ARTICLE pubs.acs.org/crt
Cytotoxicity of Al2O3 Nanoparticles at Low Exposure Levels to a Freshwater Bacterial Isolate Sunandan Pakrashi, Swayamprava Dalai, Debabrat Sabat, Suniti Singh, N. Chandrasekaran, and Amitava Mukherjee* Centre for Nano Biotechnology, School of Bio Sciences & Technology, VIT-University, Vellore-632014, India
bS Supporting Information ABSTRACT: The cytotoxicity of Al2O3 nanoparticles (NP) at very low exposure levels (1 μg/mL and less) to a dominant bacterial isolate from freshwater (lake water), Bacillus licheniformis, was examined. Sterile lake water was directly used as a test medium or matrix to simulate the freshwater environment. Exposure to 1 μg/mL Al2O3 NP for 2 h caused a 17% decrease in cell viability (as determined by plate count and MTT assay). During the test period, the particles were found to be stable against aggregation in the matrix and exerted a nano-size effect on the exposed test organisms. The decrease in cell viability was proven not to be due to the release of Al3+ ions from the nanoparticles in the dispersion. The zeta potential and FT-IR analyses suggested that the surface charge based attachment of nanoparticles on to the bacterial cell wall was responsible for flocculation leading to toxicity. The cell wall damage confirmed through SEM and the lipid peroxidation assay also contributed toward toxicity. This study warns of possible ecotoxicity of nanoparticles even at environmentally relevant concentrations. However, detailed studies need to be carried out to establish probable mechanistic aspects of this low concentration toxicity phenomenon.
’ INTRODUCTION With rising industrial applications of engineered nanoparticles, they are destined to enter the aquatic, terrestrial, and atmospheric zones in the environment, where their fate and behavior are yet to be determined.1 The unique properties and small size of the engineered Al2O3 NP have inevitably increased their application potential. Al2O3 NP has a wide range of applications as coatings and abrasives and as additives in the fields of composites and heat transfer-enhancing nanofluids.2,3 The influence of Al2O3 NP on blended concrete, in terms of its compressive strength, has recently been explored.4 Engineered nanoparticles, owing to their extensive commercial applications, have received considerable attention regarding possible environmental impacts. The fate, behavior, and transportation of nanoparticles in different water matrixes (groundwater, surface water, and seawater) are poorly understood, as is their toxicity potential. Mostly, this differential behavior depends on the nanoparticle size and stability in the dispersion medium.5 The potential release of these nanoparticles in the environment has necessitated an understanding of their stability, mobility, and reactivity.1 A recent study showed that the behavior and stability of oxide nanoparticles can differ significantly depending on the test medium.6 Exposure to nanoparticles could cause oxidative stress in cells at the cellular and subcellular levels and also releases toxic constituents (i.e., secondary toxicity).7 The toxicity of nanoscaled aluminum, silicon, titanium, and zinc oxide to different bacteria were studied in the recent past.8 Al2O3 NP demonstrated a r 2011 American Chemical Society
mortality rate of 57% to Bacillus subtilis, 36% to Escherichia coli, and 70% to Pseudomonas fluorescens at a low concentration (20 μg/mL) level using 1 g/L NaCl as the experimental medium. In a nutrient enriched test medium, we studied the antimicrobial sensitivity of Al2O3 NP to E. coli and illustrated a nominal growth inhibitory effect of NP only at very high concentrations (>1000 μg/mL).9 Another study by our group demonstrated the growth inhibitory effect of Al2O3 NP on Scenedesmus sp. and Chlorella sp. (72 h EC50 39 μg/mL and 45 μg/mL, respectively).10 A study on yeast cells demonstrated the membrane disruption property of SiO2 and Al2O3 NP at high concentrations (>1000 μg/mL), whereas at same concentrations, HfO2 and CeO2 did not show any significant effect.11 A study focused on the mechanistic aspects of TiO2 and Al2O3 NP toxicity suggested the possibilities of nonROS mediated toxicity toward certain invertebrate systems.12 In another assessment, toxicity and bioaccumulation of Al2O3 NP to a range of sediment dwelling microorganisms (such as Tubifex tubifex, Hyalella azteca, Lumbriculus variegatus, and Corbicula fluminea) were observed. Toxicity was found to be more in nanoAl2O3 as compared to micrometer-sized Al2O3 toward the survival of H. azteca in a 14-day exposure period upon direct contact with a thin layer of 625 mg or 2500 mg of Al2O3 NP.13 Experiments on Daphnia magna with nano-Al2O3 reported 48 h EC50 and LC50 values of 114 and 162 μg/mL, respectively.14 Received: June 13, 2011 Published: October 03, 2011 1899
dx.doi.org/10.1021/tx200244g | Chem. Res. Toxicol. 2011, 24, 1899–1904
Chemical Research in Toxicology Unicellular organisms like bacteria and/or algae are assumed to be more resistant to the toxic effects of NP than protists and metazoans, which possess highly developed systems for the internalization of nano and microscale particles.15 The toxicity of NP toward the bacterial species in the aquatic environment reflects the possible agitation that could impose damage to other members of the microbial communities in the biogeochemical cycles. There are also alarming reports regarding the transfer of engineered nanomaterials from prey (like Pseudomonas aeruginosa) to predator (like Tetrahymena thermophila) in a typical aquatic ecosystem.16 A recent study showed that the gold nanorods readily passed from water column to the marine food web in three laboratory-constructed estuarine mesocosms containing seawater, sediment, sea grass, microbes, biofilms, snails, clams, shrimp, and fishes.17 As the prior reports revealed that most of the ecotoxicity tests conducted with nanomaterials had two key limitations, (i) they were undertaken in defined growth media. In contrast, we have employed sterile lake water without nutrient supplements to simulate the chemical matrix of the fresh water aquatic environment. (ii) The tested concentration ranges were kept high enough to demonstrate the toxicity (atleast above 10 μg/mL). However, based on the current production rate, the environmental concentrations of different nanomaterials (TiO2, ZnO, Ag, CNT, and fullerenes) have been modeled to be below 1 μg/ mL.18 To evaluate the environmental risk of these nanoparticles, it is necessary to take up the toxicity study at a low concentration of these particles to analyze the impact (if any). This motivated us to evaluate the toxicity of Al2O3 NP at low concentration ranging from 0.05 μg/mL to 1 μg/mL. In the present study, we aim to demonstrate the differential behavior of Al2O3 NP in the lake water matrix and its reactivity toward the dominant bacterial species within a short exposure period of 2 h.
’ MATERIALS AND METHODS In this present study, surface water (collected from VIT Lake, Vellore, India) was used as a test medium with no nutrient supplements. Physicochemical characterization showed a conductance of 4.3 ( 0.13 mS/cm, pH of 7.8, dissolved oxygen content of 7.2 ( 0.46 μg/mL, and total dissolved solids content of 800 ( 74 μg/mL. The presence of metal ions in the lake water was quantified by ICP-OES (Perkin-Elmer Optima 5300 DV, USA) and other inorganic ions by the titrimetric method (Supporting Information). Primary filtration through a 20 μm sieve was done to remove the suspended solid particles. Subsequent filtrations through Whatman no.1 filter paper followed by 0.22 μm membrane filter were done to avoid the interference of large colloidal particles. The suspension thus obtained will henceforth be denoted as filtered lake water. Characterization of Al2O3 NP Dispersion. Dry Al2O3 NP (γ-phase alumina nanopowder, particle size
