1. Stable isotopes of nitrogen are useful for quantifying the trophic structure of food webs, but only if the variation in trophic enrichment (ΔN), which is the difference in δ15N between a consumer and its food, is small relative to the value of ΔN itself.
2. We examined the sources of variation in zooplankton ΔN by measuring the trophic enrichment (ΔN) of seven species of freshwater cladocerans, and by testing for an effect of age and temperature on the ΔN of Daphnia pulicaria.
3. We found that ΔN was similar among Cladocera and was not correlated with body size. Overall, the ΔN for D. pulicaria was 1.4‰ (SE = 0.69, n = 57), as was expected for the detritus diet that we used in our experiments. We found no effect of temperature (15–25 °C) on ΔN, but found that ΔN of D. pulicaria increased with increasing age (10–30 days).
4. We developed a new method to test for trophic-level variation in a group of consumers that explicitly accounts for the uncertainty in ΔN. Using this approach, we confirmed that natural assemblages of zooplankton feed at several trophic levels in lake food webs.