Overcoming matrix interference: a selective fluorescence method for the detection and quantification of PHA, PLA, and PBAT microplastics in soil

Reliable detection of biodegradable microplastics (MPs) in soil remains analytically challenging due to the complexity of terrestrial matrices and the limited specificity of conventional fluorescence approaches. Here, a selective dual-staining workflow is presented for the detection and quantification of biodegradable polyester MPs in soil, including polyhydroxyalkanoates (PHA), poly(lactic acid) (PLA), and poly(butylene adipate -co- terephthalate) (PBAT). The method combines methylene blue, used to selectively label soil organic matter, with a fluorophore, iDye Pink, that preferentially partitions into polyester matrices. Under green excitation (λ ∼530 nm), MPs exhibit strong fluorescence contrast while the background signal from organic matter is effectively suppressed. Coupled with a custom fluorescence imaging system and automated image analysis, the workflow enables rapid large-area screening and extraction of particle morphometric descriptors. Within the validated [100, 5000] μm range, spike-and-recovery experiments performed on PHA microplastics yielded an overall recovery rate of 94.2%, with a false-negative rate of 5.8% and a false-positive rate of 3.3%. μ-FTIR analysis confirmed that the staining procedure does not interfere with polymer identification. Additional tests on PLA and PBAT demonstrated the qualitative transferability of the dual-staining strategy to other biodegradable polyesters, while complementary high-resolution microscopy enabled qualitative inspection of smaller particles and fine morphological features. Complementary high-resolution microscopy further extended the analytical window to smaller size fractions. Validated in soil, one of the most complex environmental matrices, this scalable approach provides a reproducible framework for investigating the fragmentation and environmental fate of biodegradable plastics.