Is Aquaponics Beneficial in Terms of Fish and Plant Growth and Water Quality in Comparison to Separate Recirculating Aquaculture and Hydroponic Systems?

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Is Aquaponics Beneficial in Terms of Fish and Plant Growth and Water Quality in Comparison to Separate Recirculating Aquaculture and Hydroponic Systems? © 2022 the Authors Published version Atique, Faiqa; Lindholm-Lehto, Petra; Pirhonen, Juhani Atique, F., Lindholm-Lehto, P., & Pirhonen, J. (2022). Is Aquaponics Beneficial in Terms of Fish and Plant Growth and Water Quality in Comparison to Separate Recirculating Aquaculture and Hydroponic Systems?. Water, 14(9), Article 1447. https://doi.org/10.3390/w14091447 2022 Citation: Atique, F.; Lindholm-Lehto, P.; Pirhonen, J. Is Aquaponics Beneficial in Terms of Fish and Plant Growth and Water Quality in Comparison to Separate Recirculating Aquaculture and Hydroponic Systems? Water 2022,14, 1447. https://doi.org/ 10.3390/w14091447 Academic Editor: Tamas Komives Received: 4 April 2022 Accepted: 26 April 2022 Published: 30 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). water Article Is Aquaponics Beneficial in Terms of Fish and Plant Growth and Water Quality in Comparison to Separate Recirculating Aquaculture and Hydroponic Systems? Faiqa Atique 1,2, Petra Lindholm-Lehto 3,* and Juhani Pirhonen 1 1Department of Biological and Environmental Science, University of Jyvaskyla, P.O. Box 35, FI-40014 Jyvaskyla, Finland; [email protected] (F.A.); [email protected] (J.P.) 2Institute of Bioeconomy, JAMK University of Applied Sciences, Tuumalantie 17, FI-43130 Tarvaala, Finland 3 Aquatic Production Systems, Natural Resources Institute Finland, Survontie 9A, FI-40500 Jyvaskyla, Finland *Correspondence: [email protected] Abstract: Aquaponics is a technique where a recirculating aquaculture system (RAS) and hydroponics are integrated to grow plants and fish in a closed system. We investigated if the growth of rainbow trout (Oncorhynchus mykiss) and baby spinach (Spinacia oleracea) would be affected in a coupled aquaponic system compared to the growth of the fish in RAS or plants in a hydroponic system, all systems as three replicates. We also investigated the possible effects of plants on the onset of nitrification in biofilters and on the concentration of off-flavor-causing agents geosmin (GSM) and 2-methylisoborneol (MIB) in rainbow trout flesh and spinach. For the fish grown in aquaponics, the weight gain and specific growth rates were higher, and the feed conversion ratio was lower than those grown in RAS. In spinach, there were no significant differences in growth between aquaponic and hydroponic treatments. The concentration of GSM was significantly higher in the roots and MIB in the shoots of spinach grown in aquaponics than in hydroponics. In fish, the concentrations of MIB did not differ, but the concentrations of GSM were lower in aquaponics than in RAS. The onset of nitrification was faster in the aquaponic system than in RAS. In conclusion, spinach grew equally well in aquaponics and hydroponic systems. However, the aquaponic system was better than RAS in terms of onset of nitrification, fish growth, and lower concentrations of GSM in fish flesh. Keywords: biological filtration; integrated aquaculture; muscle lipids; off-flavors; salmonids; soilless culture 1. Introduction Partly due to the tightened demands for environmental permissions, especially in the land-based aquaculture, recirculating aquaculture systems (RAS) are gaining popularity in producing fish for human consumption. The main advantage of RAS is highly decreased water use compared to traditional flow-through systems. Consequently, the nutrients released by the cultured animals are highly concentrated in the limited amount of effluent, which can offer cost-efficient opportunities for nutrient reuse and wastewater treatment [ 1 ]. In RAS, the maintenance of the microbial environment in biofilters is essential because the microbes responsible for nitrification convert harmful ammonia excreted by fish, first to nitrite and then to nitrate [ 2 ]. Exposure of fish to even low concentrations of ammonia and nitrite can be harmful and affect the fish welfare and survival, while nitrate is a rather safe compound for the fish at concentrations <100 mg/L [ 3 ]. The start-up of the nitrification process using intact biofilter media can take up to two months [ 2 ], after which the levels of ammonia and nitrite should remain at levels that are safe for fish [ 1 ]. Several studies have been conducted to increase the efficiency and speed up the onset of nitrification in RAS [ 2 , 4 , 5 ]. For example, the nitrification efficiency in RAS has been studied by investigating the biofilter configuration and relationship between the heterotrophic and Water 2022,14, 1447. https://doi.org/10.3390/w14091447 https://www.mdpi.com/journal/water Water 2022,14, 1447 2 of 18 nitrifying bacteria, nitrification efficiency of the submerged biological filter, total ammonia nitrogen (TAN) concentrations and varying C/N ratios [ 4 ], biofilter media types and their effects on the efficiency of trickling filters [ 6 , 7 ] and the effects of the design of the biofilter on the oxidation of ammonia [2]. Hydroponics refers to the soilless cultivation of plants where the nutrients for the plant’s growth are provided in a solution [ 8 ], and the plants get the nutrients from the water instead of soil [ 9 ]. Hydroponics is an efficient method for producing vegetables with minimal water and space [ 10 , 11 ]. Aquaponics refers to a system where RAS and hydroponics have been combined, and the RAS effluent with concentrated nutrients is utilized to grow plants [ 12 , 13 ]. The ammonia excreted by the fish is converted in the biofilter to nitrate, which is easily absorbed by the plants [ 14 ]. The fish feed contains macroand micronutrients essential for fish growth but that are also important for the plants [ 15 ]. While absorbing the nutrients from the RAS wastewater, the plants also clean the water from compounds potentially harmful to the fish due to low water exchange [ 15 ]. However, plants differ in their demand for nutrients, and their availability in RAS effluent may not be enough for all plant species. To cope with this situation and provide enough nutrients for the plant’s growth, some nutrients can be provided in a solution [ 12 ]. Aquaponics has been regarded as a sustainable and environmentally-friendly method for producing plants and fish [ 12 , 13 ]. In addition, it supports the idea of a circular economy as the wastes produced by fish are turned into a resource for the plants. The presence of bacteria like Cyanobacteria, Actinomycetes, and Myxobacteria in RAS can produce off-flavor compounds geosmin (GSM) and 2-methylisoborneol (MIB) [ 16 – 18 ] which easily accumulate in fish flesh and cause earthy and musty flavor. GSM and MIB are semi-volatile terpenoid compounds that accumulate in the lipid-rich tissues of fish. The main route of uptake is through the gills, but also via the skin and gastrointestinal tract, and the uptake proceeds fast, typically within hours [ 18 – 20 ]. The concentrations of GSM and MIB in fish flesh seek equilibrium with their concentrations in water. However, factors such as water temperature and flow rate, fish age, size, and species, along with the exposure time, have been shown to affect their concentrations [ 18 , 19 , 21 , 22 ]. The removal of the off-flavor compounds from fish flesh is essential for it to be marketable. Depuration in clean water has been proved to be the only reliable method for off-flavor removal. Unfortunately, the removal of off-flavors is a slow process, and even in the optimal conditions, it can take from days to weeks [ 17 , 19 , 22 ]. The off-flavor compounds are typically removed by keeping the fish without feed in flow-through tanks until no off-flavor can be perceived by organoleptic testing. Other approaches have been examined to decrease the off-flavors in water and in fish, and reduce the time of depuration. These approaches include addition of peracetic acid and hydrogen peroxide (H 2 O 2 ) [ 23 ], and the ozonation of circulating water or depuration water [18,24,25], and photocatalysis [26]. Due to the increasing demand for sustainable food production, including eco-friendly seafood and vegetables for the growing human population, more research is needed to understand the potential benefits of aquaponic systems. One of the problems with RAS is the long start-up time for a fully functioning biofilter. It appears that no attention has been paid to the possibility of using plants to shorten the duration of the onset of the nitrification process in RAS or to buffer the sharp increase of ammonia and nitrite caused by the maturing biofilter. On the other hand, in our unpublished organoleptic tests, rainbow trout (Oncorhynchus mykiss) reared in an aquaponic system tasted rather normal as compared to those reared in RAS, which possessed a very strong muddy taste. This suggests that the plants could potentially be used as absorbers of compounds causing off-flavors in fish, and bacteria from the genus Streptomyces have been found to be absent in aquaponics but not in RAS [27]. Consequently, our study hypothesized (1) that the onset of the nitrification process is faster in the aquaponics treatment compared to RAS, (2) that rainbow trout grown in an integrated system with baby spinach (Spinacia oleracea) have lower concentrations of off-flavor compounds compared to those reared in RAS, (3) that the plants in an aquaponic Water 2022,14, 1447 3 of 18 system contain a higher concentration of GSM and MIB than plants grown in hydroponics, and (4) that the plants and fish grown in an aquaponic system grow equally well than in hydroponics and RAS, respectively. 2. Materials and Methods 2.1. 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 deepwater 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 × L1 × D0.35 m) were made from high-density polyethylene containers, and the rafts were made of extruded polystyrene foam (XPS) Styrodur ® 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 ◦ 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 ◦ 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. The water quality was recorded for total ammonia nitrogen (TAN), nitrite, nitrate (API ® Freshwater master test kits, Mars Fish Care Inc, Chalfont, PA, USA), pH, temperature (Digital PH/Temp Meter AD 12, ADWA instruments, Szeged, Hungary) and oxygen saturation (ExStik ® DO600 dissolved oxygen, Extech, Water 2022,14, 1447 4 of 18 Waltham, MA, USA ). In the DWC, the humidity was checked with a humidity meter (Prego, Helsinki, Finland). For the hydroponic plants, Substral ® (Transmeri Ltd., Espoo, Finland) nutrient solution was used. The Substral solution was prepared according to the manufacturer’s instructions (7 mL of Substral in 6 L of water), i.e., 408 mL of Substral was added to 350 L of water for each hydroponic DWC. This solution was added once in two weeks in hydroponics DWC when compensating for the evaporated water. The hydroponic plants were sprayed with the Substral solution (approximately 1 mL of Substral in 1.5 L of water) every day during the experiment, excluding the first week. Plants were also sprayed with water every day, excluding the first week. For the aquaponic plants, modified micronutrients solution (Fe, B, Zn, Mo) and potassium were added in the form of a solution prepared by dissolving salts of Fe (NO 3 ) 3× 9 H 2 O (101.2 g), Mn (NO 3 ) 2× 4 H 2 O (36.52 g), Zn (NO 3 ) 2× 6 H 2 O (2.7368 g), Na 2 MoO 4× 2 H 2 O (0.3533 g), K 2 B 4 O 7× 4 H 2 O (28.26 g) in 1 L water [ 28 ]. This nutrient solution (10 to 15 mL) was added into the aquaponic system whenever water was added to the system and whenever plants showed any deficiency symptoms such as a change in leaf color or growth. The plants were also sprayed with water and this nutrient solution (1 mL in 1.5 L) every day, excluding the first week. 2.2. Sampling The start point samples of spinach were taken just before the transplantation of spinach seedlings to the aquaponics system (5 May). The start point samples of fish were taken at the time of fish stocking (4 May). The endpoint samples were taken after six weeks on the day of the harvest of fish and spinach on (14–15 June). For the measurement of change in spinach biomass, 15 seedlings were sampled in the beginning, while at the end of the experiment, 20 plants were sampled from each DWC. The length and dry weight of the shoots and roots were recorded separately. The plants were dried at 60 ◦ C for 72 h. For the GSM and MIB analyses, six fresh spinach seedlings were taken at the start, and three fresh spinach plants at the end from each tray, shoots, and roots were separated, cut into small pieces, and mixed into one homogeneous sample, i.e., one sample for each tray. The dry matter content of spinach was determined by the ISO 638:2008 standard method. The final samples from spinach shoots from each DWC were also analyzed for macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Fe, Cu, Mn, Zn, B) at Eurofins Agroscience Services, Mikkeli, Finland. B, Ca, Cu, Fe, K, Mg, Mn, P, and Zn were measured with an ICP-OES method as reported by Eurofins. Nitrogen was determined with Kjeldahl-method while sulfur with ICP-OES method. Limits of detection (LOD) and limits of quantification (LOQ) for each nutrient are given in Supplementary Table S1. For estimating the fish growth, the fish were weighed in the beginning (in batches) and at the end (individually) of the experiment. For the measurement of lipid content and off-flavors (GSM and MIB) in the fish muscle, three randomly selected individuals were sampled in the beginning. At the end, three individuals were sampled from each fish tank, i.e., nine fish per treatment. The sampled fish were killed with a sharp blow on the head, gutted, and filleted. From the lateral part of the fillet [ 29 ], 500 mg of muscle was taken from each fish, and the three samples from each tank were pooled. Water samples (500 mL) were taken from each DWC, each fish tank, and tap water at the beginning and the end of the experiment for the analysis of the off-flavor compounds (GSM and MIB) and anions (chloride, phosphate, sulfate, and nitrate, and nitrite). All samples were stored at − 20 ◦ C before the analyses. 2.3. Off-Flavor Analyses The off-flavor-inducing compounds GSM (trans-1, 10–dimethyl–trans-9–decalol) and MIB (1-R-exo-1,2,7,7-tetramethyl-bicyclo [2.2.1] heptan-2-ol) were quantified by the method reported in Lindholm-Lehto [ 30 ]. In short, the sample extraction was performed by an automated SPME procedure (PAL3 autosampler, CTC Analytics, Zwingen, Switzerland) with an Water 2022,14, 1447 5 of 18 SPME Arrow fiber made of DVB/carbon WR/PDMS (divinylbenzene/carboxene/polydime thyl siloxane). The pretreatment cycle included mixing, heating, adsorption and desorption of analytes, injection into the GC port, and conditioning of the fiber. The samples were analyzed by a GC-QQQ (7000 Series Triple Quadrupole mass spectrometer, Agilent, Santa Clara, CA, USA). It was operated with a Phenomenex Zebron ZB-5MSi (Torrance, CA, USA) capillary column (30 m × 0.25 mm × 0.25 µ m) for the separation and with an electron ionization (EI) ion source, and MassHunter 10.0 software. The detection was performed in multiple reaction monitoring (MRM) mode. Levels of quantification (LOQ)s were (0.2 ng/L GSM; 0.4 ng/L MIB) for aqueous and (65 ng/kg GSM; 107 ng/kg MIB) for solid samples. The full method description and validation have been reported in [30]. 2.4. Lipid Content The total fat content was determined by the accredited in-house method JOK3008 which is based on AOAC Official Methods 920.39 (Fat (Crude) or ether extract in animal feed and) and 954.02 (Fat (crude) or ether extract in pet food; Association of Official Analytical Chemists, USA) and AACC method 30–25 (Crude fat in wheat, corn, and soy flour, feeds, and mixed feeds; Approved Methods of the American Association of Cereal Chemists, USA). The used equipment was Foss Soxtec/Hydrotec 8000 ™ System for total fat analysis, consisting of Soxtec ™ 8000 extraction unit and Hydrotec ™ hydrolysis unit (FOSS Analytical, Hillerød, Denmark). The test laboratory in Jokioinen, belonging to the Natural Resources Finland, holds FINAS (Finnish Accreditation Service) accreditation number T024 and follows the standard SFS-EN ISO/IEC 17025:2017. Muscle lipid contents have been reported as g/kg wet weight (ww). 2.5. Anions Anion chloride (Cl − ), nitrite-N (NO 2− ), nitrate-N (NO 3− ), sulfate (SO 43− ), and phosphate (PO 34− ) were studied from the water samples taken at the end of the experiment. The pretreatment of samples by solid-phase extraction (SPE) has previously been reported in Lindholm-Lehto et al. [ 30 , 31 ]. The chromatographic analysis was conducted on Thermo Scientific Dionex Integrion HPIC ion chromatography equipment (Dionex, Sunnyvale, CA, USA) with the Cromeleon 7.2 software. The equipment consisted of a gradient pump (0–6000 psi), eluent generator (EDC 500 KOH), a guard column Dionex IonPacTM NG1 ( 2×50 mm ), a pre-column (Dionex IonPacTM AG19 (2 × 50 mm–4 µ m), and an analytical column Dionex IonPacTM AS-19 (2 × 250 mm–4 µ m at 30 ◦ C). The full description of the analysis method and validation data have been reported by Lindholm-Lehto et al. [ 30 ]. The LODs ranged between 0.018–0.131 mg/L and LOQs from 0.020 mg/L to 0.175 mg/L (Supplementary Table S2). 2.6. Calculations and Statistical Analyses The specific growth rate (SGR) for each fish tank was calculated as Ln (W2) − Ln (W1) × 100/t, where W1 and W2 are the tank’s average fish weights (g) in the beginning and at the end of the experiment, and t is the experimental period in days (42 d). Feed conversion ratio (FCR) was calculated as the weight of feed eaten (kg)/fish weight gain (kg). For analyzing the spinach biomass, dry weights were recorded at the start and the end of the experiment. Shoot and root lengths were recorded for each plant at the end of the experiment. The total individual plant weight (g) on each raft was calculated using the total end dry weight (root + shoot). The starting dry weight of spinach seedlings ( 0.003 ±0.0005 ,n= 3) was negligible, and therefore biomass change during the experiment was not calculated separately. Statistical analyses were run with IBM SPSS Statistics 26. Independent samples t-test was used to compare the means between treatments for fish and plant data analysis. The mean concentrations of macronutrients (g/kg) and micronutrients (mg/kg) in spinach shoots were also compared between the treatments by the independent samples t-test. Water 2022,14, 1447 6 of 18 Homogeneity of variance was checked by Levene’s test. The variances of the means of all variables were equal. The observational unit was always the tank or tray (i.e., n= 3). The daily means of ammonia (TAN), nitrite, nitrate, and pH were compared between treatments by repeated measures ANOVA (n= 3). Mauchly’s test of sphericity p-value was always < 0.15; thus, the Greenhouse-Geisser adjustment was applied. The selected anions (chloride, nitrite-N, nitrate-N, sulfate, and phosphate) were analyzed at the end of the experiment and compared between treatments by repeated measures ANOVA ( n= 3 ). A Huynh–Feldt adjustment was applied because Mauchly’s test of sphericity p-value was always one. The values for nitrate were Ln transformed before the statistical analysis. The values for nitrite were zero on the start and end day of the experiment and were not included in the analysis. The MIB and GSM in spinach shoots and roots and lipid content in fish muscle between treatments were analyzed by independent t-test, while the start values were compared with the end values by one sample t-test. For assessing MIB and GSM in water samples and fish muscles repeated measures ANOVA was performed. A Huynh–Feldt adjustment was applied because Mauchly’s test of sphericity p-value was always one. 3. Results 3.1. Fish Performance and Plant Growth During the experiment, one fish died in one of the RAS tanks, but in aquaponics, there was no mortality. The SGR of the fish was significantly higher in aquaponics ( 1.95 ±0.12 ) than in RAS (1.67 ± 0.08) (Table 1). The FCR in aquaponics was significantly lower ( 0.85 ±0.08 ) than in RAS (1.06 ± 0.03) (Table 1). Weight gain was significantly higher for the fish grown in aquaponics than in RAS. Total feed consumed by individual fish did not differ between the treatments (Table 1). Table 1. Initial and final wet weight, fish weight gain, specific growth rate (SGR), feed consumed, and feed conversion ratio (FCR) of rainbow trout (Oncorhynchus mykiss), grown in RAS and aquaponic systems for 42 days. In the aquaponics treatment rainbow trout was grown in a coupled aquaponic system with spinach (Spinacia oleracea). RAS Aquaponics Sig. Initial weight (g) 107.7 ±6.42 108.2 ±1.26 ns Final weight (g) 217.0 ±7.24 245.3 ±10.32 ns Fish weight gain (g) 109.3 ±3.05 137.1 ±11.29 * SGR 1.67 ±0.08 1.95 ±0.12 * Feed consumed (g/fish) 112.0 ±0.03 110.0 ±0.01 ns FCR 1.06 ±0.03 0.86 ±0.08 * Values are means ± SD, n= 3. Statistical difference (Sig.) in the values between aquaponics and RAS treatments is shown by an asterisk * (p < 0.05), ns = not significant. The mean dry weights for shoot, root, total dry weights, shoot to root ratio, mean shoot length, and root length of spinach were not significantly different between aquaponics and hydroponics treatments (Table 2). 3.2. Spinach Nutrient Analysis The concentrations of macronutrients N (p< 0.005), P ( p< 0.05 ), S ( p< 0.05 ), and K ( p< 0.05 ) were significantly higher in hydroponically grown spinach while Ca ( p< 0.0001 ) and Mg (p< 0.005) were significantly higher in spinach grown in aquaponics. The micronutrients Fe (p< 0.05), Zn (p< 0.05), and B (p< 0.0001) were significantly higher in spinach grown in the aquaponics than in hydroponics, while Cu and Mn were at similar level in both systems (Table 3). Water 2022,14, 1447 7 of 18 Table 2. Dry weights for shoots and roots, plant total dry weight, shoot and root length, and shoot to root ratio for weight and length of spinach (Spinacia oleracea) grown in hydroponic and aquaponic system for 42 days. In the aquaponics treatment spinach was grown in a coupled aquaponics system together with rainbow trout (Oncorhynchus mykiss). Hydroponics Aquaponics Shoot weight (g) 0.88 ±0.27 1.23 ±0.34 Root weight (g) 0.18 ±0.08 0.30 ±0.16 Total weight (g) 1.07 ±0.29 1.53 ±0.48 Shoot length (cm) 12.15 ±1.32 14.50 ±1.69 Root length (cm) 29.23 ±4.63 37.77 ±5.73 Shoot to root ratio weight 5.54 ±2.77 4.50 ±1.46 Shoot to root ratio length 0.44 ±0.02 0.40 ±0.03 Values are means ± SD of one plant at the end of the experiment from three replicated rafts, n= 3, average start weight for total weight = 0.003 ± 0.0005. There were no statistically significant differences between the treatments. Table 3. Micronutrients (mg/kg) Fe, Cu, Mn, Zn, B and macronutrients (g/kg) N, P, K, Ca, Mg, S in spinach (Spinacia oleracea) shoots grown in hydroponic and aquaponic system for 42 days. For aquaponics treatment spinach was grown in a coupled aquaponics system together with rainbow trout (Oncorhynchus mykiss). Aquaponics Hydroponics Sig. Fe (mg/kg) 523.3 ±75.05 143.3 ±15.25 * Cu (mg/kg) 37.30 ±12.70 49.30 ±9.60 ns Mn (mg/kg) 403.3 ±40.41 366.6 ±246.84 ns Zn (mg/kg) 526.6 ±142.9 206.6 ±55.07 * B (mg/kg) 120.0 ±0.00 32.30 ±8.08 * N (g/kg) 38.70 ±3.00 57.50 ±2.61 * P (g/kg) 6.06 ±1.10 9.40 ±1.55 * K (g/kg) 64.60 ±5.68 83.30 ±8.96 * Ca (g/kg) 36.60 ±3.51 7.26 ±0.35 * Mg (g/kg) 16.60 ±1.52 5.80 ±1.01 * S (g/kg) 3.56 ±0.41 5.63 ±0.47 * Values are means ± SD from three replicated rafts (n= 3). Statistical difference (Sig.) in the values between aquaponics and hydroponics treatments is shown by an asterisk * (p < 0.05), ns = not significant. 3.3. Onset of Nitrification The mean concentration of total ammonia nitrogen (TAN) varied over days (p< 0.05) but not between treatments while the mean concentrations of nitrite (p< 0.0001), nitrate (p< 0.0001) and pH (p < 0.0001) differed significantly between treatments and over days. The maximum TAN concentration in the aquaponic treatment (2.00 ± 0.00 mg/L, n= 3) was reached on day 6 and it gradually decreased to zero by day 11. In RAS the maximum TAN (2.67 ± 0.58 mg/L, n= 3) was reached on day 9, and it decreased to 0 by day 18 (Figure 1a). From day 18 the concentration of TAN stayed at nearly zero in both treatments until the end of the experiment. The mean nitrite concentration decreased close to zero in the aquaponics treatment on day 11 while it took 39 days in RAS treatment (Figure 1b). The highest mean nitrite concentrations were recorded (4.83 ± 0.28 mg/L, n= 3) in aquaponics on day 7 but on day 11 in RAS treatment (Figure 1b). During the experiment, the highest mean nitrate concentration was recorded on day 9 in aquaponics (81.67 ± 2.88 mg/L, n= 3) while on day 14 (80 mg/L) in RAS treatment (Figure 1c). The mean concentration of the nitrate followed a gradual decline and stayed lower in aquaponics compared to RAS treatment during the experiment until day 39 but became almost equal on day 42 (Figure 1c). The pH of the circulating water was significantly different between the treatments over the course of the experiment (p< 0.0001), and it gradually decreased during the experiment. The mean daily pH in the RAS treatment varied between 7.79 ± 0.00 (n= 3) and 6.43 ± 0.05 (n= 3) while in aquaponics it varied between 7.76 ±0.05 (n= 3) and 6.83 ±0.05 (n= 3) (Figure 1d). Water 2022,14, 1447 8 of 18 Water 2022, 14, x FOR PEER REVIEW 8 of 18 aquaponics treatment on day 11 while it took 39 days in RAS treatment (Figure 1b). The highest mean nitrite concentrations were recorded (4.83 ± 0.28 mg/L, n = 3) in aquaponics on day 7 but on day 11 in RAS treatment (Figure 1b). (a) (b) (c) (d) Figure 1. The mean concentration (mg/L) ± SD (n = 3) of (a) total ammonium nitrogen (TAN) (b) nitrite, (c) nitrate, and (d) pH in RAS and aquaponics treatments during the 42-day experiment. For the aquaponics treatment spinach was grown in a coupled aquaponics system together with rainbow trout (Oncorhynchus mykiss). During the experiment, the highest mean nitrate concentration was recorded on day 9 in aquaponics (81.67 ± 2.88 mg/L, n = 3) while on day 14 (80 mg/L) in RAS treatment (Figure 1c). The mean concentration of the nitrate followed a gradual decline and stayed lower in aquaponics compared to RAS treatment during the experiment until day 39 but became almost equal on day 42 (Figure 1c). The pH of the circulating water was significantly different between the treatments over the course of the experiment (p < 0.0001), and it gradually decreased during the experiment. The mean daily pH in the RAS treatment varied between 7.79 ± 0.00 (n = 3) and 6.43 ± 0.05 (n = 3) while in aquaponics it varied between 7.76 ± 0.05 (n = 3) and 6.83 ± 0.05 (n = 3) (Figure 1d). 3.4. Water Quality Selected anions (chloride, nitrite-N, nitrate-N, sulfate, and phosphate) were analyzed and quantified at the end of the experiment. Additionally, the concentrations in tap water were analyzed containing 7.7 mg/L Cl−, 0.21 mg/L NO3-N, 0.75 mg/L SO42-, and below limits of detections for NO2-N and PO43− (Supplementary Table S2). There was no significant difference (p > 0.05) in the concentrations (mg/L) of chloride, phosphate, sulfate, or nitrate between hydroponics and aquaponics treatments (Table 4). Figure 1. The mean concentration (mg/L) ± SD (n= 3) of ( a ) total ammonium nitrogen (TAN) (b) nitrite , ( c ) nitrate, and ( d ) pH in RAS and aquaponics treatments during the 42-day experiment. For the aquaponics treatment spinach was grown in a coupled aquaponics system together with rainbow trout (Oncorhynchus mykiss). 3.4. Water Quality Selected anions (chloride, nitrite-N, nitrate-N, sulfate, and phosphate) were analyzed and quantified at the end of the experiment. Additionally, the concentrations in tap water were analyzed containing 7.7 mg/L Cl − , 0.21 mg/L NO 3 -N, 0.75 mg/L SO 42− , and below limits of detections for NO 2 -N and PO 43− (Supplementary Table S2). There was no significant difference (p> 0.05) in the concentrations (mg/L) of chloride, phosphate, sulfate, or nitrate between hydroponics and aquaponics treatments (Table 4). Table 4. Concentrations of chloride Cl − , nitrate-N NO 3 -N, sulfate SO 42− , and phosphate PO43−(mg/L) in water samples taken on the last day (day 42) of the experiment in aquaponics and hydroponics deep water culture units. For aquaponics treatment spinach (Spinacia oleracea) was grown in a coupled aquaponic system together with rainbow trout (Oncorhynchus mykiss). Element (mg/L) Aquaponics Hydroponics Chloride 24.80 ±14.68 14.24 ±6.19 Phosphate 0.10 ±0.11 8.07 ±6.20 Sulfate 69.06 ±49.74 85.32 ±57.03 Nitrate-N 3.44 ±1.96 4.51 ±2.74 Values are means ± SD, n= 3. There were no statistically significant differences between the treatments (independent sample ttest, p> 0.05). Nitrite-N was below the LOD (0.13 mg/L) in both treatments. The concentration (mg/L) of chloride was higher (p< 0.05) in the aquaponics circulating water than in RAS water, but the concentration of other anions did not differ between the treatments (Table 5). Water 2022,14, 1447 15 of 18 Acknowledgments: We would like to thank Sisä-Suomen kalatalousryhmä and JAMK University of applied Sciences for covering the cost of aquaponics facility construction, facility management and providing facility for the experimentation and all kind of assistance during the project. We will extend our thanks to Juha Ahonen for transporting the fish, Markku Paananen and Janne Ruokolainen for their support and encouragement throughout the project. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Timmons, M.B.; Guredat, T.; Vinci, B.J. Recirculating Aquaculture, 4th ed.; Ithaca Publishing Company LLC: Ithaca, NY, USA, 2018. 2. Pulkkinen, J.T.; Eriksson-Kallio, A.M.; Aalto, S.L.; Tiirola, M.; Koskela, J.; Kiuru, T.; Vielma, J. 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