## 4 Discussion
The off-flavor concentrations in the inlet water of a fish farm often increase in the spring and summer due to increased microbial activity, typical for warmer seasons [65], although moderate concentrations of off-flavors have been observed in the winter [66; 67]. Concentrations detected in the inlet water could partly explain the increase of MIB in the water samples. Low concentrations of GSM were detected in fish muscle (400-500 ng/kg), likely remaining below the typical limit of sensory detection of 700-900 ng/kg for GSM and MIB [68; 69]. However, even lower sensory detection limits have been suggested (250 ng/ kg GSM) [70]. Despite the low accumulation of GSM, concentrations of 1200-1800 ng/kg MIB were detected, which were clearly above the sensory detection limit (700-900 ng/kg) [68; 69]. However, there were no statistically significant differences between the concentrations of MIB or GSM in RAS and aquaponics water, which suggests that spinach cannot be used to significantly improve water quality in aquaponic systems with respect to preventing off-flavor accumulation in water and fish. However, this does not rule out the possibility that some other plant species could be used for this purpose, which would warrant further investigation. Water uptake of plant roots proceeds via hydraulic conductance. The roots control the movements of water in the root-soil interface by specific transporter proteins on root-cell membranes, hydraulic conductivity, and cell-wall structure [71]. Compounds are transferred in a plant via hydraulic conductivity. Lipophilic molecules cannot move freely in an aqueous cellular environment [72], leading to lower transfer of lipophilic compounds in shoots, including GSM and MIB. Besides the uptake of water by the roots, it is possible that microbes on the root biofilm also produce off-flavor compounds. GSM is known to form in the roots of several root crops, including beet (_Beta vulgaris_) and spinach [73]. Although GSM and MIB are lipophilic compounds (K\({}_{\text{ow}}\) 3.57 for GSM and 3.31 for MIB), they are still sparsely soluble in water (solubility GSM 160 mg/L, MIB 305 mg/L) and therefore transferrable to the plant shoots in minor proportions [19]. This was supported by the results of this study, and higher concentrations of MIB (with higher solubility) were detected in the shoots. All this may explain the high concentrations observed in the roots and much lower concentrations in the shoots of spinach, especially in aquaponics. In this study, both GSM and MIB were found in the roots and shoots of spinach. This may suggest that bacteria producing the off-flavor compounds can occur or be absorbed in roots at high concentrations and be transported even to plant shoots. Several studies have examined differences in the bacterial composition between RAS and aquaponic systems with some key differences [74; 75]. [PERSON] et al. [27] reported substantially more bacterial diversity in the aquaponic system than in RAS and detected bacteria closely associated with plant roots, including Rhodobacterales, Rhizobiales and [PERSON] et al. [76] in _Lysobacter_sp. Additionally, [PERSON] et al. [27] detected _Streptomyces_ in RAS but not in the aquaponic system, and both GSM and MIB are known to be secondary metabolites of _Streptomyces_[77]. So far, the functional significance of GSM is unknown in bacteria and plants. [PERSON] and [PERSON] [73] studied GSM concentrations in beet and found 43000-17300 ng/kg. This was higher than the concentrations found in this experiment, although differences between different vegetables can be expected. [PERSON] et al. [78] detected GSM in spinach roots, but they did not quantify its content. [PERSON] et al. [27] detected bacteria _Streptomyces_ only in RAS reared largemouth bass (_Microterus salmonides_) but not in aquaponics with lemongrass and spring onion. _Streptomyces_ is known to be associated with off-flavor production, but [PERSON] et al. [27] did not measure the off-flavor concentrations.