The Synthetic Antioxidant Ethoxyquin and its Effect on Fish Products

Content

1.Abstract

2.Introduction

3.Chemical structure

4.Mechanism of Ethoxyquin as an antioxidant

5.Toxicity

6.Regulations and requirements

7.Limits of ethoxyquin residuals in human food

8.Natural alternatives

9.Discussion

10.Conclusion

11.References

Abbreviations

1.Abstract

The synthetic antioxidant Ethoxyquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) is widely used, specifically in the fishmeal industry to prevent the fishmeal from lipid oxidation. New implemented regulations of EU do not allow to incorporate any traces of EQ on feed in some countries within the Europe. But in many countries, it is used in some spices like rosemary and chilli to preserve their color. The toxicity of EQ is the major problem which creates some harmful effects on human and animal when animal are fed with the feed containing EQ. At the same time human are affected indirectly by other means. Here we tried to present the physical and chemical properties of EQ, its working mechanism as an antioxidant in fishmeal, positive and negative attributes of EQ on feed, feed ingredients, human and animals, regulations and requirements of incorporation of EQ on different feed, reauthorization part, replacement of EQ by some alternative natural and/or synthetic compounds as well.

2.Introduction

Three major synthetic antioxidants are used in food industry especially in processed feed such as fishmeal. These are Ethoxyquin (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) (EQ), Butylated Hydroxytoluene (2,6-di-tert-butyl-p-cresol) (BHT) and Butylated Hydroxyanisole (tert-butyl-4-hydroxyanisole) (BHA). Other synthetic antioxidants such as octylgallate and propylgallate have no significant role, even though among all the synthetic antioxidants, ethoxyquin is mostly used because of its low production costs and higher antioxidative effect (Alina Blaszoczyk et al. 2013). Ethoxyquin was initially developed in the rubber industry to prevent cracking of rubber while manufacturing due to the formation of isoprene (A.J.de Koning, 2002).

Autoxidation of lipids can occur spontaneously in a reaction with oxygen from the surrounding atmosphere oxidizing a lipid creating an alkyl radical as seen in the first equation in figure 2. The following two steps lead to a chain reaction where more alkyl radicals are formed.

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Figure 1. Synthetic antioxidants used to prevent oxidation in fishmeal

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Figure 2. Mechanisms of lipid autoxidation. (Márquez-Ruiz et al. 2012)

The longer the carbon-chain in poly unsaturated fatty acids (PUFAs) and if the double bonds are in a cis-configuration rather than trans-configuration the more prone they are to oxidation. (Márquez-Ruiz et al. 2012)

PUFAs are highly unsaturated hence they contain a lot of bis-allylic C-H bonds. Because of the double bonds on the carbon chains these bonds are weaker than allylic C-H bonds in saturated fatty acids. The bond dissociation energy (BDE) is 65 kcal/mol in PUFAs compared to 100 kcal/mol in saturated fatty acids.

Fish oil is highly unsaturated hence it has a tendency to undergo autoxidation. Autoxidation decreases energy- and nutritional value is decreased. So the stabilization of long chain polyunsaturated fatty acids (PUFA) (contains Omega-3 (Eicosapentaenoic acid) and Omega-6 (Docosahexaenoic acid) with ethoxyquin helps to maintain all the fatty acid characteristics of the fishmeal in a transportation and storage point of view. There is a probability to get overheated and spontaneously combust when fishmeal is stored due to heat produced during lipid autoxidation. (Olcott et. al., 1962)). Stabilization of fishmeal is an integral part to avoid spontaneous combustion all through overseas transport and storage by the addition of between 400 and 1000 mg kg-[1] ethoxyquin, or of between 1000 and 4000 mg kg-[1] butylated hydroxytoluene at the period of production according to the International Maritime Organisation (IMO). This application should take place not more than 12 months prior to shipment, and the antioxidant application in fishmeal must be at least 100 mg kg-[1] at the time of shipment to preclude explosions (IMO 2003).

The autoxidation rate is also affected by the preprocessing history of the meal. For instance, when a poor quality raw material is used, then a faster oxidation can be seen. In many food systems, it is found that heme and nonheme iron promote or catalyse autoxidation. (Rhee, 1978, Toyoda et al., 1982) Iron containing fish processing equipment are contacted fish meat part enhances lipid oxidation. (Lee et al., 1977, Silberstein et al., 1978). Ethoxyquin is the most common antioxidant used in fishmeal which is more efficient to prevent lipid oxidation and methionine oxidation rather than other antioxidants. (Gulbrandsen et al., 1983).

3.Chemical structure

The molecular structure of ethoxyquin is a quinoline which is having a 1,2-dihydroquinoline bearing three methyl substituents at location 2, 2 and 4. In addition to that there is an ethoxy substituent at location 6. (6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) The molecule is polar and the boiling and melting points are 123-125 °C and 0°C respectively (Blaszoczyk et al. 2013).

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Figure 3 Chemical structure of ethoxyquin (EQ) and of some new compounds synthesized on ethoxyquin backbone with promising antioxidant properties (Blaszczyk et al. 2013).

4.Mechanism of Ethoxyquin as an antioxidant

The antioxidant capacity of the molecule ethoxyquin lies within the ability of the N-H group to scavenge radicals. The bond dissociation energy (BDE) is the enthalpy it takes to cleave the homolytic bonds (Berton-Carabin 2014). The higher the value of BDE, the better the ability to scavenge radicals. The BDE of the N-H bond in EQ is 84 kcal mol-[1] in water and 78 kcal mol-[1] in gas phase. (Najafi et al. 2013)

The arylamines and secondary alkyl groups in ethoxyquin has antioxidant capacities but furthermore, it forms an oxidation product, an aminyl radical, which also have antioxidative properties when oxidized. (Scott, 1985), (Berger et al., 1983), (Adamic et al., 1969), (Adamic et al., 1970),(Olcott et al., 1970), Brownlie et al., 1967). A simplified reaction mechanism of the formation of the antioxidative products of EQ-oxidation can be seen in figure 4. These oxidation products lead to the electron accepting chain-breaking antioxidants.

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Figure 4. A schematic drawing of the radical scavenging by ethoxyquin and the products that also have antioxidative properties. Made from the reactions described by Blaszczyk et al. (2013)

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Figure 5. An illustration of a possible inhibition mechanism by a secondary aryl-amine (Berger et al., 1983), (Denisov et al., 1980)

Besides this reaction mechanism, a radical addition on the aromatic amine radical is also possible. The product of this reaction is the quinone-imine compound.(Adamic et al., 1969). Formation of quinone-imine compound is shown in Figure 3).

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Figure 6. Quinone-imine formation (Adamic et al., 1969)

It has been observed that after oxidation of phenolic compounds, there are some oxidation products, that have stronger oxidation effects. These compounds are formed by the abstraction of hydrogen from the phenol or from different quinones like vitamin K, tocopherylquinone, rosmaryquinone or benzoquinone as shown in Figure 6. (Scott et al., 1985), (Lindsey et al., 1985), (Houlihan et al., 1985).

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Figure 7. Different Quinone compounds, those are responsible for oxidation and producing oxidative compounds(Scott et al., 1985), (Lindsey et al., 1985), (Houlihan et al., 1985).

5.Toxicity

Table 1: Effects of ethoxyquin detected after its oral intake in various animals or in humans (Contact exposure) (Alina Blaszczyk et al. 2013).

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The adverse effects of ethoxyquin was first reported in 1988, where dogs are the most susceptible to the detrimental effects of EQ which was reported by FDA Observed symptoms were dysfunction of the kidney, liver, reproductive system and the thyroid gland system (Dzanis D. A. 1991). Major symptoms like kidney and liver damage, alimentary duct alterations and weight loss are observed when animals are treated with a dosis of EQ above the maximum permitted concentration (Table 1).

Reyes et al. (Reyes J. L . et al., 1995) and Hernandez et al. (Hernandez M. E. et al., 1993) point out that EQ interaction on biosystem is dose dependent, they found inhibition of renal sodium, potassium ions and ATPase activity involved in ion transport when they conducted experiment in bovine kidney and heart. (Hernandez M. E. et al., 1993). Experiments conducted in different strains of Salmonella typhimurium to assess the mutagenicity by using Ames test gave some positive results. (Rannug A. et al., 1984) & (Reddy B. S. et al., 1983). It was observed in animals exposed to EQ that i might promote or inhibit carcinogenic activity of known carcinogens (Alina Blaszczyk et al. 2013). Programmed cell death called apoptosis or necrosis by the effects of EQ may further damage genetic material such as DNA and chromosomes (Alina Blaszczyk et al. 2013). Apoptosis in lymphocytes while in-vitro culture was observed by Blaszczyk (Blaszczyk et al., 2005). Overall, serious biological consequences such as chromosome aberrations were observed in Chinese hamster ovary cells and human lymphocytes (Rabbitts T. H., 1994).

Some case studies reveal that the use of EQ on animal feed has both direct and indirect impact on human on a larger scale. Van Hecke (Van Hecke, 1977) and Savini et al., (1989) studied that people handling EQ had an itchy, scaly, erythematous dermatitis with irregular oozing eruptions. Zachariae (1978) found that people handling EQ was suffering from exfoliative dermatitis after ten days of EQ exposure. (Brandao 1983), (Burrows 1975) and (Wood and Fulton 1975) point out that some workers handling feed material with EQ, began suffering from chronic and acute dermatitis which spread throughout their body parts.

Table 2 Effects of ethoxyquin intake of rats (Arthur. D. 1990)

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6.Regulations and requirements

According to International Maritime Organisation (IMO), stabilization of fishmeal is an integral part to avoid spontaneous combustion all through overseas transport and storage by the addition of between 400 and 1000 mg kg-[1] ethoxyquin, or of between 1000 and 4000 mg kg-[1] butylated hydroxytoluene since ethoxyquin has the ability to stabilize the long chain polyunsaturated fatty acids in the fishmeal to prevent them from oxidizing. The stabilization process was initiated in 1970s. Almost 66% of world’s fishmeal production was preserved using ethoxyquin.

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