Development of a quadruple isotope spiking methodology to study mercury methylation and methylmercury demethylation in environmental and biological media.

A quadruple isotope spiking methodology has been developed and applied for the study of Hg-species-specific transformations in incubation experiments. The method enables for the first time discrimination between interconversions reactions occurring during the incubation step from those occurring during the analytical procedure. Two different mathematical models for quadruple spiking were developed and applied here in model incubation experiments to track inorganic Hg(II) methylation and methylmercury demethylation. The results showed identical Hg compounds concentrations and interconversion reactions using both mathematical approaches so that an internal validation of the calculation procedure could be achieved. The methodology was also applied to determine Hg(II) methylation, methylmercury demethylation to Hg(II) and methylmercury loss rate in incubations performed with biofilms, sediments, freshwaters and pure phytoplankton cultures with Hg concentrations ranging from pg L-1 to μg L-1. At the initial incubation time, analytical and undesired Hg(II) methylation and/or methylmercury demethylation was fully corrected and eliminated after applying the quadruple spiking methodology. Methylation during analytical procedure was found negligible for all matrices (<0.2%) whereas significant demethylation during analytical procedure (from 3 to 10%) was observed for all samples. Natural Hg(II) methylation was only found in biofilms and sediments and, the discrimination of methylmercury demethylation and loss was achieved for all samples. The overall analytical uncertainty of the newly formed compounds was clearly affected by the concentration range. Specifically, the uncertainty of the experimental peak integration and the isotopic abundances of the isotopically enriched tracers were the main parameters contributing to the overall analytical uncertainty of the newly-formed Hg compounds concentration in natural samples. This work establishes a novel approach to study transformations of Hg compounds in biological and environmental matrices. The mathematical framework, based on multiple linear regression, is in principle transferable to other poly-isotopic elements with at least five stable isotopes and commercially available isotopically enriched species standards (e.g., tin), although such extension remains to be demonstrated experimentally.