2.5.1 Total Cd and Cu content in moss and soil
We dried moss tissue from all populations and treatments at 50 ºC, including field samples, and ground them to a fine powder in a tissue lyser (n=3 replicates per sample). Then, we digested between 1-17 mg of plant tissue in small Teflon vessels with 2 ml of HNO3(100%, TraceMetal Grade, Fisher Chemical) in a hot plate at 175ºC. After 2 h, we added 0.8 ml of H2O2 (ACS grade) to each sample and incubated them again for 2h at 175ºC. We also prepared one sample of certified reference material (M2,Pleurozium schreberi moss tissue, Steinnes, Rühling, Lippo, & Makinen, 1997) in the same way, and analyzed it along with analytical blanks after every 12 samples to control for instrumental precision and contamination.
Similarly, we dried soil samples from the field (n=3 replicates per sample), sieved them to separate the 2 mm fraction, and ground them to a fine powder before analysis. Between 9-30 mg of dried soil were digested in small Teflon vessels with 2 ml of HNO3 (100%) for 8h at 175ºC. This acid dissolves only a very small fraction of structural minerals in the soil recovering mainly the most labile/reactive fraction of heavy metals (Melo, Batista, Gilkes, & Rate, 2016). We used one certified reference material prepared in the same way as the samples, once every 12 samples, to control for instrumental precision (marine sediment PACS-2, National Research Council of Canada).
Total contents of Cd and Cu were determined in both matrices at the College of Marine Sciences (University of South Florida) by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Agilent 7500cx). The percentage recovery averaged 82% and 86% for Cu in plant and soil respectively, and 102% and 131% for Cd in plant and soil respectively.