Ecotoxicity of nanomaterials in relation to the freshwater microalga Raphidocelis subcapitata
Curry, Jonathan Michael
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Silver is the most frequently represented nanomaterial in available products, ranging across a variety of commercial and medical goods. Their main justification for inclusion in these products relates to their unique properties compared to both their bulk (larger particulate) and ionic equivalents. It is established that silver is highly toxic to a range of aquatic organisms, and despite its existence in low concentrations in the environment, concerns have been raised over products containing silver nanomaterials as a potential emerging pollutant. Within the EU the environmental risk assessment of chemicals is regulated by the Registration, Evaluation and Authorization of Chemicals (REACH) regulation, and toxicity testing deployed according to a series of tiered production/import thresholds. At the lowest regulatory production/import threshold of one tonne, the Organisation for Economic Co-operation and Development (OECD) 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test can be deployed to evaluate the toxicity to the aquatic environment via assessment of the impact of chemicals on algal species, over a 72 hour test duration. Due to the unique physico-chemico properties of nanomaterials, there is considerable uncertainty over the suitability of such test protocols for these materials. Thus, there may be a requirement for alternative test systems. In addition, the proliferation of the nanotechnology industry has created a diverse range of materials whose safety needs to be assessed. There is therefore an immediate need for rapid environmental risk assessment in order to keep pace with commercial activities. This thesis addresses such considerations in four stages. First, the OECD 201 test was applied to evaluate the toxicity of a representative silver nanoparticle (20-150 μg/l Ag as NM300K, at 24-72 hours) and AgNO3 (0.2-30 μg/l Ag, at 24-72 hours), following dispersion in two different growth media, and the influence of different medium conditions such as humic acid (through the use of environmentally relevant concentrations of Suwannee River humic acid, [5 and 50 mg/l) and pH (6 and 8) on silver nanoparticle toxicity was investigated. In addition, a miniaturised version of the OECD 201 test was developed and compared to the standard test method, as a means to increase the rate and efficiency at which laboratories can generate toxicity data. Finally an alternative, short term sublethal photosynthetic endpoint was investigated in order to identify an alternative measure of toxicity to algae and inform the toxic mechanism of action of silver to algae, and correlations with the standard growth inhibition endpoint presented. It was found that the OECD 201 test was suitable to evaluate toxicity in NM300K and enabled identification of a 72 hour growth inhibition effective concentration (EC50) of 54-130 μg/l Ag. Of interest was that silver nanoparticle toxicity decreased with exposure duration and were affected by test medium composition. AgNO3 toxicity was higher than NM300K (72 hour EC50 = 5-7 μg/l Ag)., but subject to the same time and media effects Humic acid decreased the toxicity of both forms of silver in a concentration dependent manner, and pH 8 test medium increased toxicity relative to pH 6. The proposed miniaturised test was found to be as sensitive as the standard method (for NM300K) across a similar range of test conditions. Finally, the short-term photosynthetic sublethal endpoint indicated that silver may act in a similar way to known phototoxic substances, and by measuring this endpoint a high correlation was found with later 72 hour growth inhibition. Obtained data therefore demonstrate that the OECD 201 test can be used with R. subcapitata as a tool to assess nanoparticle toxicity, and that there are opportunities to increase the efficiency of testing via miniaturisation of the test system and the use of additional toxicity endpoints.