron transfer and other material transports. When irradiated TiO2 nanoparticles were present with bacteria, the cell membrane was the primary target of the initial oxidative attack. The polyunsaturated phospholipids component of the cell membrane was oxidized by the ROS (reactive oxygen species) and hydroxyl radicals generated by TiO2 nanoparticles and forms MDA. To study the effect of cell type on MDA production under light condition, E. coli strainsand B. subtilis were used. E. coli TB1, a strain of E. coli K12 host cell, has a plasmid and antibiotic resistance genes. The MDA formations from lowest TiO2 concentration (1 mg/L) and highest TiO2 concentration (1000 mg/L) showed that B. subtilis was more resistant to nanoparticles than E. coli strains. It was also seen that a high concentration of TiO2 nanoparticles (1000 mg/L) has the greatest effect on membrane damage. Bacteria exposed to nano-TiO2 particles exhibited an initial, fast inactivation rate in the viable population followed by a slower rate in the population. As the concentrations of particles increased, the die-off of cells decreased. At small concentrations of 0.1 mg/L of P25, TiO2 nanoparticles were the most effective on the inactivation of E. coli, whereas the highest concentration of 1000 mg/L of P25 TiO2 was more damaging to the B. subtilis at the same exposure time. Further, an optimal particle concentration of 100 mg/L was observed for the survival of E. coli under dark conditions; no such observation was obvious in the presence of light, however. Generally, in the presence of light, the viable cell counts decreased with an increase in the concentrations of TiO2. An LC50 of approximately 10 and 100 mg/L was observed for E. coli exposed to P25 for 5 and 30 min, respectively. Further exposure of E. coli to photocatalytic TiO2 significantly improved the survival of the microorganism. The results indicated that a primary size of ca. 17 nm was the most biocidal under both dark and light conditions. Generally, in the absence of light, the decrease in cell viability was less drastic than that in the presence of light.
Table 1. Summary of Particle Size Determined by BET | |||
Name | Given (nm) | BET (nm) | Sources |
P25 | 30 | 34.2 | Degussa |
R10 | 6.5 | 4.8 | Reade |
Aero200 | 12 | 14.3 | Degussa |
9-780 | 73.48 | 111.1 | UD1 |
9-780 | 73.48 | 120.1 | UD1 |
7-660 | 43.87 | 45.8 | UD1 |
T1 | 20.3 | 16.2 | UD2 |
T40 | 16.4 | 13.4 | UD2 |
SL2 | 15 | 17.5 | UD3 |
S2 | 27.3 | 30.3 | UD2 |
S3 | 25.7 | 27.5 | UD2 |
S3 | 25.7 | 22.7 | UD2 |
10-600 | 34.64 | 42.0 | UD1 |
SL1 | 23 | 42.5 | Sammy |
T50 | 15.9 | 15.5 | Allie |
11-970 | 244.9 | 635.7 | UD1 |
Wo4 | 21.8 | 13.7 | UD2 |
Wo6 | 16 | 12.3 | UD2 |
U100 | 6.5 | 4.8 | UD3 |
Y350 |
| 13.4 | UD1 |
Y11-590 |
| 809.1 | UD1 |
8-840 | 122.47 | 204.1 | UD1 |
R5 | 4 | 5.2 | Reade |
ST-21 | 17.8 | 23.3 | UD1 |
ST-01 | 4.8 | 5.3 | UD1 |
Wo3 | 22 | 21.4 | UD2 |
T6 | 13.7 | 17.8 | UD2 |
UV100 | 6.5 | 4.7 | UD1 |
Y1100 | 387 | 1466.6 | UD1 |
Cryo-SEM images were taken in order to see the physical damage of the cells under dark and light conditions. Figure 1 shows the contract in appearance among the E. coli cells treated with nano-TiO2 particles in dark and under light. Results demonstrated that there was attachment of nano-TiO2 particles in the cell surface in dark conditions. However, there was obvious surface erosion on the cells exposed to light in the presence of nano-TiO2 particles.
Figure 1. SEM images of E.coli (a) with no TiO2 treatment, (b) with 100 mgL-1 P25 TiO2 treatment under dark, and (c) with 100 mgL-1 P25 TiO2 treatments under light (c).
The confocal images were taken in order to assess the physical damage of the cells under dark and light conditions. Figure 2 shows the healthy bacteria (Figure 2a) and bacteria treated with TiO2 under dark (Figure 2b) and light (Figure 2.c, d) conditions. The results show that the effect of irradiated TiO2 on the cell membrane is conspicuous relative to that of TiO2 under dark condition. As shown later, our studies on algae suggested that the photokilling reaction is initiated by a partial decomposition of the outer membrane, followed by disordering of the cytoplasmic membrane, resulting in cell death.
Figure 2. Confocal images E. coli with no treatment (a), with 100 mg/ L P25 TiO2 treatment under dark (b), light-only (c), with 100 mg/L P25 TiO2 treatment under light (d).
Exposure and rsponses of algae to photocatalytic TiO2 nanoparticles
During the project period, the goals of the research were to determine 1) the effect of titanium dioxide on the toxicity of copper to algae; 2) the effect of light intensity on the response of cell population to Nanoparticles; 3) the effect of aluminum oxide particle size on cell population; and 4) the effect of chemical composition of nanoparticles on the responses of algae.
Pseudokirchneriella subcapitata, was purchased from Aquatic Biosystems (Fort Collins, CO) for this study. The algae were in the dark at 4ºC in the dark until used in continuously stirred tank reactors (CSTR). The CSTRs were used to culture algae to be used in the experiments. Two CSTRs were used to grow algae, with a hydraulic residence time of approximately 3 days. Prior to adding samples to using the algae in the tests, approximately 1.5 L from each reactor was mixed together as samples were analyzed for absorbance, cell densities, and chlorophyll a. Titanium dioxide (P25) and silica dioxide (Aerosil 200) Aluminum oxide (Alon) were purchased from Degussa Corp. (Teterboro, NJ). All particles were analyzed for surface area by the nitrogen adsorption BET method. Stock suspensions of nanoparticles were made at 2000 mg/L in algae growth media and autoclaved prior to use.
Bioaccumulation of phtocatalytic TiO2 on algae: An effort was made to assess the uptake of nano-TiO2 particles by algae cell. This is an extra effort not included in the original proposal. We did this study because during the course of this research, we realized that the particles had become attached to the cell surface. We believe that this particle uptake process has a bearing on the response of the testing organisms. Results shown in Figure 3 indicated that nano-TiO2 particles became attached onto the algal cell surface. The amount of TiO2 particles attached was a significant function of pH. As a result of the TiO2 uptake, the surface charge of the algae was modified to match that of the TiO2 nanoparticles.
Figure 3. SEM images of algae Pseudokirchneriella subcapatitata in the absence of TiO2 particles.
The effect of titanium dioxide on the toxicity of copper to algae: Results indicated that at low Cu(II) concentration, the TiO2 nanoparticles controlled the response of the algae. As nanoparticle concentration increased, the cell growth increased from the control level to approximately 100 mg/L. At TiO2 concentration greater than 100 mg/L, there was a decrease in cell growth. This is true for Cu(II) concentration in the range between 0.05 and 20 mg/L. At 20 mg/L Cu(II) the Cu(II) dominates the toxicity. Overall, sorption of Cu(II) onto nanoparticle decreases the toxicity of Cu(II) to algae.
The effect of light intensity on the response of cell population to nanoparticles: Results indicate that as TiO2 increased, the cell growth decreased under different light intensity. The highest light intensity (2692 lux) showed the least toxic response. A mid-level light intensity (1601 lux) showed the highest toxic response. Based on the above results, it can be concluded that the high light intensity overcame the shading effect of the particles. The lowest light intensity has enough light for the algae to grow, but not sufficient intensity to kill the algae from photocatalytic activity. At 1601 lux, the light is at an optimum to cause the algae cells to have a negative growth.
The effect of aluminum oxide particle size on cell population: Results showed that the smaller particles of Al2O3 decreased the cell growth was by more than approximately 50% at 100 mg/L, while larger Al2O3 (e.g. 67.5 nm) had no effect at the same concentration. At 1000 mg/L all particle sizes caused about 65 – 80% decrease in cell growth. The growth was limited due to turbidity changes and light-limited growth.
The effect of chemical composition of nanoparticles on the responses of algae. Results indicate that an increase in nanoparticle concentration caused a decrease in cell growth for all particles tested. Nanoparticles of TiO2 and SiO2 have similar results, with an EC50 of approximately log 2.9. Al2O3 was slightly more toxic with an EC50 of log 2.5. Particle type did not play a significant role in the toxicity of nanoparticles to Pseudokirchneriella subcapitata; however, particle size played a much more important role.
Exposure and Responses of Daphnia to Photocatalytic TiO2 Nanoparticles
A total of eleven particle sizes were applied in a 24-hr acute test for assessing the particle size effect of nanoparticles on Ceriodaphnia dubia. Other experiments were conducted to assess factors such as secondary particle size, photo period, and sedimentation, which might affect the mortality rate of C. dubia. Results show that the LC50 values could be expressed mostly as a function of the primary particle size or the secondary particle sizes. Photoperiod was not a significant parameter affecting the responses of daphnia. Physical factors, such as contact time or suspension stability, had a greater effect on the mortality rate of C. dubia.
SEM images of C. dubia neonate in dilution water after 12 hr. (l) Whole body; (c) general view of carapace; (r) closer view of carapace.
SEM images of > 24-hr C. dubia neonate in the presence of TiO2 (100-mg/L P25 ) after 12 hr. (l) Whole body; (c) general view of carapace; (r) closer view carapace
To better understand the interaction between C. dubia and particles, SEM images were taken. Figure 4 shows images of C. dubia in suspension of P25 for 12 (top) and >24 hr (bottom). Results revealed the adsorption and accumulation of TiO2 on C. dubia carapace surface. Compared to the control group, C. dubia in suspension has particles on its body. The shell of C. dubia in suspension was not as smooth as that of the control group, and it seems that the particles became embedded in the shell and caused wrinkles on the carapace. Results shown in Figure 4 demonstrate that particles not only adsorbed on the carapace surface but also accumulated around the joints and in the ripples of the carapace.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 9 publications | 2 publications in selected types | All 2 journal articles |
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Erdem A, Metzler D, Cha D, Huang C. The short-term toxic effects of TiO2 nanoparticles toward bacteria through viability, cellular respiration, and lipid peroxidation. ENVIRONMENTAL SCIENCE AND RESEARCH 2015;22(22):17917-17924 |
R831721 (Final) |
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LI M, Czymmek K, Juang C. Responses of Ceriodaphnia dubia to TiO2 and Al2O3 nanoparticles:a dynamic nano-toxicity assessment of energy budget distribution. JOURNAL OF HAZARDO MATERIALS 2011;187(1-3):502-508. |
R831721 (Final) |
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Supplemental Keywords:
Ecotoxicity, nanoparticles, TiO2, photocatalysis, E. coli TB1, E. coli K12, B. subtilis, Pseduokirchneriella subcapatitata, Ceriodaphnia dubia, bacteria, algae, zooplankton,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Environmental Chemistry, Aquatic Ecosystem, Environmental Monitoring, algal blooms, Ecological Risk Assessment, Ecology and Ecosystems, bioassessment, nanotechnology, bacterial biomass, nanophotocatalysts, aquatic ecosystems, nanoparticles, water quality, aquatic ecotoxicity, ecosystem responseProgress 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.