Samples for BOD5 analyses were obtained from ponds on the Auburn University
Fisheries Research Unit, Auburn, AL, and on channel catfish Ictalurus punctatus farms in
Hale County, Alabama. Grab samples of surface water were put in 2-L plastic bottles and
held on ice in insulated chests for transport to the laboratory. The maximum time between
collection and initiation of analysis was 6 h, but most analyses were started within 2 or 3 h
after collection. Primary standard solutions for BOD5 were prepared from 1:1 mixtures of
glucose and glutamic acid (Clesceri et al., 1998).///////Standard BOD5 analyses were in accordance with Section 5210B of Standard Methods
for the Examination of Water and Wastewater (Clesceri et al., 1998). Water temperature
was adjusted to 20 1 8C by placing samples in a water bath. Each sample was treated
with sulfuric acid or sodium hydroxide solution to provide a pH between 6.5 and 7.5.
Samples were agitated to equilibrate dissolved oxygen concentration between 8.5 and
9 mg/L. Depending upon turbidity, aliquots of 50–200 mL from each sample were transferred to duplicate flasks and diluted to exactly 400 mL with BOD dilution water. The
dilution water was prepared from dry, nutrient premix (BOD Nutrient Buffer Pillows, Hach
Chemical Company, Loveland, CO, USA). A 2-mL aliquot of bacterial seed inoculum
(Polyseed1, Polybac Corporation, Bethleham, PA, USA) was introduced into each flask
before the samples were diluted. A nitrification inhibitor was not used. A BOD bottle
(305 1 mL) was filled from each flask, and the dissolved oxygen (DO) concentration was
measured immediately with a polarographic DO meter with BOD bottle probe and
mechanical stirring boot (Yellow Spring Instrument Company, Yellow Springs, OH, USA).
After removing the probe, a few drops of sample were added to replace water adhering to
the probe so that the bottle could be stoppered without creating air bubbles inside. The well
around the stopper was filled with sample, and a plastic cap was placed over the stopper and
well to prevent evaporation. Three dilution water blanks and three bacterial seed controls
were also prepared with each run. Samples, blanks, and seed controls were incubated in the
dark at 20 0.5 8C. After 5 days, the DO concentration in each bottle was measured with
the BOD bottle probe. The BOD5 was calculated with the following equation:@@@@@@@@@@@ where D1, initial DO concentration of sample, mg/L; D2, DO concentration of sample after
5 days, mg/L; B1, initial DO concentration of seed control, mg/L; B2, DO concentration of
seed control after 5 days, mg/L; P, decimal volumetric fraction of sample used; F, volume
of seed in diluted sample/volume of seed in seed control.///////////Samples for the alternate BOD5 method (direct method) were adjusted to 20 1 8C, pH
6.5–7.5, and DO of 8.5–9.0 mg/L as described above. Two BOD bottles were filled from
each sample, DO concentration was measured, a few drops of sample were added to the
bottle after removing the probe, and bottles were stoppered, capped, and incubated at
20 0.5 8C in the dark. In addition, a 50-mL bottle was filled with each sample, capped,
and held in the incubator with the BOD bottles. After 24 h, DO concentrations in BOD
bottles were measured with the BOD probe. The DO concentrations were recorded and the
samples were aerated by an oil-free aquarium air pump with plastic tubing and small air
stone until the DO concentration was above 8 mg/L. No attempt was made to saturate the
sample because of the slow rate of oxygenation between 8 mg/L DO and saturation.Water
that adhered to the aeration device and BOD bottle probe was replaced with sample from
the 50-mL bottles, and bottles were placed back in the incubator. Dissolved oxygen was
measured again after 48 h of incubation for those samples that contained less than 4 mg/L
DO after 24 h of incubation. After 72 h of incubation, DO was measured and all samples
were re-aerated. These samples with less than 4 mg/L DO after 72 h incubation were
checked again at 96 h. The final DO measurement was made after 5 days (120 h). The
BOD5 concentration was calculated by summing the losses of DO during the 5-day
incubation as follows:@@@@@@@@@@@@where Di, initial DO concentration or DO concentration after each re-aeration; Df, DO
concentration before each re-aeration or after 5 days (120 h).