Supplementary material SM 1

Supplementary material SM 1 Characterization of the selected NMs. Media dependent physico-chemical parameters determined in deionized water (Table 1), ISO medium (fish embryo test; Table 2), ADaM (test with daphnids; Table 3), and OECD medium (test with algae, Table 4).

Abbreviations: BET

Brunauer-Emmett-Teller method

Reactivity - CPH

Measurement of (surface) reactivity using the spin probe 1-hydroxy-3-carboxy2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) mixed with the chelator desferroxamine (0.1 mM) according to Papageorgiou et al. (2007).

Reactivity - DMPO

Detection of hydroxyl radicals generated via Fenton-type reactions using DMPO (5,5dimethyl-1-pyrroline-N-oxide) (Shi et al., 2003)

DI

Deionized water

IEP

Isoelectrical point

PC

Physico-chemical parameters

PDI

Polydispersity index

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Table 1:

PC - parameters of the different nanomaterials in deionized water. (NM concentration 100 mg/L, for solubility experiments 1g/L, 10 g/L NM-300K; n ≥ 2)

Nanomaterial

Agglomerate size – z.average [nm] (±SD)

PDI

Zeta potential [mV]

pH 1

IEP

Solubility 24h [µg/L] 2


Reactivity CPH (sample to blank ratio)

Reactivity DMPO (sample to blank ratio)

Reactivity DMPO irradiation (sample to blank ratio)

3570 ± 378

1.1

-34.16

5.3

/

182 ± 0.39

101 ± 98

1.11 ± 0.39

1.22 ± 0.08

-3

1522

0.4

-0.62

6.6

/

15633 ± 777

13325 ± 5334

1.77 ± 0.38

1.11 ± 0.1

-3

Ag – NM300K

149

0.2

-15.7 ± 2.4

7.4

10

140 ± 26

11 ± 10.1

173 ± 38

18.1 ± 12.7

-

Ag – SRM 110525 Ag – 1340

Cu

/ = measured but not detected; - = not measured; 1 as no validated solubility measurement of NM in aquatic media was available, a filter with a pore size off 200 nm was used to differentiate between particles and ions, as it is expected that most NMs will show much larger hydrodynamic sizes after being dispersed in aquatic media, due to agglomeration. However, it cannot be excluded that a low amount of smaller particles can be found in the supernatant. Therefore the mentioned solubility values have to be interpreted as worst case values; 2 Photocatalytic activity of the chemical composition described in the literature, but not measured in the project due to the limited budget.

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Table 4:

PC - parameters of the different Nanomaterials in OECD medium used for the test with algae. (NM concentration 100 mg/L, for solubility experiments 1g/L, 10 g/L NM-300K; n ≥ 2)

Nanomaterial

Agglomerate size – z.average [nm] (±SD)

PDI

Zeta potential [mV]

pH

IEP



Reactivity CPH (sample to blank ratio)

Reactivity DMPO (sample to blank ratio)

Reactivity DMPO irradiation (sample to blank ratio)

Ag – SRM 110525

4531 ± 2355

0.9

-23.7 ± 2.6

7.2

/

49 ± 10

38 ± 12

1.14 ± 0.07

1.07 ± 0.09

-2

Ag – 1340

1354 ± 41

0.4

-2.8 ± 2.2

7.0

/

1020 ± 849

849 ± 263

1.46 ± 0.13

1.12 ±0.15

-2

Ag – NM300K

146 ± 95

0.2

-13.5 ± 6.5

7.6

/

413333 ± 118462

453333 ± 170392

6.53 ± 2.03

1.08 ± 0.08

-2

TiO2 undoped

2664 ± 1089

0.4

-22.8 ± 3.3

7.0

6-7

-

< 0.5

0.66 ± 0.15

0.71 ± 0.09

1.48 ± 0.11

TiO2 Eu doped (5%)

1612 ± 384

0.4

-23.1 ± 1.5

7.2

6-5

-

0.7

0.85 ± 0.30

0.7 ± 0.1

1.41 ± 0.09

TiO2 Fe doped (5%)

1866 ± 106

0.4

-21.3 ± 1.5

7.2

/

-

9.1

0.83 ± 0.11

1.0 ± 0.1

1.38 ± 0.07

ZnO – NM110

1975 ± 427

0.5

-18.9 ± 3.4

7.4

/

2050 ± 50

2675 ± 1520

0.72 ± 0.07

1.0 ± 0.2

-2

ZnO – NM113

1920 ± 610

0.5

-21.4 ± 1.3

7.4

8-7

1777 ± 225

2750 ± 686

0.87 ± 0.21

0.99 ± 0.11

-2

ZnO – NM111

445 ± 245

0.2

-25.2 ± 1.8

7.5

7.5-6.5

1433 ± 322

1667 ± 503

0.79 ± 0.20

1.01 ± 0.18

-2

CeO2 Eu doped (5%)

1251 ± 32

0.4

-21.0 ± 0.9

7.7

/

Not determined

Not determined

Not determined

Not determined

-2

CeO2 – NM211

442 ± 85

0.3

-19.8 ± 1.5

7.3

5-6

-

3.9

0.78 ± 0.17

0.81 ± 0.05

-2

CeO2 – NM212

831 ± 209

0.3

-20.4 ± 1.9

7.4

7-6

-

< 0.5

0.74 ±0.23

0.96 ± 0.15

-2

CeO2 – NM213

1042 ± 178

0.4

-25.9 ± 2.4

7.4

/

-

0.67

0.85 ± 0.07

1.07 ± 0.12

-2

Cu

1789 ± 964

0.6

-16.9 ± 2.5

7.4

7.4

363 ± 5.8

142 ± 93

307 ± 46

3.5 ± 1.8

-

/ = measured but not detected; - = not measured; 1 as no validated solubility measurement of NM in aquatic media was available, a filter with a pore size off 200 nm was used to differentiate between particles and ions, as it is expected that most NMs will show much larger hydrodynamic sizes after being dispersed in aquatic media, due to agglomeration. However, it cannot be excluded that a low amount of smaller particles can be found in the supernatant. Therefore the mentioned solubility values have to be interpreted as worst case values; 2 Photocatalytic activity of the chemical composition described in the literature, but not measured in the project due to the limited budget.

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Supplementary material SM 2 Methods applied to determine the physico-chemical properties of the NMs in deionized water and the test media. Dynamic light scattering (DLS) Colloidal particles dispersed in a liquid show an undirected movement due to Brownian motion. By using Dynamic Light Scattering (DLS) the effective hydrodynamic diameter of particles in a suspension can be measured based on fluctuation of light, which is scattered by a liquid dispersion after radiation with a laser. The signal fluctuation is detected via a time correlation function - the method of cumulants (Koppel, 1972) and an average particle size (z.average) and a polydispersity index (PDI) can be calculated. Alternatively, the correlation can be numerically analyzed in terms of the particles size distribution. In this project the CONTIN Algorithm was used. This method was developed for the characterization of stable more or less spherical homogeneous monomodal samples. Problems may occur if the samples are unstable, multimodal, show a high background of natural occurring particles or if non spherical particles were measured. Here the values have to be interpreted with care. Further information can be found elsewhere (Nickel et al., 2014). Electrophoretic light scattering (ELS) The zeta potential (ZP) is a characteristic parameter of the electric double layer, which is formed at any charged surface in a liquid. It is defined as the electric potential at the shear plane, which separates the mobile oppositely charged counter-ions (ion cloud) from solvent molecules and ions that adhere to the particle surface. By imposing a relative motion between bulk solvent and particle e.g. induced by an electric field, the ZP can be detected (Delgado et al., 2007). The velocity of the electrophoretic motion is proportional to the strength of the electric field and to the ZP. The particle size has only a second order impact. In this study the ZP was measured with a DELSA-NANO C (Beckmann Coulter). This instrument measures the phase shift (Doppler effect) of a light signal that is scattered at all moving particles. From the spread of the phase shift one can derive an intensity weighted distribution of the ZP. Electron Paramagnetic Resonance (EPR) Spectroscopy The detection of particle induced reactive oxygen species (ROS) and/or “surface reactivity” was done by spin trap/probe based electron paramagnetic resonance (EPR) spectroscopy technique (EPR Spectrometer Mini Scope 400, Fa Magnettech, Berlin). Two different complementary approaches were used a) sensitive to metal (Fenton-like) induced hydroxyl radical generation (OH∙) and b) kind of surface reactivity (redox-activity). Additionally to these two approaches the photo catalytic activity of TiO2 nanomaterials was studied as impact of UV irradiation by EPR spectroscopy. Reactivity measured with spin trap DMPO In the presence of hydrogen peroxide (H2O2) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) hydroxyl radicals (OH∙) generated via Fenton-type reactions are detected (Shi et al., 2003). Briefly, 50 µL of the particle suspension was mixed with 100 μL DMPO (0.05 M) and 50 µL of H2O2 (0.5 M), incubated in a dark, shaking water bath for 15 min at 37 °C before analyzed by EPR. Reactivity measured with spin probe CPH A possible (surface) reactivity of the material, caused by particle surfaces bound components and / or physicochemical particle properties, was established by measurements using the spin probe 1-hydroxy3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) mixed with the chelator desferroxamine (0.1 mM) according to Papageorgiou et al. (2007). The (surface) reactivity is expressed by splitting of the H+ of the CPH molecule or by generating an electron delocalization via binding. This effect is driven probably by directly active surfaces of the material. The preparation was done by mixing 50 μL of particle suspensions with 50 μL CPH (1 mM) and incubating for 10 min at 37 °C before analyzing by EPR.

Reactivity measured with spin trap DMPO after UV irradiation (photo catalytic activity) Hydroxyl radical generation after UV-irradiation was measured according to Lipovsky et al. (2009) and Lipovsky et al. (2012) in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). This method is especially sensitive for the detection of hydroxyl radicals (OH∙) after UV-irradiation. For the measurement 30 µL of the particle suspension (final conc. 5 mg/L) is mixed with 30 μL DMPO (final conc. 0.05 M) and analyzed by EPR after irradiation with UV-light (Exo Terra natural light 25 Watt E27) for 10 min. Scanning electron microscopy (SEM) Scanning electron microscopy (a JEOL 7500F with a nominal resolution of 2 nm was used) was used to determine the morphology and size of the primary particles as well as their agglomeration status in media and size for selected materials. Either single crystalline silicon substrates or nucleopore membrane filters were applied as substrates. The particles were applied onto the substrates by using a defined amount of a particle suspension which was dried prior to the SEM investigations. In case of the nucleopore filters a gold layer of approximately 10 nm had to be evaporated onto the filters to make them electrically conductive. The nucleopore filter was used if the particles/agglomerate size (distribution) of the NM in the media should be analyzed. For this, the suspension was filtered with a vacuum pump to minimize the contraction of the NM during the preparation. The particle and agglomerate morphology was investigated by SEM images obtained at different magnifications. Size analysis was conducted on a series of images obtained at the same (high) magnification by means of an image analysis tool (ImageJ v1.41). Size analysis of the primary particles (near spherical particles) was conducted by measuring the particle diameter for 500 particles. For the agglomerate size the diameter was measured in two nearly perpendicular axes roughly representing the particle area (i.e. for the maximum diameter a Feret diameter is used whereas the minimum diameter of the perpendicular axis does not represent the minimum Feret diameter). These measurements were done for 300 agglomerates per material. The obtained data for both primary particle size and agglomerate size were statistically analyzed with regard to the mean and mode value of the size distribution. Solubility experiments The NM was weighted in a vial with a target concentration of the suspension of 1 mg/mL and 10 mg/mL for the silver NMs with a volume of at least 40 mL. This resulted in a test sample of 40 mg (± 1 %) of the solid NM and 40 mL (± 1 %) of the medium. The mixture was shaken for a defined time period (24 h, 72 h) using an overhead shaker at 60 revolutions / minute. The room temperature was recorded (1 hourvalues), since the temperature also influences the solubility. After shaking, the sample was immediately centrifuged for 45 minutes at 5.000 G. After centrifugation the vials were carefully removed from the centrifuge and transported in an upright position until filtration, to minimize a mixing of the solid and aqueous fraction. For the following filtration the supernatant (3 x 10 mL) of each sample was taken with a 10 mL pipette and filled into a disposable syringe (B. Braun Inject 10 mL), equipped with a nylon syringe filter (pore size of 0.22 µm). The filtered supernatant was filled in a labelled vial for quantification of the soluble fraction. The total concentration was detected by ICP-MS in the supernatant without filtering.

References Delgado, A. V., Gonzalez-Caballero, F., Hunter, R. J., Koopal, L. K., & Lyklema, J. (2007). Measurement and interpretation of electrokinetic phenomena. Journal of Colloid and Interface Science, 309, 194-224.

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Koppel, D. E. (1972). Analysis of Macromolecular Polydispersity in Intensity Correlation Spectroscopy: The Method of Cumulants. The Journal of Chemical Physics, 11, 4815- 4820. Lipovsky, A., Levitski, L., Tzitrinovich, Z., Gedanken, A., & Lubart, R. (2012). The different behavior of rutile and anatase nanoparticles in forming oxy radicals upon illumination with visible light: An epr study. Photochemistry and photobiology, 88, 14-20. Lipovsky, A., Tzitrinovich, Z., Friedmann, H., Applerot, G., Gedanken, A., & Lubart, R. (2009). Epr study of visible light-induced ros generation by nanoparticles of zno. . The Journal of Physical Chemistry C, 113, 15997-16001. Nickel, C., Angelstorf, J., Bienert, R., Burkart, C., Gabsch, S., Giebner, S., et al. (2014). Dynamic lightscattering measurement comparability of nanomaterial suspensions. Journal of Nanoparticle Research, 16(2), 2260. Papageorgiou, I., Brown, C., Schins, R., Singh, S., Newson, R., Davis, S., et al. (2007). The effect of nanoand micron-sized particles of cobalt–chromium alloy on human fibroblasts in vitro. Biomaterials, 28(19), 2946-2958. Shi, T., Schins, R. P. F., Knaapen, A. M., Kuhlbusch, T., Pitz, M., Heinrich, J., et al. (2003). Hydroxyl radical generation by electron paramagnetic resonance as a new method to monitor ambient particulate matter composition. J Environ Monit, 5, 550-556.

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