Quantification of microphytobenthos biomass in intertidal sediments: layer-dependent variation of chlorophyll a content determined by spectrophotometric and HPLC methods

We assessed the difference between chlorophyll a (Chl. a) values estimated by spectrophotometer using either 90% aceton or N.N-dimethylformamide (DMF) as an extractant, and by HPLC using DMF, in intertidal sediment samples from the Seto Inland Sea, Japan. This was conducted both at the surface(0-0.5 cm) and subsurface(0.5-2 cm) layers and through the vertical profile of the sediments (each next cm to 10 cm depth). The Chl. a content determined using the three different procedures gave similar results at the surface. In contrast, the Chl. a content at the subsurface layer was 94.5±13.7 and 70.1±19.4% that found at the surface when spect ophotometrically determined using 90% aceton and DMF, respectively, while it was 47.7 ±16.9% when determined by HPLC. Such difference between procedures was consistent with depth. At the 6-7, 8-9 and 9-10 cm layers, Chl. a determined spectrophotometrically varied from 1.1 to 2.1 ?g g-1, while it was not detected by HPLC. These results indicate that the more practical spectrophotometric method can be reasonably employed to quantify the living fraction of microphytobenthic biomass in the surface layer of these sediments. At lower layers, only relying on either aceton or DMF extraction of pigments and spectrophotometric determination is likely to lead to an overestimation of the Chl. a content up to >50%. This can be related to the progressive increase and interference with depth of photosynthetic degradation products whose reliable estimates can only be obtained by chromatographic quantification.