A test of the ashߚfree dry weight technique on the developmental stages of Patiriellaspp. (Echinodermata: Asteroidea)

Abstract

Determination of the ashߚfree dry weight (AFDW) of marine specimens requires samples to be rinsed, soaked, and centrifuged. Problems associated with this technique were examined with the developmental stages of seastar species (Patiriella) with different modes of development. The influence of three rinsing solutions (ammonium formate [AF], filtered seawater [FSW], and reverse osmosis water [RO]) was assessed. The hypothesis that the AFDW technique is a measure of organic material was addressed by drying inorganic salts. Developmental stages of Patiriella calcar rinsed in FSW were twice as heavy as those rinsed in RO or AF, indicating that samples should be rinsed in RO or AF before weighing. Soaking treatments had a significant effect on the AFDW of samples of P. calcar (planktonic developer), indicating that the rinsing period should be brief. Zygotes of Patiriella regularis (planktonic developer) were significantly heavier than ova or gastrulae, regardless of treatment. In contrast, there were no significant differences in the AFDW of any stages or treatments of Patiriella exigua (benthic developer). This may be due to the presence of a modified fertilization envelope, which protects these benthic embryos. Inorganic salts with water of crystallization and FSW lost 20–75% and 14% of their dry weight, respectively, after ashing. We propose that salt ions may retain water, which does not evaporate during drying but is lost during ashing, resulting in the overestimation of sample AFDW. If a similar process occurs in the developmental stages of marine invertebrates, changes in the intracellular ionic composition through development may result in inaccurate estimates of biomass.

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“…Estimating AFDW is simple, but this method contains a number of potential sources of error. Sample preparation is one; salts are hygroscopic and, if not removed from the exterior of marine organisms prior to drying, can cause samples to gain weight after ashing (Moreno et al, 2001), leading to an underestimate of AFDW. This phenomenon can be minimized by rinsing marine samples with ammonium formate (isotonic with seawater) or in some cases with distilled water to remove surface salts.…”

“…This phenomenon can be minimized by rinsing marine samples with ammonium formate (isotonic with seawater) or in some cases with distilled water to remove surface salts. Disturbingly, though, Moreno et al (2001) found that some inorganic salts retain water at normal drying temperature (80°C) but lose it at ashing temperatures (450°C); thus, even in well-rinsed samples, water may be retained by intracellular salts in "dried" samples only to be burned away at 450°C, leading to an overestimate of AFDW. Finally, AFDW measurements do not capture the differing energetic values of lipids, carbohydrates, and proteins (Podolsky, 2002), so AFDW may not be suitable for comparing samples in which biochemical composition is likely to differ.…”