2. Materials and methods
2
.
1
. Ponds and fish culture Ponds used in this study are located on the Auburn University Fisheries Research
Unit FRU about 10 km north of Auburn, Alabama, USA. They are rectangular with vertical concrete walls around edges to assure that no area is less than 40 cm
deep when ponds are full. Ponds have a water surface of 400 m
2
, average depths of 0.80 – 0.90 m, and maximum depths of 1.2 – 1.5 m.
On 30 May 1997, channel catfish averaging 17 g each were stocked at 550 per pond. A floating feed guaranteed by the manufacturer to contain at least 28 crude
protein was offered to fish on a satiation basis once daily for 133 days. Ponds were aerated from 00.00 h until 08.00 h with 0.37 kW, vertical pump aerator in each
pond. Dissolved oxygen DO concentration and water temperature were monitored daily between 06.30 and 08.00 h with a polarographic oxygen meter Yellow Spring
Instrument Co., Yellow Springs, Ohio, USA. The pH of pond water was measured 1 day weekly between 06.30 and 08.00 h and between 17.30 and 19.00 h. The ponds
were one treatment in a study on effects of protein concentration in feed on water quality. Soluble reactive phosphorus SRP, total phosphorus TP, nitrite nitrogen
NO
2
N, NO
3
N, TAN, and TN concentrations in the water column were
measured for the other study and presented by Gross et al. 1999b. Ponds were drained and fish were harvested between 7 and 9 October 1997. Fish were counted,
and the total weight of fish per pond was measured.
2
.
2
. Water budgets A water budget was prepared for each pond Boyd, 1982. Inflows included water
to fill ponds, rainfall into ponds, and additions to maintain water levels. Runoff into ponds was insignificant because watersheds comprise only 50 of pond areas
Boyd, 1982. Outflows included seepage, evaporation, overflow, and water drained at harvest.
Rainfall samples were captured through a large funnel in a plastic bottle. Samples also were collected when water was added to fill ponds or to replace evaporation
and seepage, when overflow occurred after rains, and during draining for harvest and analyzed for total N TN Gross and Boyd, 1998.
2
.
3
. Nitrogen gains A sample of 15 catfish fingerlings collected at stocking and samples of each new
batch of feed were analyzed for moisture by drying at 60°C and for TN by the Kjeldahl method Lovell, 1981. Inputs of N in fish stock and feed were estimated
by multiplying N content by amount of feed applied during the study and dry weight of fish stock. Inputs in water were estimated from TN concentration and
volume of each source.
Nitrogen fixation was estimated twice a month by the acetylene block method Capone, 1993. Water column samples 12 ml were placed in 20 ml, gray, butyl
rubber-capped, glass vials and treated with 2.5 ml of acid-washed acetylene. Vials were incubated in ponds, and head space gas was sampled with a syringe after
0, 1, 2, and 4 h. Head space gas was stored in 1-ml sealed vials until analyzed for ethylene with a gas chromatograph Varian Model Star 3600, Sugarland, Texas,
USA equipped with hydrogen flame ionization detector and a 2-m Porapak N column Capone, 1993. Courier gas was N
2
with a flow rate of 30 ml min
− 1
.
2
.
4
. Nitrogen losses Three fish collected from each pond at harvest were ground mechanically and
mixed aliquots dried at 60°C for moisture analysis. Dried fish was analyzed for TN by the Kjeldahl method Lovell, 1981.
Pond bottom soil samples were collected with a 5-cm diameter core tube from the upper 5-cm stratum at five points in each pond on 26 May and 5 October. Samples
were dried at 60°C, pulverized to pass a 0.25-mm screen, and analyzed for total carbon TC and TN with a Leco CHN 600 Analyzer Leco Corporation, St.
Joseph, Minnesota, USA. Aliquots of soil samples also were extracted with 2 M KCl Keeney and Nelson, 1982, and extracts were analyzed for NO
3
N by the NAS reagent method Gross and Boyd, 1998, for NO
2
N by the diazotation method, and for TAN by the phenate procedure Eaton et al., 1995. Nitrogen
accumulation in pond soil between 26 May and 6 October was calculated by the following equation
DSN=
SN
f
− SN
i
100
BDV
s
, 1
where DSN=N accumulation in soil g pond
− 1
, SN
f
= TN in soil in October g
kg
− 1
, SN
i
= TN in soil in May g kg
− 1
, BD = soil bulk density kg m
− 3
, V
s
= soil volume in upper 5-cm layer m
3
. Bulk density of the top 5-cm soil layer averages about 300 g cm
− 3
in ponds on the FRU Masuda and Boyd, 1994a. Losses of N in seepage were estimated from the average monthly seepage rate
calculated from water budgets and the amount of N in seepage. To estimate the latter, 15 undisturbed, 5-cm diameter
×
20-cm long bottom soil cores and overlying water were brought to the laboratory and pond water was replaced with a solution
containing 10 mg l
− 1
each of NO
3
N and TAN. Gauze sponges were attached to the bottoms of the cores, and after the original pore water had been replaced by N
solution, soil column leachates were collected during five, consecutive 12-h periods. The leachates were filtered and analyzed for NO
3
N with the NAS reagent method and for TAN with the phenate method. The percentage of NO
3
N and TAN that passed through the soil column was used to calculate NO
3
N and TAN losses in seepage as illustrated in the following equation for TAN
S
TAN
= C
TAN
s A X
100 ,
2
where S
TAN
= TAN lost in seepage g pond
− 1
mo
− 1
, C
TAN
= TAN concentration
in pond water g m
− 3
, X = percentage of TAN lost in seepage from laboratory study, s = average monthly seepage rate m mo
− 1
, and A = pond area m
2
. Ammonia losses to the atmosphere by diffusion were measured with oxalic acid
traps mounted on rotating weathervane samplers. This effort was part of a separate study on ammonia volatilization, and the methodology and estimates of ammonia
losses over time are provided by Gross et al. 1999a. Denitrification was estimated in situ twice a month by measuring NO
3
N disappearance from water confined in tubes within each pond Capone, 1993. This
method assumes that there is no denitrification in the water column at night when DO concentration declines. This assumption was accepted because ponds were
aerated at night and DO concentrations below 3 mgl have seldom been recorded in aerated ponds on the FRU. Pairs of 10-cm diameter PVC pipes were inserted
into pond bottoms. One pipe was sealed at the bottom to prevent water from contacting bottom soil. The other pipe had an open bottom to permit contact
between soil and water. Sodium nitrate was added to water in the pipes to increase NO
3
N concentrations by 1.0 mg l
− 1
. Water samples were collected immediately after and 8 h after NaNO
3
introduction for NO
3
N analysis by the NAS reagent method. Nitrate-N loss from the pipe with the sealed bottom represented uptake by
phytoplankton, and NO
3
N loss from the pipe with the open bottom resulted from both denitrification and phytoplankton uptake. The difference in NO
3
N loss between the open pipe and the sealed pipe represented denitrification. The percent-
age decrease in NO
3
N concentration during 8 h was multiplied by three to estimate the loss in 24 h. This allowed estimation of NO
3
N loss as illustrated below for denitrification
N
d
= C
NO
3
V
p
m
X 100
, 3
where N
d
= denitrification g N pond
− 1
mo
− 1
, C
NO
3
= average monthly NO
3
N concentration in pond water g m
− 3
, V
p
= pond volume m
3
, X = percentage decline in NO
3
N concentration in 24 h, and m = number of days in month. A similar approach was used to estimate NO
3
N uptake by phytoplankton. Nitrification in the water column was measured biweekly. The procedure was
based on the difference in DO consumption between pairs of samples in which one was treated with nitrification inhibitor and the other was not Boyd and Gross,
1999. The amount of DO consumed in nitrification was converted to its N equivalent as follows
N
eq
= DO
n N
2O
2
, 4
where N
eq
= N equivalent of DO consumed in nitrification mg N l
− 1
d
− 1
, DO
n
= DO consumed by nitrification mg l
− 1
d
− 1
, and N and O
2
= molecular
weights of N and O
2
. The daily nitrification rate in terms of NO
3
N produced was calculated as follows
N
n
= N
eq
V
p
, 5
where N
n
= nitrification g N pond
− 1
d
− 1
. Values of N
n
were averaged to provide monthly estimates.
Net mineralization of feed N was estimated from the difference between feed N and in fish N at harvest, ON accumulation in bottom soil, and ON in pond water
at draining. The appropriate equation is M = FN − RN + ON
s
+ ON
w
, 6
where M = net feed N mineralization g pond
− 1
, FN = feed N g pond
− 1
, RN = N recovered in fish g pond
− 1
, ON
s
= ON accumulation in soil g pond
− 1
, ON
w
= ON in water at draining.
2
.
5
. Nitrogen budget Budgets were prepared for each pond by summing the gains and losses of N.
Inputs were fish stock, feed, intentional water additions, rainfall, and N
2
fixation. Losses resulted from fish harvest, outflow, accumulation in soil, seepage, NH
3
volatilization, and denitrification. Data for individual ponds were averaged to provide a single N budget.
3. Results and discussion
Kategori