Article pubs.acs.org/est
Systematic and Quantitative Investigation of the Mechanism of Carbon Nanotubes’ Toxicity toward Algae Zhifeng Long,† Jing Ji,† Kun Yang,†,‡ Daohui Lin,†,‡,* and Fengchang Wu§,* †
Department of Environmental Science, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Zhejiang University, Hangzhou 310058, China § State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China ‡
S Supporting Information *
ABSTRACT: Concurrent with the increasing production and application of carbon nanotubes (CNTs) comes an increasing likelihood of CNTs presenting in the aquatic environment, and thereby potentially threatening aquatic organisms via toxic mechanisms that are, at present, poorly understood. This study systematically investigated the toxicity of three multiwalled CNT (MWCNT) samples toward a green alga (Chlorella sp.), focusing on examining and quantifying the contributions of five possible mechanisms to the algal growth inhibition. The results showed that the MWCNTs significantly inhibited the algal growth. The contribution of metal catalyst residues in the MWCNTs to the algal growth inhibition was negligible, as was the contribution from the MWCNTs’ adsorption of nutrient elements. The algal toxicity of MWCNTs could mainly be explained by the combined effects of oxidative stress, agglomeration and physical interactions, and shading effects, with the quantitative contributions from these mechanisms depending on the MWCNT size and concentration. At MWCNT concentrations around 96 h IC50, the oxidative stress accounted for approximately 50% of the algal growth inhibition, whereas the agglomeration and physical interactions, and the shading effects each took approximately 25% of the responsibility.
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INTRODUCTION As a versatile nanomaterial, carbon nanotubes (CNTs) are increasingly produced and used in a variety of industrial areas;1,2 they are therefore likely to find their way into the aquatic environment during their life-cycle.3 More and more evidence has been provided, showing that CNTs are associated with hazardous effects on cells, tissues, and organisms.4−8 However, because of inconsistent experimental conditions including CNT properties, test medium chemistry, and organism speciesresults from CNT toxicity studies are often contradictory. According to previously published CNT toxicity studies,4−8 five principal toxic mechanisms can be identified, as follows: 1. Metal Catalyst Residues. CNTs contain residual metal impurities, even after prolonged periods of purification using concentrated nitric acid.9 Leachates of low-purity as-produced fullerenes and metallofullerene waste solids were reported to induce acute toxicity in Pimephales promelas and Ceriodaphnia dubia; the toxicity could be eliminated via the addition of EDTA to the leachates, implying that divalent transition metals were the source of the toxicity.10 However, Shvedova et al. observed the toxicity of single-walled CNTs (SWCNTs) with © 2012 American Chemical Society
low iron content (0.23%) toward mice, and thereby ruled out the residual iron as a main mechanism driving the toxicity.11 2. The Adsorption of Nutrient Elements. CNTs are superb adsorbents for both organic12 and inorganic13 compounds, and therefore can likely sequester nutrient compounds from the culture medium, and thus exhibit apparent toxicity. It was reported that the clustering of CeO2 nanoparticle aggregates around algal cells might cause toxicity through a local direct effect, or through local nutrient depletion.14 However, to date, very few studies have specifically examined the possible contribution of nutrient sequestration to the apparent CNT toxicity. 3. Oxidative Stress. The connection between nanotoxicity and excessive oxidative stress is widely accepted.15 There is evidence suggesting that CNTs can provoke oxidative stress by producing reactive oxygen species (ROS)16−22 that can threaten organisms through a variety of interrelated effects, Received: Revised: Accepted: Published: 8458
May 6, 2012 June 29, 2012 July 3, 2012 July 3, 2012 dx.doi.org/10.1021/es301802g | Environ. Sci. Technol. 2012, 46, 8458−8466
Environmental Science & Technology
Article
including lipid peroxidation and DNA damage.15 Therefore, the toxicity of CNTs likely involves the formation of ROS. However, there have also been studies indicating that oxidative stress was not a major factor in the CNT toxicity.23−25 4. Agglomeration and Physical Interactions. CNTs may agglomerate with microbe cells through hydrophobic interactions, and/or hydrogen bonds formed between the cell surfaces and the oxygen defects of CNTs.25 CNTs closely attached to the cell surfaces would partition/penetrate and disrupt the cell wall/membrane, interact with the biomolecules, and physically and/or chemically inhibit physiological cell behavior, and thus present toxicity.15,24 For example, it was shown that the algal toxicity of CNTs was strongly correlated with CNT agglomeration with algal cells.25 Individually dispersed SWCNTs can also act like moving “nano-darts” in solution, constantly attacking and killing bacteria through physical puncturing.23 The contribution of agglomeration and physical interactions with organisms to the CNT toxicity merits more specific investigation. 5. Shading Effects. As carbonaceous nanomaterials are opaque, it is often speculated that shading could explain the reduced viability of phototrophic organisms exposed to these nano-objects.26−28 Previous studies have reported that nanoparticles can adhere to algal surfaces, and hence restrict light accessibility to the cells, resulting in the inhibition of growth.29−31 Schwab et al. ascribed 85% of the total toxicity of CNTs toward a green alga to the shading effects of the CNTs, and the agglomeration of the CNTs with the algal cells.25 However, a different study indicated that the effect of the external light-blocking properties of the CNTs on algal growth was insignificant.31 To date, there has been no research systematically and quantitatively examining the toxicity of CNTs from all of these five aspects; previous studies have tended to explain the phenomena as the result of only one or two mechanisms. This study therefore aimed to systematically investigate the mechanisms of the toxicity of CNTs toward a green alga. The alga was used as a model organism because alga plays an important role in the aquatic ecosystem, not only in producing biomass that forms the basic nourishment for food webs, but also in contributing to the self-purification of polluted water.32 Moreover, very limited information is available on the algal toxicity of unmodified CNTs.25 The specific objectives of this study were to (1) determine the inhibitory effects of CNTs on algal growth, (2) prove or disprove the five potential toxic mechanisms, and (3) quantify the contributions of the main mechanisms to the inhibition of algal growth. To the best of our knowledge, this is the first study to systematically and quantitatively examine the underlying mechanisms of CNT toxicity toward aquatic organisms. We believe that the findings will be helpful in ecological risk evaluations of CNTs.
representing bulk carbon materials. A portion of the pristine MWCNTs were also thoroughly washed in a 1 mol L−1 HCl solution (HCl-washed MWCNTs), or in algal cell culture medium (CM-washed MWCNTs), to allow a comparison with the pristine MWCNTs. The methods used for the AC purification and MWCNT washing are detailed in the Supporting Information (SI). The leachates from 100 mg L−1 pristine MWCNTs were collected from the culture medium after the samples had been shaken (110 rpm) for 5 days and filtered though 0.22 μm PTFE membranes; these leachates were used to assess the potential release of MWCNT-contained catalyst residues, and their effects on the algal growth. The morphology of the MWCNTs was examined using transmission electron microscopy (TEM, JEM-1230, JEOL, Japan) and scanning electron microscopy (SEM, SIRION-100, FEI Corp., Hillsboro, OR) operated at 120 kV and 25 kV, respectively. The surface areas and pore volumes were determined using the multipoint Brunauer−Emmett−Teller (BET) method.33 The ash contents were assayed using a thermogravimetric and differential thermal analyzer (SDTQ600, TA, New Castle, DE). The surface elemental contents were analyzed using an X-ray energy dispersion spectroscope (EDS, GENESIS 4000, EDAX Inc., Mahwah, NJ). The metal concentrations in the leachates were determined using an elemental inductively coupled plasma mass spectrometer (ICP-MS, G3271A, Agilent, Japan). The electrophoretic mobilities and hydrodynamic sizes were measured with a ZetaSizer (Nano ZS90, Malvern Instrument, UK). Before the ZetaSizer measurements, the MWCNTs and AC (100 mg L−1) were dispersed in the culture medium at pH 8.0, using sonication (100 W, 40 kHz, 25 °C) for 15 min. Algal Growth Assays. The unicellular green algae (Chlorella sp.) used were obtained from the Institute of Wuhan Hydrobiology, Chinese Academy of Sciences. The Organisation for Economic Co-operation and Development (OECD) recommended algal cell culture medium (the composition is detailed in the SI) was used for the algal growth assay, which was conducted following OECD guideline 201 “Alga, Growth Inhibition Test”. The algal cells were cultured in 100 mL of the OECD medium in the presence or absence of the test materials, in 250 mL Erlenmeyer flasks; the flasks were kept on an incubation shaker at 25 ± 0.5 °C, with or without illumination by white incandescent lights (100 ± 5 μE m−2 s−1, light:dark of 14:10 h). Before the inoculation of the algal cells, the media with the carbon materials (0, 5, 10, 20, 50, and 100 mg L−1) were autoclaved at 0.1 MPa for 20 min, and bath-sonicated (100 W, 40 kHz, 25 °C) for 15 min. The initial cell densities were approximately 1 × 105 and 1 × 107 cells mL−1 in the assays with and without illumination, respectively. The flasks were wrapped in opaque plastic to keep the algae in dark in the experiments without illumination. All of the flasks in the experiments were shaken by hand three times per day, except for one group, where a series of shaking speeds of 0 (shaking by hand, three times per day), 25, 110, and 180 rpm were used to examine the potential effects of physical collision on the algal toxicity of CNTs (using MWCNT100 as a representative sample). Algal cells were counted daily, using a counting chamber under a light microscope (LM, Olympus, CX21, Japan). Algal growth inhibition was calculated using the average specific growth rate during exposure for 96 h, according to OECD guideline 201. IC50i.e., the concentration of test materials leading to a 50% reduction in algal growth compared with the
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MATERIALS AND METHODS Carbon Materials and Characterization. Three multiwalled CNT (MWCNT) samples with nominal MWCNT outer diameters of
