Carbon film

To estimate how both AgNPs behave during the chronic test, particles were characterized by measuring both particle size, as hydrodynamic diameter, and zeta potential in the test medium using a Zetasizer Nano ZS (Malvern Instruments Ltd.) with 173° backscattering angle at 25 °C with an equilibration time of 60 s and three repeats per sample. Samples were prepared to represent the conditions in the chronic test as closely as possible using the same volume of Elendt M7 medium, temperature and light intensity as in the chronic test. However, no food could be added to the samples, as the algae would have disturbed the particle measurements.
In addition, the used silver concentration of 1 mg Ag L -1 was higher than what was used in all further experiments, but being at the lower limit of detection for particle characterization with the used device. Samples for size and surface potential measurements were taken at 0, 1, 2, 4, 6, 24 and 48 h in duplicates.
To visually compare the shape and the behavior of the two AgNP, Transmission Electron Microscopy (TEM) images of the stock dispersions were made using a Tecnai T20 G 2 (FEI). The samples were prepared by drying a 4 µL drop of each AgNP dispersion on a copper grid covered with carbon film (Agar Scientific).

CM12 electron microscope operated at 80 kV. A drop of the solution at 3-5%w/w was deposited on a copper grid covered with a carbon film (Agar Scientific). After blotting off the excess liquid, the material was stained with a 2% uranyle acetate solution or a 1.5% ammonium molybdate solution depending on the buffer.
X-ray scattering and x-ray diffraction. SAXS-WAXS experiments were performed at the European Synchrotron Facility (ESRF), on the ID02 beamline. The wavelength of the incident beam was 0.0995 nm with exposure times of the order of 0.1-0.5 s. The scattered x-ray were collected on two Rayonix 2D-detectors, covering respectively the [0,075 -6,408 nm -1 ] and [5,307-43,323 nm -1 ] q-ranges. The samples were inserted in 1.5 mm diameter quartz capillaries sealed with paraffin wax. The spectra were manipulated and analyzed using the SaxsUtilities software. The spectra were treated with SaxsUtilities (www.sztucki.de/SAXSutilities/) and fitted with SasView (http://sasview.org/).

To characterize the SNC, transmission electron microscopy (TEM), elemental analysis, and dynamic light scattering (DLS) were performed. TEM was carried out with a probe-corrected microscope (ARM200F cold-FEG, JEOL) operated at an acceleration voltage of 80 kV. The samples were prepared for TEM by placing a few drops of the SNC dispersion onto 200 mesh copper TEM grids that were coated with a carbon film (Agar Scientific). Elemental analysis (C, H, N, S) data were collected on a calibrated Thermo Flash 2000 elemental analyzer. The content of carbon, hydrogel, nitrogen, and sulfur was reported as a mass percentage of the sample. DLS was measured on a Brookhaven NanoBrook Omni instrument with the detection range limited from 5 nm to 800 nm to avoid measurement of large (> 800 nm) dust particles.

Transmission Electron Microscopy (TEM) was used to do the imaging of the Ag NPs released from the toothbrushes. Because tap water creates CaCO 3 particles when drying, making it more difficult to find Ag particles, the samples for TEM analysis were prepared in Milli-Q water. Specifically, the toothbrushes were immersed in Milli-Q water for 24 h, and thereafter approximately 3.5 µL of the sample was transferred to a copper grid covered with carbon film (Agar Scientific) and dried. TEM (Tecnai G2, FEI) was operated at 200 keV accelerating voltage and the presence of Ag particles was confirmed by energy dispersive X-ray spectroscopy (EDS) detector (Oxford Instruments).