Appendix F. Laboratory protocol for processing samples.
Nutrient loading rates were determined from differences between initial and post-experiment fertilizer weights (Wear et al. 1999). Undissolved fertilizer from each nutrient tube was placed in a Thelco model #17 drying oven for 72 h at 60°C to obtain a dry weight to the nearest 0.0001 g.
Ruppia samples were individually thawed and washed with filtered study site water in a 1.0-mm mesh sieve to remove extraneous sediment. The total number of Ruppia shoots was noted, and up to ten shoots were randomly selected from each core sample. Roots were excised from each shoot. The shoot was then spread on a cutting board so that leaf and stem material could be clearly distinguished. Epiphytic material was scraped from each of the shoots and collected in a covered watchglass containing 3 mL of filtered study site water. The epiphyte sample was then placed in a refrigerator (8°C) while the remaining Ruppia sample was processed.
To normalize epiphyte biomass, total surface area of the scraped Ruppia from each sample was determined. The length of each blade was measured to the nearest 0.01 mm using a dial calipers and then each blade was multiplied by its width, and then doubled. Shoot surface area comprised the accumulated surface areas for the individual blades. Total scraped SAV surface area (cm2) for each sample was the sum of individual shoot values. Pre-measured shoots were placed in a labeled pre-weighed aluminum pan, dried at 60°C for 72 h, and weighed to the nearest 0.0001 g with an Ohaus model AS120 analytical balance. Dried samples were then combusted in a Thermoline model 62700 muffle furnace for 5 h at 500°C, and then reweighed to obtain ash-free-dry-weights (AFDW) to the nearest 0.0001 g. Total biomass for core samples containing more than 10 shoots was determined by scaling per shoot AFDW values up to the actual number of recovered shoots.
Epiphyte suspensions were filtered on Whatman GF/F 42mm filters using a Fisher three-way filtration manifold powered by a General Electric 1/6 hp pump. Filters were folded in half and blotted with a paper towel to remove excess moisture, wrapped in aluminum foil, labeled, and frozen at -70° C, until processed using High Performance Liquid Chromatography (HPLC; Shimadzu system). Epiphyte chl [a] as a proxy of the amount of epiphytic algae was determined to the nearest 0.0001 ng using HPLC (Pinckney and Micheli 1998).
LITERATURE CITED
Pinckney, J. L., and F. Micheli. 1998. Microalgae on seagrass mimics: does epiphyte community structure differ from live seagrasses? Journal of Experimental Marine Biology and Ecology 221:59–70.
Wear, D. J., M. J. Sullivan, A. D. Moore, and D. F. Millie. 1999. Effects of water-column enrichment on the production dynamics of three seagrass species and their epiphytic algae. Marine Ecology Progress Series 179:201–13.