Extraction and quantification of microphytobenthic Chl a within calcareous reef sands

Calcareous reef sands are characterized by high concentrations of photosynthetic pigments that extend well below the sediment surface, as well as by high variability in concentrations between study sites. An important contributor to the observed variability may be differences in extraction protocols, further complicated by variability in calcareous sand characteristics. We tested the effects of freeze-drying, grinding, sonication, extraction temperature, and extraction time on quantification of Chl a content within calcareous sands. The resulting optimized extraction protocol consists of freeze-drying, grinding with a mortar and pestle for 30 s, and extracting with 100% acetone at -20 degrees C for at least 20 h, yielding a 39% increase in Chl a content over frozen, unground samples. Using this protocol, we measured and compared ten sedimentary Chl a profiles taken in close proximity to test for relationships between surface and sub-surface concentrations. Sedimentary Chl a content at a backreef location on the south shore of O'ahu varied between 4.33-14.25 mu g g(-1) dw, with distinctly higher values occurring in a relatively enriched surface layer (0-1 cm). Surface Chl a concentrations varied between 86-307 mg m(-2), depending on the depth of integration (0.5-2 cm), with 73% of the full-core (0-8 cm) Chl a concentration occurring below 2 cm. The concentrations of surface and subsurface layers were significantly correlated between cores, allowing for the use of plug sampling when profile generation is not feasible or necessary to determine the magnitude of the subsurface microphytobenthic biomass and its variability over scales of meters.