Beyond the transect: an alternative microchemical imaging method for fine scale analysis of trace elements in fish otoliths during early life

Microchemical analysis of otolith (calcified 'ear stones' used for balance and orientation) of fishes is an important tool for studying their environmental history and management. However, the spatial resolution achieved is often too coarse to examine short-term events occurring in early life. Current methods rely on single points or transects across the otolith surface, which may provide a limited view of elemental distributions, a matter that has not previously been investigated. Imaging by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) permits microchemical analyses of short-term events in early life with high (<10μm) resolution, two-dimensional (2D) visualization of elemental distributions. To demonstrate the potential of this method, we mapped the concentrations of Sr and Ba, two key trace elements, in a small number of juvenile otoliths of neon damselfish (Pomacentrus coelestis) using an 8μm beam diameter (laser fluence of 13.8±3.5Jcm^-2). Quantification was performed using the established method by Longerich et al. (1996), which is applied to 2D imaging of a biological matrix here for the first time. Accuracy of >97% was achieved using a multi-point non matrix-matched calibration of National Institute of Standards and Technology (NIST) 610 and 612 (trace elements in glass) using Longerich's calculation method against the matrix-matched standard FEBS-1 (powdered red snapper [Lutjanus campechanus] otolith). The spatial resolution achieved in the otolith corresponded to a time period of 2±1days during the larval phase, and 4±1days during the post-settlement juvenile phase. This method has the potential to improve interpretations of early life-history events at scales corresponding to specific events. While the images showed gradients in Sr and Ba across the larval settlement zone more clearly than single transects, the method proved sample homogeneity throughout the structure; demonstrating that 2D scanning has no significant advantage over line scans.