There is a growing interest in the development of reliable analytical methods for characterizing tire and road wear particles (TRWP). The current research extends the use of single particle analysis techniques to various experimental biota samples. TRWP and cryogenically milled tire tread (CMTT) were identified using a weight of evidence framework including density separation, optical microscopy, and chemical mapping (scanning electron microscopy coupled with energy dispersive X-ray spectroscopy). Our techniques successfully identified CMTT particles in laboratory earthworms exposed to soil spiked with CMTT. A river biota sample (bivalves) collected from the Seine with no detectable TRWP was spiked with road dust containing TRWP. Particle identification was performed after a biota digestion protocol and density separation of particles > 1.5 g/cm 3 and < 2.2 g/cm 3 which resulted in sufficient TRWP for identification and characterization. The average TRWP particle size from the road dust spiked biota sample was 126 μm by number and 220 μm by volume (range: 9-572 μm). The size distribution overlay of TRWP identified from spiked biota were consistent with TRWP identified from the original road dust sample suggesting that the current method for biota digestion, dual density separation, and TRWP characterization is feasible for similar samples.