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Raphidocelis subcapitata is a freshwater algae species that constitutes the basis of many aquatic trophic chains. In this study, R. subcapitata was used as a model species to investigate the kinetics of uptake and elimination of silver nanoparticles (AgNP) in comparison to silver nitrate (AgNO3) with particular focus on the Ag sized-fractions in solution. AgNP used in this study were provided in a suspension of 1 mg Ag/l, with an initial size of 3-8 nm and coated with an alkane material. Algae was exposed for 48 h to both AgNP and AgNO3 and sampled at different time points to determine their internal Ag concentration over time. Samples were collected and separated into different sized fractions: total (Agtot), water column Ag (Agwater), small particulate Ag (Agsmall.part.) and dissolved Ag (Agdis). At AgNO3 exposures algae reached higher bioconcentration factor (BCF) and lower elimination rate constants than at AgNP exposures, meaning that Ag is more readily taken up by algae in its dissolved form than in its small particulate form, however slowly eliminated. When modelling the kinetics based on the Agdis fraction, a higher BCF was found. This supports our hypothesis that Ag would be internalised by algae only in its dissolved form. In addition, algae images obtained by Coherent Anti-stokes Raman Scattering (CARS) microscopy demonstrated large aggregates of nanoparticles external to the algae cells with no evidence of its internalisation, thus providing a strong suggestion that these AgNP were not able to penetrate the cells and Ag accumulation happens through the uptake of Ag ions.

Nanomaterials (NMs) are used in an extremely diverse range of products and are increasingly entering the environment, driving a need to better understand their potential health effects in both humans and wildlife. A major challenge in nanoparticle (eco)toxicology is the ability to localise NMs post exposure, to enable more targeted biological effects analyses. A range of imaging techniques have been applied to do so, but they are limited, requiring either extensive processing of the material, staining or use of high intensity illumination that can lead to photo damage and/or have limited tissue penetration. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique, providing contrast based on the intrinsic molecular vibrations of a specimen, circumventing the need for chemical perturbation by exogenous labels. CARS uses near infra-red excitation wavelengths which allow microscopy at depths of several hundred microns in intact tissues and minimises photo-damage to live and delicate samples. Here we provide an overview of the CARS process and present a series of illustrative examples demonstrating its application for detecting NMs within biological tissues, ranging from isolated cells to whole organisms and including materials spanning metals to polymers. We highlight the advantages of this technique which include chemically selective live imaging and substantial depth penetration, but we also discuss its limitations when applied to nanotoxicology, which most notably include the lack of resolution for studies on single nanoparticles.

Manufactured metal (oxide) nanoparticles are entering the aquatic environment with little understanding on their potential health impacts for exposed organisms. Adopting an integrative approach, we investigated effects of particle size and coating on biological responses for two of the most commonly used metal (oxide) nanoscale particles, silver (Ag) and titanium dioxide (TiO₂) in zebrafish embryos. Titanium dioxide nanoparticles (nominally, 4 nm, 10 nm, 30 nm and 134 nm) had little or no toxicity on the endpoints measured. Ag both in nano form (10 nm and 35 nm) and its larger counterpart (600-1600 nm) induced dose-dependent lethality and morphological defects, occurring predominantly during gastrula stage. Of the silver material tested 10 nm nanoparticles appeared to be the most toxic. Coating Ag nanoparticles with citrate or fulvic acid decreased toxicity significantly. In situ hybridisation analysis identified the yolk syncytial layer (YSL) as a target tissue for Ag-nano toxicity where there was a significant induction of the heavy metal stress response gene, metallothionein 2 (Mt2) at sub-lethal exposures. Coherent Anti-stroke Raman Scattering (CARS) microscopy provided no evidence for silver particles crossing the chorionic membrane in exposed embryos. Collectively, our data suggest that silver ions play a major role in the toxicity of Ag nanoparticles.