Unravelling complexities in benthic food webs using a dual stable isotope (hydrogen and carbon) approach

P>1. Stable carbon isotope studies have been an essential component of research regarding the contribution of methane (CH4)-derived carbon to freshwater food webs and results have suggested that benthic macroinvertebrates in billabongs, streams and lakes may be partially, and in some instances, significantly 'fuelled' by methanotrophic biomass. However, the singular use of carbon isotopes can lead to ambiguous interpretations concerning the origin of carbon, especially in systems where phototrophs are likely to be using carbon respired sources and hence show more 13C-depleted values. 2. These uncertainties can be further resolved by the inclusion of additional isotopic data. Stable hydrogen isotopes are being increasingly used in food web studies with a marked advantage that sources may be isotopically distinct by one or two orders of magnitude greater than stable carbon or nitrogen, the isotopes most commonly used to delineate trophic interactions. By using hydrogen as a second biogeochemical tracer we provide further supportive evidence for the assimilation of methanotrophic microbial biomass by chironomid larvae. 3. Moreover, the hydrogen and carbon isotope values we found in chironomid tissues appear to reflect the original substrate used during methanogenesis; either acetate fermentation or carbonate reduction. Use of the former tends to result in relatively heavy carbon and light hydrogen isotope values due to kinetic isotope effects, whereas use of the latter results in relatively lighter carbon and heavier hydrogen isotope values. 4. We provide preliminary evidence to suggest that hydrogen and carbon isotope values in macroinvertebrates may be used to distinguish between CH4 formation pathways and help to explain inter-depth and inter-specific differences between co-existing chironomid species found in the same lake.