Analysing energy content: a new micro-assay and an assessment of the applicability of acid dichromate assays

This study describes a micro-assay, based on acid dichromate oxidation, wit h a resolution of at least 0.5 mu g organic carbon and an upper limit of le ss than or equal to 20 mu g C. We also document several important propertie s of acid dichromate assays and establish their effectiveness for quantifyi ng organic carbon and energy content of marine and freshwater organisms. Bo th the micro-assay and the previously described standard assay are highly s ensitive to chloride: absorbance readings were significantly depressed by t he presence of only 0.5-1.0 mu l of seawater, and the effect of seawater wa s shown to be due to its chloride content. The amount of chloride contained within the bodies of very small marine organisms may therefore be sufficie nt to interfere with the assay. Contrary to previous claims, we found that incubating samples with phosphoric acid did not prevent chloride from inter fering with the assays. The micro- and standard assays were not sensitive t o inorganic carbon and were therefore specific to organic carbon. The assay s were effective in estimating total energy content of carbohydrate and lip id material, but underestimated the energy content of protein material by 4 7-69%. This limitation can be overcome by using a protein micro-assay to co rrect for underestimation by the acid dichromate assays. Based on our findi ngs, the reliability of acid dichromate oxidation assays for analysing samp les of marine organisms is questionable. The assays are effective, however, for analysing chloride-free tissues or extracts. In addition, the assays h ave considerable potential for determining energy content of small freshwat er organisms. In particular, the micro-assay is at least an order of magnit ude more sensitive than the standard assay, and constitutes a relatively si mple way of measuring energy content of very small samples, such as individ ual embryos or early juveniles of aquatic animals and plants.