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Measurement of 5-day
biochemical oxygen demand
without sample dilution or
bacterial and nutrient
enhancement

Article in Aquacultural Engineering October 2005

DOI: 10.1016/j.aquaeng.2005.02.005

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 Aquacultural Engineering 33 (2005) 250257
www.elsevier.com/locate/aqua-online

Measurement of 5-day biochemical oxygen

demand without sample dilution or bacterial
and nutrient enhancement
Jiang Xinglong a, Claude E. Boyd b,*
a
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China
b
Department of Fisheries and Allied Aquacultures, Auburn University, AL 36849, USA
Received 28 October 2004; accepted 14 February 2005

Abstract

A direct method for measuring the 5-day biochemical oxygen demand (BOD5) of aquaculture
samples that does not require sample dilution or bacterial and nutrient enrichment was evaluated. The
regression coefficient (R2) between the direct method and the standard method for the analyses of 32
samples from catfish ponds was 0.996. The slope of the regression line did not differ from 1.0 or the Y-
intercept from 0.0 at P = 0.05. Thus, there was almost perfect agreement between the two methods.
The control limits (three standard deviations of the mean) for a standard solution containing 15 mg/L
each of glutamic acid and glucose were 17.4 and 20.4 mg/L. The precision of the two methods, based
on eight replicate analyses of four pond water samples did not differ at P = 0.05.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Aquaculture effluents; BOD5; Water quality monitoring

1. Introduction

Water pollution by aquaculture effluents has become an environmental issue (Lee and
OBryen, 2003). In response to this concern, several nations, including the United States, have
developed aquaculture regulations, and effluent monitoring may be mandated in discharge
permits (Boyd, 2003a). Lending agencies are beginning to demand environmental

* Corresponding author. Tel.: +1 334 844 4078; fax: +1 334 844 5933.
E-mail address: ceboyd@acesag.auburn.edu (C.E. Boyd).

0144-8609/$ see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaeng.2005.02.005
 J. Xinglong, C.E. Boyd / Aquacultural Engineering 33 (2005) 250257 251

accountability from their clients, and effluent monitoring may be required. Aquaculture
associations are encouraging their members to be more environmentally aware through
voluntary adoption of best management practices that may include effluent monitoring. There
is a growing interest in environmental certification of aquaculture facilities, and effluent
monitoring is a standard in some programs (Boyd, 2002). Aquaculture facilities also may
monitor source water in order to detect changes in water quality that could influence
production (Teichert-Coddington, 1995). Moreover, the influence of aquaculture effluents on
receiving water bodies may be studied through water quality monitoring (Ward et al., 1999;
Silapajarn et al., 2004). Such programs may include numerous stations extending several
kilometers above or below farm effluent outfalls (Boyd and Green, 2002).
Water quality variables of most concern in aquaculture effluents are 5-day
biochemical oxygen demand (BOD5), nutrients, and total suspended solids (Boyd
and Tucker, 1998). Although these variables can be measured on-farm by aquaculture
personnel, determination of standard BOD5 is more difficult and time consuming than
the analyses of the other variables. Waters for aquaculture facilities seldom have BOD5
concentrations above 30 mg/L (Boyd and Gross, 1999). Nevertheless, the BOD5 of most
samples is high enough to require dilution with nutrient solution and addition of bacterial
seed. Dilution is particularly problematic for samples from coastal aquaculture facilities
where salinity may vary widely. Waters may have low salinity during the wet season and
high salinity in the dry season. Also, in water quality monitoring programs for coastal
aquaculture, salinity usually varies among stations on each sampling date, and it is often
necessary to have dilution water of several salinities. Salinity is not a factor in
determining BOD5 in freshwater, but a method that does not require dilution would
simplify the analysis for on-farm use.
Boyd (2003b) suggested a modification of the ultimate BOD method for determination
of BOD5 in undiluted samples. The purpose of the present study was to determine if BOD5
concentrations measured by the alternate method compare favorably with those
determined on the same samples by the standard technique (Clesceri et al., 1998).

2. Materials and methods

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 50200 mL from each sample were
252 J. Xinglong, C.E. Boyd / Aquacultural Engineering 33 (2005) 250257

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:
D1 D2 B1 B2 F
BOD5 P (1)
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
6.57.5, and DO of 8.59.0 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:
X
n
BOD5 Di  Df 1    Di  Df n (2)
1

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).
 J. Xinglong, C.E. Boyd / Aquacultural Engineering 33 (2005) 250257 253

3. Results and discussion

The BOD5 concentration did not differ between methods for samples with mean
concentrations of 9.69 and 13.77 mg/L (Table 1). The means were different (P < 0.05)
for samples with means of 16.28 and 24.46 mg/L. Although the magnitude of the values
was greater with the standard method for all four samples, the relative error of the direct
method against the standard method was low 0.1 to 2.6%. Variation of replicate
measurements of individual samples was low with standard deviations (S.D.) of 0.21
0.43 mg/L (coefficients of variation (CV) of 1.32.5%) and 0.140.26 mg/L (CV = 0.9
2.8%) for the standard and direct methods, respectively. Variances (s2) for the two
methods were homogenous for the four samples indicating that the methods did not
differ in precision.
Using the standard method, the mean and S.D. of BOD5 for eight replicate analyses of
a solution containing 150 mg/L each of glucose and glutamic acid were 192.6
2.5 mg/
L. According to Clesceri et al. (1998), a study involving 112 laboratories provided a
mean and S.D. of 198
30.5 mg/L for this solution. Therefore, our results were well
within the acceptable range for the BOD5 analysis of the primary standard. Samples from
aquaculture facilities and receiving waters seldom have BOD5 concentrations above
30 mg/L, and values below 20 mg/L are most common (Boyd and Gross, 1999; Boyd and
Gautier, 2000). Thus, the glucoseglutamic acid standard was diluted 10 times and
analyzed 25 times over several weeks by the two methods (Fig. 1). The concentrations
ranged from 18.0 to 19.8 mg/L for the standard method and from 18.0 to 19.9 mg/L for
the direct method. Values were numerically higher for the standard method in 21 of the
25 analyses. The mean (t = 1.898) and s2 (F = 1.075) did not differ (P > 0.05) for the
two methods (Table 2), and the relative error of the direct method against the standard
method was 1.4%. The BOD5 concentration for the diluted glucoseglutamic acid
standard should be 19.8
3.0 mg/L. Thus, all individual analyses of BOD5 by the two
methods fell within the acceptable range.
For quality control purposes, Clesceri et al. (1998) recommended that an individual
laboratory conduct at least 25 analyses of the BOD5 standard over time and use the
mean
3 S.D. as upper and lower control limits. For our results, the control limits for the
direct method were 17.4 and 20.4 mg/L. To verify control over accuracy in the future,

Table 1
Comparison of the standard and direct method for measuring 5-day biochemical oxygen demand
Standard method Direct method t Percentage relative error
X S.D. X S.D.
9.69 0.25 9.44 0.26a 1.960 2.6
13.77 0.29 13.76 0.23a 0.076 0.1
16.28 0.21 15.87 0.14a 4.556b 2.5
24.46 0.43 23.83 0.23a 3.654b 2.6
Eight replicate determinations of the same sample were made by both methods. A t-test was used to compare
means and an F-test was used to determine if variances of the two methods were different.
a
Variances (s2) were homogenous for the two methods.
b
Means of two methods differed at P = 0.05.
254 J. Xinglong, C.E. Boyd / Aquacultural Engineering 33 (2005) 250257

Fig. 1. Analysis of 5-day biochemical oxygen demand (BOD5) by direct method and standard method of a
standard prepared from 15 mg/L each of glucose and glutamic acid. Analyses were conducted over a 4-week
period.

analyses of the standard (15 mg/L each of glucose and glutamic acid) should fall between
these limits.
The relationship between the direct method (X) and the standard method (Y) for the
analyses of 32 samples from catfish ponds is depicted in Fig. 2. The regression equation
was as follows:
Y 0:107 0:991X; R2 0:996 (3)
where Y = BOD5 by standard method (mg/L); X = BOD5 by direct method (mg/L).
The slope of the regression line did not differ from 1.0 (t = 1.080; P > 0.05) and the Y-
intercept did not differ from 0.0 (t = 0.937; P > 0.05). Thus, agreement between the two
methods was almost perfect for this series of samples.
Data presented in Table 1 and Fig. 1 suggest that the numeric values for BOD5 tended to
be slightly greater for the standard method. However, for the series of 32 samples (Fig. 2),
the standard method gave a greater value than the direct method for only 15 samples. The
relative error of the direct method against the standard error ranged from 0.1 to 15.4% with
a mean of 3.7%.
The findings of this study revealed that the direct method for BOD5 provided accurate
and precise results. Moreover, for an individual sample, BOD5 concentrations measured by

Table 2
Results of 5-day biochemical oxygen demand analyses of a primary standard solution containing 15 mg/L each of
glucose and glutamic acid by the standard method and the direct method
Variable Standard method Direct method
N 25 25
Mean (mg/L) 19.2 18.9
Standard deviation (mg/L) 0.51 0.49
Coefficient of variation 2.6 2.6
Relative error (%) 1.4
 J. Xinglong, C.E. Boyd / Aquacultural Engineering 33 (2005) 250257 255

Fig. 2. Regression between direct method and standard method for 5-day biochemical oxygen demand (BOD5).
Water samples from 32 channel catfish ponds each were analyzed in duplicate by both methods.

the two methods were similar. The direct method is more feasible for use at aquaculture
facilities, and especially at brackishwater aquaculture facilities, than the standard BOD5
method that requires dilution with nutrient solution and inoculation with bacterial seed.
The methodology for conducting the direct method is presented in Appendix A.
The chemical oxygen demand (COD) is sometimes used to estimate BOD5, however, this
procedure often is not accepted as an alternative to BOD5 by regulatory agencies. Moreover,
chloride interferes strongly with COD measurement, and it is difficult to make reliable COD
measurements in brackishwater or seawater (Ruttanagosrigit and Boyd, 1989).
This study was conducted for freshwater samples. However, Clesceri et al. (1998) does
not suggest modifying the standard BOD5 procedure for use in saline water. Thus, the
direct method for BOD5 should be suitable for use with brackishwater or marine water.

Appendix A. Direct measurement of 5-day biochemical oxygen demand

A.1. Apparatus
Water bath or basin with hot and cold water for adjusting temperature of samples to 20
1 8C
pH meter
Dissolved oxygen meter and BOD bottle probe with mechanical stirring boot
Aquarium air pump (oil-free)
Incubator (20
0.5 8C and darkness)
Plastic tubing (compatible with air pump outlet)
Air stone (small enough to fit inside BOD bottle)
Sample bottles (2 L)
Beakers (500 mL)
BOD bottles (300 mL)
Plastic covers for BOD bottles (can use aluminum foil as substitute)
Screw-cap bottles (50 mL)
256 J. Xinglong, C.E. Boyd / Aquacultural Engineering 33 (2005) 250257

Medicine dropper
Mercury thermometer (050 or 100 8C)
Magnetic stirrer and stirring bars

A.2. Reagents

Acid solution, 1NAdd 28 mL concentrated sulfuric acid to distilled water and dilute
to 1 L.
Alkali solution, 1NDissolve 40 g sodium hydroxide in distilled water and dilute to
1 L.
A.3. Procedure

(1) Adjust temperature of sample to 20
1 8C by placing it in water bath or basin of water

at this temperature.
(2) Agitate sample by pouring back and forth between two beakers to assure DO
concentration near saturation (8.59 mg/L).
(3) Put 400 mL of sample in beaker, insert pH probe and adjust pH between 6.5 and 7.5 by
adding acid or alkali solution dropwise while stirring and monitoring pH.
(4) Fill BOD bottle and 50-mL bottle with sample.
(5) Measure and record DO concentration in BOD bottle with probe. Remove probe and
add sample from 50-mL bottle dropwise to replace loss. Insert glass stopper in BOD
bottle, fill well around the stopper with sample, and place cap or aluminum foil over
well to prevent evaporation. Incubate BOD bottle and 50-mL bottle of sample at
20
0.5 8C in the dark.
(6) After 24 h, measure and record DO concentration, and re-aerate the sample using air
pump and air stone. Also, re-aerate sample in 50-mL bottle (DO measurement not
necessary). Measure and record DO concentration in BOD bottle again. Add sample
from 50-mL bottle to replace loss, re-stopper, fill well, replace cap, and incubate BOD
bottle and 50-mL bottle again.
(7) If the DO concentration fell below 4 mg/L after 24-h incubation, measure DO after
48 h, re-aerate sample, and return to incubator as described above. Otherwise, measure
DO concentration in sample after 72 h, re-aerate and return it to the incubator.
(8) If the DO concentration was below 4 mg/L after 72 h, measure DO after 96 h, re-aerate
sample, and return it to the incubator. Otherwise, leave sample in incubator.
(9) Make final DO measurement after 5 days (120 h) of incubation.

A.4. Calculation

Compute the DO loss between each re-aeration event and sum the losses to provide the
BOD5 using Eq. (2).

References

Boyd, C.E., 2002. ACC updates effluent standard. Global Aquaculture Advocate 5 (6), 1012.
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Boyd, C.E., 2003a. The status of codes of practice in aquaculture. World Aquaculture 34 (2), 6366.
Boyd, C.E., 2003b. Alternate measurement of biochemical oxygen demand. Global Aquaculture Advocate 6 (6),
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Boyd, C.E., Gautier, D., 2000. Effluent composition and water quality standards. Global Aquaculture Advocate 3
(5), 6166.
Boyd, C.E., Green, B.W., 2002. Coastal water quality monitoring in shrimp farming areas: an example from
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