All the methods and principles for partition of the total community respiration were applied at FARTC. An antibiotic mixture, neutralized formalin treatment of the intact water and sediment samples permitted quantification of the bacterial respiration in the water column and in the sediment and the chemical oxygen uptake of the sediment. The partition of the total sediment respiration has a special importance in the case of the old undrainable perennial rural ponds where a very thick sediment accumulates over long periods. This oxygen deficient, nutrient and organic-bound energy-rich sediment creates an environment adversely affecting the fish production processes. A quantification of the different types of decomposition in these thick sediments is necessary before suitable measures can be introduced. A sediment sampler was constructed at FARTC to take the undisturbed sediment column for sediment respiration experiment. The oxygen consumption was monitored by Beckman electrodes and the respiration in the different treatments were conducted by the following formula:
| where R | = | oxygen consumption in the glass test tube with sediment core and overlying water during the incubation period, mg d m-3 |
| 1 | = | the height of the overlying water column in the glass test tube, cm |
| 10 000 | = | conversion to m2 |
| 24 | = | conversion to day |
| 1 000 | = | initial conversion to cm2 |
| t | = | incubation time, h |
A simple but informative and highly practical method was developed to measure directly the rate of decomposition of a number of model substrata in the whole water and sediment column of the undrainable rural fish pond with thick sediment. A 2 m long and 5 cm diameter bolting net bag was divided into 5 cm compartments and packed with the model substrate. The long net bag attached to a bamboo rod was pushed into the sediment so that about 0.5 m of the test bag was positioned in the sediment and 15 m exposed in the water column. A series of test bags were exposed and after successive removal, weight loss was measured. The decomposition of cellulose silk protein, intact and dried water hyacinth were monitored.
Sediment-water interface is of far more importance in the nitrogen and phosphorous nutrient supply of shallow water ecosystems than in deep, stratified lakes, since the nutrients, due to the lack of metalimnion, can reach the water column and the surface without any obstacle via diffusion or other processes. With the determination of various nitrogen and phosphorous compounds in the water and in the interstitial water of the sediment, and with reliable measurement of the diffusion surface by applying Fick's equation (dn/dtq= -Dgrade C), important basic data can be provided on the mineral cycling of the shallow fish pond ecosystem.
A reliable sediment squeezing apparatus has been constructed at FARTC to collect the interstitial water from the different sediment layers in order to measure the vertical gradient of the nitrogen and phosphorous nutrients in the thick sediment of the undrainable ponds.
Amino acids form a permanent component in the water column and in the interstitial water of the sediment. No work has so far been carried out in subtropical and tropical fish ponds on the presence and abundance of the free dissolved amino acids which serve as a nutrient source for the primary production and bacterial production. In the case of a limited inorganic nitrogen supply in fish ponds, the phytoplankton need more amino acid to cover the daily requirement for protein biosynthesis. Simultaneously with this process the bacterioplankton compete for uptake of the amino acids and also mineralize them into ammonia. During this process, the energy liberated is used for the formation of valuable bacterial protein. With regard to the maximal uptake velocity (of both the groups), Vmax ranged between 1 and 13 μg dm-3H-1 individual amino acids.
The concentration of amino acids can be easily measured by the automatic amino acid analyser shortly to be available at the fish nutrient department. For the measurement of amino acid uptake a liquid scintillation counter is required. The classical Wright and Hobbie system is applied, enriching a series of bottles with an increasing concentration of (labelled) amino acids, and the Michaelis-Menten enzyme kinetics method is used to calculate the uptake parameters.
Apparatus/equipment
sampler
membrane filters, 0.2 μm pore size
membrane filter holder
filter pump
Chemicals
14 C labelled amino acids
Bray scintillation mixture
Except where complete pelleted food is given to the fish, the fish yield in the fish ponds depends primarily on the quantity and availability of plant nutrients mainly in the form of ammonia, nitrate and phosphate dissolved in the water. For ammonia, the maximal uptake velocity (Vmax) reached the value of 261 μg dm-3 h-1 compared to the range of 23–72 for Hungarian fish ponds. Different macrophyte species have an even higher uptake velocity, successfully competing for nutrients with the phytoplankton in fish ponds.
To determine the ammonia and nitrate uptake velocity the use of the N15 stable isotope is the most convenient and reliable method. However, for this procedure, a special N15 analyser or a Mass spectrometer is required. In the absence of Mass spectrometer, a simple approach was adopted at FARTC to measure the plant nutrient uptake in fish ponds. The procedure is based on the Michaelis-Menten enzyme kinetics method. Twelve 250-ml bottles are filled with lake water and enriched in duplicate with ammonia or nitrate of five increasing concentrations and treated with N-serve to a final concentration of 5 mg dm-3. One bottle from each enrichment is incubated in situ for 4 hours and the other bottle serves for immediate measurement of the initial ammonia or nitrate concentration. After incubation the final concentration in the bottles is measured and the differences between the initial post-incubation concentrations give the uptake rate for each enrichment level. The plot of this uptake value (V) versus concentration values (Sn + Sa) represents a typical curve of saturation type following the classical Michaelis-Menten uptake model. An (Sn + Sa)/v versus Sn + Sa plot gives a linear form which simplifies the calculation of all the important enzyme kinetic parameters accordingly where v = uptake, μg dm-3 h-1, Sn = natural substrate concentration μg dm-3, Sa = added or enriched concentration μg dm-3, KM = Michaelis constant or half saturation constant μg dm-3, Vmax = maximal uptake velocity μg dm-3 h-1, T = turnover time in hours.
Apparatus/equipment
glass bottle of 25 ml volume
water samplers
kits for nitrate and ammonia determination
Chemicals
N-serve as a special nitrification inhibitor
The nitrogen fixation in the water column, mainly by the blue-green algal species of the phytoplankton community and in the sediment by anaerobic bacterial population, enriches the fish ponds with nitrogen and counterbalances the nitrogen loss through denitrification. The annual amount of nitrogen entering the water body through biological nitrogen fixation reaches the value of 150 kg ha-1 in shallow lakes. The magnitude of nitrogen fixation in subtropical and tropical fish pond ecosystems is not known. The quantification of this cheap nutrient source and the research and analysis of the influencing factors under these conditions are of paramount importance in these nutrient-deficient regions.
The acetylene reduction method (discovered by Schollhorn and Burris and simultaneously by Dilwarth) for rapid, sensitive and cheap measurement of nitrogen fixation accelerated research to quantify the rate of nitrogen fixation in the nitrogen budget of aquacultural and agricultural agroecosystems and in the biosphere and water environment. The method was applied first by Stewart in 1967 for measurement of nitrogen fixation of the water column in the year immediately after its discovery. Following the results of earlier years, the original procedure was modified at the Fisheries Research Institute in Hungary to maintain as strictly as possible the in situ conditions during incubation. It has been proved that acetylene reduction in sediments is definitely an enzymatic process accomplished by the nitrogenase enzyme. The sediment sample should be shaken before and after incubation to ensure saturation with acetylene and to recover the ethylene quantitatively. However, shaking destroys the in situ pattern and arrangement of the physico-chemical and biological gradients in the samples, and consequently the activity thus measured can differ significantly from the real in situ activity. To maintain the in situ physico-chemical and biological gradients and to obtain complete acetylene saturation, a perforated plastic tube sampler was developed to sample an intact sediment core. The PVC plastic tube, 40 cm long and 4 cm in diameter, is punched with 2 cm holes on its lower part and these holes closed with tight-fitting teflon corks. The whole sediment is saturated with acetylene through the teflon corks, and after incubation in situ the whole column well shaken for quantitative recovery of ethylene. The whole procedure for water and sediment measurements is similar to that described for denitrification measurement, except that the gas collection is carried out by simple manual shaking instead of the tedious gas purifying process. This is because the product of the acetylene reduction, the ethylene solubility in water is very low compared to the solubility of the nitrous oxide in the denitrification method.
Apparatus/equipment
Gaschromatograph with 2.1 m long and 0.4 cm diameter glass column packed with PARAPAK-Q, able to operate at 80°C in pure nitrogen gas as carrier with a flow rate of 40 ml/min and supplied with hydrogen flame ionization detector.
Hargrave sediment sampler or similar developed and manufactured during this consultancy at the FARTC able to take an undisturbed sediment column.
Reaction vessels. For water measurement a 300-ml baby bottle volume and for the sediment measurement a perforated plastic tube described above.
Football bladder closed with glass plug suitable for field work as a gas container.
Syringes with a volume of 1, 20, 50 cm3 and supplied with needles.
Gas storing vessel of 5–10 ml closed with teflon plug.
Chemicals
Acetylene gas, high purity
Ethylene gas, gaschromatographic purity
Nitrogen gas, high purity
Saturated HgCl2 solution
According to Hungarian data, tonnes of ammonia are converted annually to nitrate under a hectare of fish pond water surface by highly specialized bacterial populations. Although considerable attention has been given to nitrifyers from the viewpoint of agricultural and sewage treatment, few studies have investigated the process of nitrification in lake water and there have been no investigations except in Hungary with the reliable new methods evolved last year revolutioning the quantification of this important process. The new method is based on the phenomenon that N-serve (2-chloro-6/trichloromethyl/pyridine) inhibits specifically the autotrophic nitrofying bacteria in oxidizing ammonia. N-serve has been used to measure C14-bicarbonate incorporation by nitrifying bacteria, and the incorporation rate is related to the rate of ammonia oxidation.
A 250 ml water sample is collected and poured into an Erlenmyer flask with glass stopper, and 25 μ Ci(925 kBq) NaH14CO3 is added. The sample mixed without re-aeration is directly divided into two 100 ml and poured into two dark bottles. One of them contains 0.05 ml of an alcoholic solution of N-serve to obtain a final concentration of 5 mg dm-3; the other has the same volume of pure ethanol. The two bottles are incubated at in situ temperature and in the dark. After an incubation period of 4 hours, three 10 ml samples from each bottle are filtered through membrane filters (pore size: 0.2 μm). The filters are immediately treated with a 0.5 NHCl solution to eliminate any radio-activity associated with precipitated carbonate. The radio-activity retained on the filters is measured by liquid scintillation after dissolution of the filters in Bray scintillation mixture. Radioactivity of an aliquot of non-filtered samples are also counted as a standardization.
Bicarbonate concentration in the samples are determined by acid titrations. Bicarbonate incorporation is then calculated from each bottle by routine dark H14CO3 uptake procedures. For each measurement, the differences between the averages of N-serve treated and untreated samples are calculated. This is the N-serve sensitive bicarbonate incorporation. The community of nitrifying bacterial populations fixes 0.12 μ mole bicarbonate for each μ mole ammonium oxidized to nitrate. It is thus possible to estimate the rate of nitrogen oxidation from the value of N-serve sensitive bicarbonate incorporation by multiplying the latter by the factor of 8.3.
Apparatus/equipment
Water sampler
Erlenmeyer flask of 250 ml with glass stopper
Membrane filters, 0.2 μm pore size
Membrane filter/holder
Filter pump
Liquid scintillation counter
Chemicals
14C-labelled carbonate (NaH14CO3)
N-serve
Bray scintillation mixture
A rough estimate of the nitrification in water samples is possible without this radio-active procedure by applying the Michaelis-Menten enzyme kinetics model and using the procedure described for the ammonia and nitrate uptake. The difference between the N-serve treated and untreated ammonia uptake gives an approximate value of the nitrification.
In an oxygen deficient environment, the nitrate serves as an electron acceptor for many bacterial populations and makes the mineralization of the organic matter possible under anaerobic conditions where the electron oversupply and the shortage of electron acceptors limit the rate of bacterial decomposition. During this process, the nitrate is reduced to nitrous oxide or dinitrogen gas and is lost as a nutrient. The two other nitrate reduction pathways in the nitrogen cycle, the assimilatory nitrate reduction and the dissimilatory nitrate respiration, do not cause any loss in the nitrogen supply because both reduce the nitrate to ammonia. Although the denitrification is an important pathway in nitrogen cycle, resulting in a significant loss of nitrogen in many freshwater and marine water ecosystems, the fertilization strategies in fish ponds pay no attention to this loss because in fish culture activities there are no suitable laboratories with adequate environmental monitoring research facilities. During the last four years, the Fisheries Research Institute in Hungary has developed a simple and reliable procedure of the acetylene inhibition technique for fish pond studies, applying the conventional gas-chromatographic procedure to measure the quantity of the nitrous oxide gas accumulated in the acetylene treated sample during the incubation period. Using this procedure, significant denitrification has been found both in the water column and in the sediment of the inorganic fertilized and manured fish ponds. The total loss of nitrogen through denitrification ranged from 65 to 156 kg N ha-1 per growing season which is about 150 days in Hungary. The nitrogen loss in tropical and subtropical fish ponds may be much higher due to the longer growing season. The quantification of this important nutrient loss in these fish ponds would be only the first stage of research; the final target must be to discover the influencing factors in order to minimize the loss in these nutrient-deficient waters.
The principle of the new method is based upon Fedorova's significant discovery that the acetylene inhibits the NO3-NO2-NO-N2O-N2 route of denitrification in the last reaction step converting the nitrous oxide (N2O) to dinitrogen (N2). As a result of this inhibition, the nitrous oxide accumulates in the acetylene-treated sample and can be easily measured with gaschromatography. The method is cheap, rapid and sensitive.
The undisturbed sediment column is collected by Hargrave sampler. Three undisturbed sediment cores are taken from this column by perforated plastic tube. The first tube is immediately analysed after collection or poisoned by saturated HgCl2 to prevent further microbial activity. The second sediment core is incubated without acetylene and the third with acetylene for 4–10 hours. The HgCl2 and acetylene treatments are carried out through the silicone rubber inserts of the perforated tubes. A quantity of 2 ml of acetylene-saturated distilled water (1.6 ml of C2H2 per ml of water) is injected into the sediment at each hole. Injections are taken such that the needle is slowly withdrawn through the sediment with exchanges in its horizontal and vertical directions in order to distribute the inhibitor (C2H2) thoroughly inside the sediment. The water above the sediment has to be siphoned from the core before incubation. The core stoppered from above and the acetylene gas is added (15% V/v) to the head space of the tube. The incubation must be performed at in situ temperature and in the dark. During or after incubation, gas samples are taken from the headspace of the tube to measure any N2O gas released by the sediment surface. The N2O gas accumulated in the sediment has to be recovered by a special gas purging procedure. For this, the sediment core has to be cut into 2-cm segments pistoning out of the incubation tubes. The segments are placed in a rounded bottom flask. The flask containing the sediment sample is connected to the gas purging system. Helium gas stream (20 ml minute-1) is flushed through the flask purging the sediment sample and passes through a 5-ml volume stainless steel tube loop, attached to the system and completely covered with a liquid nitrogen cooled jacket. Purging is facilitated by a magnetic stirring bar inside the flask. Absorption traps containing NaOH solution to remove CO2 and anhydrous CaCl2 to remove water vapour are also attached to the purging system. N2O gas is drawn from the loop to the gas chromatograph after 30 min of purging and the liquid nitrogen trap immediately removed.
Denitrification measurement in the water column is based on the same procedure but using a 300-ml baby bottle as reaction vessel.
Apparatus/equipment
Gaschromatograph with 2.1 m long and 0.4 cm diameter glass column packed with PARAPAK-Q, able to operate at 45°C in pure nitrogen gas as carrier with a flow rate of 30 ml/min and supplied with electron capture detector operated at 320°C.
Hargrave sediment sampler. The original plexiglass sampler was substituted by a metal modified one developed at the Institute and manufactured in the local workshop.
Reaction vessel, 40 cm long and 5 cm diameter, plastic tube perforated with 1 hole in the upper half and 10 holes in the lower part and all holes closed with silicon rubber plug. Two rubber corks to close the perforated plastic tube.
Football bladder closed with glass plug for field work as a gas container.
Syringes with a volume of 1, 20, 50 cm3 and supplied with needles.
Gas purging apparatus for nitrous oxide extraction. Normal laboratory glasswear, stainless steel tube loop of ml volume, a 2-way-valve and 3-way-valve are required to build and connect to the chromatograph.
| Chemicals | |
| Acetylene gas, high purity | |
| Dinitrogen oxide (nitrous oxide) | gaschromatographic purity |
| Nitrogen gas, high purity | |
| Liquid nitrogen for nitrous oxide trapping | |
| Saturated HgCl2 solution | |
