## 3 Discussion
Both methods gave the same result: the active compound in the VOCs and the 8-nonenoic acid standard acted the same (Figures 2 and 3). This, to our understanding, proves that the identity of the emitted compound from ABp5 is 8-nonenoic acid. A concentration assay in liquid and solid media was performed to determine the most effective concentration for full pathogen inhibition. We were also interested in determining whether the state of the medium (liquid/solid) influences the compound's activity. Due to the fact that the growth radios of control treatment (_S. parasitica_ plug with only water and PDB) in liquid was smaller than control treatment in solid media, we had to normalize the results of _S. parasitica_'s growth radius from all other treatments by presenting the percentage of growth compered to control. This allowed us to compare the efficiency of 8-nonenoic acid's inhibition between solid and liquid. Since 8-nonenoic acid was discovered as a volatile, we initially tested its ability to inhibit _S. parasitica_ as a volatile and not by direct contact (as described in [PERSON] et al., 2020). Unexpectedly, 8-nonenoic acid did not inhibit _S. parasitica_ in the whole petri-plate area. Therefore, we tested its activity by adding the standard directly into the medium (Figure 4). We think that when this metabolite is added to a solid culture it evaporates to the upper air volume of the compartment. As a volatile, this molecule is active, but the concentration needs to be higher than that in direct contact in order to affect the pathogen. Although the compound's solubility in water is very limited (284 mg/L at 30 \({}^{\circ}\)C) it demonstrates activity at a much lower concentration in water than as a volatile or in solid culture. We do not know why this is the case, but it is not the first time that we have encountered this behavior of volatile metabolites [37]. We speculate it has to do with the molecule ability to move and migrate easier in the liquid media than in the agar matrix but was not demonstrated in our study. A simple in vivo bioassay was performed by exposing tilapia eggs to _S. parasitica_ in the presence or absence of 8-nonenoic acid. We chose to use tilapia as a model for infected eggs as they are easy to treat, relatively easy to infect with the pathogen and available at the hatchery in the department of fisheries at the Volcani Institute (Rishon LeZion, Israel). We found that 8-nonenoic acid at very low concentrations can control the pathogen and prevent disease development on tilapia eggs, while exhibiting no toxicity to the eggs or larvae, which grew and developed normally. We found a significant effect of the _S. parasitica_-inoculation treatment on the eggs' mortality compared to the non-inoculated eggs (control) or the eggs treated with 8-nonenoic acid only. This means that the inoculation of _S. parasitica_ was successful and that at 5 mg/L, 8-nonenoic acid is not lethal to the eggs. Moreover, addition of 5 mg/L 8-nonenoic acid to eggs infected by _S. parasitic_ significantly reduced egg mortality, meaning that it prevents infection of the eggs and enables them to develop normally (Figure 5). As a comparison [PERSON] 1992 reported the efficiency of Malachite green oxalate against _S. parasitic_ at the 0.25 mg/L in salmonid fish hatcheries [38]. Since 1983, Malachite green oxalate was banned for use in aquaculture and food related applications in many countries due to its classification as a Class II Health Hazard and evidence to its toxicity and carcinogenicity [39]. Other compounds were screened and tested in search after a safe competent control for _Saprolegnia_ spp. and other fish pathogens, among those bronopol (2-bromo-2-nitropropane-1,3-diol) was found effective at the ranges of 50-100 mg/L against _S. parasitic_[40], Formalin at the range of 100-400 mg/L, it is needs to be pointed that as with Malachite green formalin is currently not approved as a veterinary medicine for the treatment of live fish in most of the EU countries [39]. Additional compounds known to inhibit _Saprolegnia_ spp.