A Preliminary Study on the Nutrient Sources for Fish Growth in Manured Pond as Indicated by Delta C Analysis

by

Guo Xian-zheng, Fang Yingxue, Wang Jikun, Fang Xiuzhen and Liu Zhiyun

NETWORK OF AQUACULTURE CENTRES IN ASIA
Bangkok, Thailand
September 1985

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A PRELIMINARY STUDY ON SOURCES OF FISH GROWTH IN MANURED POND AS INDICATED

BY DELTA C ANALYSIS

Guo Xian-zheng, Fang Yingxue, Wang Jikun, Fang Xiuzhen and Liu Zhiyun

Freshwater Fishery Research Centre of Chinese Academy of Fishery Science

ABSTRACT

In a manured fish pond, the conversion of manure-feeds-fish undergoes a complicated process of pond dynamics. In order to understand and manipulate this process and to help us improve farming productivity, we introduced carbon isotope analysis in our experiment. We measured delta C of organic matter, such as fish body, fish feeds, manure, etc. And with this information, we traced the flow of carbon, analysing the interactions of fish growth and the complicated food web in a manured pond. The data show that filtering and omnivorous species in this manured pond obtained carbon from both autotrophic and heterotrophic production systems. This is of practical significance in planning aquacultural activities and seeking the truth of pond dynamics.

INTRODUCTION

Organic manure, such as animal wastes, is often used in ponds of integrated fish farming. Between the input of manure and output of fish come a series of complicated processes involving food organisms, material circulation and energy conversion. This is generally termed pond dynamics. In order to actively raise the farming productivity, it is useful to understand these processes. In recent years, scientists at home and abroad have been studying pond dynamics with variant methods (Buck, 1978; Schroeder, 1978; Hu Baotong, 1983; Li Yuanshan, 1983; Guo Xian-zheng, 1983), including the carbon isotopic trace analysis. This analysis indicates the relationship between the food and fish growth and may provide information not revealed by the conversional method of microscopic analyses of fish intestine contents (Schroeder, 1983; Shan Jian, 1983).

It is well known that carbon is the major element in nutrition of organisms (e. g. plant, animal & microbe) and the principal constituent of the organisms, accounting for 50% of organism by dry weight.

The carbon of organic and inorganic matter contains two stable isotopes: ¹²C and ¹³C. ¹³C, whose atoms have atomic mass of 13, accounts for about 1% of the natural abundance; the natural abundance of ¹²C, which has a mass of 12, is approximately 99% abundance. The ratio of ¹³C; ¹²C is also naturally occurring tracers. The ratio of ¹³C: ¹²C is reported as delta C.

(Standard sample of delta C PDB is carbonate rock from South Carolina, USA.)

The distribution of these isotopes in natural carbon reflects in the composition of atmospheric CO₂ as ¹³CO₂ or ¹²CO₂. The samplings show that during photosynthesis, plants metabolize the atmospheric CO₂ at different rates and two main pathways of photosynthesis: C₃ circulation and C₄ circulation. Delta C of these two types of plants are measurably different (Smith & Epstein, 1971). Unlike plants that have a selectivity in metabolizing ¹³C or ¹²C, an animal's delta C is decided by delta C in the food. The isotope C incorporated into the fish tissues is equal to that of the metabolized food (DeNiro and Epstein, 1978; Schroeder, 1983), in other words, delta C of fish is approximately equal to delta C in food. Based on this similarity, we adopt the mass spectrographic analysis of the isotope ratios to trace the flow of carbon in our experiment by accurately sampling delta C index of organisms of fish body, food and manure concerned. With this information, we analyse qualitatively the complicated interactions between the food web in manured ponds and the carbon source for fish growth so as to help us discover and modefy the pond dynamics.

MATERIALS AND METHOD

1. Stocking species and ratio

The earthen pond area in our experimental farm is one mu (1/15ha.) with a water depth of 1.5–2 meters filled with Lake Taihu water. According to the ratio of 9:2:6:3, two-year-old fingerlings of silver carp (Hypophthalmichthys molitrix), bighead (Aristichtys nobilis), Baiji (Carassius cuvieri) and common carp (Cyprinus carpio) were stocked to a total number of 1100 individual fish per mu.

The research lasted six months from mid-April to mid-October. The details of stocking and harvesting are listed in Table 1.

Table I. Stocking and harvesting

2. Amount and methods of manure application

Throughout the period, only chicken manure was applied. No other feeds were used. The amount of fertilization was limited to 3% (in dry weight) of fingerling body weight per day by speading evenly all over the pond, two or three times a week. The body length and weight were measured regularly to adjust the application on time.

3. Sampling and preparation of delta C

In order to make delta C samples a strict representative for a precise survey, we referred to some foreign documents for information before conducting a series of preparatory tests. The method of collection and preparation of delta C samples we designed and finally chose are as follows.

Chicken manure: Fresh chicken manure that we usually applied without any leftover of feeds were baked dry at 90–100 °C for 10 hours prior to being fully ground. 2–5g sample was randomly put into a test tube for measuring.

Chicken feeds: Samples of food pellets, corn flour, soybean cake and barley meal that we usually fed were collected and baked dry at 90–100 °C for 10 hours before grinding for future use.

Bacteria: 100 ml of pond water from 8–10 points were collected and pumpfiltered through a 20μm meshed funnel to remove planktons. Under an aseptic condition, the filtered sample was transferred onto the agar plate with chicken manure infusion as its only carbon source. Colony was scraped for future use after 48 hours' culture at 25 °C. Experiment shows the carbon source is not influenced by the agar.

Organic detritus: About 100 ml of water sediment sample were collected with a plankton net at different sites. The liquid part was removed after several hours' precipitating and the sediment was repeatedly rinsed by distilled water until no plankton is microscopically visible.

Plankton: Tens of liters of pond water from various locations were collected and filtered in grades by virtue of a 120μ and 50μ meshed filter and centrifuged for 10 minutes at 4000 r/m and 5000 r/m respectively for future use.

Attachments on rocks: Attachments on rocks just emerged from water upon drainage were scraped from 8–10 points along pond side in time at the end of the experiment.

Pond bottom settlings: Settlings on the sludge surface from 8–10 spots were collected through the self-made collector, which consists glass tubes, emulsion tubes, rubber bulb-shaped sucker. The liquid part was taken away after several hours' standing. The sediment was collected and blended with H₃PO₄ till the pH value reaches 1–2 with CO₃ removed.

Sludge: Sludge out of 8–10 bottom sites was collected upon drainage and added with H₃PO₄ till pH turns to 1–2 with the CO₃ driveh away.

Fingerlings: Three individual fish of silver carp, bighead, baiji and common carp respectively were randomly taken from fingerling rearing pond before stocking. Scales and epidermis were removed. Muscles about one cm² and 0.5 cm thick apiece were scissored off form dorsal part and caudal peduncle. Blood, if any, was cleaned with distilled water. In addition, some dorsal fins and tail fins were cut for preparation.

Adult fish: At the end of the experiment, three individual fish of the four species were taken randomly to make samples as above.

4. Purification and determination of delta C samples

5–10 mg of each of samples mentioned above was put into a quariz bowl and burned at 850°C with an electrical oven in a lower vacuum system filled with oxygen. The gases produced, such as CO₂, flowed to a -50°C refrigeration piping to remove water vapour and then to a -196°C liquid nitrogen freezing separator to freeze the CO₂ while allowing surplus oxygen gas to escape. The CO₂ was warmed backed to -50°C in a higher vacuum system and appeared as gas. The purified CO₂ in the collector was frozen to solid status through -196°C. Isotopic analysis of the pure CO₂ gas was done at room temperature on a MAT-250 type isotopic mass spectrometer.

RESULTS AND DISCUSSION

Results are presented in Table II, III and IV, out of delta C sampling of fish body, natural food, chicken manure and feeds.

TABLE II Delta C in the pond fish

Note: Fingerling were sampled before stocking. Adults were sampled at the end of the experiment.

Table III Delta C value in chicken manure and chicken feeds

Table IV Delta C among the natural food and sludge

1. Three tables show that delta C of measured samples range from -13.83 to -28.76 ‰, while delta C of growout of organisms in the pond is between -19.38 and 28.76 ‰. Carbon composition during the growth of all kinds of organisms can be distinctively identified in line with delta C index.

Error of duplicate data randomly measured from some samples was with the range of ± 0.01–0.20 ‰, much lower than the permitted error scope of ±1.0 ‰ in terms of foreign record. (Schroeder, 1983). This implies the accuracy of our own delta sampling system and approves the feasibility of our delta C sample collection and preparation.

2. Table II shows that delta C in the fins of the four tested fingerlings was about 1 per mil less negative than that of fish flesh. The difference may be related to the carbonate content of the bones of the fin. Delta C in fish flesh of the fingerlings is 4–5 per mil less negative than that of the adults and indicates a difference in available feeds or feeding habita between adult and fingerling. These are in conformity with the results reported abroad.

The four species were reared in the same pond before the experiment starts. They were mainly fed with soybean juice, soybean residues and soybean cake. Therefore, their delta C index was -25.13 to -28.76 ‰, quite close to that of soybean cake (-26.36 ‰). During the growth period of 180 days, feeding habit of the fishes changed and all the fishes gained weight several times because of the variation of diets. (See Table I).

The difference in carbon assimalated and accumulated in adult stage of fishes as compared with the fingerling stage indicates the objective changes of pond dynamics.

3. Organic fertilizers in manured ponds are a main source of carbon input. Green algae, which fix CO₂ in the atmosphere by utilizing sunlight, also provide carbon to fish ponds. When manures are decoposed by a large quantity of aquatic bacteria and mineralised into nutritive salts, they can be used by photoplankton for growth. Based on this, both heterotrophic production system predominated by bacteria and autotrophic production system predominated by algae are thus formed gradually through a series of autotrophic and heterotrophic production activities. The crisscross development and mutual promotion and interaction of these two systems form a complicated food wed in the pond and breed many kinds of food organisms to enable fish to get carbon for their growth. The delta C values indicate a relationship between feedstuffs and the fish involved. (See Figure 1)

As the typical strainers, silver carp and bighead living in the middle and upper water layers filter photoplankton and zooplankton. The target plankton that these two fishes filter are not the same. This is also proved by delta C. Their delta C values are -23.56 ‰ and -24.25‰ respectively, close to that of plankton (See Table IV). Silver carp mainly filter the plankton smaller than 50μm in size, the delta C of which is -23.02‰ so there is only 1‰ less of difference between silver carp and the plankton. Bighead largely filter the plankton between 50 and 120 m, the delta C of which is -22.36‰, so there is only 2‰, less of difference between bighead and the plankton. These facts are in conformity with the research results of histological anatomy.

Baiji and common carp are omnivorous species, but with different living behaviour and foraging characteristics. The results of our experiment shows that their delta C are -20.89‰ and -19.43‰, which are quite similar. Baiji living in the mid-layer of the pond water, swallow bacterial groups, organic detritus and manure residues. Baiji involved in our experiment prefers plankton. Delta C sampling has proved that the Baiji delta C is blassed toward the delta C value of the above-mentioned feed (Tables III & IV, Figure L). There is a small difference only about 0.3–1.5‰ between the two fishers; 0.27% difference between Baiji and bacteria (-20.63‰), 0.23‰ difference between Baiji and organic detritus (-21.12‰), and the Baiji delta C is also close to those of chicken manure and some planktons.

Common carp delta C is biased-toward the delta C value of their feed. The common carp delta C value of -19.42‰ is very close to -19.38‰, that of the attachments on rock, mainly composed of epiphytic algae, settling algae and benthos as well as to that of bacteria (-20.62) (See Tables III & IV, Figure 1) with only 0.05–1.2‰ difference. The delta C of acidificated bottom settling and sludge is 1–2.5‰ lighter than that of the unacidificated substances. The link between the acidificated substances and carbon source of common carp is not difficult to identify.

The delta C Value of common carp and their feed objectively reflect the feeding habit of common carp, which live in lower layer of water, digging the bottom, scraping the rocks and searching for benthos and precipitates.

4. The analysis of the feeds and the four species helps us understand an interesting fact that feed for fishes is composed of more than two sources. Since their delta C values are different, carbon provided by these diets, their function and contribution in fish growth can be further determined by the following equation for equilibrium and calculation.

If the feed is composed of two diets which have different delta C assuming X for the heavier delta C (more positive), and 1-X for the lighter delta C (more negative), equation can be simplified to:

Then equation (1) may be rewritten as

Thus, in manured pond, the heterotrophic activities initiated by bacteria supplied about 40–70% of the carbon incorporated into the fish body during the growth period of Baiji, while the autotrophic activities of plankton supplies approximately 30–60%.

Therefore, carbon sources of other species could be calculated in the same way. (Table V)

Table V Sources of fish growth (%)

We can see that the delta C-of chicken manure (-18.22) is close to that of feed pellets (-16.87), with only 1.35‰ difference. With the help of the equation mentioned above and the delta C value of chicken manure and chicken feeds, proportions of chicken feeds could be figured out: feed pellets accounts for about 60%, other feeds about 40% (out of which, corn flour 43%, soybean cake 10%). These figures primarily agrees with the data we investigated on chicken farms.

CONCLUSION

In China, we are just beginning to apply the isotopic mass spectrographic analysis to fisheries research aspect, though it is not new to scientific study of geology, pedology, biology and medicine. (ut preliminary delta C study on the conversion of diet into fish body in a manured pond implies that in the organic manured pond, the growth carbon of filter feeders (silver carp and bighead) include from the autotrophic production system. More than ⅓ to ½ growth carbon of omnivorous species Baiji is provided by autotrophic production system; Nearly ½ to ⅔ from heterotrophic production system. Approximately ½ to 2/3 of the growth carbon of omnivorous specirs common carp comes from autotrophic production system; and nearly ⅓ to ½ are from heterotrophic production system.

The classification of these facts has a rather practical significance both on guiding aquaculture activities toward developing pond management that will encourage growth of useful natural foods and exploration of pond dynamics law.

ACKNOWLEDGEMENTS

This work was sponsored by Mr. Hu Baotong, Deputy Director of Freshwater Fisheries Research Centre of Chinese Academy of Fisheries Sciences. We greatly appreciate the finding support from International Development Research Centre, the help from Dr. Davy, IDRC and the concerns from Mr. Chen Foo Yan, NACA Coordinator. And special thank to Mr. Shan Jian, RLCC Director, Mr. Hu Baotong, and Associate Professor Zhu Lingen for reviewong this paper. In the meantime, we are grateful that Dr. Schroeder read this paper and offered us precious suggestions.

REFERENCES:

Buck, H., Baur, R. & Rose, C., 1978 Polyculture of Chinese carps in ponds with swine wastes. In: R. Smitherman, W. Shelton & J. Grover, (Editor) Culture of Exotic Fish Conf. Proc. Am Fish Soc., Auburn, AL, USA, pp. 144–155

Schroeder, G. L., 1978 Autotrophic & heterotrophic production of microorganisms in intensively manured fish ponds and related fish yields. AQUACULTURE 14: pp. 303 – 325

Hu Baotong, 1982, Ecological base as multispecies polycultured in fertilized pond. (SYMPOSIUM COLLECTION OF HIGH YIELD OF POND FISH CULTURE IN CHINA) ECOLOGY JOURNAL 3: pp 17 – 21

Li Yuanshan, 1983, Preliminary study of fish pond ecosystem and the effect of silver carp Freshwater Fisheries 3: pp 8 – 11

Guo Xianzhen, et al. 1983 Preliminary study on bacterial population and its growth - and - disappearance in animal manured pond water FRESHWATER FISHERIES 1984, 1: pp. 31 – 35

Schroeder, G. L., 1983 Stable isotope ratios as naturally occurring tracers in the aquaculture food web AQUACULTURE, 30; pp. 203 – 210

Shan Jian and Schroeder, G. L., et al 1985. Observations on feeding habits of fish in ponds receiving green and animal manures in Wuxi, People's Republic of China. AQUACULTURE, 46; pp. 111 – 117

Smith, B. and Epstein, S., 1978. Two categories of ¹³C/¹²C ratios for higher plants PLANT PHYSIOLOGY, 47: pp. 380 – 384

DeNiro, M., and Epstein, S., 1978. Influence of diet on the distribution of carbon isotopes in animal. GEOCHIM COSMOCHIM ACTA, 42: pp. 495 – 506

Schroeder, G. L., 1983 Sources of fish and prawn growth in polyculture ponds as indicated by delta C analysis. AQUACULTURE 35: pp. 29 – 42

Figure 1. Corresponding delta C(δC) values of the four carp species in relation with food intake