The 15N content (15N/14N ratio) can be used to test the diet estimates determined by gut contents analysis, since the 15N content directly reflects the what the fish actually digest, not what they might have in their stomachs when caught. In particular, 15N analysis suggests that although algae often is a major component of the stomach content of fish, it is only a minor component of what is actually digested, as suggested by the large differences in the 15N content in algae compared to fish (as shown).  This is an important difference since the meHg content of algae can be high in some environments.

The nitrogen isotopic compositions (delta 15N, usually written as d15N) of organisms in the Everglades range from -10 to +20 permil. These delta 15N values are in good agreement with suspected trophic position; primary producers have lower values than herbivores while omnivores and carnivores have successively higher values.  Plants show an extremely large range of values. Most of the variability is in the macrophytes, and these almost always have lower values than algae.  Among the invertebrates, insects show considerable overlap with crustaceans and small fish but generally have lower delta 15N values. Crustaceans have lower delta 15N values than most fish. Small omnivorous fish have lower delta 15N values than larger carnivorous fish. In general, the delta 15N values of gambusia (mosquitofish) are 7 to 9 permil higher than the values for co-existing periphyton mats.

The carbon isotopic compositions (delta 13C, usually written as d13C) of organisms range from -40 to -15 permil. In general, the delta 13C values of algae, invertebrates, and fish show considerable variability with little or no consistent increase in delta 13C with increasing trophic level. Hence, bulk carbon isotopes are not very useful for determining trophic position. However, there is usually very good separation of the delta 13C values of macrophytes and algae; most macrophytes have higher delta 13C values than algae and other organisms.  The generally high delta 13C values of the macrophytes are inconsistent with their being a major food source in most locations.

In general, organisms collected in high-nutrient sites near the Everglades Agricultural Area (EAA) have higher delta 15N values than ones collected in more pristine areas to the south. Near the EAA, organisms in the canals generally have higher delta 15N values than samples from adjacent marshes, and the delta 15N values decrease with distance from the canals. This difference probably reflects a persistently higher value of  the delta 15N of dissolved inorganic nitrogen (DIN) at the canal sites than at marsh sites.  One likely explanation for this pattern is denitrification in anoxic waters and sediments in stagnant parts of the canals, which would cause the delta 15N of the resultant DIN to increase. Marsh sites near canals show a much larger range of isotopic compositions than more pristine marsh sites because of the periodic influxes of eutrophic canal waters.