# Chemical Compositional Changes in Over-Oxidized Fish Oils
## Discussion
### Hoki Liver Oil Over-Oxidation
Other small differences in experimental conditions may cause relatively large differences over the long and sustained oxidation period, but their influence is not possible to estimate. Differences in oxygen tension in the oil might also be considered to be a factor, since the rate of lipid peroxidation depends on oxygen tension, as shown for rapeseed oil [62]. Air-saturated water at 20\({}^{\circ}\)C contains an oxygen concentration of \(\approx\)0.29 mM [63], whereas at full saturation, dissolved oxygen is \(\approx\)1.1 mM. The solubility of oxygen in marine triglyceride fish oils is in the range of 1.22-2.44 mmol/kg oil at 20 \({}^{\circ}\)C, and it is expected to be higher at 50 \({}^{\circ}\)C under Condition B [64]. With continuous oxygen delivery to the oils, oxygen tension is likely maximal, suggesting that a small deviation in the precise rate of continuous delivery of oxygen gas to the oils should not be expected to affect the oxidation rate. It is clear that a very high level of oxidation was achieved in the hoki liver oil by the photooxidative conditions at high oxygen tension after one month (condition A). The attained TOTOX number of 296 is \(\approx\)11 times larger than the maximum limit of 26 set voluntarily by industry and with which many fish oil producers and retailers comply [18]. With the employed antioxidant-free hoki liver oil, this limit was exceeded already within 1 day of incubation. Furthermore, the marked rise in oligomers indicates that the oil was severely damaged, exceeding the pharmacopeial monograph limit of 1.5% that is considered acceptable for refined EPA/DHA omega-3 triglyceride oils [65]. The present study also confirms that massive oxidative damage to an oil, exceeding regulatory quality limits many-fold, is needed to achieve a relatively modest 10% reduction in EPA and DHA content in an unprotected oil, as exemplified by the hoki liver oil in this study. It has previously been shown that the minor oxidative changes that can occur during normal shelf-life conditions of fish oil finished products do not lead to measurable decreases in EPA and DHA content [19]. The content of saturated and monounsaturated fatty acids was not affected, which was expected, since these fatty acids are insensitive, or much less sensitive, to oxidation than PUFA. Other LCPUFA, such as AA, DPA n-3, DPA n-6, and AdA, did not display a decrease in content, but small amounts were nevertheless converted by lipid peroxidation to the corresponding isoprostanoids. The sustained oxidation of oils and biological membranes rich in PUFA is understood to occur in three stages: initiation, propagation, and termination [13; 66]. Initiation involves the formation of fatty acid peroxyl radicals, propagation involves an unimpeded chain reaction of further free radical-mediated lipid peroxidation, and termination forms nonradical products. Once formed, a fatty acid peroxyl radical can abstract a bis-allylic hydrogen atom from another PUFA, thereby forming the corresponding fatty acid hydroperoxide, the major primary oxidation product, and a new PUFA radical. The hydroperoxides can further react with radicals to form alkoxyl radicals or fragment by chain scission, leading to the formation of an array of hydroxylated derivatives, saturated and unsaturated aldehydes, ketones, and short-chain hydrocarbons, which are collectively called secondary oxidation products. The acid value is sometimes used as an indicator for oxidative quality in fish oils. However, the absence of detectable changes in acid value did not support the hydrolysis of esterified fatty acids to increased free unesterified fatty acids to occur under the experimental conditions. Possibly, the low water content in the oils precluded changes in the acid value. However, hoki liver oil had a three-fold higher baseline acid value (1.49 \(\pm\) 0.01 mg KOH/g oil) than the anchovy oil (0.49 \(\pm\) 0.00 mg KOH/g oil), which may be attributable to a higher level of free fatty acids.