Nile Red Staining Technique for Microplastics Detection in Sardines (Sardinella lemuru) Using Fluorescence Stereomicroscopy

Nile Red (NR) fluorescence is an emerging method for microplastics (MPs) detection due to its efficiency in distinguishing MPs from non-plastic materials. However, drawbacks to the use of NR include plastics that cannot be stained, whereas others show changes in physical and chemical characteristics at higher NR concentrations. As a practical approach, this study optimizes MP staining concentration combining NR fluorescence (approx. 170 million a.u.) in an inexpensive stereomicroscope adaptation with UV-LED at an excitation wavelength of 365 nm. The effects of NR staining concentration were then measured in terms of polymer recovery, polymer surface area, fluorescence intensity, and infrared spectra. Our findings revealed that the tested stain concentrations did not significantly alter MP recovery counts and surface area. At 10 μg/mL NR concentration, only polyethylene terephthalate (PET) and polystyrene (PS) fluoresced, indicating polypropylene’s (PP) resistance to NR staining. The potential functional groups involved in the excitation are mainly alkenes (for PET, PS, and PP) and aldehydes (for PET), as confirmed by the FTIR analysis that could be attributed to polymer structural rearrangement, aggregation, or depolymerization. This study also demonstrates that NR staining, when paired with an adapted stereomicroscope, can be effectively used to detect and quantify MPs in sardines (Sardinella lemuru)