Analysis of EPA + DHA and omega3/omega6 ratio in 17 edible wild fish species in Bahrain

Abstract Fish are an important part of the Mediterranean diet, which recommends consuming more fish (at least twice a week) than meat (on a monthly basis). Over the past decade there has been a significant promotion of fish intake because of its high-quality protein content, low fat content, and because it is a good source of vitamins and minerals, but overall because it contains large amounts of omega 3 polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The aim of this study is to determine the fatty acid contents of ten edible wild fish species sold at the Bahrain market along with twelve previously studied edible wild fish species in Bahrain to find out the best of them in terms of EPA, DHA, and n-3/n-6 ratio. Ten edible wild fish species in which their fatty acid contents were not previously studied were selected from the Bahrain market. Lipid extraction and fatty acid methylation were performed. Gas chromatography analysis was conducted to determine the fatty acid compositions of the selected fish species. The result showed significant differences (p < 0.05) in the fatty acid compositions among all ten fish species studied. Edible wild fish species from the present study were given numbers (number 1 is the one with the best fatty acid contents, whereas number 17 is the one with poorest fatty acid contents) based on the sum of EPA and DHA and the n-3/n-6 ratio. The number one fish based on EPA + DHA was Doubleber Bream (Faskir) 26.63 ± 0.30%, while the lowest sum of EPA + DHA was observed in White Sardinella (Oom) 5.45 ± 0.35%. On the other hand, fish with the highest n-3/n-6 ratio is Doubleber Bream (Faskir) with a ratio of 2.62 ± 0.29%, whereas Haffara Bream (Gorgofan) has the lowest ratio 0.60 ± 1.45%. The sum of EPA and DHA and the n-3/n-6 ratio of the 17 fish species from Bahrain were compared with fish species that have high amounts of omega-3 around the world and fish species from the Arabian Gulf Sea and the Mediterranean Sea based on the sum of EPA + DHA and n-3/n-6 ratio. The result has shown that EPA + DHA and the n-3/n-6 ratio were similar to those from the Arabian Gulf Sea but lower in the fish species in Bahrain compared to the Mediterranean Sea and those with high amounts of omega-3 around the world. In conclusion, consuming only wild fish species from Bahrain will not be adequate to gain the recommended daily consumption of 500-1000 mg EPA + DHA.

Omega-3 precursor alpha-linolenic acid (ALA) and omega-6 precursor linoleic acid (LA) are two important types of essential polyunsaturated fatty acids (EPUFAs) that cannot be synthesized by humans in sufficient quantities and must be supplied from the diet (Kruse et al., 2020).
EPA and DHA omega-3 PUFAs are found mostly in fatty fish like salmon and sardines and in other marine organisms like algae, while the n-3 precursor ALA is found primarily in chia seeds, walnuts, rapeseed, and flaxseeds oils.Similarly, beef, poultry, and eggs are good sources of the omega-6 (x-6, n-6) PUFAs arachidonic acid (AA), but the omega-6 precursor LA is mostly found in sunflower, soybean, and corn oils (Sanchez et al., 2020;Santos, Price, & Bueno, 2020).
Several studies have investigated the effect of the omega-3 and omega-6 ratio on human health.The two families of omegas (omega-6 and omega-3) are necessary for a person's regular growth and development.During the evolution, the intake of omega-6 and omega-3 was balanced with a ratio of omega-3 to omega-6 is 1:3.Currently, in Western civilizations, the ratio is roughly 1:16 omega-3/omega-6 due to the high consumption of vegetable oil such as soybean, safflower, corn oil and linseed oil (Feng et al., 2015;Sanchez et al., 2020;Simopoulos, 2011).As a result, omega-3 fatty acids will be deficient, and an increase in the amount of omega-6 fatty acids, which have a significant impact on human health and are linked to several disorders, including heart disease, cancer, inflammatory diseases, and autoimmune diseases (Sanchez et al., 2020;Simopoulos,1999;Simopoulos & Cleland, 2003;Simopoulos & Ordovas, 2004).
This study aims to determine the fatty acid contents of ten edible wild fish species in Bahrain and compare them with previously studied fish species in Bahrain in order to find out the best fish species in Bahrain based to their EPA, DHA, and omega-3/omega-6 ratio.

Sample preparation
A total of 10 fish species that were not studied before (Diamond Mullets, Dory Snapper, Blackfin Mojarra, Talung Quenfish, Blue Barred Parrot, Haffara Bream, Spotted Half Beak, Peraly Goat, Black Banded Trevally, and Cephalopholis Argus) were selected from the edible wild fish available at the Kingdom of Bahrain local markets (Table 1).Wild fish samples were purchased separately and freshly from local fish markets in Bahrain during November-December 2021.The collected fish samples were placed in an icebox and transported immediately to the laboratory.Fish length (cm) and weight (g) were measured (Table 1).Fish samples were washed with distilled water and dissected.The two sides' fish fillets were homogenized and prepared for immediate further analysis.Five specimens were used from each fish species, and two replicas from each fish were prepared.

Lipid extraction
Lipid extraction was carried out following the procedures of Bligh and Dyer (1959) with some modifications as described by Freije and Awadh (2009).
Raw fish muscle was ground, and one gram of the ground fish muscle was transferred into a large Pyrex reaction tube and homogenized with 6 ml of saline solution (0.85%).The homogenate extraction (6 ml) was done by adding 15 ml methanol, followed by 7.5 ml chloroform twice, and 10 ml of water with vigorous shaking for 30 s after each addition.
The mixture was centrifuged for 10 min at 2500 rpm and filtered to discard the muscle plug.The filtered solution was transferred to a test tube and allowed to separate into two layers.The top layer was discarded, and the lower layer that contains the lipid extract in chloroform was transferred to another pre-weight test tube and weighed again to determine the total lipid content.

Fatty acid methylation
Fatty acid methylation was carried out following the method of AOAC (1990) with some modifications as described by Ozogul, Sims ¸ek, Balikc ¸i, and Kenar (2012).Twenty-five mg (25 ml) of the extracted fish oil was transferred into a test tube, and 1.5 ml of 0.5 M methanolic sodium hydroxide was added and mixed.The mixture was heated at 100 C for 7 min and cooled at room temperature.
Two ml of 14% borontrifloride-methanol were added, and the mixture was heated at 100 C for 5 min and cooled to 30-40 C. One ml of isooctane was added, and the tube was shaken for 30 s. Five ml of saturated sodium chloride was added immediately, followed by shaking the tube for 30 s, and the content was allowed to separate.
The upper isooctane layer that contains fatty acid methyl esters (FAMEs) was removed and placed into another test tube.The lower layer was extracted again by the addition of one ml of isooctane and shaken for 30 s. Finally, the two extracted isooctane layers were combined and dried over tiny amounts of anhydrous sodium sulphate and concentrated to one ml under nitrogen gas.

Fatty acid analysis
The fatty acid analysis was performed using Perkin Elmer, Clarus 500 GC-FID equipped with Flam Ionization Detector (FID) and Thermal Conductivity Detector (TCD).The individual FAMEs were separated using a fused carbon-silica column (Stabilwax, Crossbond, Carbowax, Polyethylene glycol) with 30 m length, 0.25 mm of internal diameter (width), 0.25 mm film thickness, and a temperature range of 40 C to 260 C. The oven temperature was set at 200 C and held constant for 80 min.The FID temperature was set at 300 C, and TCD temperature was set at 150 C. The injector temperature was maintained at 250 C with a split ratio of 1:20.Nitrogen gas was used as a carrier gas at a flow rate of 0.76 ml/min, and the other two gases were air with a flow rate of 450 ml/min and hydrogen with a flow rate of 45 ml/min.The sample injected volume was 5 ml with three pre and post-injection sample washes.A sampling rate of 12.5 Hz was used.The FAMEs were identified based on the comparison of their peak area against authentic standards (PUFA No.1 and PUFA No.2) supplied by SUPELCO USA.

Results
The fatty acid compositions of ten wild fish samples are summarized in Table 2. Total lipid content varies between the studied fish species except for four species, Dory Snapper (Naiser), Blackfin Mojarra (Badeh Raiash), Black Banded Trevally (Hamam), and Cephalopholis Argus (Balol) that have the same amount of total lipid contents equal to 0.06 ± 0.03 g/g, 0.06 ± 0.02 g/g, and 0.03 ± 0.01 g/g respectively.The fish that had the highest total lipid content was Haffara Bream (Gorgofan) (0.35 ± 0.28 g/g), while the fish that had the lowest lipid content were Blue Barred Parrot (Gain) (0.03 ± 0.01 g/g), Black Banded Trevally (Hamam) and Cephalopholis Argus (Balol) (0.03 ± 0.01 g/g).
There were significant differences (p < 0.05) in saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs), which include monounsaturated fatty acids (MUFAs) and PUFAs between all the fish samples.Total PUFAs were higher than the total MUFAs with different concertation among the selected wild fish species.Diamond Mullets (Maid) had the highest percentage of total MUFAs (25.46 ± 1.60%), while Spotted Half Beak (Sels) had the lowest percentage of total MUFAs (8.67 ± 1.06%).On the other hand, Blue Barred Parrot (Gain) and Black Banded Trevally (Hamam) have the highest percentage of total PUFAs, which are equal to 40.48 ± 2.47%, and 40.31 ± 3.34%, respectively, whereas Diamond Mullets (Maid) had the lowest percentage of total PUFAs (22.23 ± 1.08%).
Table 3 shows the arrangement of seventeen edible wild fish species in Bahrain, twelve of them were previously studied, and ten of them from the present study based on their sum of EPA and DHA.The fish with the highest sum of EPA þ DHA was given the number one.Doubleber Bream (Faskir) was fish number one with EPA þ DHA equal to 26.63 ± 0.30%, whereas White Sardinella (Oom) was given the last number (seventeen) with a sum equal to 5.45 ± 0.35%.Therefore, fish species with a low sum of EPA þ DHA tend to have a low concentration of omega-3, a high concentration of omega-6, and a low n-3/n-6 ratio.
Table 4 shows the arrangement of seventeen edible wild fish species in Bahrain, twelve of them were studied previously, and ten of them from the present study based on the n-3/n-6 ratio.Fish with the highest ratio of n-3/n-6 was given the number one.Doubleber Bream (Faskir) was the fish number one with an n-3/n-6 ratio equal to 2.62 ± 0.29%, and Gorgofan was fish number seventeen with an n-3/n-6 ratio equal to 0.60 ± 2.40%.

Discussion
The present study was done to find out the fatty acid compositions of ten selected edible wild fish species.These fish and other previously studied edible wild fish species in Bahrain were ranked based on their sum of EPA þ DHA and their n-3/n-6 ratio.Based on the ranking presented in Tables 3  and 4, a comparison was made between edible wild Table 4. Fish arrangement based on an n-3/n-6 ratio.Fish with the highest n-3/n-6 ratio was given the number one, whereas fish with the lowestest n-3/n-6 ratio was given the last number (seventeen).

Fish Number Fish common name Fish local name
Fatty acids (%) fish species in Bahrain and the fish that contain a high amount of omega-3 around the world, such as Tuna, sardines, salmon, mackerel, herring, trout, cod, and haddock (Table 5) (Mayo Clinic, 2019).
According to Table 5, based on the sum of EPA þ DHA, the fish that contains a high omega-3 around the world have a higher sum of EPA þ DHA.Among the fish presented in Table 5, cod has the highest sum of EPA þ DHA equal to 55%, while among the edible fish species in Bahrain Doubleber Bream (Faskir) has the highest sum of EPA þ DHA (26.63 ± 0.30%).Haddock has the lowest sum of EPA and DHA (23.00%, whereas edible wild fish species in Bahrain White Sardinella (Oom) has the lowest sum of EPA þ DHA (5.45 ± 0.35).Although, haddock has the lowest sum of EPA þ DHA (23%), this value of EPA þ DHA considered as one of the highest value among the edible wild fish species in Bahrain as seen in Blue Barred Parrot (Gain) (23.06 ± 1.58%).
The present study's results are consistent with the results of several studies regarding EPA þ DHA and n-3/n-6 ratio in fish species from the Arabian Gulf Sea (Table 6).EPA þ DHA values ranged from 7.1 to 27.82%, similar to the values found in the present study (5.45 ± 0.35 À 26.63 ± 0.3%).The only exception was the value of theYellowfin Tuna (177.63%), and Scomberomorus commerson (40.75%).Similarly, the n-3/n-6 ratio in fish species from the Arabian Gulf Sea ranged from 0.41to 5.91% in comparison to fish species from Bahrain (0.66 ± 2.40 À 2.62 ± 0.29%).
Fatty acid profiles vary in fish, and one of the important causes of this variation is the differences found among the fish species.It is important to note that the nutritional components vary based on the aquatic environment in which the fish lives, and living in an aquatic environment with different ecological conditions affects the fatty acid profile in fish.The reproduction status, size, and fishing seasons are all considered factors that affect the fatty acid compositions in fish (Tas ¸bozan & G€ okc¸e, 2017).
Based on their habitat, fish are split into two classes; the first is marine fish, and the second is freshwater fish.Water temperature and salinity are considered environmental factors that play an important role in the fatty acid compositions of fish.In addition, fish are also divided into two classes based on water temperature, warm-water fish and coldwater fish.Warm-water species prefer temperatures of roughly 25-30 C, while cold-water species, on the other hand, prefer temperatures below 20 C. It is important to note that some fish species are diadromous species, meaning they migrate from freshwater to sea water or vice versa, such as salmonids and eels.Therefore, those different habitats affect the fatty acid compositions of fish (Tai, Chirala, & Wakil, 1993;Tas ¸bozan & G€ okc ¸e, 2017).
acid to EPA and DHA is possible in much freshwater fish.In contrast, this is more difficult in marine fish because the enzymes elongases and desaturases responsible for the conversion of linolenic acid (18:3n-3) to EPA and DHA, stop working due to high water temperature and salinity.That is way marine fish require EPA and DHA directly from the photosynthetic organisms which are the primary producers of PUFAs such as macroalgea, seaweeds, and phytoplankton.Those organisms are rich in omega-3 PUFAs and have the ability to convert linolenic acid (18:3n-3) to EPA and DHA, even if the water  temperature and salinity are high.Thus, freshwater fish require the precursor linolenic acid (18:3n-3) to convert it to EPA and DHA by the enzymes elongases and desaturases which are not affected by environmental factors especially the water temperature and salinity since the freshwater has low water temperature and low salinity.This can also be considered as a reason for the high amount of omega-3 in freshwater fish species in comparison to the marine fish species (Chuang, Chang, & Hwang, 2011;Grunnet & Knudsen, 1979;Johns & Kubow, 2006;Tas ¸bozan & G€ okc ¸e, 2017).
A study investigated the amount of omega-3 PUFAs in ten fish that are regularly consumed in Bahrain and found that their omega-3 PUFAs were low compared to fish found in American markets and Mediterranean fish, and the reason behind this was attributed to the warm water temperature and high salinity of Bahrain seawater.These two environmental factors can play an important role in decreasing phytoplankton density and the amount of omega-3 PUFAs in the phytoplankton that fish depends on as a source of food, so this can affect the fatty acid compositions in Bahrain fish (Al-Arrayed, Al Maskati, & Abdullah, 1999).Lipid content, fatty acid compositions, and nutritional characteristics of 22 commercially important marine fish species from the Pearl River Estuary in the south China sea were studied.The study found that changes in the number of phytoplankton, such as dinoflagellate and diatoms, may have affected the concentration of fatty acids in fish (Zhang et al., 2020).
The fatty acid profile of raw and cooked fish species commonly consumed in Bahrain, including Pearl-spotted Rabbitfish (Safai), Narrow-barred Spanish mackerel (Kanad), and Diamond Mullet (Maid) was investigated by Musaiger and D'Souza (2011).Total PUFAs level was highest in Narrowbarred mackerel (Kanad) and lowest in the other two fish species, which are Diamond Mullet (Maid) and the Pearl-spotted Rabbitfish (Safai).Omega-3 fatty acids were predominant in all fish species as the Narrow-barred Spanish mackerel (Kanad) has the highest amount of DHA, whereas the fish with the lowest amount of omega-6, especially the AA, which is the predominant fatty acid among the omega-6 family, was in Diamond Mullet (Maid).According to EPA and DHA, Pearl spotted Rabbitfish (Safai) has the lowest value of EPA, Diamond Mullet (Maid) has the lowest value of DHA, whereas the Narrow-barred Spanish mackerel (Kanad) has the highest value of DHA (Musaiger & D'Souza, 2011).Comparing these three fish species with the present study Rabbitfish (Saffy) is considered as one of the fish that has the high level of total PUFAs, followed by the Spanish Mackerel (Kanad); in contrast, Diamond Mullets (Maid), has the lowest value of total PUFAs.
Several studies investigated the recommended optimal n-3/n-6 ratio for human health, which is 1:5 (0.2) (Begum et al., 2019;Hossain, Almatar, Al-Abdul-Elah, & Yaseen, 2012;O'Neill, Le Roux, & Hoffman, 2015;Usydus, Szlinder-Richert, Adamczyk, & Szatkowska, 2011).All the studied edible wild fish species in Bahrain have an n-3/n-6 ratio higher than the optimal (0.2) (Table 4).As a result, all selected edible and other studied wild fish species play an important role in supporting human health and providing nutritional value (Hossain et al., 2012;O'Neill et al., 2015;Simopoulos, 2006).However, the recommended daily intake of 500-1000 mg of EPA þ DHA is required as the primary prevention of CHD (Harris, 2004;Smith et al., 2006;Van de Werf et al., 2003).This amount cannot be met by consuming wild fish species from Bahrain.EPA and DHA content of five fish types of important commercial values in Bahrain reported by Al-Arrayed, Al Maskati, and Abdullah (1999) were used to calculate the amount of fish required to provide 1 g of EPA þ DHA.It was shown that the level of EPA þ DHA in local fish from Bahrain is very low (0.030-0.239 g/3 oz), and that the amount (0.36-2.7 kg fish/day) required to provide $1 g of EPA þ DHA seems to be difficult to attain (Freije, 2009).
Adequate amounts of roughly 220 to 240 g of fatty fish, such as salmon, must be ingested weekly to meet a daily intake of 500 mg EPA þ DHA (Opperman, Marais, & Benade, 2011;Opperman & Benade, 2013).Therefore, only the consumption of wild fish from Bahrain will not be sufficient to meet such criteria.

Conclusion
Based on the fatty acids analysis done in this study, it was found that the fish that has the highest sum of EPA and DHA and the n-3/n-6 ratio was the Doubleber Bream (Faskir), and the lowest sum was found in White Sardinella (Oom), whereas the lowest ratio of n-3/n-6 was observed in Haffara Bream (Gorgofan).The result showed that all studied edible wild fish species in Bahrain have a low concentration of omega-3 compared to fish species with a high amount of omega-3 around the world.However, when comparing the n-3/n-6 ratio of the edible wild fish species in Bahrain with the suggested optimal ratio (0.2), these wild fish species have a higher n-3/n-6 ratio than the optimal ratio.As a result, they are nutritionally beneficial for human health.However, it is difficult to attain the required daily amount of 500-1000 mg of EPA þ DHA through the consumption of local fish species from Bahrain; therefore it is recommended to consume imported fish species that contain a high amount of EPA þ DHA such as salmon and sardines in addition to the consumption of local fish species.Fish oil supplements can also be considered as an alternative option.

Table 1 .
Local, common, and scientificnames, number of samples, weight (mean ± SD), length (mean ± SD), and date of collection of fish samples studied.

Table 2 .
Total lipid and fatty acid compositions of ten edible wild fish species in the Kingdom of Bahrain.Total lipid is expressed as mean weight (g/g) ± SD.Fatty acid values are expressed as % of total fatty acid methyl esters; mean ± SD.EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; SFAs: saturated fatty acids; UFAs: unsaturated fatty acids; MUFAs: monounsaturated fatty acids; PUFAs: polyunsaturated fatty acids.

Table 3 .
Fish arrangement based on EPA and DHA values.Fish with the highest sum of EPA þ DHA was given the number one, whereas fish with the lowestest sum of EPA þ DHA was given the last number (seventeen).

Table 5 .
EPA, DHA, and n-3/n-6 ratio in fish species that have the highest amount of omega-3 around the world.

Table 6 .
EPA, DHA, and n-3/n-6 ratio in fish species from the Arabian Gulf Sea.

Table 7 .
EPA, DHA, and n-3/n-6 ratio in fish species from the Mediterranean Sea.