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

Abstract

This study describes a micro-assay, based on acid dichromate oxidation, with a resolution of at least 0.5 μg organic carbon and an upper limit of ≤20 μg C. We also document several important properties of acid dichromate assays and establish their effectiveness for quantifying organic carbon and energy content of marine and freshwater organisms. Both the micro-assay and the previously described standard assay are highly sensitive to chloride: absorbance readings were significantly depressed by the presence of only 0.5–1.0 μl of seawater, and the effect of seawater was shown to be due to its chloride content. The amount of chloride contained within the bodies of very small marine organisms may therefore be sufficient to interfere with the assay. Contrary to previous claims, we found that incubating samples with phosphoric acid did not prevent chloride from interfering with the assays. The micro- and standard assays were not sensitive to inorganic carbon and were therefore specific to organic carbon. The assays were effective in estimating total energy content of carbohydrate and lipid material, but underestimated the energy content of protein material by 47–69%. This limitation can be overcome by using a protein micro-assay to correct for underestimation by the acid dichromate assays. Based on our findings, the reliability of acid dichromate oxidation assays for analysing samples of marine organisms is questionable. The assays are effective, however, for analysing chloride-free tissues or extracts. In addition, the assays have considerable potential for determining energy content of small freshwater organisms. In particular, the micro-assay is at least an order of magnitude more sensitive than the standard assay, and constitutes a relatively simple way of measuring energy content of very small samples, such as individual embryos or early juveniles of aquatic animals and plants.