Proximate, amino acid, fatty acid and mineral composition of Grey mullet, Mugil cephalus L: A comparative study between the culture and wild resources in different size groups and potential contribution to nutritional security

Mugil cephalus is widely distributed across all oceans and it is a potential candidate species for aquaculture. Nutrient proling in terms of proximate, amino acid, fatty acid and mineral composition was analyzed in muscle tissue of M. cephalus sourced from culture and wild with four different sizes (100–150 g, 151–250 g, 251–500 g and > 500 g). Results of proximate composition revealed that both the factors (resource and size) had a signicant (P < 0.05) effect on moisture, crude lipid, total ash and gross energy but not on crude protein and crude bre. Though the amino acid composition was not inuenced by resource and size, the level of essential amino acids of ideal protein was higher than the recommended level by FAO/WHO for phenylalanine (43.29–45.60%), tryptophan (36.60-39.02%) and lysine (33.61–41.51%). Fatty acids like C14:0, C16:0, C18:0, C18:1c, C18:2c and γC18:3 was signicantly (P < 0.05) high in cultured species compared to wild caught sh, irrespective of the size groups and the reverse was true for αC18:3, C20:4 and C20:5. The amount of calcium, phosphorus, sodium and potassium was signicantly (P < 0.05) higher by 10.68, 5.82, 9.31 and 6.93% in wild caught sh than its counterpart. The potential contribution of this sh to nutritional security in terms of its daily value (DV) was calculated for one serving of 100 g sh to adult human being. Results revealed that lysine, methionine, threonine, tryptophan and EPA + DHA were considered as outstanding nutrients in this sh, irrespective of their source and size as their DV crossed > 70%. Similarly, isoleucine, leucine, phenylalanine and valine could also able to meet 50–70% of the daily needs of adult human indicating the nutritional richness of both wild and cultured M. cephalus regardless of its size. and C20:5, whereas there was no signicant difference for C22:6 between culture (120.07 mg/100 g) and wild (118.45 mg/100 g) collected sh. The size of the sh had a direct proportion to the muscle fatty acid composition, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, the ratios of n-6/n-3 and DHA/EPA were found to be high in 151–250 g and 251–500 g groups, respectively. Fatty acids like C14:0, γC18:3, C20:4 and C20:5 were signicantly (P < 0.05) increased with increasing sh size, whereas there was no signicant difference for C16:0, C18:0, C16:1, C18:2c and C22:6 between the sh categorized into 100–150 g and 151–250 g groups. Similarly, no signicant difference was observed for C18:1c and C18:2c between the groups had a body weight of 151–250 g and 251–500 g. Among the presented fatty acids, the difference was very conspicuous for C16:0 and was increased by 1103.61 mg/100 g in cultured sh compared to the wild one. Among the size groups, the increase was 1187.39, 1105.80, 730.23 mg/100 g in sh with a body weight of > 500 g compared to the groups of 100–150 g, 151–250 g and 251–500 g, respectively. groups. The proportion of the increase was almost similar between 100–150 g and 151–250 g groups as well as between 151–250 g and 250–500 g groups, while the increase was almost doubled in the sh categorized under > 500 g. The similar variations in fatty acids in relation to sh size have been reported in gold-spot mullet 14 , rainbow trout 47 and gilthead sea bream 48 . Kiessling and Kiessling 49 suggested that the relative variations on fatty acid composition with varied body weights could be attributed to the selective aerobic phosphorylation of fatty acids into the mitochondria of muscle tissue. The selective mobilisation of fatty acids to the reproductive organs in larger sh would also be a reason for the same 50 . In contrast, Ghomi and Nikoo 51 observed higher level for C18:1, C18:2 and C18:3 in small size sh of sturgeon roe than the larger one. of the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), of Fisheries, Animal Husbandry and Dairying, Govt. of India, on care and use of animals in scientic research. This study was undertaken with approval of the Institute Animal Ethics Committee (IAEC), ICAR-Central Institute of Brackishwater Aquaculture, Chennai, India (F.No. SPA/Dir/5–90. Dated: 07.04.2014). Each group has been designed to contain six shes with three replications. After dressed, skin and bone were removed from all the shes. Boneless muscle tissues were collected from both dorsal and ventral areas and stored at -20° C until analysis. Proximate composition of was determined by AOAC 54 methods. The gross energy of samples was tested using a semi bomb calorimeter (Parr-1425). Amino acid proles were analysed using pre-column HPLC gradient system (Shimadzu Corp, LC ‐ 30AD) after hydrolysing the samples with 6 N hydrochloric acid in a sealed tube lled with nitrogen for 22 h at 110° C in a vacuum oven 55 . Individual amino acids were separated by YMC ‐ Triart C18, RRH (1.8 µm, 2.1 × 100 mm) column after derivatization with mercaptopropionic acid, O ‐ phthalaldehyde and uorenylmethoxycarbonyl chloride. A gradient elution using phosphate buffer (20 mmol as mobile phase A) and combination of acetonitrile:methanol:water (45:40:15 as mobile phase B) at the ow rate of 0.3 ml/min was used. The gradient was changed by increasing mobile phase B concentration at the rate of 11–13% at 3 min, 31% at 5 min, 37% at 15 min, 70% at 20 min and 100% at 25 min. Amino acids were qualied and quantied by a uorescent detector (RF ‐ 20AXS) using the amino acid mixer as an external standard (Sigma Aldrich, Cat. No: AAS18) and nor-leucine as an internal standard. Tryptophan, being labile to acid hydrolysis, was measured after alkali hydrolysis using a spectrophotometric


Introduction
Fish is a relatively cheaper food material with higher quality than the meat sources, which plays a crucial role in global Food Nutrition Security (FNS), especially in low and middle income countries 1 . Fish serves a healthy, safe, nutritious and balanced diet to human, as it is rich in valuable nutrients (protein with essential amino acids (EAA), long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), minerals mainly micro elements). Therefore, its consumption plays an important role in reducing global burden of disease. Alasalvar et al 2 stated that LC n-3 PUFA cannot be synthesized in human and should be supplemented by diet. As this is rich in sh, helps in neural development in both utero and rst few years after birth in infants 3 . In addition, consuming PUFA plays a vital role in controlling various diseases and disorders like aggression, arrhythmias, cancer, coronary heart failure, depression, hypertension, in ammation, psoriasis, etc 4 . WHO 5 and FAO/IFAD/WFP 6 reported that around 870 million peoples are affected by protein-energy malnutrition globally and it is nearly 80% in developing countries alone. As sh is an important protein source, its prolonged use can reduce a lethal form of malnutrition 7 . Likewise, certain functional amino acids involved in the process of wound healing and act as an anti-mutagenicity and anti-oxidant 8 . Similarly, element content of sh would be most bene cial to human, as they play vital roles in various metabolic pathways 9 .
The capture sheries production has become static (96.4 million tonnes in 2018), which could not meet the demand for sh due to the geometric growth of global population 10 . However, the expansion of aquaculture production (114.5 million tonnes in 2018) could able to bridge the gap between the demand and supply worldwide. FAO 10 reported that the aquaculture sector grown by 527% in 2018 compared to 1990, which alone contributes by 54.46% of the total global sh production with currently valued at USD 263.6 billion 11 . As in the world, Indian aquaculture sector has also been evolved with considerable diversi cation in terms of species. Though the Indian major carps have dominated, certain other shes have also been cultured in India traditionally. One among them is Grey mullet (Mugil cephalus L), and it is predominant in the state of West Bengal, Andhra Pradesh, Tamil Nadu and Kerala in India. M. cephalus has a very wide distribution in all the oceans, estuaries and brackishwater of tropical and sub-tropical regions from 42° N to 42° S. Their catches have increased as a result of a growing market in the south-eastern United States, Gulf coast, Asian and Mediterranean markets with good nutrient pro les 12 . Jannathulla et al 13 reported that the nutrient composition of aquatic animals greatly varies, not only between the species and even within the groups, which is mainly due to the variation in feed and biological resource present in the waterbody. In addition, various factors would also in uence the nutrient pro ling of sh, the most important are age and sources of collection 14 . In this context, various methods have been developed by Nutrition Labelling and Education Act (NLEA) in the United States and Association of Analytical Chemist (AOAC) for documenting fundamental knowledge on the nutritional quality of aquatic species. Several studies have already been reported the nutrient compositions of various n-shes collected from wild and culture [14][15][16][17][18] . However, the available data on M. cephalus is scarce yet to date. Hence, the present study is aimed to investigate the nutrient composition of muscle tissue in M. cephalus collected from two different sources with four varied size groups. This baseline data would help to facilitate the consumers to realize about the nutritional quality of this sh, by which they could assure about their health, immunity and tness against the lifestyle disease, which is of great signi cance to increase the market value of this sh in India and around the globe in the future.

Results
Proximate composition of M. cephalus sourced from both culture and wild with different size groups (Table 1) revealed that both the factors (resource and size) had a signi cant (P < 0.05) effect on moisture, crude lipid, total ash and gross energy but not on crude protein and crude bre level. The moisture content was signi cantly (P < 0.05) high in wild caught sh (74.23%) compared to culture one (72.32%) and was gradually decreased from 75.08-70.57% with increasing size. Wild caught sh had 2.54% of crude lipid and was more than doubled in cultured species (5.44%). Though the lipid content was almost similar in the groups categorized under 100-150 g and 151-250 g (2.44-2.75%), its level was higher by 36% and 141% in 251-500 g and > 500 g groups, respectively. Total ash content found to be high (P < 0.05) in wild caught sh compared to the cultured one, whereas, its level was not affected in the groups had a weight beyond 151 g. Gross energy was signi cantly (P < 0.05) high in cultured sh (133.03 Kcal/100 g) than wild sh (110.12%) and was gradually (P < 0.05) increased from 107. 46-145.27 Kcal/100 g with increasing size. Among the essential amino acids (EAA), leucine found to be high followed by lysine and arginine, while the least values were noticed with histidine and tryptophan, whereas in non-essential amino acids (NAA), the highest values were observed for glutamic acid followed by aspartic acid (  Both resources and size have highly in uenced (P < 0.05) the tissue fatty acid composition as depicted in Table 3. Among the fatty acids presented, C16:0 was abundant and accounted about 35-40% of total fatty acids, while the least was noticed with both γ and α C18:3. Fatty acids like C14:0, C16:0, C18:, C18:1c, C18:2c and γC18:3 was signi cantly (P < 0.05) high in cultured species compared to wild caught sh and the reverse was true for αC18:3, C20:4 and C20:5, whereas there was no signi cant difference for C22:6 between culture (120.07 mg/100 g) and wild (118.45 mg/100 g) collected sh. The size of the sh had a direct proportion to the muscle fatty acid composition, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, the ratios of n-6/n-3 and DHA/EPA were found to be high in 151-250 g and 251-500 g groups, respectively. Fatty acids like C14:0, γC18:3, C20:4 and C20:5 were signi cantly (P < 0.05) increased with increasing sh size, whereas there was no signi cant difference for C16:0, C18:0, C16:1, C18:2c and C22:6 between the sh categorized into 100-150 g and 151-250 g groups. Similarly, no signi cant difference was observed for C18:1c and C18:2c between the groups had a body weight of 151-250 g and 251-500 g. Among the presented fatty acids, the difference was very conspicuous for C16:0 and was increased by 1103.61 mg/100 g in cultured sh compared to the wild one. Among the size groups, the increase was 1187.39, 1105.80, 730.23 mg/100 g in sh with a body weight of > 500 g compared to the groups of 100-150 g, 151-250 g and 251-500 g, respectively. The mineral composition of M. cephalus sourced from both culture and wild with different size groups is presented in Table 4. Regardless of resource and size, the highest values were found in potassium followed by phosphorus, while calcium, sodium and magnesium were in the range of 10-40 mg/100 g. The level of calcium, phosphorus, sodium and potassium in cultured sh was 18.66, 185.49, 35.76 and 231.78 mg/100 g, respectively, and was signi cantly (P < 0.05) increased by 10.68, 5.82, 9.31 and 6.93% in wild caught sh. The increasing trend with increasing body weight was noticed for all the four macroelements as mentioned above. However, the amount of magnesium did not vary between the source as well as size. The level of all the micro-elements analysed was < 0.5 mg/100 g and was not in uenced by both resources and size. The daily value (DV) of lipid, cysteine, n-6 + n-3 fatty acids and calcium was < 25% and protein, valine, phosphorus and selenium were between 25-50%. Isoleucine, leucine, lysine, phenylalanine and threonine showed a range of 50-100%, whereas it crossed > 100% for methionine and EPA + DHA (Fig. 3). The DV was increased by 3.71, 3.03, 1.04, 0.78, 5.63 and 2.78% for lipid, phenylalanine, threonine, tryptophan, valine and n-3 + n-6 fatty acids, respectively, and was decreased by 0.57, 1.01, 0.45, 2.97, 7.12, 10.18, 0.26, 1.8 and 3.21% for protein, cystine, leucine, lysine, methionine, EPA + DHA, calcium, phosphorus and selenium in wild caught sh compared to the cultured species, whereas no difference was noticed for isoleucine between the resources (Fig. 4). The result of DV of sh with the varied size has shown that the sh with a body weight of > 500 g had a higher DV for lipid, lysine, methionine, EPA + DHA, n-3 + n-6 fatty acids, calcium and phosphorus, while cysteine and selenium were high in 251-500 g group. The DV of protein, isoleucine, leucine, phenylalanine, threonine and tryptophan found to be high in sh with 151-250 g weight and the group of 100-150 g showed a higher value for valine.

Discussion
Nutrient composition of sh, collected from wild and culture, is greatly varied in general. Because in intensive culture, sh are provided with nutrient dense compounded feeds that enables them to deposit large reserves of nutrients, in particular lipids. In contrast, considerable changes occurred in environmental condition uctuates the availability and composition food that would affect the nutrient composition of wild sh 20 . In addition to food/feed, Piggott and Tucker 21 listed some other factors in uencing the nutrient composition of sh such as species, genetics, size, reproductive status and environmental characteristics. In our study, the moisture content of wild sh was observed to be the highest compared to the cultured ones and was drastically reduced with increasing body size. This result is in agreement with the ndings of Alasalvar et al 2 , who found higher moisture content in the wild caught sea bass than the cultured sh. In the present study, muscle protein content of M. cephalus did not vary between the resources. Similarly, size variation had no effect on the protein level. This result is corroborated with the nding of Nettlon and Exler 22 in channel cat sh, coho salmon and rainbow trout. In contrast, a slight variation in protein content was observed between wild and cultured yellow perch 17 . The difference in protein content might be due to the variation in environmental conditions, species, size, sex of the individual animals and their reproductive status 14 .
In the present study, the muscle tissue of the cultured M. cephalus contained higher lipid content than the wild caught sh. This is in agreement with earlier reports that have shown that the level of lipid tend to be lower in sh collected from wild compared to cultured individuals of the same species, such as yellow perch 17 , turbot 23 , sea bream 24 , and silver pomfret 25 (Table 1).
It is essential to have adequate knowledge on amino acid content of sh protein to establish its nutritional value, as the nutritional quality of protein is mainly depending on EAA. Fish protein has a greater satiety effect than other animal proteins like chicken, beef, etc., with cheaper price 7 35 reported that lysine is one of the most limiting amino acids in cereal-based diets given to children and is extensively required for optimal growth, hence it is suggested sh as an optimal supplement for cereal-based diets. Methionine plays a vital role in treating Parkinson's disease, liver disorder and schizophrenia, which would also help in the treatment of alcoholism, allergies, asthma, drug withdrawal, poisoning, particularly eliminating copper, radiation side effects, etc., 14 and was ranged from 0.74-0.81% in M. cephalus. Arginine was the second highest EAA in the muscle portion of M. cephalus, which is mainly considered as a precursor for the biological synthesis of nitric acid and play an important role in neurotransmission, blood clotting and blood pressure maintenance. The disease of sepsis, preeclampsia, erectile dysfunction and anxiety was much recovered with the supplementation of arginine. Isoleucine, phenylalanine, threonine and valine had a variety of roles in human nutrition mainly in chronic renal failure and nervous system disorders 26 and were found to be around 1% in both wild and cultured M. cephalus. Liao et al 37 documented the necessity of histidine in the growth and repair of tissues, myelin sheaths maintenance and to remove heavy metals from the body. Similarly, tryptophan acts as a precursor for different neurotransmitters like serotonin, dopamine and nor-dopamine and was in the range of 0.28-0.29% in our study.
Though NAA are synthesized de nova, they would also play a crucial role metabolically as in EAAs, especially in the regulatory process of gene expression, micro-RNA levels, metabolism, innate and cell mediated response 38 . Deutz et al 39 reported that during critical illness, the e uxed glutamic acid from muscle serves as an important carrier of nitrogen as ammonia in the splanchnic area and immune system, which alone contributed more than 30% in total of NAA in our study. However, no signi cant difference was observed in muscle composition of both EAA and NAA, indicating that the protein content in M. cephalus was well balanced in amino acid composition, in particular EAA and is of high quality, irrespective of the resources and size. This result is corroborated with the ndings of Gonzalez et al 17 40 found a marginal decrease in the IP value for histidine, leucine and threonine in golden mahseer, common snow trout and common carp, respectively as compared with the recommended level. Though the IP level of valine reduced than the recommended level in our study, the calculated EAAI has shown no signi cant difference among the EAA, including valine in both wild and cultured sh and also showed a positive correlation for EAAI between cultured and wild caught M. cephalus.
As a source of membrane constituents, energy, metabolic and signaling mediators, fatty acids, particularly n-3 PUFAs are recognized as essential nutrients for life. Aquatic species, primarily sh are generally characterized by high level of n-3 PUFAs, however, its composition in uenced by many intrinsic and extrinsic factors. In our study, the level of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) was signi cantly high in cultured M. cephalus than in wild caught sh, while the reverse was true for PUFA except C18:2c and γC18:3 and as re ected in n-6/n-3 ratio, which shown that signi cantly lower n-6/n-3 ratio in wild compared to cultured sh, indicating that marine environment would have an excellent source for n-3 rich foods and which might be lower in intensive culture system. This result is greatly in agreement with those previously reported by Zhao et al 4 . In contrast, Alasalvar et al 2 found the reverse trend in sea bass, who reported about 29.2 and 33.4% of SFA in cultured and wild collected sea bass and the remaining quantity was shared by MUFA and PUFA. But in the present study, SFA was about 50.7% in cultured sh and almost similar level was observed for wild sh (49.6%). C16:0 was about > 70% of the total SFA content of both cultured and wild M. cephalus and was signi cantly higher in cultured than in wild sh, which might be attributed to the usage of supplementary feed containing higher C16:0. Similar results have also been reported in sea bass 2 , crappie 16 , rohu 31 and sturgeon 41 . In MUFA, C18:1 was found as a major one in the both the sh and was signi cantly higher in cultured sh than that of wild ones. Similarly, C18:2c found to be ten-times higher in cultured M. cephalus compared to its wild counterpart, which is due to its dominance in compounded feed used for intensive culture 26 . Among n-3 series, both EPA and DHA had no much variation like other fatty acids between the wild caught and cultured sh, indicating that both the sh are of good sources for these fatty acids. However, a marginal increase was noticed in wild sh than the cultured ones, but the difference was insigni cant for DHA. A great variation was noticed in C20:4 and was much higher in wild sh (170.36 mg/100 g) than in cultured ones (60.44 mg/100 g). A similar result was found by Gonzalez et al 17 in yellow perch and who suggested that this might be due to the dietary effect and saturation and/or elongation mechanisms. Similarly, the highest concentration of αC18:3 was observed in wild sh than in culture, which could be attributed to the type of food, in which M. cephalus is exposed in the wild such as insect larvae, algae, crustacean that are rich in αC18:3 42 .
Simopoulos 8 documented that the ratio of 1-2:1 for n-6/n-3 fatty acids is being considered as an ideal level for bene cial health, while the Department of Health of United Kingdom recommends that this level might be up to 4:1. Whereas the Western diets provide n-6/n-3 ratio of around 15-25:1, which would be a reason for occurring various common health disorders like coronary heart disease and cancer. McDanniel et al 43 suggested an approach, whereby consuming a higher dietary quantity of n-3 PUFAs, in particular C20:5 and C22:6 would be helpful in normalizing n-6/n-3 ratio. In our study, this ratio was 2.21:1 in cultured sh and was signi cantly low in wild caught sh (0.76:1), while it was in the range of 1.35-1.81:1 among the sh containing different body weight. All the values in our study were within the recommended values, indicating that M. cephalus could be considered as an optimal food source. Similarly, the DHA/EPA ratio seems to be lower in wild caught sh compared to cultured sh and the same was found in halibut sh 44 , sea bream 45 and cat sh and tilapia 46 . In our study, all the fatty acids were gradually increased with increasing body size. However, the rate of increasing was not same among all the size groups. The proportion of the increase was almost similar between 100-150 g and 151-250 g groups as well as between 151-250 g and 250-500 g groups, while the increase was almost doubled in the sh categorized under > 500 g. The similar variations in fatty acids in relation to sh size have been reported in gold-spot mullet 14 , rainbow trout 47 and gilthead sea bream 48 . Kiessling and Kiessling 49 suggested that the relative variations on fatty acid composition with varied body weights could be attributed to the selective aerobic phosphorylation of fatty acids into the mitochondria of muscle tissue. The selective mobilisation of fatty acids to the reproductive organs in larger sh would also be a reason for the same 50 . In contrast, Ghomi and Nikoo 51 observed higher level for C18:1, C18:2 and C18:3 in small size sh of sturgeon roe than the larger one.
Mineral content of sh is in uenced by both diet and environment, especially in intensive culture and would also have an in uence on avor. In addition, is considered as poor. According to this, lysine, methionine, threonine, tryptophan and EPA + DHA were considered as outstanding nutrients in both wild and cultured M. cephalus. Similarly, isoleucine, leucine, phenylalanine and valine were grouped under the excellent category. Protein, phosphorus and selenium were categorized as very good. A similar trend was noticed among the size groups. Cultured M. cephalus contained a higher DV for lipid, phenylalanine, threonine, tryptophan, valine and n-3 + n-6 fatty acids, whereas protein, cysteine, leucine, lysine, methionine, EPA + DHA, phosphorus and selenium were high in wild caught M. cephalus.
In conclusion, muscle of both cultured and wild M. cephalus is of good sources of protein with balanced amino acids, in particular EAA irrespective of the size groups. However, an important difference is found in some of the quality of lipid, fatty acid and macro mineral composition and they are in uenced by both resources and size. In addition, the results of the present study indicate that M. cephalus contains higher ideal protein compared to FAO/WHO/UNU recommended level with a good daily value for most of the nutrients. Our results suggest that despite of the changes in nutrient content, both wild and cultured M. cephalus fall under the category of nutrient rich sh and would be an effective and healthy food material.

Methods
Different size group (100-150 g, 151-250 g, 251-500 g and > 500 g) of M. cephalus specimens of cultured and wild were sourced from brackishwater sh farm at Nagayalanka (15.945  15 min, 70% at 20 min and 100% at 25 min. Amino acids were quali ed and quanti ed by a uorescent detector (RF-20AXS) using the amino acid mixer as an external standard (Sigma Aldrich, Cat. No: AAS18) and nor-leucine as an internal standard. Tryptophan, being labile to acid hydrolysis, was measured after alkali hydrolysis using a spectrophotometric method at 500 nm 56 . Essential amino acid index (EAAI) was calculated based on the ideal protein/amino acid level recommended by FAO/WHO/UNU 19 .
Lipid was extracted using a combination of organic solvent (chloroform and methanol at 2:1 ratio) and the process of saponi cation followed by esteri cation was done according to Metcalfe et al 57 . FAMEs were subsequently analysed using a gas chromatograph (GC-2014 Shimadzu) on a RTX-Wax Capillary Column (100 m length × 0.25 mm I.D × 0.2 µm lm thickness). Nitrogen was used as the carrier gas at a linear velocity of 20.9 cm/s with 3 ml/min of purge ow. The oven temperature was held at 100º C for 4 min and increased to 225º C at the rate of 3º C/min and held for 5 min and further increased to 240º C at the rate of 1º C/min. The operating temperature for injection ports and ame ionization detector was 225 and 250ºC respectively. Individual fatty acids were identi ed by comparing with the retention times of 37 Component FAME Mix (Supelco-Sigma). Tridecanoic acid methyl ester (C13:0, Supelco-Sigma, USA) was used as an internal standard to calculate fatty acid content in the sample (mg/100 g). Mineral composition was estimated according to the method of Jannathulla et al 58 . Brie y, a gram of sample was digested with 6 ml of HNo 3 and 2 ml of H 2 O 2 in microwave digestion system (Anton Paar). All digested samples were analysed in triplicates using inductively coupled plasma-optical emission spectrometry (ICP-OES) in an instrument of Agilent-5100, using the 5.2 software. The analytical measurements were made with an autosampler equipped with a peristaltic pump, across-ow nebulizer (coupled to a double-pass spray chamber) and Quartz central torch tube injector with an internal diameter of 2 mm. Certi ed reference material, ICP multi-element standard solution (10 mg/l Merck), was used for calibration.
The statistical package of SPSS ver.16.0 for Windows was used to assess the signi cant differences between the means of experimental data according to a two-way analysis of variance (ANOVA) using a 2 × 4 factorial design with two different factors such as resources (culture and wild) and size (100-150 g, 151-250 g, 251-500 g and > 500 g). The descriptive statistical measures were calculated for the two main factors and their interactions. For this, post-hoc was done with Tukey's test and differences were considered signi cant when P < 0.05. Regression analysis was performed to nd a correlation on EAAI between the resources irrespective of the size groups. Prior to statistical evaluation, data were checked for homogeneity of variances after ascertaining normal distribution.