River phytoplankton growth responses to organic nutrients with associated nutrient data from 25 rivers in GB

Raw water samples were collected at each site on a single sampling occasion and kept at 4°C in the dark and transported to the laboratory within 48 hours. They were acclimatised in a constant temperature room at 20°C. Water was filtered through a 100 µm mesh to remove any zooplankton or large detritus present, 35 mL of sample was transferred to 50 mL boiling tubes. 12 different nutrient treatments were applied at approximately Redfield ratio [N: 90 µmol L-1 (1.261 mg N L-1) and P: 6 µmol L-1 (0.186 mg P L-1)] in triplicate to quantify growth responses to both organic N and P. These included: 1) a control with no addition of nutrients, 2) inorganic N only, 3) inorganic P only and 4) inorganic N and P, 5-8) four organic N additions and 9- 12) four organic P additions. Tubes were stoppered with foam bungs enabling gas exchange and incubated for 14 days at 20°C under a photon irradiance of 80 – 120 µmol m-2 s-1 (photosynthetically available radiation, Macam Q102) and an 18 h light, 6 h dark cycle. After the incubation period, tube contents including biofilm were resuspended by scraping and vortex mixing before being filtered onto a Whatman GF/C glass fibre filter (nominal pore size 1.2 µm) and frozen. Filters were later defrosted, and chlorophyll a extracted in hot methanol and measured spectrophotometrically. Growth response was determined as a change in the concentration of chlorophyll a in the treatment relative to the relevant control calculated as a natural log response ratio. For the water chemistry data (measured in mg/L), a Skalar++ multi-channel continuous flow autoanalyzer (Skalar Analytical B.V., The Netherlands) was used to carry out inorganic nutrient analysis, including total and total dissolved N and P (TN or TP, TDN or TDP) following persulphate digestion of unfiltered and filtered (pre-leached 0.45 µm cellulose nitrate) samples respectively, and dissolved inorganic N (DIN), including total ammoniacal nitrogen (NH3 and NH4), or soluble reactive P (SRP) on filtered samples. Dissolved organic nitrogen (DON) and dissolved organic phosphorus (DOP), were determined by difference between TDN and DIN or TDP and SRP, respectively.

Data from these analyses were checked and entered into spreadsheets for later analysis.