Rates of fluorescein diacetate hydrolysis, phenol oxidation and potential denitrification in sand and gravel sediments from an agriculturally-impacted stream

Sediment samples were collected from the streambed in June 2015 using an AMS slide hammer with a 5 cm diameter and a trowel to collect sediment from between 0 and 10 cm depth. Sediment was collected from two sites and five pseudo-replicates were collected from each site, and subsequently homogenised and sieved (2 mm) within 36 hours of collection, then stored cold until the beginning of the experiments. Sediment was incubated in three separate incubation experiments to determine rates of FDA hydrolysis, extracellular phenol oxidase activity and potential denitrification. FDA hydrolysis was determined by mixing air-dried sediment with 1 M tris-hydroxymethyl-aminomethane (THAM) buffer and 0.5 ml of FDA substrate, closing the flask and incubating at 37 °C for 3 hours. 2 ml of acetone was then added to the sediment to prevent further reaction. The samples were filtered and fluorescein concentration was determined using a spectrophotometer (Varian Cary UV-Vis). The concentration of fluorescein in the control was subtracted from that in the sample, and then normalised by the mass of soil used for that sample, and the number of hours of the incubation. Extracellular phenol oxidase activity was determined by mixing dried sediment with deionised water, and then adding 10 mM dihydroxy-phenylalanine. The samples were then shaken at 25 °C for 30 minutes, before centrifuging to terminate the reaction. The solution was then filtered and dopachrome concentration was measured using a spectrophotometer (Varian Cary UV-Vis). The potential denitrification was determined by adding stock solution to field-moist sediment in dark bottles. The stock solution used was ultrapure water for control experiments, nitrate solution for nitrate-spiked experiments, glucose solution for carbon-spiked experiments and nitrate+glucose solution for nitrate-carbon-spiked solutions. Sample bottles were made anoxic, acetylene gas was added to prevent the conversion of N2O to N2, and samples were shaken during incubation at 22 °C. Gas samples were taken from the headspace at zero, three and six hours. N2O concentrations in the gas samples were determined on an Agilent 7890A gas chromatograph – micro electron detector. Concentrations were converted to production per mass of sediment per hour by normalising the concentration of fluorescein and dopachrome by the mass of sediment used and the length of the incubation period, and calculating the difference in N2O concentration between zero and six hours and normalising by the mass of sediment used and the length of the incubation period. Results from the experiments were entered into an Excel spreadsheet and exported as a comma separated value file (.csv) for ingestion into the EIDC.