## 4 Discussion
Given the significant effect the silver treatments had on the growth metrics of the hydroponic-grown lettuce plants, the uptake of silver by the lettuce plants was investigated next. No silver was detected in the leaves of lettuce grown in the substrate-based arcillite system; however, silver was detected in the substrate-less hydroponic setup in the 31 ppb, 125 ppb, and 500 ppb silver water treatments. These amounts resulted in 7 ug/g dry mass (7000 ppb) under 125 ppb treatment and 4 ug/g dry mass (4000 ppb) silver under 500 ppb treatment. In food items, the European Chemicals Agency (ECHA) has established an acceptable daily intake (ADI) of 0.9 ug silver ions/kg (0.9 ppb) of body weight per day [34]. According to this guideline and given that 0.2 g of shoot dry biomass (or 5 g of shoot fresh biomass) yields an average of 1.1 ug of silver, a 50 kg individual could consume up to \(\sim\)40 of the silver stunted plants without exceeding this daily limit. Yet these plants were so severely stunted that it is impractical to recommend growing them hydroponically with such levels of silver in the water. Elemental analysis of the control and silver-treated plants in the hydroponic setup showed an inverse relationship between silver uptake and the uptake of calcium, potassium, iron, phosphorus, and sulfur in the lettuce leaves, as previously observed in radial [14]. The stunted growth of the lettuce plants demonstrates how toxic silver can be in an unbuffered hydroponic system. The effect on the lettuce microbiome was also assessed for both the arcillite and hydroponic systems in response to increasing concentrations of silver in the irrigation water. It was observed that the alpha diversity, or diversity within each sample, was similar for the root control and silver-treated root samples in the substrate-based arcillite setup. The root samples from the arcillite setup had an overall higher alpha diversity than the roots from the hydroponic setup. The shoots from the substrate-based arcillite setup and the substrate-less hydroponics setup had the lowest diversity when compared to the diversity of the microbes associated with roots and the irrigation water from both setups. Furthermore, the hydroponic and arcillite shoot samples reflected the microbial genera that were most abundant in root samples from the respective silver treatments. The irrigation water for the substrate-less hydroponics setup had a higher diversity in the control samples than in the three silver water treatments. Among the three biocide concentrations, the alpha diversity is highest at the 500 ppb silver concentration. Taking a closer look at the roots treated with the three silver water concentrations of 31 ppb, 125 ppb, and 500 ppb in the hydroponics setup, the alpha diversity is lowest in the control and progressively increases as the silver concentration increases. Similarly, the alpha diversity increases in the hydroponic water trays over time for all silver treatments. This trend persisted despite weekly water changeouts of the water trays done to deliver fresh biocideand nutrients. The trend signifies that silver has a microbial biocide effect in the hydroponic water tray but is also selecting for potentially resistant microbes over time. Little is currently known regarding the microbial diversity and response to ionic silver exposure in hydroponic systems. The observed increase in microbial diversity for the plant roots treated with increasing silver concentrations stood out in this dataset because the existing literature on heavy metal contamination in the soil environment reports a decrease in microbial diversity of the root zone microbiome upon exposure [35]. More studies are necessary to understand the intersecting trends between environmentally distinct soil-based outdoor agriculture and indoor controlled environment agriculture.