Aquatic and Their Element and Fatty Acid Profiles G. Kumar1, R. K. Goswami1, A. K. Shrivastav2, J. G. Sharma2, D. R. Tocher3 and R. Chakrabarti1 1Aqua Research Lab, Department of Zoology, University of Delhi, Delhi 110 007, India 2Department of Biotechnology, Delhi Technological University, Delhi 110042, India 3Institute of , Faculty of Natural Sciences, University of Stirling, Stirling FK9 4LA, Scotland, UK.

Abstract The present study aims to evaluate the elements and fatty acids composition of twelve aquatic plants. Freshwater plants Azolla microphylla, A. pinnata, Enhydra fluctuans, Hydrilla verticillata, Ipomoea aquatica, minor, Marsilea quadrifolia, stratiotes, Salvinia molesta, S. natans, and arrhiza were cultured using organic manures, cattle manures, poultry wastes and mustard oil-cake (1:1:1). Among various aquatic plants significantly (P<0.05) higher crude protein and lipid were found in L. minor and S. polyrhiza. The ash content was significantly (P<0.05) higher in H. verticillata, W. arrhiza and P. stratiotes compared to others. Highest Na, Mg, Cr and Fe levels were recorded in P. stratiotes. H. verticillata was the rich source for Cu, Mn, Co and Zn; Ca, Mg, Sr and Ni contents were highest in S. polyrhiza; Se and K contents were higher in S. natans and W. arrhiza, respectively. The n-6 and n-3 polyunsaturated fatty acids (PUFA) levels were significantly (P<0.05) higher in W. arrhiza and I. aquatica, respectively compared to others. Linoleic acid (C18:2n-6) and alpha linolenic acid (C18:3n-3) were dominant n-6 Fig.1. Culture of Lemna minor in outdoor tanks. and n-3 PUFA, respectively. Highest (4.04) n-3/n-6 was found in I. aquatica. The ratio ranged from 0.61-2.46 in other macrophytes. Keywords: Alpha linolenic acid, Element profile, Fatty acids, Aquatic plants

1. Introduction Aquatic plants are those plants which grow on water and they uptake the nutrients directly from the water. At present the content of fish feeds at commercial level is consist of fish meal (as a source of protein), so it is clear that dry fish powder is required for fish feed formulation. The growing aquaculture industry needs higher amount of fish meal to formulate the feeds for fish culture in culture fisheries system. Human population is also increasing so we need to save the fish for human consumption and other allied activities related to human welfare development. The new era begins to use the freshwater aquatic plants as fish feed ingredients to replace this fish meal with new source of nutrition (Protein, Lipid). Present study aims to get the nutritional profile (Proximate, fatty acids and minerals profile) of twelve freshwater macrophytes, Azolla microphylla, A. pinnata, Fig. 2. Culture of Spirodela polyrhiza in outdoor cemented tanks. Enhydra fluctuans, Hydrilla verticillata, Ipomoea aquatica, Lemna minor, Marsilea quadrifolia, Pistia stratiotes, Salvinia molesta, S. natans, Spirodela polyrhiza and Wolffia arrhiza. Macrophytes Sodium (Na) Potassium (K) (Ca) (Mg)

2. Materials and Methods Azolla microphylla 5.36± 0.043d 28.79± 0.251e 0.98± 0.008h 2.85± 0.081d 2.1. Culture of macrophytes Macrophytes were cultured in outdoor cemented tanks (1.2 × 0.35 m) with clean dechorinated tap Azolla pinnata 4.94±0.089f 30.71±1.041d 0.90±0.070h 2.22±0.060f water (Chakrabarti et al., 2018). A 10 cm layer of soil was used for the culture of H. verticillata, M. Enhydra fluctuans 6.63±0.031b 37.34±0.563c 0.77±0.104i 3.59±0.242b quadrifolia and E. fluctuans. All other macrophytes were cultured without soil base. The depth of water was 30 cm in all culture tanks. A combination of organic manures viz. cattle manure, poultry Hydrilla verticillata 5.96±0.203c 32.74±1.003d 1.85±0.023e 3.91±0.073b droppings and mustard oil cake was used (1:1:1) at the rate of 1.052 kg/m3. Ipomoea aquatica 6.11±0.149c 22.11±0.368g 1.24±0.063g 3.41±0.158c 2.2. Proximate and biochemical composition analysis Crude Protein - Micro-Kjeldhal method Lemna minor 2.56±0.073h 40.28±1.086b 2.39±0.055c 2.88±0.065d Crude Lipid - Folch et al., 1957 Marsilea quadrifolia 1.13±0.021i 29.45±0.323e 0.51±0.002j 1.96±0.027g Minerals - ICP-MS, Agilent 7900, USA Carbohydrates - Subtraction method Pistia stratiotes 7.04±0.229a 32.19±1.096d 2.87±0.019b 5.29±0.136a Fatty Acids - Gas Chromatograph, PerkinElmer, USA Salvinia molesta 3.85±0.006f 18.69±0.073h 1.47±0.041f 2.89±0.023d 3. Results and Discussion 3.1. Proximate composition Salvinia natans 6.63±0.103b 26.83±0.284f 1.44±0.025f 2.52±0.010e Significantly higher crude protein levels were found in two duckweeds - L. minor and S. polyrhiza Spirodela polyrhiza 3.45±0.155g 28.45±1.618e 3.17±0.058a 5.30±0.094a compared to the other macrophytes (Fig. 1.). Highest lipid content was also recorded in L. minor, followed by S. polyrhiza. Lowest lipid content was found in E. fluctuans (Fig. 2). Ash content was Wolffia arrhiza 5.15±0.108e 47.09±0.597a 2.01±0.075d 2.58±0.036e significantly higher in H. verticillata, W. arrhiza and P. stratiotes compared to other macrophytes. There was no significant difference in ash content among these three macrophytes. The ash content Table 1 Macro elements (mg/g) composition of twelve freshwater macrophytes. was minimum in M. quadrifolia. Carbohydrates levels were minimum and maximum in L. minor and M. quadrifolia, respectively. 40 a a

3.2. composition 3.2.1. Macro minerals 35 b c Among these twelve freshwater macrophytes, significantly higher level of sodium (Na) was found in d d d P. stratiotes compared to others (Table 1). This group was followed by S. natans and E. fluctuans. 30 Significantly higher level of potassium (K) was found in W. arrhiza in the present study. This e e duckweed was followed by L. minor, E. fluctuans, H. verticillata and P. stratiotes. Calcium (Ca) 25 content was highest in S. polyrhiza, followed by P. stratiotes. Significantly higher magnesium (Mg) f level was found in P. stratiotes and in S. polyrhiza compared to others. There was no significant 20 h g difference between these two macrophytes. Lowest amount of Na and Mg were found in M. 15 quadrifolia. 10 3.2.2. Trace minerals In the present study, total nine trace minerals were analyzed in twelve macrophytes. Molybdenum 5 (Mo) contents were significantly higher in A. microphylla, A. pinnata and P. stratiotes compared to other macrophytes. There was no significant difference among these three macrophytes. g) (g/100 Content Protein 0 Manganese (Mn), (Zn), copper (Cu) and cadmium (Cd) contents were significantly higher in H. Azolla Azolla Enhydra Hydrilla Ipomoea Lemna Pistia Marsilea Salvinia Salvinia Spirodela Wolffia verticillata compared to other plants. In P. stratiotes, significantly higher level of iron (Fe) and microphylla pinnata fluctuans verticillata aquatica minor stratiotes quadrifolia molesta natans polyrhiza arrhiza aluminium (Al) were found compared to the other plants. Among these macrophytes, A. pinnata Freshwater Macrophytes ranked second for both Fe and Al; A. microphylla ranked third for iron and fourth for aluminium Fig. 3. Protein composition of twelve freshwater macrophytes. contents. Maximum strontium (Sr) level was recorded in S. polyrhiza followed by P. stratiotes. In all 9 these macrophytes lead (Pb) was found. a

8 c b 3.2.3. Ultratrace minerals Five ultra-trace minerals were assayed for these macrophytes . Significantly higher level of selenium 7 d (Se) was found in S. natans compared to other plants. This macrophyte was followed by H. e f verticillata and P. stratiotes. Selenium was absent in S. molesta, E. fluctuans, I. aquatica and S. 6 g j i h polyrhiza. Chromium content was significantly higher in P. stratiotes, A. microphylla and A. pinnata k compared to others. Cobalt (Co) content was significantly higher in H. verticillata compared to 5 l others. This macrophyte was followed by P. stratiotes and S. natans. Nickel (Ni) and tin (Sn) levels were significantly higher in S. polyrhiza and A. microphylla, respectively compared to other plants. 4 Among these macrophytes, P. stratiotes ranked third for nickel content. 3 3.3. Fatty acid profiles The fatty acid profiles of twelve freshwater macrophytes were documented in the present study. The 2 saturated fatty acids (SFA) content was significantly higher in W. arrhiza compared to others. This g) (g/100 Content Lipid 1 was followed by A. pinnata, L. minor and I. aquatica. SFA content was minimum in P. stratiotes. Among, SFA, palmitic acid (C16:0) was the dominant one in all these plants. 0 Monounsaturated fatty acids (MUFA) content was significantly higher in M. quadrifolia compared to Azolla Azolla Enhydra Hydrilla Ipomoea Lemna Pistia Marsilea Salvinia Salvinia Spirodela Wolffia other plants. Among various MUFAs, oleic acid (C18:1n-9) was present in most of the plants and the microphylla pinnata fluctuans verticillata aquatica minor stratiotes quadrifolia molesta natans polyrhiza arrhiza amount was also higher compared to others. Like SFA, MUFA content was also minimum in P. Freshwater Macrophytes stratiotes. Though in small amount two other monounsaturated fatty acids like palmitoleic acid (C16:1n-9) and nervonic acid (C24:1) were present in all macrophytes, except E. fluctuans and A. pinnata. Another isomer of palmitoleic acid (C16:1n-7) was absent in two species of Azolla and S. Fig. 4. Crude lipid composition of twelve freshwater macrophytes. natans. The n-6 PUFA content was significantly higher in W. arrhiza compared to others. This plant was followed by L. minor and A. pinnata. The minimum level was found in A. microphyla. Among n-6 1800 PUFA, linoleic acid (C18:2n-6) was the dominant one and present in all macrophytes. Arachidonic a acid (C20:4n-6) was the second dominant n-6 PUFA found in all macrophytes, except in L. minor. 1600 a Alpha linolenic acid, ALA (C18:3n-3) was the only member of n-3 PUFA present in these macrophytes. 1400 1200 Conclusions P. stratiotes was the best among these cultured macrophytes in terms of Na, Mg, Cr and Fe 1000 b contents; it ranked second for Co, Sr and Ca. H. verticillata was the rich source for Cu, Mn, Co and 800 c Zn and it ranked second for Se. Ca, Mg, Sr and Ni contents were higher in S. polyrhiza compared to d d other macrophytes. S. natans and W. arrhiza were rich sources for Se and K, respectively. All these 600 e macrophytes were rich sources of n-6 and n-3 fatty acids. This study provided variety of freshwater e macrophytes in the food basket as rich source of minerals and PUFA. This is also clear from the 400 f study that the culture of plants following a standard protocol helps in the quality production. ALA Content (mg/100 g) Content ALA g h 200 i Acknowledgements 0 This study has been supported by Department of Biotechnology, Government of India, New Delhi, Azolla Azolla Enhydra Hydrilla Ipomoea Lemna Pistia Marsilea Salvinia Salvinia Spirodela Wolffia India (Dy. No. 102/IFD/SAN/4678/2015-2016, dated 28.3.2016) and the Biotechnology and microphylla pinnata fluctuans verticillata aquatica minor stratiotes quadrifolia molesta natans polyrhiza arrhiza Biological Science Research Council (BB/N005031/1). GK is thankful to Indian Council of Medical Freshwater Macrophytes Research (ICMR) for providing him fellowship (3/1/3/JRF-2017/HRD-LS/55340/01). Fig. 5. Alpha Linolenic Acid (ALA) content of twelve freshwater macrophytes.