## 2 Materials and Methods  
### Experimental Setup  
A 42-day experiment was conducted from 4 May to 14 June 2021 at the Tarvaala Bioeconomy campus of the JAMK University of Applied Science, Finland, where three replicated RAS, aquaponic, and hydroponic systems were set up (3 + 3 + 3) in an industrial hall without temperature control. In the RAS and aquaponic systems, each of the six fish tanks was stocked with 20 rainbow trout of c. 90-110 g on 4 May, purchased from a RAS farm (Finnforel Ltd., Varkaus, Finland). Two hundred and fifty ml of filter starter (Easystart, Easy-Life International BV, Duiven, Netherlands) was added to each biofilter tank one week before (27 April) and six days after (10 May) the fish stocking. Each of the six deep-water culture (DWC) rafts (three rafts for aquaponics and hydroponics) were transplanted with 25 baby spinach plants on 5 May. Spinach seeds were germinated and grown in a greenhouse of the University of Jyvaskyla for three weeks before transplantation. The DWC tanks (W1 \(\times\) L1 \(\times\) D0.35 m) were made from high-density polyethylene containers, and the rafts were made of extruded polystyrene foam (XPS) Styrodur(r) with 25 drilled holes for 5 cm hydroponic pots filled with expanded clay. Each DWC was continuously aerated through air stones. In DWCs, the air temperature ranged from 15 to 20 \({}^{\circ}\)C. Light was provided to plants with LED lights (Kinwua bright, 215-watt, light intensity c. 1000 lux) for 16 h per day, and the scattering light from the DWCs provided illumination for the fish tanks which did not have separate lamps. Each of the six dual-drain fish tanks (500 L) was connected to a settling tank (500 L), bead filter (SuperBead small, Air-aqua BV, Staphorst, Netherlands, filled with 37.5 kg of beads), a moving bed biofilter filled with 300 L helix floating bio media (Sibo Fluidra, Doornhoek, Netherlands), and a UV light (AquaForte UV-C lamp 18 watt, Sibo Fluidra, Doornhoek, Netherlands). In the aquaponic systems, water was pumped from the DWC back to the fish tanks (i.e., coupled aquaponics). The oxygen saturation in the fish tanks was maintained at 80-85% throughout the experiment using air pumps and air stones. The water temperature depended on the hall temperature and increased during the experiment from 12 to 19 \({}^{\circ}\)C due to the lack of a temperature controller. The RAS and aquaponics water exchanges in the fish tank with tap water were done using the following percentages at each water change: first week 50% four times, second week 20-30% four times, third and fourth week 10% three times. No water was changed in the fifth week, and in the sixth week, 40-50% water of the system was changed twice in RAS while 20 to 30% in aquaponics. An equal amount of water was changed from RAS and aquaponics treatments (except week 6) which meant relatively more water change in RAS because the water volume for aquaponics was bigger (RAS + DWC). The fish were fed with dry pellets (EFICO Enviro 923 Advance 4.5 mm, Biomar, Brande, Denmark). According to the manufacturer, crude protein and fat contents of the diet were 43% and 51%, respectively. The fish were fed by hand twice per day for the first week and thereafter with automats three times per day. Feed intake was monitored every day, and the quantity of feed was changed depending upon the uneaten amount of feed on the tank bottom. Uneaten pellets were siphoned out of the tanks and counted. The number of uneaten pellets was converted to the weight of dry feed, knowing that 14 dry pellets equaled 1.00 g. The amount of daily feed intake was calculated as the difference between the fed and uneaten feed. The fish were not fed on the day of the harvest. The water quality in fish tanks was recorded daily during week one and 3-4 times a week from week two to onward.