## 1 Introduction  
In 2016, the production of plastic around the world, mainly used for packaging, exceeded the 320 million tons. This production rise was accompanied by an unprecedented exponential increase in the occurrence of plastic waste in both soil and aquatic environments [1]. Once in the environment, plastics may breakdown through several processes, for instance mechanical forces and photo-oxidative processes, into microplastics (MPIs; smaller than 5 mm) and nanoplastics (NPIs; sized between 1 to 1000 nm), changing their physicochemical characteristics, and thereby their bioavailability and potential impact on the organisms [2]. MPIs and NPIs occur in soil at higher levels--at least a factor of four--than in marine environments [3]. However, the presence of MPIs and NPIs in terrestrial ecosystems and their toxic effects to the biota have received little attention [4]. In fact, plastic debris remaining on the soil surface are more exposed to UV (ultraviolet) light than the ones present in the marine compartments, leading to a high presence of MPIs and NPIs on the terrestrial ecosystems [5]. For instance, around 4000 MPIs particles.kg\({}^{-1}\) of dry sludge (\(\sim\)0.04%) were found at European agricultural and landfill sites [6]. Although it is known the widespread presence of NPIs into the environment, data concerning their environmental levels are still missing due to the limitations of the analytical techniques associated with the NPIs small size [2]. NPIs concentrations \(<15\)\(\upmu\)g/L have been predicted to be environmentally relevant concentrations [7]. Presenting a much higher surface area to volume ratio than MPIs, NPIs may be more easily ingested (depending e.g., on their aggregation state) and may induce greater toxic effects to the organisms [8]. Despite the increasing number of studies regarding the toxicity of NPIs to aquatic invertebrates (e.g., _Mytilus galloprovincialis_[2, 9] and _Daphnia magna_[10, 11, 12, 13]), concerning soil invertebrates only one study was found to the model species _Enchytraeus crypticus_[5]--Table 1. Therefore, it is crucial to increase the knowledge about the toxicity of NPIs to terrestrial organisms, contributing to a better environmental risk assessment of NPIs to the soil ecosystems. Due to the urgent need to prioritize research dealing with this topic, the aim of this study was to assess the toxicity of polystyrene NPIs to the standard and ecologically relevant soil model _Enchytraeus crypticus_. Toxicity was measured using survival and reproductive outputs as endpoints. Additionally, the avoidance behaviour of _E. crypticus_ after the exposure to NPIs was also evaluated. Enchytraeids live in the litter layer and the upper mineral soil where they feed with fungal hyphae, microorganisms and dead organic matter (OM) [17]. Moreover, they vigorously contribute to the acceleration of OM decomposition and nutrient recycling processes. Being a soft-bodied soil invertebrate, uptake is made via ingestion (e.g., food and soil particles) as well as via the body surface or dermis (used for gaseous exchange and water uptake) [18]. Many studies already demonstrated _E. crypticus_ as a valuable bioindicator of contaminated environments from a wide range of anthropogenic sources (e.g., metals and nanoparticles) [19, 20, 21].