# Effects of Invasive Watermilfoil on Primary Production in Littoral Zones of North-Temperate Lakes
## Materials and Methods
### Bottle Production Estimates  
Production estimates for phytoplankton, epiphytes, and benthic periphyton were performed by placing collected primary producers suspended in lake water into 300-mL BOD (biological oxygen demand) bottles and sealing without any air bubbles. For each primary producer group, 3 bottles were filled to determine initial concentrations of dissolved gasses (hereafter called \"initial bottles\"). In addition, 3 to 5 pairs of bottles were filled; then, one in each pair was tightly wrapped in heavy- duty aluminum foil (hereafter, \"dark bottle\"), while the other was left unaltered (hereafter, \"light bottle\"). Initial bottles were sampled at the start of the incubation period, while light and dark bottles were suspended for in situ incubation from a horizontal bar at sample collection depth [49; 50]. Phytoplankton and epiphytes were suspended at a depth of 0.5 m below the surface. Benthic periphyton bottles were set on the bottom on hard lake bottoms, whereas on soft lake bottoms bottles were suspended 0.1 m above the bottom to prevent immersion in sediments. To account for production of phytoplankton in lake water used to suspend benthic periphyton in BOD bottles, we collected a second set of phytoplankton samples, hereafter referred to as \"blanks,\" that were incubated with benthic periphyton bottles. Incubation durations were based on pre-study trials to determine the optimal length that would allow detection of change in dissolved oxygen while avoiding large changes in internal bottle conditions. Based on the results of these trials, bottles with phytoplankton and blanks were incubated for 6-9 h, while bottles with epiphytes and benthic periphyton were incubated for 2-4 h.  
To measure dissolved gas concentrations in all BOD bottles, triplicate water samples were collected by siphoning into 12-mL Exetainers (Labco, Lampeter, Wales, UK) and preserving with zinc chloride (0.67 g L\({}^{-1}\) final concentration). Oxygen to argon ratios (O\({}_{2}\):Ar) from each Exetainer were determined using membrane inlet mass spectrometry (MIMS) [51], and triplicates were averaged to calculate mean O\({}_{2}\):Ar per bottle. For each primary producer, production rates were calculated as the change in O\({}_{2}\):Ar ratios from the average of all initial bottles to the end of the incubation period. Net primary production (NPP) was determined from the change in light bottles, and respiration (R) from the change in dark bottles. Calculations were adapted from [49], with the addition of Ar saturation values calculated from [52] using the water temperature and barometric pressure at the time of sample collection. All rates were scaled for collection area, determined as described above, and reported in mg O\({}_{2}\) m\({}^{-2}\) h\({}^{-1}\).  
For each primary producer, gross primary production (GPP) was calculated as mean NPP subtracted from the mean R. Benthic periphyton GPP rates were adjusted for phytoplankton in lake water by subtracting blank GPP rates, while epiphyte GPP rates were adjusted by subtracting phytoplankton GPP rates. Propagation of standard errors from these GPP calculations followed [53; 54].