# Quantifying Moss Response to Metal Contaminant Exposure Using Laser-Induced Fluorescence
## 2 Materials and Methods
### Laboratory Treatment of Moss with Copper  
For the experiment, we selected _Thudiium plactile_, which is an endemic front-like moss species found in Hawai'i [28]. We collected moss samples from the same mat found along the Wa'ahila Ridge Trail and State Recreational Area (21.307\({}^{\circ}\), \(-\)157.797\({}^{\circ}\)) on 1 June 2020. The area is a relatively uncontaminated environment that can be found downstream of the Honolulu Watershed Forest Reserve, transitioning into a forest on the southeastern part of the Ko'olau mountain range. Samples were separated into four patches of moss, referred to as moss masses, and placed on trays covering an area of 316 cm\({}^{2}\) (7 in \(\times\) 7 in). The moss was then placed in a grow tent for a week before any experimental treatment to allow the samples to acclimate to their new environment. Growth conditions were kept at 50-60% relative humidity, an average temperature of 18-20 \({}^{\circ}\)C, and given 14-17 W/m\({}^{2}\) of ambient light (1400-1800 lux) with a day length of 10 h. Samples were only removed from the grow tent for about 0.5-1 h in total per day during the experiment for Cu treatment and image collection. In the literature, metal absorption and resulting physiological change in moss were studied using aqueous solutions of copper sulfate (CuSO4) or copper chloride (CuCl2) with concentrations between 0.05 and 2 mmol/m\({}^{2}\)[1,29,30,31]. However, these experiments often did not collect physiological observations of changes in moss over time, making it difficult to predict the behaviors of moss after exposure. We explored a full range of concentrations close to, but not reaching, those inducing death in the moss. Upper limits of Cu dosing were taken from commercial landscaping applications using CuSO4 in a solution of \(\sim\)1 to 5 mmol/m\({}^{2}\)[32], which has been shown to significantly inhibit growth. At the higher end of dosing levels, various moss species change color (browning), which can be used as a clear visual confirmation of physiological change. To test moss response, we used three treatment regimes, which from here on are referred to as trials, ranging from levels tested previously in laboratory experiments [29; 30; 31] up to the commercially used level. Cu concentrations were chosen to document the effects of a single dose versus reaching the same concentration level in multiple doses. The three trials used doses at levels of 0.5 mmol/m\({}^{2}\), 1 mmol/m\({}^{2}\), and 1.5 mmol/m\({}^{2}\), respectively. Within each trial, the respective doses were applied five times (every 48 h) to reach cumulative Cu concentrations of 2.5 mmol/m\({}^{2}\), 5 mmol/m\({}^{2}\), and 7.5 mmol/m\({}^{2}\). These concentrations are equivalent to 2.690 mmol/cm\({}^{2}\), 5.385 mmol/cm\({}^{2}\), and 8.075 mmol/cm\({}^{2}\) for the moss area of 316 cm\({}^{2}\). We administered Cu as CuCl\({}_{2}\) every odd day at 1/5 th of the cumulative Cu dose (0.538 mmol/cm\({}^{2}\), 1.077 mmol/cm\({}^{2}\), and 1.615 mmol/cm\({}^{2}\)) dissolved in 30 mL of de-ionized water, reaching the desired cumulative Cu concentration by the 10 th day of the experiment (Table 1). Trial 1 was a trap with a control moss mass. Every morning before imaging, 30 mL of de-ionized (DI) water was applied to the control trap, while the treatment trays received 30 mL of DI or DI + Cu. Application of solutions (DI or DI + Cu) was carried out using a spray bottle to simulate wet deposition via the atmosphere. The effectiveness of the Cu treatment was assessed by the direct measurement of Cu accumulation in moss. After imaging each day, 3 pairs of fronds (6 total per trial per day) were removed from three sections (left, center, and right) across the moss masses. The fronds were weighed and then leached using a sequential elution technique (SET) to extract metal from the surface of moss as well as its extra- and intracellularly bound Cu content [33; 34]. Front pairs were shaken in 10 mL of DI for 30 s to remove any unbound metals.