Plasma vitellogenin (VTG) concentration is one of the primary apical endpoints of regulatory test methods using fish models where estrogen/androgen/steroidogenesis modalities are evaluated (e.g. OECD 229/230/234 and OCSPP 890.1350). With current European Union regulations mandating that all biocides and plant protection products be evaluated for potential endocrine disrupting effects—and the prevalence of VTG as an apical endpoint in such studies—there has recently been concern regarding the high intra- and inter-laboratory variation in reported plasma VTG concentrations and the potential impacts of this variation on the robustness of associated assays. One potential source of variation is sample storage. It is currently unclear how storage methodology and storage duration influences the stability and subsequent analysis of fathead minnow plasma VTG.
While relevant guidelines outline a storage procedure for plasma samples (i.e., store with aprotinin at -80 °C), it is clear from the literature that not all laboratories are using the described procedure. In addition, these guidelines provide no clear indication of the stability of samples under this storage regime. The paucity of information regarding the effects of storage method and duration makes it difficult to determine if these factors may be influencing the previously noted variation in VTG concentrations.
Therefore, the objective of this project was to evaluate the impacts of storage method and storage duration on plasma VTG concentrations. To achieve this objective, composite plasma samples from male and female fathead minnows were stored under multiple storage scenarios (i.e., with/without aprotinin at -20°C or -80°C) and sample VTG concentrations were measured via ELISA at time points over an extended period. The results of this project will provide valuable information regarding the influence of storage method and duration on measured plasma VTG concentrations in fathead minnows.
Dr. Julie Krzykwa, Biologist, presented the research at the SETAC North America 43rd
Annual Meeting, November 13-17, 2022.