Study area and sample collection

Ten wild adult brown trout samples (> 10 cm TL) were collected in June 2018 from a headwater tributary of Flumendosa River (Ermolinus Stream, 39°52ʹN, 9°23ʹE) located in the Montarbu Forest (Sardinia, Italy) (Fig. 1). The number of fish was determined following the indications in the USEPA Guidelines (USEPA, 2000).

Rio Ermolinus, Montarbu Forest, and anthropogenic pressure classes in Sardina, Italy

The stream is characterized by clear, well-oxygenated water, a moderately fast current, and the presence of waterfalls jumps, riffles runs, and pools. Moreover, the stream bed consists of sand, gravel, stones, and carbonate rocks. The carbonated part of the stream is characterized by travertine deposition (calcium carbonate) that occurs in various forms, cementing substrate particles in small dams. The abiotic parameters, water temperature, pH, water dissolved oxygen concentration, and water conductivity were recorded using a multi-parameter probe (In Situ Inc. Smart Troll MP). Trout fish were captured using low-frequency, pulsed DC electrofishing. Stunned fishes were immediately killed by spiking the brain (WOAH, 2019), held in iceboxes and transported to the laboratory. Finally, samples were frozen until analysis.