Analysis of the C, N, and S stable isotope compositions of plants, invertebrates, and vertebrates can be used to establish relative trophic levels among various organisms because at each ascending trophic level, there is an increase of about 0 to 1 permil in d13C and 2- 3 permil in d15N. Traditional methods for determining food web relations include direct observation and gut content analysis; both of these methods are tedious, time consuming, and emphasize food which is not digested and may not constitute important contributors to body mass. Stable isotope analysis provides a complementary and perhaps alternative method for testing hypotheses about animal diets. Its major strength is that the isotope compositions provide relatively easy and precise information about what was actually assimilated over the life-time of the organism. This information is critical to evaluating how methyl mercury (MeHg) gets into food chains. As part of several investigations, some 600 fish, periphyton, and invertebrate samples have been analyzed for d13C and d15N; a smaller subset has also been analyzed for d34S. Most of these samples have also been analyzed for MeHg and gut contents.

For C and N isotopic analysis, samples are dried, ground to a fine powder, 0.2 to 2 mg of the powder is weighed into tiny tin or silver foil boats, the boats are crimped, and they are loaded into an autosampler. If the samples contain calcite, silver boats are used instead of tin, and the weighed-out samples are acidified by exposure to HCl vapor for 18 h in a desiccator prior to crimping. Samples are combusted and the resultant N2 and CO2 gases separated chromatographically on an elemental analyzer. These gases are then automatically analyzed for C and N abundances and isotope ratios on the attached Optima continuous flow stable isotope mass spectrometer. Analysis for S isotope ratios requires combustion of a second larger aliquot and different processing. The analytical precision is about 0.15 permil for C, N, and S isotope ratios of solid samples >0.5 mg. The reproducibility of duplicate aliquots of samples analyzed on different days is better than 0.3 permil; much of the variability is due to inhomogeneity of the samples.

Surveys of organisms at USGS sites in the Water Conservation Areas (WCAs) generally show that samples from canals have higher d15N values (+9 to +18 permil) than samples from marshes (+1 to +14 permil). This difference probably reflects a persistently higher value of the d15N 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 resultant DIN to increase in d15N. The compositions at individual sites show some seasonal variability in composition. In general, the d15N values of gambusia are 7 to 9 permil higher than co-existing periphyton. If periphyton is an important component of the gambusia food chain, this is equivalent to 2 to 3 trophic levels above the periphyton.

The d13C values for samples range from about -17 to -36 permil; these values provide valuable clues about trophic relations among consumers. Different sites appear to have different food chains. At many sites, the d13C values of omnivorous gambusia are 1 to 4 permil lower than co-existing periphyton and some herbivores. This apparent lack of isotope enrichment at higher trophic levels suggests that carbon from bulk periphyton is not the base of the gambusia and many other consumer food chains. This is puzzling because periphyton constitutes a major component of gambusia gut contents, and had been considered a major source of carbon to consumers. Likely candidates for bottom of the food chain include plankton (zoobenthos), partially degraded detrital organic matter, or some more readily digestible component of the periphyton community that has a lower d13C value than the bulk total. Organisms such as diatoms and green algae have been positively identified in Everglades periphyton through pigment analysis. The few samples of utricularia, which feeds on plankton, that have been analyzed have low d13C values consistent with plankton being an important carbon source. Additional sampling will be required to resolve this issue.

Approximately 10 samples of large-mouth bass were collected at each of 10 sites across the Everglades to determine local variability in diet within a population. Bass at some sites (for example, the ENR outlet, L-7, and a mid-marsh site in WCA1) have narrow and distinctive ranges in isotopic compositions. These compositions suggest that the bass at the WCA1 do not migrate in or out of L-7, and that L-7 and the ENR have significantly different environmental conditions. The larger and overlapping ranges in d13C and d15N values at sites in WCA 2 and 3 are consistent with movement of bass between canal and marsh sites, probably in response to fluctuations in water levels. These data indicate that for adjacent canal and marsh sites where the primary producers have distinguishable isotope compositions, the isotope compositions of fish can be used to determine whether the fish migrate in and out of the marshes in response to changes in water levels or food availability. These findings should prove useful for determining where some populations of game fish are acquiring elevated levels of MeHg.

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