Brackish-Water Aquaculture: A New Horizon in Climate Change Matrix

Abstract

Brackish-water aquaculture has become an important source of seaweed, shellfish and finfish, especially for human food and production, which is likely to expand well in the next century if sea-level rise maintains its present pace. It has both direct and indirect impacts on biodiversity through the consumption of natural resources and the production of wastes. Most of the brackish-water aquaculture (particularly the shrimp farms) has developed in the mangrove ecosystem as the water has congenial parameters and tidal actions.

Appendices

Annexure 8A.1: Sustainable Freshwater Aquaculture in Mangrove-Dominated Indian Sundarbans Using Floral-Based Feed

• Abhijit Mitra,
• Rajrupa Ghosh,
• Ajoy Mallik,
• Kunal Mondal,
• Sufia Zaman &
• Kakoli Banerjee 

1. R. Ghosh • A. Mallik • K. Mondal • K. BanerjeeDepartment of Marine Science, Universityof Calcutta, 35, B.C. Road, Kolkata, West Bengal, 700 019, India

   Rajrupa Ghosh, Ajoy Mallik & Kunal Mondal

2. Department of Marine Science, University of Calcutta and Department of Oceanography, Techno India University, Salt Lake Campus, Kolkata, West Bengal, 700 091, India

   Abhijit Mitra, Sufia Zaman & Kakoli Banerjee

Abstract

Indian aquaculture is gradually evolving from the level of subsistence activity to that of an industry. This transformation has been made possible with the development and standardization of many new technologies. In the early 1990s, aquaculture created great enthusiasm and interest among entrepreneurs especially for shrimp farming in coastal areas. However, shrimp farming is a capital-intensive activity and uncontrolled mushrooming growth of it has led to outbreak of diseases and attributed environmental issues calling for closure of shrimp farms.

Although India has vast freshwater resources, they are not fully exploited except for carp culture in limited scale. Freshwater fish culture employing composite fish culture technology has become popular for use in large number of tanks and ponds in the country. To meet the raw material required by the processing units for export demand, there is urgent need to expand production base of India. In addition, it is always stressed that there is a need to utilize our natural resources productively to ensure the much needed food security.

Considering the high export potential, the giant freshwater prawn, Macrobrachium rosenbergii, the scampi, has immense potential for culture in India. About four million ha of impounded freshwater bodies in the various states of India offer great potential for freshwater prawn culture. Scampi can be cultivated for export through monoculture in existing as well as new ponds or with compatible freshwater fishes in existing ponds. Since the world market for scampi is expanding with attractive prices, there is great scope for scampi production and export. However, the quality of the product must be of high grade to keep the export chain sustainable. To fulfil this objective, the present pilot programme was initiated at P.S.-Swarupnagar, Sub-Div:Basirhat, Dist-North 24 Parganas in West Bengal. In order to maintain the quality of the product as well as of the culture environment (pond), attempt was undertaken to replace the animal (trash fish) ingredient of prawn feed with floral components, and for this the salt-marsh grass of Sundarbans, Porteresia coarctata, was used as protein source to the culture species. The technology of feed preparation has been highlighted in the report. The formulated feed from the salt-marsh grass not only improved the aquatic health of the pond but at the same time increased the growth and protein level of the prawns. In the present venture, the overall production picture in the experimental ponds speaks in favour of the efficacy of the herbal-based prawn feed. The investigation also shows that prawn farming in the freshwater system of Indian Sundarbans is an economically feasible project and the return can be enhanced if specially formulated herbal feed is provided to the culture species instead of the traditional one.

Table 24 List of Acronyms

8.1.1 8A.1.1 Introduction

In the Indian subcontinent, aquaculture contributed over one-third of the country’s total fish production of 6.1 million tonnes in 2003. The total aquaculture production of 2.2 million tonnes was valued at US$ 2.5 billion of which carp alone was responsible for as much as 1.87 million tonnes. On the other hand, the production of high-valued shellfish species, namely, giant river prawn, produced 30,000 tonnes, while shrimps from brackish water, mainly P. monodon, produced 1,15,000 tonnes (FAO 2005). Aquaculture plays an important role in Indian economy and is greatly related with the socio-economic condition of the fish farmer. Apart from supplying quality protein to the consumer, this sector plays a major role in providing employment opportunities and earning foreign exchange. After the high risk of disease outbreak and environmental pollution, the aquaculturists have shifted their interest from shrimp (Penaeus monodon) culture towards scampi culture. The giant freshwater prawn (Macrobrachium rosenbergii) known as scampi in commercial parlance is a highly valued delicious food and commands very good demand in both domestic and export market. Macrobrachium rosenbergii culture is gradually gaining momentum in the present era owing to its price, taste, fast growth rate, less susceptibility to diseases and its compatibility to grow with carps. It is now farmed in many countries; the major producers (>200 million tonnes) are Bangladesh, Brazil, China, Ecuador, India, Malaysia, Taiwan Province of China and Thailand (FAO 2002). About four million ha of impounded freshwater bodies in the various states of India offer great potential for freshwater prawn culture. Scampi can be cultivated for export through monoculture in existing as well as new ponds or with compatible freshwater fishes in existing ponds. Today freshwater aquaculture has witnessed diversification through the incorporation of high-valued species like scampi and has increased its production from 455 tonnes in 1992 to over 30,000 tonnes in 2003. Freshwater aquaculture activity is prominent in the eastern part of the country, particularly the states of West Bengal, Orissa and Andhra Pradesh with new areas coming under culture in the states of Punjab, Haryana, Assam and Tripura. The state of Andhra Pradesh dominates the sector with over 86 % of the total production in India with approximately 60 % of the total water area dedicated to prawn farming, followed by West Bengal. In the state of West Bengal, the existing culture system includes both monoculture and polyculture with Indian major carps in ponds. Grow-out stocking densities range from 0.5 to 2.5 scampi/m² in polyculture and 1–5/m² in monoculture. The culture period is 6–8 months at the beginning of southwest monsoon (June/July with temperature around 27–30 °C), and the scampi are fed with farm-made or commercial feeds (Mitra et al. 2005). In Indian Sundarbans, freshwater aquaculture, in general, is practised with the utilization of low to moderate levels of inputs, especially organic-based fertilizers and feed. The concept of using balanced diet or feed rich in protein for boosting the production is still in an embryo stage. In many pockets of North 24 Parganas district, meat/flesh of live mussels and several gastropod species (mainly Telescopium telescopium) is used with the aim to increase the quantum of final harvest.

In Indian Sundarbans, standard practices in freshwater prawn culture include:

• Pond fertilization with organic manures from cattle or poultry as well as inorganic fertilizers like urea and single super phosphate.

• Provision of supplementary feeds mainly in the form of a mixture of rice bran/wheat bran and groundnut/mustard oilcake in equal ratio.

• Meat/flesh of molluscs is often used as supplementary feed.

It is however documented from several literatures that in a successful Macrobrachium culture, the main thrust is generally given to the dietary protein content. The retention of dietary protein for growth is the goal of nutritionists for the development of cost-effective diets. It has been observed from a series of pilot projects that successful and sustainable aquaculture is a direct function of proper feed which should be nutritionally balanced, eco-friendly and economically viable (Kaushik 1990). For producing cost-effective diet and maintaining an ecologically sound culture system environment, nowadays, artificial feeds particularly of floral origin are extensively used.

Studies conducted on the proximate analysis of green seaweeds, salt-marsh grass and mangrove litter revealed considerable percentage of protein in these flora (Chakraborty and Santra 2008; Banerjee et al. 2009a, b; Hoq et al. 2002). The biomass of these floral species, as documented from the field study, also indicates the viability of using these species as ingredients for mass production of prawn feed. On this background matrix, the present programme was initiated to develop a sustainable scampi culture on the foundation of mangrove floral resources. Another important objective of the present study is to maintain the stability of aquatic health, which deteriorates mostly due to use of traditional feed (having trash fishes as the main ingredients) and flesh of molluscs.

8.1.2 8A.1.2 Objectives

Mangroves and their associate halophytes are unique vegetations with multiple ecological benefits, but linking these endemic vegetations directly with the livelihood of the Sundarban people is the primary aim of this programme. To reach this goal, we designed our proposal to undertake the following objectives:

• To screen the candidate flora (salt-marsh grass) for protein content (quantitative estimation) with the aim to develop eco-friendly nutritive feed for freshwater prawn (Macrobrachium rosenbergii)

• To develop location-specific fish feed preparation technology (through incorporation of floral pulp rich in protein as the major feed ingredient)

• To investigate the impact of formulated feed on the prawn biomass, survival rate, condition index (a function of length and weight) and FCR

• To investigate the interrelationship between protein-rich fish feed and protein level in prawn tissues, through regular monitoring of protein content of culture species

• To investigate the impact of formulated feed on water quality in terms of salinity (as salt water often penetrates the pond from adjacent brackish-water canal), pH, dissolved oxygen (DO), BOD, COD, nutrient concentrations, phytopigment level and organic carbon of bottom soil of ponds

This experimental venture was designed not only to upgrade the ecological health of the culture pond but at the same time to understand the responses of the culture species towards the feed prepared from mangrove associate floral species. It is expected that such venture may lead to economic upliftment of the poverty-stricken island dwellers and open window of alternative livelihood for the local community.

8.1.3 8A.1.3 Physiography of the Study Area

The study area is located at P.S.-Swarupnagar, Sub-Div:Basirhat, Dist-North 24 Parganas in West Bengal. The geographic location of the study area is 22°48′19″ N latitudes and 88°54′21.5″ E longitudes. Physically it is a wetland (Bilballi) along both sides of Sonai canal. The area of the Bilballi is around 15 km². The Sonai canal is connected to the Ichhamati River (Fig. 8A.1.1). The wetland gets submerged under water during monsoon (July–October) and postmonsoon (November–February) and remains exposed during premonsoon (March–June).

Fig. 8A.1.1

Map showing the culture site in the Bilballi wetland (red mark)

The area around the Bilballi wetland is dominated by the fishermen community, who are mostly engaged in wild catch from the canal. However, a section of this fishing community practises polyculture in ponds.

8.1.4 8A.1.4 Materials and Methods

8.1.4.1 8A.1.4.1 Selection of Pond

The selection of ponds was done on the basis of water availability and water quality. The study area Bilballi is a low-lying land, and adequate water is available throughout the culture period (February 2010–September 2010) which is one of the prime requisites for scampi culture. The salinity of the culture area is also low (3–6 psu) and the pH ranges from 6.80 to 8.10. Water is drawn from the adjacent Sonai canal to fill the culture ponds. The pond availability throughout the entire culture period was ensured after negotiating with the pond owner (also the beneficiary) on the basis of the following terms and conditions:

• Feed has to be supplied (as per the requirement) by the University of Calcutta.

• The university will have no claim on the final harvest (except for research related data).

• Day-to-day monitoring of the cultured species and feeding will be done by the beneficiary as per the instruction and training of the researchers of Calcutta University.

• The university research group will be monitoring the water quality and cultured species in every fortnightly interval, and the beneficiary will be required to assist the team during this monitoring phase.

8.1.4.2 8A.1.4.2 Pond Preparation and Water Filling

Two ponds were selected, of which one was experimental and the other was treated as control. The area of the experimental pond is 7,500 m², and the control pond is 10,000 m². At the very initial stage of the experiment, attention was given on scientific pond preparation. For this purpose, ponds were dried sufficiently in order to decompose all organic matters, to oxidize different toxic compounds present in the soil of pond bottom and also to eliminate undesirable filamentous algal mat and eggs of different predatory fishes, crab, etc. Then lime was applied accordingly to maintain soil pH and neutralize the organic acid, pyrite, etc. present in the pond bottom. After the preparation of the pond, water was filled which was mainly done by storing the rainwater and by pumping the water from the adjacent Sonai canal.

8.1.4.3 8A.1.4.3 Stocking of Seeds

Prawn seed collection is a common practice in coastal West Bengal, which is presently discouraged by all sections of the society due to its linkage with several environmental issues like ecological crop loss (mass destruction of several fish juveniles), uprooting of mangrove seedlings and health problems of seed collectors. To step aside all these dark environmental issues, seeds were procured from a commercial hatchery of Nellore district of Andhra Pradesh and stocked on 5 February 2010 at a rate of two individual/m² with initial size 0.80 cm and 0.01 g body weight in each pond. The mean stocking weight was determined from a sample of 100 prawn seeds that were blotted to free from water. Before stocking all the prawn seeds are well acclimatized to avoid temperature and pH shocks (Sarver et al. 1982).

8.1.4.4 8A.1.4.4 Feed Preparation and Feeding Rate

The feed preparation was initiated since the beginning of the programme from the first week of February 2010. The feed composition of the experimental pond (Table 8A.1.1) shows the presence of 30 % dust of salt-marsh grass Porteresia coarctata, which is a mangrove associate species of deltaic Sundarbans.

Table 8A.1.1 Feed ingredients

The Porteresia powder was prepared by drying the pulp taken out from the plant material. The pulp was dried in hot air oven at 45–50 °C and then it was powdered. All the ingredients were weighed and mixed well. Dough was prepared with warm water which was passed through a pelletizer to obtain the desired feed pellets. All of these were carried out in the laboratory of Department of Marine Science, University of Calcutta.

The commercial feed (mostly available from the local market) was provided to the control pond whose ingredient composition is given below in Table 8A.1.2.

Table 8A.1.2 Feed ingredients of locally used commercial feed

As a part of scientific culture, feed chart (Table 8A.1.3) was maintained on the basis of DOC during the culture period in the experimental pond.

Table 8A.1.3 Feed chart

8.1.4.5 8A.1.4.5 Analysis of Physico-chemical Parameters

The success of prawn culture and production of prawn is a direct function of water quality, and the most relevant aquatic parameters in context to aquaculture are listed below along with detailed methodology:

• (a) Surface water temperature

• (b) Surface water salinity

• (c) Surface water pH

• (d) Soil pH

• (e) Organic carbon content of the soil

• (f) Dissolved oxygen

• (g) Nutrient (nitrate, phosphate, silicate) concentration

• (h) BOD

• (i) COD

• (j) Total coliform

• (k) Phytopigment concentration

All these parameters were regularly monitored at an interval of 15 days (fortnightly interval) following the standard protocols (Strickland and Parsons 1972; American Public Health Association (APHA), American Water Works Association, Water Pollution Control Federation 1998) as discussed here.

• (a) Surface water temperature

  Surface water temperature was recorded in both experimental and control ponds by a Celsius thermometer at an interval of every 15 days.

• (b) Surface water salinity (psu)

  Surface water salinity was checked in the field by refractometer and cross-checked in the laboratory by argentometric method.

• (c) Surface water pH

  Surface water pH was recorded in the field by a portable pH meter (sensitivity = ±0.1) during each field trip.

• (d) Soil pH

  Sediment was collected from pond bottom during each field trip.10 g of the collected sediment sample was dried in hot air oven. The dried sample was stirred with 100 ml of distilled water, and it was kept for few hours. Then the pH of the supernatant water was measured by using portable pH meter (sensitivity = ±0.1)

• (e) Organic carbon content of the soil

  The organic carbon content of the collected pond sediment was estimated using the procedure of Walkey and Black (Walkley and Black 1934). 1 g of dried soil sample was taken in a conical flask, and 6 ml of distilled water and 1 ml of phosphoric acid were added. It is kept in hot air oven for 10 min. Then 10 ml K₂Cr₂O₇ and Ag₂SO₄ was added and it is kept in dark for 30 min. After that 200 ml distilled water, 10 ml phosphoric acid and 1 ml DPA indicator were added. Then it was titrated against Mohr salt which was taken in the burette. Finally organic carbon is calculated as % of carbon using the following formula:

  $$ \%\ \text{of}\ \text{carbon}=\left(3.95÷g\right)\times \left(1-T/S\right)$$

  • where G = weight of sample in g

  • S = Mohr salt solution for blank

  • T = Mohr salt solution for sample

• (f) Dissolved oxygen

  The sample was taken from the surface water of the pond in the Winkler bottle of capacity to approximately 150 ml. The water was allowed to flow at a moderate speed avoiding air bubbles. 1 ml of Winkler-I solution was added followed by 1 ml of Winkler-II solution. The bottle was carefully closed with a stopper avoiding the trapping of air bubbles and the bottle was vigorously shaken; after the precipitation settled, 1 ml (1:1) H₂SO₄ solution was added. The bottle was kept in a dark place and titrated in a maximum time of 1 h. The dissolved sample was quantitatively washed down into the conical flask and was titrated with standard thiosulphate solution till the very pale straw colour remained. Then 1 ml of starch solution was added, and the titration was continued to get colourless solution. The volume of thiosulphate was noted and DO of the pond water was calculated using the following formula:

  $$ \text{DO}\left(\text{mg}/\text{l}\right)=\left({V}_{1}\times N\times 32,000\right)/4\left({V}_{2}-\frac{2}{125}\right)$$

  • where N = strength of sodium thiosulphate

  • V ₁ = volume of sodium thiosulphate

  • V ₂ = volume of the sample taken

• (g) Nutrient (nitrate, phosphate, silicate) concentration

  Surface water for nutrient analysis was collected in clean TARSON bottles and transported to the laboratory in ice-freezed condition. Triplicate samples were collected from the same collection site to maintain the quality of the data. The standard spectrophotometric method of Strickland and Parson (1972) was adopted to determine the nutrient concentration in surface water. Nitrate was analyzed by reducing it to nitrite by means of passing the sample with ammonium chloride buffer through a glass column packed with amalgamated cadmium fillings and finally treating the solution with sulphanilamide. The resultant diazonium ion was coupled with N-(1-napthyl)-ethylene diamine to give an intensely pink azo dye. Determination of the phosphate was carried out by treatment of an aliquote of the sample with an acidic molybdate reagent containing ascorbic acid and a small proportion of potassium antimony tartrate. Dissolved silicate was determined by treating the sample with acidic molybdate reagent. The resultant silico-molybdic acid was reduced to molybdenum blue complex by ascorbic acid and incorporating oxalic acid prevented formation of similar blue complex by phosphate. SYSTRONIC UV–VIS spectrophotometer (Type117, Sr.No.690) was used for nutrient (NO₃, PO₄ and SiO₃) analysis at their respective wavelengths.

• (h) BOD (Biochemical oxygen demand)

  • 1. Two 300 ml BOD bottles were half-filled with dilution water. With a large-tipped pipette, the pre-calculated amount of sample was dispensed into each of the two 300 ml of BOD bottles. Then each bottle was filled with dilution water and the stopper was inserted and all air bubbles were excluded.

  • 2. An additional two 300-ml BOD bottles with only dilution water were filled, and the stopper was inserted as in step 1.

  • 3. At 20 °C, one bottle containing diluted samples and one containing only dilution water were incubated.

  • 4. A DO determination on the remaining BOD bottles from step1 and step 2 was run and the initial DO content was recorded.

  • 5. After 5 days, DO determination tests were done with the incubated bottles. The DO content of the incubated bottles was recorded. There should not be an increase or decrease of more than 0.2 mg/l of DO between initial dilution water and final dilution water. Large changes may be caused by improper techniques or contaminated dilution water.

  $$ \text{BOD}=\left({D}_{1}-{D}_{2}\right)-\left({B}_{1}-{B}_{2}\right)\text{}f/P\ \text{mg}l^{-1}$$

  where D ₁ is the dissolved oxygen of diluted sample immediately after preparation, mg/l; D ₂ is dissolved oxygen of diluted sample after 5 days incubation at 20 °C, mg/l; P is decimal volumetric fraction of sample used; B ₁ is dissolved oxygen of seed control before incubation, mg/l; B ₂ is dissolved oxygen of seed control after incubation, mg/l; and f is ratio of seed in sample to seed in control = (% seed in D ₁)/(% seed in B ₁).

• (i) COD (chemical oxygen demand)

  Organic substances in the sample were oxidized by potassium dichromate in 50 % sulphuric acid solution at reflux temperature. Silver sulphate was used as a catalyst, and mercuric sulphate was added to check chloride ion interference.

• (j) Total coliform

  For the microbial analysis of surface water in terms of total coliform load, the most probable number (MPN) procedure by Multiple Fermentation Technique (5 test tube method) was followed as stated in APHA (1998). The technique involves inoculating the sample and/or its several dilution in a liquid medium of lauryl tryptose broth. After expiry of the incubation period, the tubes were examined for gas and acid production by the coliform organisms. This test is known as presumptive test. Since the organisms other than the coliforms may also produce this reaction, the positive tubes from the presumptive test were subjected to a confirmatory test using Brilliant Green Lactose Bile Broth. The density of bacteria was calculated on the basis of positive and negative combination of the tubes using MPN table. In the case of water samples, the results were expressed in MPN/100 ml.

• (k) Phytopigment (chlorophyll a) concentration

  Phytopigment concentration of the ambient aquatic phase was analyzed in order to monitor the productivity of the water. For pigment analysis, 1 l of surface water, collected from each of the pond, was filtered through a 0.45-μm Millipore membrane fitted with a vacuum pump. The residue along with the filter paper was dissolved on 90 % acetone. And it was kept in a refrigerator for about 24 h in order to facilitate the complete extractions of the pigment. The solution was centrifuged for about 20 min under 5,000 rpm, and the supernatant solution was considered for the determination of the chlorophyll pigment by recording the optical density at 750, 664, 647 and 630 nm with the help of SYSTRONICS UV–VIS spectrophotometer (Type177, Sr. No. 690). All the extinction values were corrected for a small turbidity blank by subtracting the 750 nm signal from all the optical densities, and finally phytoplankton pigment was estimated as per the following expressions of Jeffrey and Humprey (1975):

  $$ \text{Chl}\ \text{}a=11.85\ {\text{OD}}_{664}-1.54\ {\text{OD}}_{647}-0.08\ {\text{OD}}_{630}$$

  The values obtained from the equation were multiplied by the volume of the extract (in ml) and divided by the volume of the water (in litre) filtered to express the chlorophyll content in mg/m³.

8.1.4.6 8A.1.4.6 Monitoring of Zootechnical Parameters

Individual weights and lengths of prawns (n = 100) were taken at fortnightly interval during the entire culture period, and the relevant response variables were determined for each control and experimental ponds.

The length–weight relationship of the cultured species was determined to evaluate the proportionality in growth for both control and experimental ponds. Length–weight relationships have been extensively used for estimation of weight from length due to technical difficulties and the amount of time required to record weight in the field, conversion of growth in length equations to growth in weight for use in stock assessment models, estimation of the biomass from length observations and estimation of the condition factors of the aquatic species. In addition to the above, length–weight relationships are useful for understanding spatial and temporal variations of life histories of cultured species in response to environmental variables, feed type, etc.

Condition index (C.I.) was analyzed at fortnightly interval during the culture period as per the expression: C.I. = W/L ³ × 100, where W = weight of the cultured species (in g) and L = length of the cultured species (in cm).

Percentage weight gain was calculated as the difference in weight from the average final weight with respect to the initial weight; weight gain = [(average individual final weight – average individual initial weight)/average individual initial weight] × 100.

Feed consumption was estimated on the basis of the total amount of feed provided to the cultured species during the culture tenure (6 months). Feed conversion ratio (FCR) was analyzed after the harvesting of shrimps as per the expression: FCR = ∆f/∆b, where ∆f = change in feed biomass and ∆b = change in body biomass of the cultured species.

The survival rate was measured as percentage of the difference of stocking number and production volume (No.) at the end of the culture period.

Body pigmentation was assessed for each treatment on prawn cooked for 5 min in boiling water and comparing the orange-red colouration with Roche SalmoFan^TM colour score. The astaxanthin content of the harvested prawn was also analyzed by the standard chemical method.

8.1.4.7 8A.1.4.7 Protein Estimation

Protein of feed and prawns were estimated by Lowry’s method as per the following procedure: Protein sample was mixed with 10 ml phosphate buffer in tissue homogenizer and was homogenized and centrifuged. The supernatant was used to estimate the proteins. 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard of bovine serum albumin were pipetted out in a series of previously cleaned test tubes and volume was made out to 1 ml. 1 ml supernatant was also pipetted out into one another test tube. A tube containing 1 ml distilled water was taken as a blank. 5 ml of reagent C was added to each tube and after well mixing kept for 10 min. 0.5 ml of reagent D was added to all test tubes and mixed well. The mixture was incubated in room temperature at dark condition for 30 min. Blue colour was developed and OD values were measured at 600 nm. Then a graph was drawn and factor was calculated. The concentration of protein was calculated using following relation:

$$ \begin{array}{l}\text{Protein}\ \text{concentration}\ (\%)=\text{Factor}\times \text{OD}\ \text{value} \\ \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \text{of}\ \text{the}\ \text{sample}\times 100\end{array}$$

8.1.4.8 8A.1.4.8 Statistical Analysis

Analysis of variance (ANOVA) was computed between all the selected parameters (indicators of our experiment) considering both control and experimental ponds to evaluate the differences caused by inclusion of Porteresia coarctata dust in the feed. All statistical calculations were performed with SPSS 9.0 for Windows.

8.1.5 8A.1.5 Results

8.1.5.1 8A.1.5.1 Physico-chemical Parameters

The results of physico-chemical parameters of the two ponds (control and experiment) are shown in Table 8A.1.4 and Fig. 8A.1.2.

Table 8A.1.4 Monthly variations of environmental parameters in experimental and control ponds during 8 months culture period of freshwater prawn (Macrobrachium rosenbergii)

Fig. 8A.1.2

Hydrological parameters and soil conditions of control and experimental ponds with trend line equations

The surface water temperature during the study period ranged from 28.3 to 36.1 °C with a mean value of 32.4 ± 2.87 °C in the control pond, while in the experimental pond the value ranged from 28.3 to 36.2 °C with a mean value of 32.4 ± 2.93 °C.

The surface water salinity ranged from 3.40 to 6.05 psu with a mean value of 4.92 ± 0.90 psu in the control pond and 3.00 to 5.67 psu with a mean value of 4.61 ± 0.82 psu in the experimental pond.

The surface water pH ranged from 6.88 to 8.10 with a mean value of 7.53 ± 0.44 in the control pond and from 7.80 to 8.02 with a mean value of 7.92 ± 0.08 in the experimental pond.

The DO values ranged from 3.35 to 5.93 mg/l with a mean value of 4.52 ± 0.87 mg/l in the control pond and from 4.58 to 5.85 mg/l with a mean value of 5.14 ± 0.4 mg/l in the experimental pond.

The nutrient (nitrate) ranged from 9.81 to 25.01 μgat/l with a mean value of 19.08 ± 5.53 μgat/l in the control pond and from 10.67 to 15.22 μgat/l with a mean value of 13.43 ± 1.68 μgat/l in the experimental pond.

The phosphate concentration ranged from 0.95 to 3.68 μgat/l with a mean value of 2.25 ± 1.04 μgat/l in the control pond and from 1.02 to 2.01 μgat/l with a mean value of 1.58 ± 0.39 μgat/l in the experimental pond.

In case of silicate the concentration ranged from 25.32 to 31.09 μgat/l with a mean value of 28.13 ± 1.71 μgat/l in the control pond and from 23.41 to 34.87 μgat/l with a mean value of 28.67 ± 3.87 μgat/l in the experimental pond.

The BOD value ranged from 3.5 to 13.6 mg/l with a mean value of 9.07 ± 3.34 μgat/l in the control pond and from 3.2 to 6.8mg/l with a mean value of 4.97 ± 1.1 μgat/l in the experimental pond.

In case of COD the value ranged from 95 to 131 mg/l with a mean value of 117.75 ± 10.85 μgat/l in the control pond and from 72 to 90 mg/l with a mean value of 80.87 ± 6.22 μgat/l in the experimental pond.

The Chl a concentration during the study period ranged from 2.93 to 4.57 mg/m³ with a mean value of 3.7 ± 0.61 mg/m³ in the control pond and from 2.90 to 3.71 mg/m³ with a mean value of 3.26 ± 0.28 mg/m³ in the experimental pond.

The total coliform load (MPN value) during the study period ranged from 359/100 ml to 1,136/100 ml with a mean value of 722.62 ± 235.33 MPN/100 ml in the control pond and from 365/100 ml to 567/100 ml with a mean value of 419.62 ± 63.53 MPN/100 ml in the experimental pond.

The organic carbon of the pond bottom soil during the study period ranged from 1.03 to 3.15 % with a mean value of 1.956 ± 0.9 % in the control pond and from 1.08 to 2.47 % with a mean value of 1.45 ± 0.45 % in the experimental pond.

The pH of the pond bottom soil ranged from 5.25 to 8.02 with a mean value of 7.13 ± 0.95 in the control pond and from 7.02 to 7.25 with a mean value of 7.13 ± 0.08 in the experimental pond.

8.1.5.2 8A.1.5.2 Zootechnical Parameters

Prawns fed with Porteresia-based diet exhibited higher final weights and better weight gain at the end of the experiment (Figs. 8A.1.3 and 8A.1.4). C.I. values of prawns were also higher in experimental ponds than control pond (Table 8A.1.5), which implies a better environment in experimental pond for the survival and growth of the species. The survival rate was found to be 71.2 % in the control pond and 76.5 % in experimental pond. The biotic indicators of the experimental approach are summarized in Table 8A.1.6. The trend line equations of length–weight relationship for experimental and control ponds during the culture period are shown in Figs. 8A.1.2 and 8A.1.3. The allometric equations reveal the proportionate increase of weight with respect to length in experimental pond. The R ² values also indicate good significance of the trend lines. On contrary, in case of control pond the allometric equations reveal completely a different picture where there is a disproportionate increase of weight with respect to length. The R ² values for control pond are insignificant showing minimum goodness of fit.

Fig. 8A.1.3

Length–weight relationship of Macrobrachium rosenbergii in experimental pond

Fig. 8A.1.4

Length–weight relationship of Macrobrachium rosenbergii in control pond

Table 8A.1.5 Monthly variation of condition index in experimental and control ponds

Table 8A.1.6 A comparative account of zoo-technical parameters (associated with M. rosenbergii culture) in control and experimental ponds

An important factor governing the consumer acceptance and market value of many cultivated fish and prawn species is the pink or red colouration of their flesh or boiled exoskeleton (Brun and Vidal 2006). In the wild, this colouration is achieved through the ingestion of carotenoid pigments particularly astaxanthin contained within invertebrate food organisms (Johnson et al. 1977; Ibrahim et al. 1984). The Porteresia-based feed in the present study resulted in higher astaxanthin values in harvested prawns of experimental pond (115.56 ppm) as reflected through darker orange-red colouration of shrimp exoskeleton in comparison to control pond (84.38 ppm). Roche SalmoFan^TM colour score showed the value of 24 in control pond, much less than experimental pond with a colour score of 29. The protein content was also higher in the prawns fed with Porteresia-based feed (Table 8A.1.7).

Table 8A.1.7 Protein level in formulated feed and prawn

The present pilot-scale study speaks in favour of healthy pond environment, better and proportionate growth, higher survival rate and low FCR values through use of Porteresia-based feed.

8.1.6 8A.1.6 Discussion

ANOVA results indicate no significant differences between surface water temperature, salinity and pH between two ponds which may be attributed to location of both the ponds in the same area. The linear distance between experimental and control pond is approximately 50 m. Significant differences with respect of organic carbon and pH of pond bottom soil, dissolved oxygen, nutrient load, BOD, COD and phytopigment concentrations of water were observed (p < 0.01) which clearly indicates the difference in water quality due to application of different types of feed. The formulated feed prepared from salt-marsh grass not only upgraded the water but also reduced the total coliform count which was higher in case of control pond (where commercial feed available in local market was applied). This commercial feed contains trash fish and shrimp dust as a source of protein. The residual commercial feed deteriorated the water quality by increasing the organic carbon, nutrient load (except silicate), BOD, COD and total coliform.

The trend lines of these variables along with their respective equations and R ² values confirm the variation of water quality through application of mangrove-based feed.

Critical analysis of the zootechnical parameters reveals better growth of the species cultured in experimental pond. In addition to increase of survival rate of the cultured species, the specific growth rate has also increased in the experimental pond (Tables 8A.1.6 and 8A.1.8).

Table 8A.1.8 A comparative picture of growth performance, survival rate, condition index and FCR (Feed Conversion Ratio) related to M. rosenbergii culture in control and experimental ponds

Analysis of the length–weight relationship reveals some interesting features like proportionate increase of length and weight of the prawns in the experimental pond throughout the culture tenure. On the contrary, such proportionality has decreased with the increase of age of the stocked individuals in the control pond (Figs. 8A.1.2 and 8A.1.3). This feature indicates that the formulated feed from Porteresia coarctata regulates the length–weight relationship throughout the culture period in a uniform pattern. The deviation from uniformity in control pond may be attributed to use of commercially available feed from local market which was fed without any regularity as stated in the feed chart (Table 8A.1.3). This may increase the load of residual feed in the pond bottom leading to less biomass production in the control pond. The FCR value is a litmus test of the situation.

Low FCR value in experimental pond indicates that majority of the feed has been converted into biomass which is also an indication acceptability of Porteresia coarctata-based feed by the species. In case of control pond, the FCR value (relatively higher than the experimental pond) reflects a major quantum of wastage with low input in the biomass sector. The maximum percentage of feed wasted in case of control pond remained as residual feed. The residual feed degrades the water quality and pond environment which is again indicated by the condition index value. The index is the reflection of the health of ambient environment of cultured species, and its lower value (as seen in case of control pond) is a reflection of degraded environment. ANOVA performed with the monthly condition index values (Table 8A.1.5) indicates significant difference (p < 0.01) between the two ponds. The Porteresia coarctata-based prawn feed seems to be the major player for such variation.

Plant proteins have been found to be relatively poorly utilized in crustaceans in terms of growth in comparison to protein of animal origin. However, digestibility studies in freshwater prawn have indicated that the species can efficiently digest both plant and animal protein sources (Ashmore et al. 1985). The omnivory of freshwater prawn permits the use of a wide variety of locally available feedstuffs including commercial by-products as ingredients in formulated diets. To create a balanced diet, it is necessary to establish the minimum protein level to provide essential amino acids (Guillaume 1997; Tacon and Akiyama 1997). However, in the present study, the amino acids for P. coarctata diet have not been determined. Hence, the protein level of the salt-marsh grass may be the factor responsible for acceleration of growth and survival percentage. Millikin et al. (1980) indicated that M. rosenbergii species attains best growth at 40 % protein level in feed. Castell et al. (1989) have concluded that protein level ranging between 30 and 38 % resulted in the best growth of the species.

The results of the present studies partially differ with the findings of Du and Niu (2003), who conducted a study in tank water on M. rosenbergii fed with diets where 0, 20, 50, 75 and 100 % of fish meal is replaced by soybean meal. They concluded that soybean meal, without supplementation of amino acids or other additives, is not suitable as a major protein source in freshwater prawn diets. However, Weidenbach (1980) reported that prawns are able to adjust to the absence of feed pellets by increasing consumption of available vegetation. Tidwell et al. (1993) indicated that animal ingredients like fish could be partially or totally replaced by soybean meal and distiller by-products in diets for the pond production of freshwater prawns. According to Tidwell et al. (1995), prawns may be able to adjust to reductions in the nutritional value of prepared diets (i.e. protein source and vitamin and mineral content) by increasing predation on natural fauna (i.e. macro invertebrates) in the pond. All these studies reflect the wide adaptability of the species to different categories of feed. With this wide range of metabolic adaptability, inclusion of salt-marsh grass in the feed ingredient list of freshwater prawn will not only upgrade the ecological environment but will also ensure a better quality product and a livelihood option for the local inhabitants.

8.1.7 8A.1.7 Summary

Aquaculture has become a peak industry in the present millennium, which involves farming with prawn, cuttlefish, squid, lobster and other such culinary delights actually ‘cultivated’ in aquatic enclosures under scientifically controlled conditions (Rajkhowa 2005). The use of nutrient-rich feed continues to gain wide acceptance in the aquaculture industry in order to boost up the quality of the aquacultural products. Such feed results in substantial reduction in the overall variable cost of an operation through improved animal performance (indicators are length–weight relationship, specific growth rate, etc.), better FCR and improved water quality due to reduction in the amount of nutrients and suspended solids (i.e. faecal matter and uneaten residual food particles) in the culture system. Porteresia-based formulated feed showed better growth performance of the cultured species with respect to condition index values and survival rate (Table 8A.1.8). Body pigmentation improved in the cultured species of experimental pond and showed significantly higher astaxanthin level than the controlled pond. A series of experiments are still needed for time testing the results and make the programme sustainable for the poor island dwellers of lower Gangetic delta.

8.1.8 8A.1.8 A Way Forward

The farming of giant river prawn (Macrobrachium rosenbergii) has gained increased interest in recent years, due to its high economic value and an annual production of over 30,000 tonnes through the use of monoculture practice. In addition, the sector has been witnessing increased interest in diversification with the inclusion of high-valued species, including medium and minor carps, catfishes and murrels. While carp and other finfishes are grown for the domestic market, a large proportion of freshwater prawn production is exported. Aquaculture in India is usually practised with the utilization of low to moderate levels of inputs, especially organic-based fertilizers and feed. India utilizes only about 40 % of the available 2.36 million hectares of ponds and tanks for freshwater aquaculture, and there is still enough room for expansion. In areas like the one where this pilot programme was carried out, there is a great potential for aquaculture. However, this is practised in a non-scientific way without any water and feed management. The inevitable results are disease outbreak and mass mortality of the cultured species particularly shrimps and prawns. Under this circumstance, the present programme and its success motivated the local aquaculturists. Suggestions came from their end to scale-up the venture, which is beyond the capacity of the academic institute like the University of Calcutta. Hence, the Forest Department, Govt. of West Bengal, being the major stakeholder of Sundarbans, was approached with the proposal of training the local people the art of prawn feed preparation from salt-marsh grass, Porteresia coarctata. The department agreed to this proposal. It is expected that this untapped service of mangroves may be used for the betterment of local people and island dwellers of Indian Sundarbans. The coordinator feels that a publication from the end of MFF, IUCN, may spread the message of such nonconventional use of mangroves to stakeholders engaged in the pisciculture sector.

The Sundarbans mangrove region is a threatened ecosystem due to a multitude of factors like prolonged overexploitation of its natural resources, its use as a sink of anthropogenic wastes, industrial and maritime wastes generated in the upstream of the rivers flowing through the region, high population pressure around the region and the resultant shrinkage of the area brought about by clearing of forest land for agriculture and tiger prawn culture and lack of proper ecological management. The present programme has immense ecological and economic relevance in connection to these threats, due to its connection to the following lanes:

• Upgradation of the freshwater system (canals, ponds, ditches, etc.) and therefore clearance of mangrove areas for the culture of Penaeus monodon may be totally avoided.

• Involvement of the local people in three livelihood tiers: preparation of fish feed, eco-friendly culture practice and nursery development of Porteresia coarctata for raw material backup to sustain the floral-based fish feed industry. It is expected that such involvement will restrict a sizable fraction of the people from intruding into the forest.

• Scientific utilization of mangrove floral resources for sustainable pisciculture practice in the area.

• Improvement of aquatic health in terms of physico-chemical parameters due to replacement of animal ingredients (like trash fish dust, shrimp dust) in the traditional fish feed with floral components.

• Introduction of a new technology be fitted to the area.

• Economic upliftment of the local people.

Annexure 8A.2: Study on the Role of Mangrove-Based Astaxanthin in Shrimp Nutrition

• Abhijit Mitra,
• Rajrupa Ghosh,
• Ajoy Mallik,
• Kunal Mondal,
• Sufia Zaman &
• Kakoli Banerjee 

1. R. Ghosh • A. Mallik • K. Mondal • K. BanerjeeDepartment of Marine Science, Universityof Calcutta, 35, B.C. Road, Kolkata, West Bengal, 700 019, India

   Rajrupa Ghosh, Ajoy Mallik & Kunal Mondal

2. Department of Marine Science, University of Calcutta and Department of Oceanography, Techno India University, Salt Lake Campus, Kolkata, West Bengal 700 091, India

   Abhijit Mitra, Sufia Zaman & Kakoli Banerjee

8.2.1 8A.2.1 Preface

In the Indian subcontinent, tiger prawn (Penaeus monodon) is the single dominant item in the export basket of marine food, which accounts for almost two thirds of the total export earnings. Although several species of crabs, lobsters, oysters, mussels, sea cucumbers and fin fishes are in the list of exportable items, it is shrimp farming which has opened the avenue of large-scale livelihood in coastal villages of our country. The backbone of this aquaculture sector, however, depends largely on proper feed and pond management, which have been very poorly progressed and neglected in terms of research, development and technology transfer.

The rearing of large number of shrimps in relatively confined conditions necessitates a detailed understanding of their nutrition in order to provide a diet that is adequate for their optimum growth and well-being. Adequate and balanced diet for cultured shrimp is the foundation on which the success of commercial shrimp farming stands. Shrimp farming, on a global scale, till a few years back, was largely dependent upon ‘natural’ food with some supplementation of the by-products of agriculture, fishing and industry, such as slaughterhouse wastes; tiny shrimps, snail meat, clam meat, mussel meat in raw conditions; grain wastes; and silkworm pupae. At very low stocking densities, these diets were adequate as most of the nutrient requirements for shrimp/prawn were satisfied from natural sources. However, at high stocking densities, shrimps are dependent on artificial feed, benefiting slightly from natural feed. Thus, at high stocking densities, inadequate supplementary feeding leads to malnutrition with the resultant consequences like poor growth, increased disease susceptibility and parasitic and bacterial infestation. This was the primary cause behind the failure of blue revolution in the mid-1990s, when disease problems totally devastated the shrimp industry.

Nutritionally complete and balanced diet helps to promote faster growth of the cultured species and resistant against diseases. Nowadays special substances are incorporated in fish feed in low dose to enhance feed intake, growth and feed conversion to biomass and resistance against diseases. These are called additives and are obtained from various natural sources. The market value of shrimp and consumer acceptability is determined on the basis of colour, which is basically the reflection of carotenoid content in the shrimp tissue. The colouration of shrimp has been observed to be a function of astaxanthin content, which is an important constituent of additives used in shrimp feed. It is a naturally occurring carotenoid pigment possessing strong biological antioxidant property. Apart from imparting attractive colour, astaxanthin exhibits strong free radical scavenging activity and protects against lipid peroxidation and oxidative damage of LDL-cholesterol, cell membranes, cells and tissues (http://www.astaxanthin.org/). Many types of fish and crustaceans including salmon, red sea bream, shrimp and lobsters accumulate astaxanthin in their body tissues. In most cases, astaxanthin has a red-orange to pinkish tinge, and in some live crustaceans, the astaxanthin molecule is bound to a protein. During the process of cooking, the bond between the protein and astaxanthin is disrupted allowing us to see the red-orange colour of astaxanthin (http://www.astafactor.com/in-nature.htm). Considering the marine source of astaxanthin, the proposed project is aimed at formulation of shrimp feed by using astaxanthin derived from coastal floral sources (Avicennia marina, Avicennia alba, Avicennia officinalis, Sonneratia apetala, Porteresia coarctata, Enteromorpha intestinalis, Ulva lactuca, Catenella repens and Sueda sp.).

8.2.2 8A.2.2 Project Task

The main tasks of the project encompass the introduction of healthy seeds from hatchery (wild catch will be avoided), their culture through scientific methods by way of water management and soil management, feed preparation technology (from floral extract rich in astaxanthin), feed management and finally the cost–benefit analysis of the beneficiaries.

8.2.3 8A.2.3 Objectives

The prime objectives of the present programme are:

• To screen the candidate flora for astaxanthin content (quantitative estimation) with the aim to develop eco-friendly nutritive feed for shrimp (Penaeus monodon)

• To develop location-specific shrimp feed preparation technology (through incorporation of floral astaxanthin) and impart the same to the local people through workshops, awareness programmes and meeting at panchayat level

• To investigate the impact of formulated feed (with astaxanthin based additive) on the shrimp biomass, survival rate, condition index and FCR

• To investigate the interrelationship between astaxanthin-rich shrimp feed and astaxanthin level in shrimp tissues, through weekly monitoring of cultured shrimp’s astaxanthin content

• To investigate the impact of formulated feed (with astaxanthin based additive) on water quality in terms of salinity, DO, nutrient load and phytopigment level

8.2.4 8A.2.4 Introduction

Aquaculture-related feed technology for the last two decades has advanced a lot, and as a result, different types of artificially made compounded feed are being used for the culture of shrimps in different countries (SEAFDEC 1981; Liu and Mancebo 1983; Shigueno 1984). However, in this context, it is very much pertinent to note that knowledge of nutrient requirements for shrimp gathered by different successful feed manufacturers throughout the world are not well documented at this time. Every company keeps such information as a trade secret. Multinational companies have greater advantage in the ability to improve its feed quality rapidly and efficiently, since sufficient funds are provided for R & D (Chaudhuri 1995). Feed cost is the major limitation to profitable aquaculture in most areas. Traditionally, formulated shrimp feed contains high levels of marine protein sources. While used mainly as a source of protein, marine meals are also valued for their content of essential fatty acids, cholesterol, attractants and other unidentified growth factors. As more information becomes available on shrimp nutrition, synthetic components such as amino acids and fatty acids were used with successful results as a means to lower feed costs. More plant protein such as soybean meal is being used to replace the expensive marine proteins. Soybean meal also has the advantage of being resistant to oxidation and spoilage and is naturally clean from organisms such as fungi, viruses and bacteria that are harmful to shrimp.

To date, limited research has been conducted to evaluate the nutritive value of soybean meal as a partial or complete replacement for marine protein sources in shrimp feeds. Levels as high as 20–40 % soybean meal give acceptable performance. Results are variable within the range, however, and depend on several factors such as water quality, salinity, age of shrimp, diet composition and inclusion of synthetic amino acids. Soybean meal has been projected and estimated to remain an abundant and economical source of feed protein relative to fish meal well into the next century. Long-term success of shrimp industry will depend on increased use of soybean meal in combination with supplemental amino acids. The feed becomes nutritionally well balanced and disease resistive with the incorporation of astaxanthin from external sources. Today farmed salmon are fed with a diet containing natural astaxanthin to achieve the same astaxanthin profile as their wild counterparts. Esterified astaxanthin, found in Haematococcus pluvialis, is a stable form and is believed to be stored in the tissue without oxidation. Although astaxanthin level is an important criterion of fish species in terms of quality, research and development in this particular branch has not yet crystallized (Ziegler 1989; Shigeo et al. 1994; Finkelstein et al. 1995; Graves et al. 1996). The production of astaxanthin from Phaffia sp. has been extensively studied. Phaffia is yeast that naturally produces astaxanthin. Phaffia cells normally produce about 300 ppm per dry mass. This level of production is too low to develop a commercially viable synthesis. Considerable research has been performed to increase the productivity of astaxanthin synthesis in Phaffia (R. Mawson, U.S. Patent, 5453565, 1995). The result of this effort has encouraged several companies in the world to produce a Phaffia product containing astaxanthin for the fish and shellfish aquaculture industry.

The single-celled alga Haematococcus pluvialis has also been extensively studied as a host to produce astaxanthin (Furubayashi 1991). Technology has been developed to take advantage of the physiology of this alga. Under growing conditions, this alga does not produce astaxanthin. However, when the culture is subjected to stress in which nutrients are eliminated from the growth medium, then the alga produces and accumulates astaxanthin. The levels of astaxanthin can be very high, and there are reports of astaxanthin accumulation of greater than 4 % per dry mass. Under most conditions, this level of astaxanthin synthesis and accumulation occurs only after several weeks of growth. Methods to reduce the time required to produce astaxanthin are currently under extensive study by several groups, and pilot-scale production is under way at several sites. As with Phaffia, cells of Haematococcus pluvialis have been found to be a suitable delivery vehicle for astaxanthin for aquaculture, and no further purification of the astaxanthin is required. A number of groups have investigated a variety of organisms and systems to produce various carotenoids. The plant Adonis aestivalis produces astaxanthin in the petals of the flower. Researchers have developed varieties of Adonis with an increased astaxanthin content (Mawson 1995). The present programme of screening the mangroves as a source of astaxanthin has not been reported yet from any part of the world.

8.2.4.1 8A.2.4.1 Astaxanthin: An Overview

Oxygen is an indispensable molecule for the growth and survival of aerobic organisms in the planet Earth. The entire mechanism of aerobic respiration resulting in the liberation of ATP is triggered by oxygen. However, this gaseous lifeline of the planet has an important demerit as it poses oxidative stress. Oxidative stress has been defined as a disturbance in the cell or organism related to pro-oxidant–antioxidant balance in favour of the former (Sies 1991). It differs from any other stresses in that its primary effectors, the reactive oxygen species (ROS), can arise largely in the course of normal cell metabolism (Marova et al. 2005). Oxidative stress is involved in several pathological problems, especially in chronic degenerative diseases as diabetes, atherosclerosis, cancer, and Alzheimer’s disease. Question arises how the organisms get rid of the oxidative stress. Till date the answer is related to the antioxidant defence mechanism of aerobic organisms. This defence mechanism is provided by integrated antioxidant system, which has three distinct components (Scheme 8A.2.1), each equipped to reduce oxidative stress and the resultant adverse effects.

Scheme 8A.2.1

Components of integrated antioxidant system

Preventative antioxidants suppress the formation of free radicals. Radical-scavenging antioxidants, such as the flavonoid compounds and vitamin C, serve to ‘mop up’ excess free radicals. Thus, scavenging antioxidants remove the ROS once formed, thereby preventing radical chain reaction (Marova et al. 2005). Repair enzymes play an important role in repairing and removing ROS damaged molecules. Vitamin E and the carotenoids are very important biological antioxidants that have both preventative and radical-scavenging roles.

Astaxanthin is a carotenoid. It belongs to a larger class of phytochemicals known as terpenes. It is classified as a xanthophyll, which means ‘yellow leaves’. Like many carotenoids, it is a colourful, fat/oil-soluble pigment. Astaxanthin can be found in microalgae, yeast, salmon, trout, krill, shrimp, crayfish, crustaceans and the feathers of some birds. Professor Basil Weedon was the first to map the structures of astaxanthin (Fig. 8A.2.1).

Fig. 8A.2.1

Chemical structure of astaxanthin

Astaxanthin, unlike some carotenoids, does not convert to vitamin A (retinol) in the human body. Too much vitamin A is toxic for a human, but astaxanthin is not. However, it is a powerful antioxidant; it is ten times more capable than other carotenoids, which is due to its pure antioxidant nature, unlike other pigments which have pro-oxidant features (Fig. 8A.2.2).

Fig. 8A.2.2

Comparative Anti- and Pro-oxidant properties of different carotenoids

In nature, a typical xanthophylls-producing unicellular microalgae is Haematococcus pluvialis, well known for its massive accumulation of ketocarotenoids, mainly astaxanthin up to 4 % of its dry mass and its acyl esters, in response to various stress conditions, e.g. nutrient deprivation or high irradiation. Also, the yeast Phaffia rhodozyma has been widely used for astaxanthin production in fed-batch fermentation processes using low-cost materials as substrates (An et al. 2001; Chociai et al. 2002; Vazquez et al. 1998). Because of antioxidative properties and the increasing amount of astaxanthin needed as a supplement in the aquaculture of salmonoids and other seafood, there is growing interest in finding out the natural reservoir of astaxanthin. The present project is a venture towards this mission in the framework of lower Gangetic region, which sustains the famous Sundarbans mangrove ecosystem.

8.2.5 8A.2.5 Physiography of the Study Area

The Indian Sundarbans is one of the most biologically productive and taxonomically diversified, low-lying, mangrove detritus-based, open, dynamic, heterogeneous coastal ecotones situated at the apex of the Bay of Bengal (between 21°13′ and 22°40′ N latitude and 88°03′ to 89°07′ E longitude) (Fig. 8A.2.3). The region is bordered by Bangladesh in the East, the Hugli river in the West, Dampier and Hodges line in the North and the Bay of Bengal in the South. The important rivers in this deltaic lobe from west to east are Hugli, Muriganga, Saptamukhi, Thakuran, Matla, Gosaba and Harinbhanga that finally end up at Bay of Bengal. This mangrove forest has been declared as the world’s largest mangrove forest and is declared the World Heritage Site by IUCN in 1987, Biosphere Reserve under Man and Biosphere Programme by UNESCO in 1989 and is a proposed Ramsar Site, which sustains 34 true mangrove species and about 50–55 mangrove associate species. It is the only mangrove forest in the planet Earth inhabited by the Royal Bengal Tiger (Panthera tigris tigris). The aquatic subsystem in and around this deltaic lobe is the cradle of several species of finfish, nursery of different variety of shell fish and reservoir of several biological resources – still untapped. The landscape of Indian Sundarbans covering an area of 9,630 km² encompasses mangrove forests, riverine, estuarine, coastal and marine habitats. On one hand, it exhibits enormous diversity based on its genesis, geographical location, hydrological regimes and substrate factors, and on the other hand, it also sustains rare endemic genetic material which demands preservation and proper sustainable utilization for the benefit of mankind.

Fig. 8A.2.3

Map showing Indian Sundarbans

The climate of the area is humid (up to 96 %) and tropical with temperature ranging from 11.8 to 34.5 °C. The climate is monsoonal with an average rainfall of 1,900 mm. During monsoon months (July–October), the estuarine system becomes dominated by freshwater resulting in strong predominance of ebb tides. From November to February, the system becomes salinity gradient dominated, and during premonsoon period (March–June) due to less freshwater discharges, effects of tide are considerably accentuated resulting in the system more or less marine dominated. The low-lying tidal flats of the quaternary period have been developed from alluvial deposits of river Hugli, Saptamukhi and Matla together with tidal incursions. The soil consists of clayey loam or different black clay; there is no rock. The areas are about 1 m above the mean sea level and are submerged under saline estuarine waters for several hours in the spring tide twice a day.

There are about 102 islands in Indian Sundarbans (54 inhabited and 48 uninhabited) which supports human habitation of about 4.2 million people. This landscape has changed remarkably due to large-scale human intervention, overexploitation, demographic pressure, loss of habitats and change of ecological condition. Several of the earlier workers reported that many species have become extinct or are in a very threatened or degraded state. However, the geographical area is a rich reservoir of biotic resources for future.

8.2.6 8A.2.6 Materials and Methods

8.2.6.1 8A.2.6.1 First Phase: May 2007–April 2008: Screening of Mangroves for Astaxanthin

The entire network of the present programme encompassed the sampling of the leaves of ten dominant mangrove species during the low tide period from the Jharkhali island during May 2007 to April 2008. Leaves of the selected species were collected from two different portions (submerged lower zone and exposed upper zone) of the same plant. The lower region of the tree gets inundated during the high tide condition and the upper region of the same plant remains unexposed to tidal water. In addition to true mangrove species, the astaxanthin level of few associates like Porteresia coarctata, Enteromorpha intestinalis, Ulva lactuca, Catenella repens and Sueda sp. was also monitored. Salinity, pH, temperature, dissolved oxygen and nutrient load of the ambient water were analyzed simultaneously to pinpoint the hydrological parameters to which the plant species are exposed in natural condition. The collected leaves were thoroughly washed with ambient water followed with deionized water and oven dried at 110 °C overnight. The extraction of astaxanthin was done in organic solvent as per the standard method and analyzed spectrophotometrically. The mean results of all the analyses (of 12 months; May 2007 to April 2008) are shown in Table 8A.2.2.

8.2.6.2 8A.2.6.2 Second Phase: May 2008–April 2009: Shrimp Nutrition

On the basis of astaxanthin concentration, Porteresia coarctata was selected for feed preparation because of its high astaxanthin content. Accordingly two types of feed (for control and experimental ponds at Jharkhali) were selected. The experimental pond was provided with Porteresia coarctata dust to replace the fish and shrimp meal component (Table 8A.2.1) of the traditional fish feed. The second phase was thus devoted for shrimp feed preparation with the aim to replace the animal ingredients of the feed with plant matter.

Table 8A.2.1 Feed ingredients for shrimp culture in control and experimental ponds

8.2.6.3 8A.2.6.3 Third Phase: May 2009–April 2010: Shrimp Tissue and Water Quality Analysis

8.2.6.3.1 8A.2.6.3.1 Water Quality Analysis

Hydrological parameters were analyzed at fortnightly interval for a 90-day culture period (2 January–2 April 2010).

The surface water salinity was recorded by means of an optical refractometer (Atago, Japan) in the field and cross-checked in laboratory by employing Mohr–Knudsen method (Strickland and Parsons 1968). The correction factor was found out by titrating the silver nitrate solution against standard seawater (IAPO Standard Seawater Service, Charlottenlund, Slot Denmark; chlorinity = 19.376 psu). This laboratory method was applied to estimate the salinity of standard seawater procured from NIO and a standard deviation of 0.02 % was obtained for salinity. The average accuracy for salinity (in connection to the triplicate sampling) is ± 0.28 psu.

Glass bottles of 125 ml were filled to overflow from collected water samples and Winkler titration was performed for the determination of dissolved oxygen.

The pH of surface water was recorded through a portable pH meter (Hanna, USA), which has an accuracy of ± 0.1.

A Secchi disk was used to measure the transparency of the water column, and the data was used to calculate the euphotic depth.

Surface waters were analyzed for nutrient concentrations (nitrate, phosphate and silicate) following the standard spectrophotometric method (Strickland and Parsons 1972).

Organic carbon content of pond bottom soil was estimated by the standard titration method (Walkey and Black 1934).

8.2.6.3.2 8A.2.6.3.2 Shrimp Tissue Analysis

Individual weights and lengths of shrimps (sample size = 50 from each pond) were taken at fortnightly interval during the 90-day culture period, and the relevant biological variables were determined for each control and experimental ponds.

Condition index was analyzed as per the expression: C.I. = W/L ³ × 100, where W = weight of the cultured species (in g) and L = length of the cultured species (in cm).

Percentage weight gain was documented by calculating the difference in weight from the average final weight with respect to the initial weight; weight gain = [(average individual final weight – average individual initial weight)/average individual initial weight] × 100.

The survival rate was measured as percentage of the difference of stocking number and production volume (No.) at the end of the culture period.

Feed conversion ratio (FCR) is the weight of feed consumed per unit of body weight gain and was analyzed after the harvesting of shrimps as per the expression: FCR = ∆f/∆b, where ∆f = change in feed biomass and ∆b = change in body biomass of the cultured species.

Astaxanthin in the shrimp tissue was estimated as per the standard spectrophotometric method (Schuep and Schierle 1995) and body pigmentation of the cultured shrimp was assessed (for each treatment) after boiling the shrimp for 5 min in water and comparing the orange-red colouration with Roche SalmoFan^TM colour score.

8.2.7 8A.2.7 Results and Discussion

8.2.7.1 8A.2.7.1 Astaxanthin Level in Mangroves

The astaxanthin level in the selected mangrove species (collected from Jharkhali region) exhibits significant variations. It is of the order Heritiera fomes > Bruguiera gymnorrhiza > Avicennia alba > Avicennia marina > Avicennia officinalis > Sonneratia apetala > Aegiceras corniculatum > Aegialitis rotundifolia > Ceriops decandra > Rhizophora apiculata (Table 8A.2.2). The relatively greater astaxanthin content in the submerged leaves of mangroves confirms the synthesis of astaxanthin content under stressful condition. However, more studies are needed to confirm the influence of tidal influx and subsequent salinity fluctuation on astaxanthin level in the mangrove floral parts. The present data may serve as baseline information on the regulatory role of tidal submergence on astaxanthin level in the estuarine and coastal vegetation. The enhancement of astaxanthin production under stressed condition of organisms is a matter of interest, and several researches are still being undertaken to pinpoint the reaction pathway of astaxanthin production by inducing stress of varied nature. Many types of yeast have been described with an increase ability to produce carotenoids when they grow under unfavourable environment (Certik et al. 2005). Several workers have reported both in the dark and light the enhancement of the accumulation of astaxanthin in cysts of Haematococcus pluvialis under salt stress conditions. The present study points to higher astaxanthin level in those leaves of the mangroves that are inundated for 10–12 h by tidal waters of Jharkhali station having typical estuarine water characteristics (salinity = 10–25.85 psu; pH = 7.98–8.28; temperature = 29.8–31.5 °C; dissolved oxygen = 5.93–6.10 mg/l; NO₃ = 15.09–21.04 μgat/l; PO₄ = 1.12–1.39 μgat/l and SiO₃ = 64.44–83.16 μgat/l). The steep enhancement of astaxanthin level in the inundated Sundari leaves (Heritiera fomes) clearly reflects the highest degree of stress posed by water salinity on this species. Heritiera fomes, being freshwater-loving mangrove species, cannot tolerate high salinity (Mitra and Pal 2002), and thus, acceleration of astaxanthin production may probably be a part of its adaptation to cope with the stenohaline condition of coastal and estuarine environment that becomes acute during high tide. The astaxanthin level of mangrove flora is thus a function of its physiological system, which is extremely species specific. Highest astaxanthin was recorded in mangrove associate Porteresia coarctata (commonly known as salt-marsh grass) in comparison to other species and therefore considered for feed preparation. The level of astaxanthin estimated in the mangrove floral species of Indian Sundarbans is less than the existing natural mega-reservoir of astaxanthin like Phaffia rhodozyma and Haematococcus pluvialis (Table 8A.2.3).

Table 8A.2.2 Mean astaxanthin content in mangrove and associate species collected from Jharkhali island of Indian Sundarbans during May 2007 to April 2008

Table 8A.2.3 Astaxanthin level in different organisms

8.2.7.2 8A.2.7.2 Shrimp Nutrition and Growth

Shrimps fed with Porteresia diet exhibited higher final weights and better weight gain (Table 8A.2.4) at the end of the experiment (27.2 g final weight) in comparison to control pond (20.5 g final weight). Condition index values of shrimp were also higher in experimental ponds (3.87 ± 0.67) than control ponds (2.98 ± 0.55) (Table 8A.2.4). The FCR value for control pond was 1.67 and for experimental pond was 1.27. The survival rate was found to be 58.4 % in the control pond and 69.2 % in experimental pond. The present pilot-scale study speaks in favour of healthy pond environment, better growth, higher survival rate and low FCR values through the use of Porteresia-based feed.

Table 8A.2.4 Success indicators of the research programme

An important factor governing the consumer acceptance and market value of many cultivated fish and shrimp species is the pink or red colouration of their flesh or boiled exoskeleton (Brun and Vidal 2006). In the wild, this colouration is achieved through the ingestion of carotenoid pigments particularly astaxanthin contained within invertebrate food organisms (Johnson et al. 1977; Ibrahim et al. 1984). The Porteresia-based feed in the present study resulted in higher astaxanthin values in shrimps of experimental pond (15.32 ± 1.22 ppm; n = 10) as reflected through darker orange-red colouration of shrimp exoskeleton in comparison to control pond (8.66 ± 0.78 ppm). The Roche SalmoFan^TM colour score showed the value of 23 and 29 for shrimps from control pond and experimental ponds, respectively (Table 8A.2.4), which confirms the variation of astaxanthin level due to different feed ingredients.

8.2.7.3 8A.2.7.3 Water Quality

Hydrological parameters of the shrimp culture ponds are a reflection of the quality of feed provided to the cultured species, and the condition index values symbolize the suitability of the environment for the species (Maceina and Murphy 1998). The hydrological parameters are recorded in Table 8A.2.5.

Table 8A.2.5 Variation in the physico-chemical parameters of the culture ponds

Surface water temperature in both the culture ponds showed more or less parallel trend of variation throughout the study period. The uniformity in temperature profile is due to the location of both the ponds in the same site that experience similar weather and climate. Water temperature plays a major role in shrimp enzyme kinetics which may have a regulatory influence on their growth (Mitra et al. 2006). It also affects the process of moulting during the post-larval stage of shrimps (WWF-India 2006).

The salinity of the Hugli–Matla estuarine complex is known to exhibit intensive variations (Saha et al. 1995). The difference in salinity between ponds may be attributed to the different soil salinity (as substratum of the pond) that leaches soluble salt to the pond water. The relatively higher C.I. values in the experimental pond with less salinity prove the efficiency of formulated feed in combating the stress posed by salinity.

Shrimp culture directly affects the pH of the pond bottom through deposition of excess feed, shrimp excreta, dead shrimps, etc. This shifts the soil and overlying aquatic pH towards acidic condition. In the present study, such condition was not observed owing to the traditional practice of liming at a regular interval of time.

Dissolved oxygen (DO) is a vital parameter regulating the aquatic life. The shrimp health is a direct function of dissolved oxygen and its diurnal variation. Excessive organic load in the system results in lowering the DO value during night/dawn posing threat to the survival of aquatic life. In the present study the DO level in the control pond showed lower value owing to deposition of organic carbon at the bottom of the pond. The significant variation of DO between ponds may be attributed to different growth rate of the culture species and also the use of different types of feed. Traditional feed contains dry fish dust and trash shrimp dust which lowers the DO due to their utilization for oxidizing the residual matter (BOD value increases under this situation). Floral-based feed, on the other hand, generates very limited residue due to which DO remains almost unaltered.

Transparency controls the phytoplankton standing stock in shrimp culture ponds due to their dependency on the solar radiation for photosynthesis. The experimental pond provided with formulated feed showed increased transparency due to its unique binding property. The ready acceptance of the Porteresia-based feed by the cultured species in the experimental pond may be the basis of reduced suspended particulate matter in the aquatic phase of the experimental pond.

Nutrients (comprising of nitrate, phosphate and silicate), budget in the aquatic phase of the culture ponds, are regulated through quantum of excretory products of the cultured species, leftover feed and also by the churning of the pond bed (due to run-off from the adjacent landmasses).

High concentration of nitrate in the control pond may be due to leaching of the feed ingredients (particularly from animal component in traditional feed) in pond water and also the faecal matter that generates ammonia (Mitra and Choudhury 1995).

The phosphate concentration during the study period showed no significant variation between the ponds owing to ban imposed on washing utensils, clothes and other daily household activities during the culture period.

The silicate level of the ponds may be attributed to substratum or pond bottom composition. In both the control and experimental ponds, no significant variation in silicate was observed.

Soil organic carbon was greater in control pond due to more generation of residual feed and excreta in the absence of any feed management. On the contrary, the lower value of organic carbon in the experimental pond is an indication of better acceptability of Porteresia-based feed by shrimp due to which wastage was a minimum.

8.2.8 8A.2.8 Looking Ahead

The mangrove ecosystem of Indian Sundarbans is one of the most biologically productive and taxonomically diverse ecotone of Indian subcontinent with a unique reservoir of bioactive substances. The detection of antioxidant astaxanthin in the floral parts of these typical estuarine and coastal vegetations adds a new dimension to these halophytes. Astaxanthin is an important feed ingredient with wide application both in pisciculture and animal husbandry sector owing to its antioxidant nature. Since the animals cannot synthesize carotenoids within their system, pigments must be supplemented to their feeds, allowing the assimilation and providing the characteristic pigmentation of the cultured aquacultural species, egg yolk, etc. for increasing the quality and consumer acceptance in the market place (Johnson and An 1991). This will not only upgrade the nutrition sector of animal husbandry and aquaculture but will also increase the immunity power of the cultured fish species and domesticated livestock. An ecologically fragile ecosystem sustaining a large fraction of poverty-stricken population like Sundarbans needs an alternative livelihood programme not only to upgrade their economic profile but also to realize the utility and application of their surrounding vegetation as a part of strengthening the root of conservation. The present programme may open an avenue of preparing fish feed, poultry feed and cattle feed by utilizing the antioxidant base of mangroves through involvement of the local people. This will defray the people from illegal entry into the forest and will also improve the animal and fish nutrition sector of the area through setting up of small-scale feed units. The antioxidant reservoir of Sundarbans mangrove ecosystem has several future applications (Table 8A.2.6), and hence, proper policy is needed to blend the biotechnological approach with the livelihood components of the island dwellers.

Table 8A.2.6 Application mangrove antioxidant property

Annexure 8A.3: Seasonal Variation of Biochemical Composition in Edible Oyster (Saccostrea cucullata) of Indian Sundarbans

• Abhijit Mitra,
• Sufia Zaman &
• Kakoli Banerjee 

1. Department of Marine Science, University of Calcutta and Department of Oceanography, Techno India University, Salt Lake Campus, Kolkata, West Bengal, 700 019, India

   Abhijit Mitra & Sufia Zaman

2. K. BanerjeeDepartment of Marine Science, University of Calcutta, 35, B.C. Road, Kolkata, West Bengal, 700 019, India

   Kakoli Banerjee

Abstract

Protein, lipid, glycogen, moisture and ash content of the edible oyster species were analyzed on a monthly basis during 2004 and 2005 from the cultured site at Chotomollakhali in eastern sector of Indian Sundarbans. The culture of edible oyster was initiated in this part of the country in order to provide alternative livelihood to the poverty-stricken population in the Indian Sundarbans, which may otherwise lead to destruction of natural resources of this mangrove ecosystem. Simultaneous monitoring of hydrological parameters (surface water temperature, salinity, pH, nitrate, phosphate and silicate) and phytopigment level of the ambient water was also carried out in this cultured site to investigate the interrelationship between the hydrological parameters and biochemical composition of the oyster tissue. The 2-year study indicates significant seasonal oscillation of the major biochemical constituents of the oyster.

Keywords Indian Sundarbans • Mangrove • Phytopigment • Saccostrea cucullata

8.3.1 8A.3.1 Introduction

Indian Sundarbans is one of the most biologically productive and taxonomically diversified, low-lying, mangrove detritus-based, open, dynamic, heterogeneous coastal ecotone situated at the apex of the Bay of Bengal (between 21°13′ and 22°40′ N latitude and 88°03′ to 89°07′ E longitude). The entire forest of this unique ecosystem acts as a potential reservoir of marine biotic resources. The lower stretch of the estuarine complex and high saline zones are extremely favourable for the survival and growth of edible oyster (Mitra and Banerjee 2005). Saccostrea cucullata is the dominant oyster species in Indian Sundarbans although Crassostrea gryphoides and Crassostrea madrasensis are also reported in the basal part of hard substrata (like sluice gates, jetties, pillars of fish landing stations, light house). Oyster is a good source of protein, vitamins, minerals and trace elements. Researchers have collected detailed information on edible oysters, largely because they can be valuable food (Tack et al. 1992; Ruwa and Polk 1994). However, many internal and environmental factors including pollutants can affect the growth and reproductive success of marine bivalves (Mac MacDonald and Thompson 1985; Steele and Mulcahy 1999). This research programme highlights the seasonal variation of biochemical composition (% lipid, % protein, % glycogen, % moisture and % ash) in the edible oyster (Saccostrea cucullata) sampled from the eastern sector of Indian Sundarbans with respect to hydrobiological parameters.

8.3.2 8A.3.2 Materials and Methods

The entire network of the present programme comprised of the monthly sampling and collection of oysters (Saccostrea cucullata) from Chotomollakhali island in the eastern sectors of Indian Sundarbans for a period of 2 years (2004–2005) along with simultaneous monitoring of hydrobiological parameters (surface water temperature, salinity, pH, nitrate, phosphate, silicate and phytopigment concentration, i.e. Chl a, Chl b and Chl c). The different phases of the programme are discussed separately.

8.3.2.1 8A.3.2.1 Phase 1: Collection of Oysters

Edible oyster species, Saccostrea cucullata (Born) were collected from the cultured site of Chotomollakhali island in the eastern sector of Indian Sundarbans. These species are highly variable in shape, growing in clusters on rocks, bricks, wooden piles or jetties, and also settle on the stems of mangrove plants and on molluscan shells. Twenty samples of almost uniform size (mean length 8.0 cm) were collected at monthly interval from the cultured site during January 2004 to December 2005. They were transported live to the laboratory after proper washing and removal of fouling organisms from the outer surface of the shells for further biochemical analysis.

8.3.2.2 8A.3.2.2 Phase 2: Analysis of Hydrobiological Parameters

Hydrological parameters around the oyster culture site like surface water temperature, salinity, pH, nitrate, phosphate, silicate and phytopigment concentration of the ambient aquatic phase were analyzed on a monthly basis as per the standard methodology outlined in Strickland and Parsons (1968), APHA (1998). Surface water temperature of the aquatic medium in the sampling station was measured by a Celsius thermometer (scale ranging from 0 to 100 °C). The salinity of the surface water was measured by means of refractometer and cross-checked in the laboratory by employing ‘Mohr–Knudsen’ method as outlined by Strickland and Parsons (1968). The correction factor was found out by titrating the silver nitrate solution against standard seawater (IAPO Standard Seawater Service, Charlottenlund, Slot Denmark, chlorinity = 19.376 ppt.). pH of the ambient water was determined by a portable pH meter (sensitivity = ± 0.02).

Surface water for nutrient analysis was collected in clean TARSON bottles and transported to the laboratory in ice-freezed condition. Triplicate samples were collected from the same culture site to maintain the quality of the data. The standard spectrophotometric method of Strickland and Parsons (1968) was adopted to determine the nutrient concentrations in the surface water. Nitrate was analyzed by reducing it to nitrite which was determined by treating the samples with a solution of sulphanilamide, and the resultant diazonium ion was coupled with N-(1-napthyl)-ethylene diamine to give an intensely pink azo dye. The reduction was then carried out by treating the sample with ammonium chloride and passing it through a glass column packed with amalgamated cadmium fillings. The determination of phosphate was carried out by treatment of an aliquot of the sample with acidic molybdate reagent containing ascorbic acid and a small quantity of potassium antimony tartrate. Dissolved silicate was determined by treating the sample with acidic molybdate reagent. The resultant silico-molybdic acid was reduced to molybdenum blue complex by ascorbic acid and incorporating oxalic acid prevented formation of similar blue complex by phosphate. Systronics Digital Spectrophotometer (Type-16S) was used for nutrient (nitrate, phosphate and silicate) analysis at their respective wavelengths.

Phytopigment concentration of the ambient aquatic phase was analyzed in order to monitor the food reservoir of the cultured species. For pigment analysis, 1 l of surface water collected in black bottles from sampling station was filtered through a 0.45-μm Millipore membrane fitted with a vacuum pump. The filter paper was transferred to a homogenizer containing acetone. The contents were grounded thoroughly and placed in refrigerator for 24 h in order to facilitate the complete extraction of phytopigment. Finally the chlorophyll density was estimated as per Jeffrey and Humphrey (1975) with the help of ‘SHIMADZU UV 2100’ spectrophotometer.

8.3.2.3 8A.3.2.3 Phase 3: Biochemical Analysis

The biochemical analyses were done on tissue samples pooled from ten individual oysters. The samples (average length of 8.0 cm) were collected from the cultured site at monthly intervals. They were washed with double-distilled water and processed for biochemical analysis.

Total protein was estimated using Lowry’s et al. (1951) method. The assay used 50 mg of the dried sample homogenized in 10 ml phosphate buffer followed by collection of supernatant after centrifuge. The supernatant was treated with complex forming reagent (2 % Na₂CO₃: 1 % CuSO₄, 5H₂O: 2 % sodium potassium tartrate = 100:1:1) followed by addition of Folin’s reagent. The optical density was determined at 750 nm using a spectrophotometer (Systronics Digital Spectrophotometer; Type-16S). BSA (Bovine Serum Albumin) was used as standard for the preparation of calibration curve.

Gravimetric method of Barnes and Blackstock (1973) was used for the estimation of lipid concentration. A sample of 0.5 g was homogenized in 5 ml of double-distilled water and allowed to stand for overnight in the refrigerator. For the gravimetric determination of lipid, aliquots of the homogenate were extracted in 5 ml of 1:2 (v/v) methanol:chloroform (Folch et al. 1957). Lipid residues were weighed using a Mettler AB 204-S microbalance after evaporation of the chloroform using liquid nitrogen.

Methodology of Hewitt (1958) was used for the determination of glycogen content. 100 mg sample was homogenized in 5 ml sulphuric acid reagent and extracted overnight at 5 °C. Then the homogenate solution was centrifuged and subdivided into two portions. One portion was incubated in a water bath at 95 °C for 4 h, and the other portion was stored at 5 °C. The optical density of both portions was determined at 340 nm using a spectrophotometer (Systronics Digital Spectrophotometer; Type-16S). d-glucose was used as standard solution for the preparation of calibration curve.

The excess water of the oyster tissue was soaked using a Whatman filter paper (No. 1). The meat was then homogenized by a tissue homogenizer. A part of the homogenized tissue was oven dried at 100 °C to determine the moisture content. The ash content in oyster tissue was determined by placing the oyster sample in the Muffle Furnace overnight at 400 °C. The ash was then weighed and expressed in percentage.

8.3.3 8A.3.3 Results and Discussion

In Indian Sundarbans region, edible oyster (Saccostrea cucullata) exhibits a unique seasonal cycle with respect to their biology and biochemical composition.

The physico-chemical variables showed significant seasonal variations in the sampling station during the study period. Surface water temperature was high during premonsoon (March–June) and monsoon (July–October) and low during postmonsoon (November–February). The surface water salinity and pH were highest in the season of premonsoon and lowest in monsoon that might be due to excessive evaporation in premonsoon and heavy precipitation and subsequent discharge of freshwater run-off from the adjacent city of Kolkata in monsoon. The quantum of discharge in the form of sewage also increases the nutrient load in the aquatic phase during monsoon (Table 8A.3.1). Report states that 1,125 million litres of wastewater is discharged per day through Hugli estuary. The lower stretch receives waste and wastewater load of 396 × 10⁸ km³/h along with the annual run-off 493 km³. The total volume of sewage discharge from the environment of Kolkata has been estimated 350 m (Mukherjee 2003). The minimum nutrient load in the water of Indian Sundarbans during premonsoon may be attributed to their uptake by the phytoplankton community that propagates in March/April in the present geographical locale. The average composition of phytoplankton is (CH₂O)₁₀₈(NH₃)₁₆H₃PO₄, and in case of siliceous diatom, it is slightly modified as (CH₂O)₁₀₈(NH₃)₁₆H₃PO₄(SiO₄)₄₀ as stated in the standard literature (Riley and Chester 1971), which justifies the incorporation of nutrients in cell system of phytoplankton leading to the reduction in nutrient concentration of ambient water. Apparently this fact has been highlighted through an inverse relationship between nutrient level and phytopigment concentration (Table 8A.3.3).

Table 8A.3.1 Hydrobiological parameters of the selected sampling station during January 2004 to December 2005

The biochemical composition of the oyster tissue showed significant seasonal variation of protein (5.49–11.87 %), lipid (5.51–10.76 %), glycogen (1.02–6.95 %), moisture (74.60–77.10 %) and ash (0.20–3.09 %) content during the 2-year study period (Table 8A.3.2).

Table 8A.3.2 Biochemical composition (in %) of edible oyster (Saccostrea cucullata) collected from the selected sampling station during January 2004 to December 2005

Analysis of biochemical composition of oysters showed significant seasonal variation. It was seen that reproductive cycle greatly influences the protein–lipid–glycogen content of the tissue. Protein value reached maximum in the months of January–February and September–October, i.e. in the pre-spawning period. In the months of April–June and December, it showed minimum value, i.e. immediately after spawning, the protein value sharply decreased. During other times of the year, protein showed average value of 9.70 %.

Considerable seasonal variations of glycogen and lipid content in oyster tissues were also observed during the study period. There were significant positive correlations of glycogen level in the oyster tissue with seawater salinity, temperature and pH, which confirms that glycogen content in oyster hiked up before spawning during the period of high salinity, temperature and pH in late premonsoon (Table 8A.3.3). Add correlation value of moisture and ash.

Table 8A.3.3 Interrelationship between the relevant hydrobiological parameters and biochemical composition of edible oyster (Saccostrea cucullata) in the selected sampling station during January 2004 to December 2005

In the pre-spawning phase when protein and glycogen content reached maximum value, lipid content exhibited minimum value indicating possible interconversion between them. As carbohydrates are recognized as the major energy source in bivalves, (Gabbott 1975) lower value of lipid concentration during pre-spawning period indicates possible mobilization of lipid towards glycogen to provide energy necessary for the spawning process. The present study reveals that biochemical composition of edible oyster (Saccostrea cucullata) in Indian Sundarbans is controlled by the seasonal influence of hydrobiological parameters, which also governs the reproductive cycle in oysters. A long-term monitoring of relevant hydrobiological parameters is therefore needed to explore the environmental potential of the region to develop large-scale oyster culture practice.

Sundarbans, being the only mangrove-dominated tiger land in the planet Earth, is presently under severe stress due to natural calamities, erosion, shrimp culture-related problems and lack of proper planning in resource management. Although the region is flooded with several species of seaweeds, edible molluscs and several organisms with biomedical values, but hardly any initiative has been taken to link these untapped biological resources with the economics of the state. Under such circumstances, promotion of edible oyster culture may be an alternative livelihood scheme for the local population.