Use of fluorescent-labelled nanoplastics (NPs) to demonstrate NP absorption is inconclusive without adequate controls

Whether nanoplastics (NPs) are able to be absorbed across epithelial membranes and accumulate within internal tissues of organisms is an important determinant of their potential toxicity. Evidence of absorption and accumulation requires detection of NPs within internal tissues, and investigations with fluorescently labelled NPs have attempted to provide this information. We hypothesize that studies that do not control for the fluorescent dye leachate and/or cellular autofluorescence are inconclusive and can be misinterpreted. Our goal was to analyse previous investigations critically and conduct further research to determine if fluorescent-labelled polystyrene NPs (nanoPS) can provide conclusive evidence of absorption and internal accumulation of NPs. We exposed zebrafish embryos and larvae to NPs (500 and 1000 nm) labelled with a green or an orange fluorescent dye, to solutions resulting from nanoPS dialysis, and to Nile-Red (a fluorescent dye used as a positive control). Previous studies have claimed that NPs cross epithelia without accounting for dye leachates and/or cellular autofluorescence. Our results demonstrate that commercial fluorescent-labelled nanoPS can leach their fluorophores, and the fluorophore alone can accumulate within internal tissues of zebrafish larvae. We further observed green autofluorescence in fish larvae not exposed to any particles. Previous claims of NP absorption based on observations of fluorescence in zebrafish tissues should thus be considered inconclusive. Although the addition of purification steps and inclusion of controls for leaching of dyes are methodological improvements, the use of fluorescent nanoPS should not be considered to provide absolute conclusive evidence of particle absorption.