Investigating the presence of nanoplastics in freshwater chironomids from glacial habitats using Raman spectroscopy

The detection of nanoplastics (NPs) in the natural ecosystems is challenging due to the size and the low concentrations of NPs. The aim of the present study is to investigate the presence of NPs in larvae of two chironomid species (Diamesa zernyi and Diamesa tonsa) colonizing two high-altitude glacier-fed streams (Mandrone and Amola streams, Trentino, Italy). The analytical method developed in this work combines enzymatic and oxidative digestion followed by a purification step in ethanol to enable on-chip identification through Raman spectroscopic analysis. To validate the extraction procedure, three pools of 100 mg (wet weight) each of Diamesa zernyi larvae from the Mandrone stream were spiked with polystyrene NPs of 500 nm in size at two different theoretical concentrations (10⁷ and 10⁹ particles/mL). Quantification of the particles in the residual matrix was performed using Single Particle Extinction and Scattering (SPES) analysis. The results demonstrate good recovery rates, respectively of 109 ± 28% and 82 ± 12% for the high and low concentration spiked samples. This methodology enabled the effective identification of plastic particles using confocal Raman spectroscopy. Successively, three pools of 100 mg (wet weight) of non-spiked specimens of Diamesa tonsa from the Amola stream were analysed revealing the presence of polystyrene particles. Despite the low number of replicates from only one analysed sampling site and the detection limits of the Raman spectroscopy, this approach represents the first reliable analytical extraction procedure to demonstrate the accumulation of NPs by aquatic insect larvae and, consequently, the potential environmental pollution of glacial streams from Italian Alps.