Incubation experiment: The experiment involved temperature-controlled batch incubations, conducted over 21 days, with sediment (15 g), algae (2 g, dry weight) and water (400 mL; MilliQ) placed in a 500 mL Pyrex glass bottle that was open to the atmosphere. Five temperatures (8, 14, 20, 26, 32oC) were selected, to bracket the environmental range of water temperatures in the Macleay River. Sediment was collected from the Macleay River (30°39'46.98"S; 151°54'36.05"E). Temperature was controlled via a water bath and water chiller in combination (+0.2oC; Grant, OLS200; Ratek, TH8000). Aqueous samples were collected at 24 h, 72 h, 7 d, 14 d and 21 d. During incubation, the Pyrex vessel was stationary, thereby allowing sediment and algae to settle at the bottom of the vessel and O2 to naturally diffuse from the atmosphere into the water column. At 1 hr prior to each sampling interval, the Pyrex vessel was sealed with a screwcap lid and inverted several times to thoroughly mix settled sediment / algae with the water column, followed by the screwcap lid being removed for the remainder of the hour. This approach of being open to the atmosphere combined with static conditions and occasional mixing (rather than a closed system with sediment-water homogenisation via constant stirring) was employed to mimic both diffusive and occasional re-suspension-induced interactions that may occur between hyporheic zone benthic sediments and the water column. Such interactions are expected in a low-flow, oxic yet eutrophic, river system comprised of pools and riffles.
Aqueous and gaseous analysis: At each sampling time (24 h, 72 h, 7 d, 14 d, 21 d), water temperature, dissolved oxygen (DO), pH and redox potential (corrected to standard hydrogen electrode) were measured in situ using HACH HQd portable meters and freshly calibrated probes. At each sampling time, a 35 mL aliquot of aqueous sample was filtered (0.45 m; enclosed syringe filter), preserved via acidification with HNO3- (to pH <1) and quantified for As, Sb, Fe, Mn and major cations (Na, Mg, Ca, K) via ICP-MS (Perkin-Elmer ELAN-DRCe). Detection limits for As and Sb were 0.15 and 0.10 µg L-1 respectively while duplicate analysis of 15% of samples were within +10% and +6%. Filtered aliquots were added to a 1,10-phenanthroline solution for determination of Fe2+. Total aqueous Fe was also determined by the 1,10- phenanthroline method after pre-reduction of Fe3+ by hydroxylamine hydrochloride. In addition, HCO3- was determined by the Bromophenol Blue method, while Cl- and SO42- were analysed via ion chromatography (Metrosep A Supp4-250 column, an RP2 guard column and eluent containing 2 mM NaHCO3, 2.4 mM Na2CO3 and 5% acetone, in conjunction with a Metrohm MSM module for background suppression).
Aqueous As speciation was determined by ion pairing chromatography (Wu and Sun, 2016) on filtered (0.45 µm; enclosed syringe filter) aliquots of water extracts that had been preserved with 10 µL of ultra-pure HCl and kept at 4 °C until subsequent speciation analysis that was conducted within 4–8 h of water extraction. In brief, the As speciation of aliquots was determined via high performance liquid chromatography (HPLC; Perkin-Elmer Flexar) coupled with inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer Nexion 350D), using a C18 (5 µm) column and mobile phase of 1 mM tetra-butyl ammonium hydroxide, 0.5 mM EDTA and 5% ethanol in de-ionized water (flow rate 1 mL min-1; 20 µL injection). Triplicate analysis on 12% of samples confirmed an analytical precision for As(III) within +6% and for As(V) within +10%. After aqueous sampling, at the 24 and 72 h intervals, the sediment-water incubation headspace was allowed to equilibrate for 30-60 min in the Pyrex vessel by enclosing the vessel with an airtight lid and the CO2 concentration was analysed via an calibrated cavity ring-down spectrometer (Picarro G2201-iCO2).
