The Indian River Lagoon, Florida, is a unique closed “bar built” estuary system that has little interchange with the Atlantic Ocean and which is home to many resident species. Three fish species were investigated to see if their isotopic and fatty acid signatures differed based on geographic location. The goal was to assess the degree of resolution of spatial variation that is possible when using stable isotope and fatty acid signature analysis to interpret feeding habits and potential linkages between feeding habits and health status. Spotted seatrout (n=40), pinfish (n=60) and white mullet (n=60) were collected over a 4 week period at sites 30 km apart in two distinct biogeographic regions of the IRL. Fish were analyzed for stable isotope (δ13C and δ15N) and fatty acid composition. All three species were significantly different from each other in both isotopic and fatty acid signatures. In the North Indian River segment, spotted seatrout, white mullet and pinfish had mean values (± SE) for δ13C of -18.00 ± 0.08, -14.59 ± 0.07 and -16.88 ± 0.04 respectively and for δ15N, mean values were 14.43 ± 0.05, 8.30 ± 0.04 and 10.43 ± 0.03 respectively. For the North-Central Indian River segment, spotted seatrout, white mullet and pinfish had mean values for δ13C of -18.98 ± 0.02, -16.25 ±0.06 and -16.94 ± 0.04 respectively and for δ15N, mean values were 14.21 ± 0.02, 8.07 ± 0.03 and 10.64 ±0.03 respectively. When species and location interactions were examined using ANCOVA, a post-hoc Tukey’s HSD test showed that δ13C was significantly affected by sampling segment only for spotted seatrout and that there was no significant effect of location on δ15N values. Spotted seatrout was the only species that differed between segments for δ13C. Classification and Regression Tree (CART) analysis was then performed on the “extended dietary fatty acids” for each individual species and collection location. In this analysis, all three species were correctly identified to segment (white mullet - MR 2/53 p~ 0.03; spotted seatrout (MR 0/40), and pinfish (MR 0/56) indicating that individual species were exhibiting significant differences in their fatty acid signature over distances of 30 km. The ability to discern fine-scale differences in potential prey allows for the possibility of better resolution of dolphin feeding habits and hence a better understanding of both habitat utilization and health impacts. Due to limited exchange of clean salt water, contaminants can theoretically become a problem and there are indications that the health of the resident population of bottlenose dolphins (Tursiops truncatus) has been declining over recent years.

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