## 4 Discussion  
The physicochemical characterization of NPIs is crucial to: i) understand the correlation between the NPIs biological effects and their characteristics and ii) study the possible characteristics alterations and behaviour of NPIs on the environmental media. The assessment of the physicochemical behaviour of NPIs in environmental matrices, such as soil, is currently a challenge due to a lack of adequate protocols, expensive techniques and the difficulty of developing suitable methods for better characterization [21; 30]. In the present study, the characterization of NPIs in the stock dispersion and diluted in ISO water showed that particles had a negative surface charge, the expected size and a low PDI, indicating that the NPIs dispersions were highly monodisperse. This characterization showed the stability of the NPIs in ISO water. However, some aggregates/agglomerates were seen in the exposure with organisms as depicted in the obtained photos of _E. cryptic_ exposed to NPIs via ISO water (supplementary materials). This can be related to the exudates produced by the organisms during the exposure that can interact with the NPIs. With the fragmentation, plastics acquire new physicochemical characteristics that enhance their potential interaction with organisms causing direct and/or indirect toxicity [4]. Due to the size of MPIs/NPIs, soil fauna may consume them when feeding [6]. Decreased reproduction and growth, and several other harmful effects have been previously detected in soil organisms exposed to MPIs and NPIs [5; 6; 15; 16]. In our study, only the two highest tested concentrations of NPIs dispersion had significant effects on the organisms survival and/or reproduction. However, these effects may be mainly associated with the toxicity of tween 20 and NaN\({}_{3}\) present in the NPIs dispersion. As tested in the present study, in general, ecotoxicological studies test commercially available formulations of polystyrene MPIs and NPIs, containing various preservatives, antimicrobials or surfactants, such as NaN\({}_{3}\), tween 20 and sodium dodecyl sulfate [31]. As previously reported, the presence of these compounds may introduce artefacts in the toxicity evaluations [31], corroborating the data found in our research. The results from an acute toxicity test on _Daphnia magna_, using commercial polystyrene NPIs (20 and 200 nm) dispersion containing 2 mM NaN\({}_{3}\), showed that the acute toxicity of the commercial formulation of polystyrene NPIs was mainly associated with NaN\({}_{3}\) and not with NPIs themselves. In our study, this is also shown by the exposure via ISO water where the dispersion of 1500 mg NPIs/L induced 100% of organisms mortality due to the presence of tween 20 and NaN\({}_{3}\). These results show the importance of removing the preservatives, antimicrobials or surfactants present in the NPIs dispersions to know only the effects induced by NPIs. An alternative could be via centrifugation of the NPIs dispersions, without compromising particle characteristics, to remove these compounds, and can be performed in further ecotoxicity tests to assess the specific effects of NPIs. On the other hand, our study highlighted the importance of considering the toxicity of other compounds present in commercial plastics that can induce harmful effects on the organisms and the interaction of NPIs with them inducing dissimilar effects than those expected. An interesting result was found in this work, it seems that NPIs alleviated the effects of tween 20 and NaN\({}_{3}\). Dispersion of 900 mg/kg of NPIs containing 174.8 mg/kg of tween 20 and 22.2 mg/kg of NaN\({}_{3}\) had no effect in terms of organism reproduction. However, exposures to the dispersant solution--tween 20 + NaN\({}_{3}\)--at the same concentrations (174.8 + 22.2 mg/kg) decreased the reproduction of _E. cryptic_.