Despite recent efforts to manage them more efficiently, estuaries are natural sinks for a wide range of metal contaminants, many of which accumulate at potentially toxic concentrations for fish populations, posing a threat to recruitment and stocks. While analysis of metal concentrations in soft tissue and water samples calls for continuous and long-term sampling operations, the use of otoliths to study metal pollution may be one way of providing a historical record of pollutant exposure. In this study, we examine the potential use of otoliths as natural tracers of metal contamination. A "cocktail" of different metals (Cd, Pb and Ni) was used to test bio-accumulation in otoliths and tissue (liver, kidney, muscle and gills) extracted from juvenile flounder (Platichthys flesus). Assessment took place under controlled conditions over a three month period, with water exposure concentrations increasing every 3 weeks. The concentrations used were natural (T1), X5 (T2), X10 (T3), and null (T4). Chemical analyses were carried out using an inductively coupled plasma optical emission spectrometer ICP-OES and atomic absorption spectrometer AAS for water and tissue, while otolith microchemistry analyses were performed using a femtosecond laser ablation-high resolution inductively coupled plasma mass spectrometer (fsLA-ICP-MS-HR). Significant differences between control and exposed individuals, as well as an increase in metal concentrations according to exposure level, were observed in all tissues except in muscle tissue. Significant increases in Pb were also detected in contaminated fish otoliths compared with control specimens, with the highest concentrations occurring in T3. Cartographies of Pb distribution in otoliths of both control and contaminated fish only showed high concentrations of Pb at the edge of contaminated fish otoliths, indicating an accumulation of metal during the experiment. Although the relationships between exposure level and Pb concentration in otoliths were complex, the concentrations were correlated with those in the water. Analysis of flounder specimens collected from 2007 to 2014 in the Gironde estuary (SW France) showed interannual variability in Pb concentrations, with higher values for fish otoliths from 2007 to 2010 than those from 2012 to 2014. This trend indicated a decrease in Pb in the Gironde estuary over the last decade, which is consistent with the results of other surveys on bivalves. Our study demonstrates that it is possible to use otolith microchemistry as a tool in assessing and retracing long-term metal pollution in estuarine systems. © 2016 Elsevier B.V.