Hiking up to the next plot…
Used unglazed ceramic tiles (5 × 5 cm) as substrata. In 2016, tiles placed in East River for 12 days to allow colonization by D. geminata and other microorganisms. Channels conditioned by flowing stream water for 6 days, then rocks from adjacent streambed placed at head of each channel for 6-day period and slurry of diatoms scraped from biofilm-covered stream rocks poured into recirculating holding tanks.
Quantities: 5 × 5 cm ceramic tiles, colonization period of 12 days in river, 6 days channel conditioningDuration: 12 days colonization, 6 days conditioning, 6 days with source rocksConditions: Natural stream water flow
Mesocosm cell inoculation
In 2017, three channels from each treatment initially given single dose of 100 μg P l⁻¹ for 1 h before experiment began. Rather than colonizing tiles by in situ stream incubation, slurry of D. geminata cells added to each channel and allowed to incubate overnight resulting in initial density of 300 cells/cm². All channels run as recirculating for 24 h to ensure even colonization.
Quantities: 100 μg P l⁻¹ dose for 1 hour, initial density of 300 cells/cm², 24 h recirculation periodDuration: 1 h phosphorus dose, overnight incubation, 24 h recirculationConditions: Controlled laboratory conditions
Mesocosm sampling for biological parameters
Three times weekly, one tile from upstream portion and one from downstream portion of each channel removed, scraped with razor blade, and scrubbed with nylon brush to remove all cells and stalk material. Resulting slurry preserved in Lugol's iodine and counted at later date. D. geminata density enumerated using compound microscope and Palmer cell.
Quantities: 2 tiles per channel, 3 times weekly sampling, 12 channels totalDuration: Three times weekly throughout experimentConditions: Laboratory processing conditions
Equipment: razor blade, nylon brush, Lugol's iodine, compound microscope, Palmer cell
Mesocosm stalk measurements
D. geminata stalks were enumerated under dissecting microscope at × 40 magnification. Total length and number of tips of stalks counted. Stalk width measured on three stalks from each channel on each date. Biovolume calculated assuming equal branching within stalks and cylindrical shape using formula v = πr²Ls.
Quantities: 3 stalks measured per channel per date, magnification × 40Duration: Each sampling dateConditions: Laboratory conditions under dissecting microscope
Equipment: dissecting microscope
Water chemistry analysis
Nutrient samples filtered through Pall Gelman AE filters and placed on ice. SRP analyzed using MAGIC method with detection limit 0.04 μg l⁻¹. Ammonium measured using fluorometric method with detection limit 0.4 μg l⁻¹. Nitrate measured using second derivative method. Additional trace nutrients analyzed on specific days.
Quantities: Weekly sampling in 2016, three times weekly in 2017, detection limits: SRP 0.04 μg l⁻¹, ammonium 0.4 μg l⁻¹Duration: Weekly to three times weekly depending on yearConditions: Samples kept on ice until analysis
Equipment: Pall Gelman AE filters, Turner Trilogy Fluorometer, Thermo Scientific GENESYS 10S UV-VIS Spectrophotometer