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Transition metals in particulate matter (PM) are hypothesized to have enhanced toxicity based on their oxidative potential (OP). The acellular dithiothreitol (DTT) assay is widely used to measure the OP of PM and its chemical components. In our prior study, we showed that the DTT assay (pH 7.4, 0.1 M phosphate buffer, 37 °C) provides favorable thermodynamic conditions for precipitation of multiple metals present in PM. This study utilizes multiple techniques to characterize the precipitation of aqueous metals present at low concentrations in the DTT assay. Metal precipitation was identified using laser particle light scattering analysis, direct chemical measurement of aqueous metal removal, and microscopic imaging. Experiments were run with aqueous metals from individual metal salts and a well-characterized urban PM standard (NIST SRM-1648a, Urban Particulate Matter). Our results demonstrated rapid precipitation of metals in the DTT assay. Metal precipitation was independent of DTT but dependent on metal concentration. Metal removal in the chemically complex urban PM samples exceeded the thermodynamic predictions and removal seen in single metal salt experiments, suggesting co-precipitation and/or adsorption may have occurred. These results have broad implications for other acellular assays that study PM metals using phosphate buffer, and subsequently, the PM toxicity inferred from these assays.