## 4 Discussion  
Food-related silver studies have largely investigated the effect of AgNPs rather than ionic silver. Space exploration water systems may use immobilized AgClNPs to deliver ionic silver (Ag\({}^{+}\)) to spacecraft potable water at a dosage between 200 to 500 ppb (0.2-0.5 mg/L). This specified amount is due to the effective dose for microbial biocide activity as well as the need to maintain a high enough silver concentration given that silver can precipitate out of solution. Therefore, the final concentration of silver that will be dispensed from the potable water dispenser is currently unknown and may vary. The recommendation from the Environmental Protection Agency (EPA) is for the concentration of silver in drinking water to not exceed 0.10 milligrams per liter of water (100 ppb) because of the skin discoloration that may occur [25]. Here, a range of residual silver concentrations of 0 ppb, 31 ppb, 125 ppb, and 500 ppb were tested to encompass the highest potential dose in an exploration water system. Here it was investigated how Ag\({}^{+}\) biocide irrigation water affected plants grown in both a substrate-based arcillite setup and a substrate-less hydroponics setup. Being a clay material, arcillite has a high cation-exchange capacity for silver capture. In a hydroponic setup with very little cation ion exchange surface, there is  
Figure 8: (**A**) Venn diagram illustrating the significantly enriched pathways for the silver-treated roots of the lettuce hydroponics samples as compared to the 0 ppb silver root control and the overlap of the 3 treatment comparisons. (**B**) The log\({}_{2}\)FC of the thirteen enriched pathways specific to the 125 ppb silver-treated roots are plotted on the x-axis and the \(-\)log\({}_{10}\) (\(p\)-values) of the 125 ppb silver-treated roots are plotted on the y-axis. Also represented on this plot are the log\({}_{2}\)FC values of the thirteen overlapping pathways also enriched for the 500 ppb silver-treated roots. This is represented as a shade between red or blue and the \(-\)log10 (\(p\)-values) represented by the size of the circle. (**C**) The log\({}_{2}\)FC of the twelve enriched pathways specific to the 500 ppb silver-treated roots are plotted on the x-axis and the \(-\)log\({}_{10}\) (\(p\)-values) of the 500 ppb silver-treated roots are plotted on the y-axis. little opportunity for the positively charged silver ion to precipitate out of the solution onto a substrate, such as in the negatively charged arcillite clay particles. Therefore, we hypothesized that the hydroponically grown plants would be more affected by silver content due to the direct interaction of the seed and root surfaces with the silver in the water. Visual observation showed that lettuce grown in arcillite under all three silver water treatments germinated at an equal rate and supported biomass growth on a scale equal to that of the control. The growth metrics of plant height, plant area, shoot fresh mass, leaf area, leaf number, and shoot dry mass supported this observation with none of the metrics from the silver-treated plants significantly different from the control plants. However, lettuce grown in a hydroponic setup with silver showed slower germination and severely stunted growth under 125 ppb and 500 ppb treatments and with variable growth defects at 31 ppb. The same growth metrics as above support this observation. A previous study evaluating lettuce exposure to AgNO\({}_{3}\) observed stunted growth at as low as 0.01 mg/L (10 ppb) in a hydroponics system [12]. In this study, anthocyanin content increased in the 125 ppb and 500 ppb silver treatments and this translated visually to a high red pigmentation in the smaller plants of the hydroponics experiments. Prior work in Arabidopsis has also observed this dose-dependent increase in anthocyanin accumulation as an antioxidant response under silver stress [33].