## 1 Introduction  
In recent years, plastic production and usage have continuously increased, and as they grow, their impact on aquatic ecosystems also increases [1]. The entrance of plastic litter into aquatic systems is due to different causes, such as ineffective waste management (deposition in landfills), wind events, littering on shorelines or at sea, loss of fishing tackle, stormwater runoff and treated wastewater discharges still containing microplastics (MPLs), among others factors [2, 3]. MPLs are known as plastic particles smaller than 5 mm, while nanoplastics (NPLs) are those below \(1\) um [4]. MPLs can be classified according to their origin, with primary ones entering the environment with particles below 5 mm, and secondary MPLs resulting from fragmentation and erosion in the environment of bigger plastic items [1, 5]. Some of the main problems that derive from micro- and nanoplastics (MNPL) pollution are their contribution to eutrophication processes [6, 7]; entrance into the aquatic food chain [8, 9]; physical toxicity [10] contributing to oxidative stress and inflammatory lesions [11]; chemical toxicity (because of leaching monomers of the plastic polymers and additives used in plastic formulation, such as plasticizers, flame retardants, fillers, UV-stabilizers, coating finishers, colourants, and metals, among others) [10]; and potential contribution to transferring other co-contaminants from surrounding waters accumulated onto their surface to biota, also known as the Trojan Horse effect [12; 13; 14]. It should be highlighted that MNPLs' toxicity is directly related to their size and shape. The smaller the particles, the bigger the active surface areas that facilitate both leaching of monomers and additives and the adsorption and transfer of other contaminants present in the surrounding environments to biota. Moreover, plastic particles cannot be digested, but NPLs can be translocated through tissues and bioaccumulated. For these reasons, NPLs are the most relevant from the toxicological point of view. Nonetheless, there is a substantial gap in information regarding MNPLs' accumulation in biota and, particularly, regarding NPLs because of technical limitations and difficulties in their analysis. Analytical techniques used for the characterisation of the size and shape of plastic particles are, in general, based on scanning electron microscopy (SEM) [15], SEM coupled to energy-dispersive X-ray spectroscopy (SEM-EDS) [15; 16; 17] and SEM with energy-dispersive X-ray spectroscopy (ESEM-EDS) [15; 17]. Nowadays, the most common techniques for the investigation of the polymeric composition of MPLs are those based on spectroscopies such as Fourier-Transform Infrared (FTIR) and Raman spectroscopies, which are combined with microscopies to assess the number of particles. However, these approaches are limited by the particle sizes [15; 16]. Using spectroscopic techniques, the smallest particle sizes that can be investigated are about 1 um for u-FTIR and for u-Raman, a bit less, with u-FTIR being the most commonly used recently to investigate MPLs in fish GITs [18; 19]. Other promising approaches are pyrolysis gas chromatography (Pyr-GC-MS) [20] and size-exclusion chromatography coupled to high-resolution mass spectrometry (SEC-HRMS) [21], which are not limited by the particle size [2; 15] and can provide qualitative and, coupled to HRMS, quantitative information in terms of mass units. Under this framework, the central goal of this study was to assess the occurrence of MNPLs in the gastrointestinal tracts (GTIs) of fish from the Ebro River (NE of Spain). The MNPL extraction from the GITs was achieved with sequential alkaline and acidic digestion. Then, the identification of the bigger particle fraction was carried out using FTIR.