**4. Conclusions**

It is encouraged to develop standardized experimental procedures into OECD test guidelines to evaluate the dissolution rate of nanomaterials in natural aquatic environments [32]. Flow-through/continuous flow dissolution experiments are well suited to study dissolution kinetics of NPs and to systematically investigate NPs dissolution rates under environmentally relevant conditions. The main advantages are (1) a continuous supply of exposure media, (2) the possibility to expose small amounts of NPs (low solid/liquid ratio), and (3) the instantaneous separation between the dissolved and the solid fractions. For ENMs of very high or very low solubility, the flow-through testing can be easily adjusted according to the fast depletion of the material in the one, and too small outlet concentrations in the other case (i.e., NPs loading, flow rate). In contrast to recent studies investigating dissolution of ENMs in physiological fluids using a continuous flow system [40,41,54], our experimental approach uses the injection of ENMs dispersions which allows to load significantly lower amount of ENMs, relevant to mimic realistic natural environmental settings [5,58]. Our results demonstrated the feasibility to run flow-through dissolution tests for ENMs loadings down to the microgram range using environmentally relevant exposure media. The influence of the flow rate on the thickness of the diffusional boundary layer, the dissolution rate [54], and the local saturation at the vicinity of the nanoparticles should be further investigated. In particular, local saturation at the vicinity of the nanoparticles may occur in natural settings, for example, in water-saturated soils and sediments where pore waters may have longer residence time. In such specific aquatic compartments, it is relevant to determine various dissolution rates from long term experiments with various flow rates. In addition, establishing a clear recommendation of media compositions that would represent a wide range of natural waters, covering the dissolution-relevant species, would bring the tests closer to environmental realism. Here, it must be differentiated between standard testing for the comparison of materials regarding only their dissolution behavior, and tests, which aim to observe realistic environmental behavior including possible ENMs transformations. In the first case, a controlled pH and a simple inert background electrolyte would be suitable, whereas in the latter case, a more complex water chemistry including phosphate, sulfate, chloride, sulfide, and NOM (and others) would be chosen. In summary,

there is a growing need for standardizing and implementing continuous flow dissolution testing in test guidelines to address a more realistic scenario of ENMs in natural aquatic systems. This would improve reproducibility and comparability of results for the wide diversity of pristine and transformed ENMs and allow their registration with international regulatory bodies.

**Author Contributions:** Conceptualization, F.v.d.K.; investigation, L.S., A.M. and N.T.; writing— original draft preparation, L.S.; writing—review and editing, A.M., N.T., F.v.d.K., L.S. and T.H.; funding acquisition, F.v.d.K. and T.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was part of the NanoFASE and Gov4Nano projects funded by the European Union's Horizon 2020 research and innovation programme under the gran<sup>t</sup> agreements 646002 and 814401.

**Acknowledgments:** The authors thank Wolfgang Obermaier for his support on ICP-MS measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.
