## 4 Discussion
The mechanisms behind the altered toxicity of tween 20 and NaN\({}_{3}\) by the presence of NPIs, found in the present study through the exposure via soil, should be further investigated but may be related with incorporation rates, sorption ability, cellular defence mechanisms and different modes of action. NPIs are capable of adsorbing to biomolecules (such as proteins, lipids and metabolites) present in the organisms [32; 33]. This adsorption may alter the dynamics of the surface ionic charges in the environmental context [34; 35] and, ultimately, resulting in altered toxicity of the complex containing NPIs and adsorbed elements [36]. The possible absorption of tween 20 and NaN\({}_{3}\) to NPIs may result in less bioavailability of these compounds to the organisms and consequently less interaction, resulting in less pronounced effects on organisms reproduction when compared with the effects induced by tween 20 and NaN\({}_{3}\) single exposures. Organisms tended to decrease the avoidance responses with the NPIs concentration increase. Indeed, the organisms' avoidance responses were only significant at the lowest tested concentration (0.015 mg/kg). The lower aggregation/agglomeration processes of NPIs at low concentrations when compared with high concentrations can explain the response pattern observed. As already reported, aggregation/agglomeration processes are likely to increase with the increase in the number of particles per volume [37]. At low concentrations, NPIs can maintain their sizes, presenting higher likelihood to be detected by the organisms inducing protective mechanisms as the avoidance responses detected. Nonetheless, the present findings highlight the importance of studying the effects of NPIs at low concentrations as previously shown for nanomaterials [38]. On the other hand, with increasing concentrations, more NPIs are present in the soil, reacting with the organisms and may induce a negative effect (e.g., a neurotoxic effect). This neurotoxic effect can induce a non-response in the organisms, not being possible for them to avoid the contaminated soil. Indeed, [PERSON] et al. [25] reported that the non-avoidance behaviour of _E. cryptic_s might be associated with the gamma-aminobutyric acid (GABA) system. The up-regulation of the GABA is known to trigger anaesthetic effects [39], hence this may at least partially explain the obtained results. Other invertebrates, such as nematodes--_Caenorhabditis elegans_ (live in the interstitial water of soil)--altered their locomotor behaviour after polystyrene (nano)microplasics exposure (3 d to 1 mg/L) [40]. Despite being related to an aquatic vertebrate, behavioural alterations were also detected in locomotion, aggressiveness, shoal formation and predator avoidance of _Danio rerio_ adults (7 d exposure to 1.5 mg/L polystyrene NPIs) [41]. Overall, this can indicate the ecological impact of NPIs in soil: organisms are not efficiently able to escape to non-contaminated soils, become intoxicated, and this can cause impact in other biological responses. The calculated percentages of avoidance (the highest was \(A=58.3\%\) for the condition 0.015 mg NPIs/kg) were not equal or higher than 80%, which can suggest that the habitat function will not be compromised after the exposure to the tested concentrations of NPIs. A NPIs dispersion toxicity dependent on the medium of exposure (i.e., soil versus water) was demonstrated in the present study. Indeed, 150 mg NPIs/kg via LUFA soil induced no effect in terms of the survival of the organisms after 21 d, although 150 mg NPIs/L via ISO water induced 100% of organisms mortality after 5 d. Toxicity via soil was comparatively lower than via water exposure, as previously reported for different environmental contaminants [26]. We hypothesize that this could be due to the interaction of NPIs with the soil particles, decreasing the bioavailability of NPIs and consequently inducing less effect on the organisms.