Innovative approach for quantitative determination of ingested microplastics by <italic>Daphnia magna</italic>: use of differential scanning calorimetry and thermogravimetry.

Due to the serious environmental impacts of plastic pollution, some conventional plastics have been replaced with biodegradable alternatives. However, these biodegradable options can also fragment and form microplastics, which can be ingested by and harm various biota. The standard methods for quantifying ingested microplastics involve digestion of the gut or the whole organism using acidic, alkaline, or oxidative processes. However, these aggressive methods may destroy biodegradable microplastics, leading to erroneous results. Therefore, in this study, we employed thermal analysis methods, specifically differential scanning calorimetry (DSC) and thermogravimetry (TG), to quantify the ingestion of poly-3-hydroxybutyrate (P3HB), a biodegradable microplastic, by the freshwater crustacean, <italic>Daphnia magna</italic>. During chronic experiments, we analysed organisms exposed to P3HB with sizes smaller than 125 and 63 μm, at concentrations ranging from 1.56–25 mg L−1. DSC identified an endothermic peak associated with the melting of semicrystalline P3HB, and its enthalpies were utilised to compute the number/mass of P3HB ingested by <italic>D. magna</italic>. Notably, shifts in melting points suggested that higher concentrations induced particle agglomeration, and these agglomerates could not penetrate deeply into the organism. The TG approach involved subtracting the mass loss between 200 and 400 °C in <italic>D. magna</italic> specimens exposed to P3HB suspensions from controls without P3HB exposure. Both methods provided comparable data, revealing that, depending on particle size, individual <italic>D. magna</italic> ingested up to 10% of their body mass. Our findings indicate that both methods effectively detect P3HB (and potentially other plastic fragments), with DSC demonstrating better sensitivity. While the suggested approach did not enable us to calculate the level of determination or quantification, we were able to demonstrate that DSC can detect P3HB in only one specimen of <italic>D. magna</italic> exposed to the lowest suspension concentration. This indicates that D. magna exposed to the 63 μm fraction ingested approximately 3 μg of P3HB, whereas those exposed to the 125 μm P3HB ingested around 4 μg of P3HB. The introduced methods expand the possibilities for detecting ingested microplastics and probably also nanoplastics, in zooplankton and possibly also other species. [ABSTRACT FROM AUTHOR]