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This dataset consists of concentration and isotopic data for a method designed to isolate and analyze methylmercury. Several matrices (lab-based, and natural) were tested to ensure that the method was capable of recovering methylmercury amendments. Additionally, natural matrices (plankton and sediment) with ambient methylmercury were also analyzed to ensure that the method could be successfully applied to natural matrices. Separation tests for sediment, water, and biotic matrices showed acceptable recoveries (98 ± 5%, n = 54) and reproducible δ202Hg isotope results (2SD < 0.15 ‰) down to 5 ng of MeHg. This dataset is part of a complimentary paper published in Analytical and Bioanalytical Chemistry (https://doi.org/10.1007/s00216-019-02277-0)...