Periphyton Zinc Exposure Culturing and Fractionation

Cobble substrate covered with periphytic algae was collected from a reach of Clear Creek (Jefferson County, Colorado, USA) which has high levels of Zn contamination from historical mining activities. In order to produce non-contaminated cultures used as control and acute exposure groups, substrate was placed in 1 cm of water containing Guillard's growth medium (Guillard 1975 ) fortified with 1.36 mM silicon using dissolved sodium meta-silicate 9-hydrate in 11.2 cm × 6 cm polyvinyl chloride (PVC) troughs. Solutions were renewed daily. Pumps (Rio 600, Taam Inc., Camarillo, California, USA) provided a recirculating flow of 757 L/hr. Periphytic algae was cultured on 6.25 cm², unglazed porcelain tiles (Cinca Tile Co., Fiães, Portugal). Tiles were arranged close together in culture trays to limit algal growth to the top surface, thereby producing uniform mats. Cultures received 12 h cycles of wide spectrum (Ecolux Plant & Aquarium Wide Spectrum, F40PL/AQ-ECO, General Electric Inc., Boston MA, USA) light. Culture tanks were positioned under two rows of the paired T12 florescent bulbs such that each tile was 120 cm and 150 cm (± 15 cm) from each pair of lights. Zn-cultured periphyton tiles (i.e., chronic exposure group) were prepared as above but with zinc sulfate (ZnSO₄) added to growth media of Zn-contaminated cultures to a concentration of 1600 µg/L in order to produce concentrations similar to those found in a survey of Colorado mountain streams (Schmidt et al. 2011 ). Surface area of culture tanks was scrubbed weekly, and a subset (20%) of tiles were replaced with new acid washed tiles to ensure periphytic algae had ample surface area to colonize. All cultures were maintained for three weeks prior to use in any exposure testing or analysis. Throughout the growth period, one water sample was taken from control and Zn treated troughs once every four days to measure aqueous Zn. Periphyton communities were dominated by Scenedesmus spp. (personal communication, Sarah Spaulding, United States Geological Survey, Boulder, Colorado, USA).
Immediately following the three-week growth period, a subset of tiles cultured in Zn (n = 4) and non-contaminated cultures (n = 4) were processed for subcellular fractionation to assess chronic exposure to Zn as well as growth in control conditions. Using a flow-through system, a subset of non-contaminated cultures was then bathed in 1600 µg/L Zn for either 15 min, 24 h, or 48 h to reflect episodic exposure likely to occur downstream of metal-contaminated landscapes as well as approximate common techniques used when preparing contaminated algae used in dietary toxicity trials (Irving et al. 2003 (link); Conley et al. 2009 (link); Xie et al. 2010 (link)). The flow-through system consisted of an 850 mL, circular artificial stream (Brinkman and Johnston 2008 (link)). Aqueous Zn samples were collected from the artificial stream at 0 min, 15 min, 24 h, and 48 h to ensure stable Zn exposure. Following acute exposure, four tiles from each group were processed for subcellular fractionation. All preparation methods/exposure regimes are listed in Table 1.Table 1

Exposure regimes/preparation methods used

Method	Description
Control	Cultured in non-contaminated Guillard’s growth media
Zn-cultured	Cultured in Guillard’s growth media contaminated with 1600 µg/L
15 min bathed	Cultured in non-contaminated Guillard’s growth media then bathed in 1600 µg/L for 15 min
24 h bathed	Cultured in non-contaminated Guillard’s growth media then bathed in 1600 µg/L for 24 h
48 h bathed	Cultured in non-contaminated Guillard’s growth media then bathed in 1600 µg/L for 48 h