When Fluorescence Is not a Particle: The Tissue Translocation of Microplastics in <i>Daphnia magna</i> Seems an Artifact

Previous research reported the translocation of nano- and microplastics from the gastrointestinal tract to tissues in Daphnia magna, most prominently of fluorescent polystyrene beads to lipid droplets. For particles >300 nm, such transfer is biologically implausible as the peritrophic membrane retains these in the daphnid gut. We used confocal laser scanning microscopy to study tissue transfer applying the setup from a previous study (neonates exposed to 20 and 1000 nm polystyrene beads at 2 µg L^-1 for 4 and 24 h), the same setup with a fructose-based clearing, and a setup with a 1000-fold higher concentration (2 mg L^-1 ). We used passive sampling to investigate whether the beads leach the fluorescent dye. Although the 1000 nm beads were visible in the gut at both exposure concentrations, the 20 nm beads were detectable at 2 mg L^-1 only. At this concentration, we observed fluorescence in lipid droplets in daphnids exposed to both particle types. However, this did not colocalize with the 1000 nm beads, which remained visible in the gut. We further confirmed the leaching of the fluorescent dye using a passive sampler, a method that can also be applied in future studies. In summary, we cannot replicate the original study but demonstrate that the fluorescence in the lipid droplets of D. magna results from leaching of the dye. Thus, the use of fluorescence as a surrogate for particles can lead to artifacts in uptake and translocation studies. This highlights the need to confirm the stability of the fluorescence label or to localize particles using alternative methods. Environ Toxicol Chem 2019;38:1495-1503. © 2019 SETAC OPEN PRACTICES: The present study has earned Open Data/Materials badges for making publicly available the digitally shareable data necessary to reproduce the reported results. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.