Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus
Introduction
Man-made nanoparticles (NPs) range from the well-established multi-ton production of carbon black and fumed silica for applications in plastic fillers and car tires to microgram quantities of fluorescent quantum dots used as markers in biological imaging (Hoet et al., 2004). At nanosize range, the properties of materials may differ substantially from respective bulk materials. As by today there is increasing scientific evidence that these physical and chemical properties of manufactured NPs lead to an increase of bioavailability and toxicity (Nel et al., 2006). NPs can cross most strong biological barriers such as blood–brain barrier (Lockman et al., 2003). Barnard (2006) summarises the problem of potential hazard of NPs:”...Nanohazards are different because nanomaterials do not behave in a predictable way. They are the Jekyll and Hyde of materials science, giving us unique chemical, electrical, optical and physical properties; as well as a new range of possible carcinogens, poisons and allergens....”. Nevertheless, nanosized materials were until recently treated as variations of the technical material or existing formulation and thus not requiring a separate registration (Oberdörster et al., 2005).
Conventional TiO2 has been studied and/or applied due to its photocatalytic biocidal/antiproliferative properties (Blake et al., 1999). ZnO and TiO2 have been used as antibacterials in dentistry (Moorer and Genet, 1982, Matsunaga et al., 1985) and CuO in wood preservation and antimicrobial textiles (Cox, 1991, Gabbay et al., 2006). NPs of TiO2 and ZnO are growingly used in cosmetics and sunscreens (Serpone et al., 2007), but also in solar-driven self-cleaning coatings (Cai et al., 2006) and textiles (Yuranova et al., 2007). Currently TiO2 and ZnO NPs are already produced in industrial amounts (The Royal Society, 2004).
The main mechanism of toxicity of NPs is thought to be via oxidative stress (OS) (Kohen and Nyska, 2002) that damages lipids, carbohydrates, proteins and DNA (Kelly et al., 1998). Lipid peroxidation is considered most dangerous as leading to alterations in cell membrane properties which in turn disrupt vital cellular functions (Rikans and Hornbrook, 1997). OS-mediated toxicity has been shown for TiO2 in in vitro studies, including brain cells (Long et al., 2006). Near-UV-light potentiates the toxicity and genotoxicity of TiO2 (Maness et al., 1999, Ashikaga et al., 2000). Xia et al. (2006) have shown that the toxicity of NPs might be predicted from their ROS generation capability in vitro. For bacteria the toxicity of NPs has been studied for fullerenes (Lyon et al., 2006), for ZnO and TiO2 (Adams et al., 2006, Zhang et al., 2007). For nano ZnO, participation of OS mechanisms leading to membrane damage and antibacterial properties has been demonstrated for Escherichia coli (Zhang et al., 2007), whereas external generation of H2O2 from bulk ZnO suspension has been considered as one of the primary factors of its antibacterial activity (Sawai et al., 1998). Also chemical toxicity of NPs based on the release of (toxic) ions should be taken into account as solubility of NPs strongly influenced the cytotoxicity (Brunner et al., 2006). Liberation of cytotoxic amounts of Cd2+ was shown for CdSe quantum dots (Derfus et al., 2004). Similarly, the solubilized metal ions from metal containing NPs may be the reason for their antibacterial effects as bacteria are largely protected against NP entry (no transport mechanisms for supramolecular and colloidal particles). For example, only <5 nm quantum dots entered the bacterial cells, probably by light-aided oxidative damage of the cell membrane (Kloepfer et al., 2005). Also, the antibacterial activity of ZnO-containing dental materials has been attributed to mobilized Zn2+ (Moorer and Genet, 1982). Some bacteria may solubilize ZnO in nature (Fasim et al., 2002). Using recombinant sensor bacteria we have also shown that bacteria can mobilize heavy metals from soils (Ivask et al., 2002).
Although there are already some studies on potential hazard of manufactured NPs, their release into the aquatic environment and their harmful effects remain largely unknown (Moore, 2006). Most of the attention has been focused on fullerens (C60). Oberdörster (2004) showed that 0.05 mg l−1 C60 induced lipid peroxidation in the brain of the fish. Relatively strong antibacterial activity of fullerenes has been found starting from 0.4 to 4 mg l−1 level (Fortner et al., 2005) whereas smaller aggregates posed higher antibacterial activity (Lyon et al., 2006). C60-fullerenes with tetrahydrofuran as solubilizing vehicle were also toxic to daphnids (48 h LC50 = 7.9 mg l−1; Lovern and Klaper, 2006). Thus, toxicity testing of NPs should be performed in an environmentally relevant mode (i.e. for solubilization) to avoid misleading information on toxicity of NPs (Oberdörster et al., 2006). The first report on the impact of manufactured NPs on soil microflora showed that C60-fullerenes at μg kg−1–mg kg−1 level, depending on the treatment, had little impact on the structure and function of the soil microbial community and processes (Tong et al., 2007).
The aim of this study was to evaluate toxicity of NPs of ZnO, CuO and TiO2 for Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus with a special emphasis on metal oxide formulations (nano or bulk) and hydrolysis of metal oxides. Crustaceans and bacteria as representatives of different food-web levels are good models for prediction of toxicity of chemicals/pollutants to ecosystems. In addition, for D. magna and V. fischeri there is a large database for toxicity of chemicals, and these organisms have been used among other invertebrate test organisms in our lab for the toxicity investigation of solvents, pesticides, phenols, heavy metals, xylidines, etc. (Rozkov et al., 1999, Kahru et al., 2005, Kahru and Põllumaa, 2006, Kahru, 2006). To our knowledge, this is the first evaluation of toxicity of nano and bulk ZnO, CuO and TiO2 to V. fischeri and T. platyurus and also a first study on toxic effects of ZnO and CuO NPs to D. magna.
Section snippets
Chemicals
All nanosized metal oxides were purchased from Sigma–Aldrich with particles sizes of 25–70 nm for nano TiO2, 50–70 nm for nano ZnO and mean ∼30 nm for nano CuO. The bulk form of TiO2 was purchased from Riedel-de Haën, ZnO from Fluka and CuO from Alfa Aesar. The stock suspensions in Milli-Q (40 g l−1) were sonicated for 30 min and stored in the dark at +4 °C. Stock solutions of ZnSO4 · 7H2O and CuSO4 (from Sigma–Aldrich and Alfa Aesar, respectively) were prepared analogously but were not sonicated. pH of
Toxicity of metal oxides to Vibrio fischeri
The toxicity ranking of metal oxides (both nano and bulk) to V. fischeri was as follows: TiO2 < CuO < ZnO (Table 1, Fig. 1). TiO2 suspension was not acutely toxic even at 20 g l −1 level. Also, bacterial growth was not impaired by further incubation in 20 g l−1 TiO2 (8 h in the dark) (Table 1). However, Maness et al. (1999) have shown that 100–1000 mg TiO2 l−1 (bulk formulation) coupled with near-UV illumination reduced survival of E. coli by 70%.
Differently from TiO2, all tested Zn compounds were very
Conclusions
Our innovative approach based on the combination of traditional ecotoxicology methods and metal-specific recombinant biosensors allowed to clearly differentiate the toxic effects of metal oxide NPs per se and solubilized metal ions. In addition, this is the first evaluation of ZnO, CuO and TiO2 toxicity to V. fischeri and T. platyurus. For nano ZnO and nano CuO this is also a first study for D. magna. For all the tested compounds T. platyurus was more sensitive than D. magna although D. magna
Acknowledgements
This work was supported by Estonian Science Foundation Project No. 6956, Estonian targeted funding project 0222601Bs03 and by NICPB basic funding. A. Ivask was also supported by the World Federation of Scientists, CERN, Switzerland.
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