903979006
U.S. ENVIRONMENTAL PROTECTION AGENCY
MIDDLE ATLANTIC REGION-III 6th and Walnut Sfeets, Philadelphia, Pennsylvania 19106
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EPA 903/9-79-006
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SIMPLIFIED N.O.D. DETERMINATION*
*Presented at the 34th Annual Purdue Industrial Waste Conference at
r Purdue University, West Lafayette, Indiana on May 9, 1979
Li
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SIMPLIFIED N.O.D. DETERMINATION
May 1979
Joseph Lee Slayton
E. Ramona Trovato
Annapolis Field Office
Region III
U.S. Environmental Protection Agency
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u
SIMPLIFIED N.0.0. DETERMINATION
^ Joseph L. Slayton and E. Ramona Trovato
U.S. Environmental Protection Agency
Region III, Annapolis Field Office
Biochemical oxygen demand (BOO) is a bioassay procedure concerned
with the utilization of oxygen in the biochemical oxidation (respiration)
of organic material. This test is one of the most widely used measures
'" of organic pollution and is applied both to surface and waste waters.
\m
The standard method of BOD measurements adopted by APHA1 is a five
I day test in which a water sample is maintained at 20°C in the dark
[jt
and oxygen depletion is monitored. The five day incubation period
'- was selected to maximize the oxygen demand associated with the
'" oxidation of carbon compounds while minimizing the oxygen demand of
L
autotrophic organisms. That portion of the BCD due to the respiration
r"
of organic matter by heterotrophic organisms is termed the carbonaceous
,., oxygen demand and that portion involved with nitrification is termed
'•" nitrogenous oxygen demand. The desire to separate the NOD and CBOD
results not only from the fact that the organisms responsible for these
. at
components have different nutrient requirements, but also because
< "»
,_f they differ in reaction rates, A02/Atime; temperature coefficients;
and tolerance to toxic materials. Nitrifying bacteria are in general
ijt slower growing2; more drastically affected by temperature3; and are more
The mention of trade names or commercial products in this report is
for illustration purposes and does not constitute endorsement or
recommendation by the U.S. Environmental Protection Agency.
r*
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sensitive to materials as1*: phenols; cresol ; halogenated solvents;
heavy metals; and cyanide. The organisms involved in the CBOD and
NOD processes would therefore be expected to react differently to the
same aquatic environment. The determination of the BOD components
would better define the BOD test results and aid in extrapolating these
results to the prediction of dissolved oxygen profiles in a body of water.
The purpose of this paper is to demonstrate that a simple
procedure involving an inhibitor to nitrification, N-serve, could
provide an accurate and precise measurement of nitrification occurring
in the BOD test while not affecting the carbonaceous oxygen demand.
Nitrification
Nitrification is the conversion of ammonia to nitrate by
biological respiration. This type of respiration is employed by
seven genera of autotrophic nitrifyers.5
It should be noted that heterotrophic nitrification can also
produce N0£ and N0§ by reactions that do not involve oxidation.3
However, only Nitrosomonas spp and Nitrobacter spp are regularly
reported by in situ nitrification studies.2 Therefore, the treatment
of nitrifying river samples with inhibitors specific to Nitrosomonas
and Mitrohacter can be expected to stop all appreciable nitrification.7
The reactions involved in nitrification are as follows:
Nitrosomonas
NOjf + h 02
The stoichiometries of the nitrification reactions dictate that the
conversion of 1 gram of nitrogen from ammonium to -nitrite utilizes
-------
P 3.43 grams of oxygen and the conversion of 1 gram of nitrite-nitrogen
in
to nitrate-nitrogen involves the utilization of 1.14 grams of oxygen.
^ Mowever, nitrifying bacteria are autotrophic and as such utilize
,,„ a portion of the energy derived from nitrogen oxidation to reduce CO?,
'•" their primary source of carbon. T^e net result is a reduction in
P the amount of oxygen actually consumed. Short term, zero to five
L*
day, laboratory experiments8/9'10 employing cultures of Nitrosomonas
r
>« and Nitrobacter have related the depletion of oxygen to the production
l» of nitrite and nitrate with the corresponding oxygen to nitrogen ratios
'"" of 3.22 and 1.11. However, in long term experiments, the decay of
' these organisms would be expected to exert an oxygen demand approximately
equivalent to the oxygen originally generated, resulting in an
!'*
,- overall relation not significantly different from 4.57.11
!" The equation used to calculate the NOD from the changes in
nitrogen states upon incubation was:
MOD = 3.43 (ANOg-P + ANOa-N) + 1.14 (ANOs-N) Equation 3
'. M
where A = final - initial.
k.- The potential NOD was calculated as:
potential MOD = 4.57 (TKN) Equation 4
L 4*
where TKN = (NH3-N + Norg-N)and N02-N was insignificant.
The NOD was also measured by the difference in oxygen depletion
,„ in an unaltered sample and in a sample altered by the addition of
^ the nitrification inhibitor, nitraoyrin.
^ Nitrification Inhibitor
Ul
The inhibitor used was formula 2533 Nitrification Inhibitor,
a product of the Hach Chemical Company. The product consists of
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2-chloro-6-(trichloromethyl) pyridine known as TCMP or nitrapyrin.
This compound is plated onto a simple inorganic salt which serves as
a carrier and is soluble in water. The DOW Chemical Company, Midland,
Michigan, markets this chemical under the name N-Serve as a fertilizer
additive.
Studies12^1 Vs using nitrapyrin suggest that it acts as a
"biostat" at moderate concentrations to delay nitrification and
aids in the retention of ammonia or urea fertilizers on crops by
retarding conversion to the more highly Teachable MQ3~. TCMP is
slowly biodegraded to 6-chloropicolinic acid which leaves the fields
in their original state, with no further inhibition to nitrification.
The advantage of this is that 20 to 30 day NOD assays may be performed
without significant inhibitor contribution to the carbonaceous
demand. :1'ls
Because of concern for the potential environmental impact
resulting from extensive farm use, studies were performed on the
toxicity of this material. These studies have revealed the inhibitor
to be very selective and effective at stopping nitrification when
used at a concentration of 10 mg TCMP/1 ,1W7
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r» Experimental
LJi
A. NOD Svnthetic Ammonia Exoeriment
r, . 1
L* 1. 300 ml BOD bottles were weighed before and after the addition
'"« of water and found to be reliable to within 1%. They were
used as volumetric flasks for all experiments.
2. Two ml of a solution of O.lSOg glucose/1 plus 0.150g glutamic
acid/1 were spiked into BOD bottles using a repipet.
'* 3. Stale settled sewage was filtered through Kiiiwipes18 and
' * diluted. One ml was dispensed into each SOD bottle.
4. MHs-N spikes were made using a ^4.5 mg Nr^Cl-N/l stock solution.
5. The BOD bottles were then filled with APHA standard dilution
P, water.1
^ 6. Ammonia was assayed using a Technicon automated colorimetric
phenate method.19 Nitrate was determined using a Technicon
. m
automated cadmium reduction method and nitrite was assayed
using a Technicon automated NEDA-diazotizing method.19
7. Dissolved oxygen (DO) was monitored ucing a YSI Model #57
* meter and #5720 probe. DO measurements were made before
and after incubation which was carried out in the dark at 20°C.
tf
8. The nitrification inhibitor (Hach Chemical Co. #2533) was
i
, dispensed, using a powder dispenser, directly into the BOD
bottles. This allowed quick and uniform additions of the
inhibitor. Two sets of bottles were filled with each sample;
one received the inhibitor and represented CBOD and the
uninhibited bottle expressed total BOD. The NOD was determined
by difference.
Ul
r*
L*
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B. NOD Synthetic Nitrite Experiment
This experiment was identical to the synthetic ammonia
experiment except spikes of NaN02 were substituted for NH4C1 .
C. Synthetic Glucose Samples-Respiration Experiment
1. BOD bottles were spiked with approximately 3.0 ml of a 3.0g/l
stock glucose solution using a repipet. Raw sewage influent
was filtered through Kimwipes and diluted with distilled
water. One ml of this seed was spiked into each bottle. TCMP
was added to one-half of the bottles using the Hach powder
dispenser and all bottles were filled with standard BOD
dilution water.1
2. Oxygen was bubbled through the bottles using a Fisher gas
dispersion tube and purified oxygen. The samples were then
incubated in the dark at 20°C.
3. Initially and after different periods of incubation, samples
were placed in a refrigerator at 4°C to stop bacterial activity.
At the conclusion of the experiment bottles were assayed for
glucose.20 The samples were first filtered through a 0,45u
Mi Hi pore filter to remove bacteria. Four ml of each filtrate
were placed into 125 ml Erlenmyer flasks; which had been
chromic acid washed and muffle furnaced for 24 hrs. at 550°C.
Repipets were then used to dispense 4 ml of phenol solution
(25.0 gms/500 ml deionized water) and 20 ml of acid reagent
(2.5 g hydrazine sulfate/500 ml cone. H?SOd). The acid
reagent was added with swirling and the flasks were placed in
a refrigerator at 4°C for 2 hours to cool. The absorbanca
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was read on a Varian 535 spectrophotometer using 5 cm quartz
r"
u< cells at 490 nru. A 500 mg/1 glucose stock solution was
r, prepared and appropriate volumes were diluted with deionized
UJ water to generate standard curve solutions. The resultant
standards were filtered and assayed as samples.
t.j
Calibration Curve Data
r »
u- Glucose (mg/1) Absorbance
, - 0 0
2.5 0.125
5.0 0.252
L *
10.0 0.435
r »
15.0 0.560
20.0 0.332
""* 25.0 1.068
30.0 1.230
, M
35.0 1.459
,' «*
t,- slope = 0.0402
intercept = 0.0484
correlation coefficient = 0.999
i **
4. Dissolved oxygen was measured directly in the BOD bottles
,„ using the YSI 5720 probe and the pH was determined using a
^ Corning 110 research meter and electrode.
r" 0- TCMP and the Measurement of Dissolved Oxygen
HJt
1. Electrode and Winkler Methods
m
^ a. A 20 liter carboy of deionized water was stirred with
r» a magnetic stirring bar as water was slowly siphoned into
^ 16 sets of four 300 ml BOD bottles and capped. This
r*
u
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procedure was repeated to generate 32 sets of 4 bottles.
b. TCMP was added to two bottles from each set using the
Hach powder dispenser.
c. Two bottles (one with TCMP) were analyzed for DO via
the Winkler azide modified method1 using a Fisher Model 41
potentiometric titralyzer. An incubation period of 2 to 3
hours after the addition of the inhibitor and Winkler
reagents was allowed prior to titration to enable potential
reactions, which may have resulted in interferences, to occur
d. The remaining two bottles of each set (one with TCMP) were
analyzed by a YSI 5720 DO probe and £57 meter. This
meter had been previously calibrated against the Winkler
method as outlined in Standard Methods.1
2. Starch End Point - Azide Modified Winkler DO
a. Fourteen potassium biiodate standards, each with 3 ml
of Fisher SO-P-340 stock biiodate solution (0.0250 N),
were prepared as outlined in APHA Standard Methods1
for Winkler Dissolved Oxygen measurements.
b. To seven of these TCMP and starch (Fisher T-138 thyodene)
were added.
c. The samples were titrated with sodium thiosulfata solution
using a Fisher Model 41 titralyzer in the manual mode
and titrating to the disappearance of the blue color.
Potomac River Study
1. The BOD test employed was that outlined in Standard Methods
APHA 14th edition.1 The river water samples were stored at
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r1"
t_»
4°C until analysis. Three-hundred ml of each sample was
r»
^ placed in each of two BOD bottles. The bottles were purged
r» for 15 seconds using purified oxygen and a Fisher gas dispersion
*"* tube to obtain an initial DO of 10 to 15 mg/1 . One bottle of
each pair was dosed with the Hach Co. £2533 Nitrification
(..«
Inhibitor.
r»
L. 2. Dissolved oxygen was measured immediately using a YSI 5720 DO
<» probe and again after 20 days of incubation in the dark at 20°C.
3. TKN was analyzed on the unaltered river samples using a
Technicon automated phenate method.19
r »
t .
F. Lehigh River Study
i- 1. Samples were prepared in six replicate BOD bottles and two
r" bottles of each set were spiked with TCMP using the Hach
i.«
powder dispenser.
r *»
2. Dissolved oxygen was analyzed immediately and after several
periods of incubation in the dark at 20°C using a YSI 5720
u. DO probe.
r"* 3. One bottle was sacrificed after each DO reading and assayed
i«
for NOj?-N and N03-N by the automated methods previously described
r'*
^ 4. Three classes of sample preparation were employed to allow
P, for differences in sample character:
*••* a. River samples were unaltered.
*"* b. Industrial effluents with low level Nh'3-N were seeded with
Ul
1 ml of stale settled sewage per 300 ml BOD bottle and
r»
u correction blanks were carried through the experiment.
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c. Sewage treatment plant effluent samples and industrial
effluents with high levels of ammonia were diluted.
Samples of October 4 were diluted by a factor of 30 and
those of October 5 and 6 were diluted by a factor of 15
with seeded APHA diluted water. Correction blanks
were carried through the experiment.
Results and Discussion
NOD Synthetic Ammonia Experiments
Initial experiments were performed on synthetic samples to
establish the accuracy of the NOD determinations made using TCMP.
The experiment consisted of spiking samples of APHA dilution water1
with a glucose-glutamic acid solution, bacteria, and ammonia. The
concentrations of ammonia, nitrate, and nitrite were then determined
before and after incubation. The changes (A) in the states of nitrogen
were determined and used to calculate the actual MOD wich had occurred
(Equation £3).
The dissolved oxygen initially and finally present was determined
in all bottles. The oxygen utilized in the inhibited bottles was
taken as CBOD where as the depletion in the uninhibited bottles was
taken as NOD plus CBOD. This NOD, signified as NOD-TCMP, was
determined by the average difference observed between these sets.
The results of these experiments are presented in Table 1. A
paired student's t-test of the nitrogenous oxygen demand established
(t=1.41, n=32) at a=.05 that there was no significant difference between
these two methods of NOD determination. The average difference
between the two methods was 0.3 mg/1 NOD.
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Fable 1. NOD of synthetic ammonia samples as determined by analysis
of nitrogen conversions and by measurement with TCMP
i_*
•"•NHs-Ni N02-Ni
LJ mg/1 mg/1
.361 .053
m
tut
™ .637 .052
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The oxygen depletion was monitored over time for several of the
samples and the DO data is presented in Figure 1. This work
illustrates the potential use of the inhibitor in establishing
deoxygenation constants for NOD separate from CBOD.
The seed source for these experiments was stale sewage. The
sporadic growth of the nitrifyers observed during these experiments
was largely corrected in later work by filtration and the use of
more seed material.
NOD Synthetic Nitrite Experiment
The effect of TCMP upon the growth of nitrifying bacteria
was tested using spikes of sodium nitrite into seeded APHA dilution water
containing glucose/glutamic acid (Table 2). The calculated nitrogenous
oxygen demand based on the measured changes in the states of nitrogen
was significantly higher than that predicted by the use of TCMP
when compared by a paired t test (t=7.3 at 2=0.05 & n=15). The changes in
nitrite and nitrate were also measured in the TCMP spiked bottles,
which allowed the calculation of the NOD occurring despite the presence
of TCMP. This calculated error matched favorably (correlation
coefficient = .92) with the average error actually observed between the
calculated NOD in the samples and that measured using TCMP. The
inhibitor had little inhibitory effect uoon aitrobacter spp,
since all of the NO^-N in the spike was converted to MO§-N after
30 days of incubation.
Although the mechanism of its action is unclear, the inhibitory
effect of nitrapyrin is apparently restricted to Nitrosomonas. This
selectivity is advantageous in that it stops the procass of nitrification
at ammonia with little or no effect on urea hydrolysis21, thus assuring
an adequate nitrogen source for the heterotrophic bactaria contributing
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C 3 C 2
C 3
£2
C 1
Figure I. Oxygen depletion of synthetic glucose/glutamic acid samples spiked with ammonium chloride
o>
e
a
o
OQ
10
9
8
7
6
5
4
3
2
0
Untreated Sample*
• O.24 mg/l NH4-N added
• 0.60 mg/l NH^-N added
A 1.63 mg/l NH4 - N added
Treated Samples
D Average of TCMP treated samples
spiked with 0.24, 0.80 and 1.63
mg/l NH^-N
NOD29= 6.7 mg/l
NOD29= 4.0mg/l
NOD2fl= 0.6mg/l
Q-
0
8 10 12 14 16 18 20 22 24 26
28 30 32
Days of Incubation
-------
.
Table 2. MOD of synthetic nitrite samples as determined by analysis
of nitrogen conversions and by measurement with TCMP
Uninhibited Samoles
3.43
N 1.14 NOD MOD
UNOs-N) calc TCMP ot
ma/1 mg/1 mg/1 en
T99 2.4 1.9
1.00 2.5 2.0
.98 2.4 1.9
1.00 2.5 2.0 .'
1.55 3.0 1.5
1.12 1.3 0
1.56 3.1 1.5
1.60 3.2 1.8 1.
2.03 3.3 1.9
2.09 3.5 2.1
- .05 0.1 **(0) 1 .4
2.01 3.2 1.9
2.29 3.1 0
2.33 3.3 2.1 1.7
2.34 3.3 2.1
2.02 2.0 0.9
NOD Ave.
(calc. calc.
err.) err.
.53 .6
.53 .6
.53 .6
.53 .6 .6
1.12 1.3
1.11 1.2
1.11 1.2
1.12 1.3 1.3
1 .57 1 .4
1.62 1.6
1.61 1.6
1.79 2.3 1.7
2.03 2.0
2.03 2.0
2.02 2.0 2.0
* initial NH3-N value is an average of 24 values with s.d. = 0.02
** omitted from calculation
i - initial reading; initial nitrogen values are the average of thrae measurements
f = final reading; after 30 days of incubation
i = final-initial
NH3-Ni* N02-N1
mg/1 ma/1
.436 .456
.456
.456
.456
.934
.934
.934
.934
1.408
1.408
1.408
1.408
1.759
1.769
1.769
1.769
.436 .459.
.459
.459
.459
.942
.942
.942
.942
1.419
1 .419
1.419
1.419
1.787
1.737
1.787
N02-Nf
mq/1
0
0
0
0
0
0
0
0
0
0
1.50
0
0
0
0
0
a
0
Q
Q
0
0
0
0
Q
0
0
0
0
0
0
ANOg-N
mg/1
-.456
-.456
-.456
-.456
-.934
-.934
-.934
-.934
-1 .408
-1.403
.092
-1 .408
-1 .769
-1 .769
-1 .769
-1.769
- .4sa
- .'459
- .459
- .459
- .942
- .942
- .942
- .942
-1.419
-1 .419
-1.419
-1 .419
-1.787
-1 .737
-1.787
N03-N1
mq/1
0
0
0
0
0
0
0
.045
.045
.045
.045
.061
.061
.061
.061
TCMP
0
0
0
Q
0
0
0
0
.045
.045
.045
.045
.056
.056
.056
N03-Nf
mg/1
.870
.380
.864
.880
1.363
.984
1.370
1.401
1 .828
1.880
0
1.807
2.068
2.101
2.117
1.835
£N03-N +AN
Q3-i*
mg/1 mg/1
.870 1.42
.880 1
.864 1
.880 1
1.363 1
.984
1.370 1
1.401 1
1.783 1
1.835 1
- .045
1.762 1
2.007
2.040
2.056
1.774
.45
.40
.45
.47
.17
.50
.60
.29
.46
.16
.21
.32
.93
.98
.04
Inhibited Samples
.463
.468
.468
.468
.984
.974
.974
.984
1.424
1.467
1.455
1.614
1.835
1 .335
1 .829
.463
.468
.468
.468
.984
.974
.974
.984
1 .379
1 .422
1.410
1.569
1.779
1 .779
1.773
.03
.03
.03
.03
.14
.11
.11
.14
.14
.01
.03
.51
.03
.03
.04
-------
to the CBOD. The disadvantage of this selectivity is that Nitrobacter
Ur
are not inhibited and NOJjj will be oxidized to NOs. This limitation
r«
u generally represents a small error since the concentration of nitrite-
pi nitrogen is generally much smaller than Total Kjeldahl Nitrogen in
** river water. Further, the demand associated with the N02-N initially
*"* present is 1.14/4.57 or one-quarter that associated with the TKN-N
I*
initially in the sample.
r»
Synthetic Glucose Samples-Respiration Experiment
To directly determine the effect of TCMP on the rate of heterotrophic
i* respiration, synthetic samples of APHA dilution water were spiked with
r» glucose and seed bacteria. Several bottles were immediately assayed
U
for glucose, dissolved oxygen, and pH, while others were incubated
r«
and later analyzed for these parameters. The results, compiled in
i*
Table 3, indicate that TCMP did not appreciably decrease the rate at
i»
(- which glucose was utilized. The potential problem with this
,» interpretation is that these results may have been at steady state
L* and therefore may not actually represent the rate at which steady
'* state was achieved.
ui
This experiment was again performed with the emphasis placed on
r»
determining when steady state occurred in bottles in which growth
,„ was observed. Glucose concentration, pH, and dissolved oxygen level
lj were measured initially and periodically during incubation. The
'"* final levels determined were similar to those in the previous
L*
experiments. The results, compiled in Table 4 and Figure 2, indicate
r»
that: the glucose respiration rate was not significantly affected
u
by TCMP; steady state was not established after 4 days of incubation;
r»
L* and suggested that the interpretation of the first experiment was valid.
U
*
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Table 3. Effect of TCMP on the utilization of glucose in
synthetic samples
Day 0
TCMP
Inhibited
Sample
Uninhibited
Sample
Day 0
TCMP
Inhibited
Sample
Uninhibited
Sample
'A Ave.
Glucose Glucose Ave. D.O.
mg/1 ave. mg/1 pH mg/1
27.3 0 5.8 15.5
27.7
28.0
29.8
29.6 0 6.7 15.5
28.9
29.6
28.6
29.1
28.0 0 6.5 13.2
26.2
26.9
27.6
26.7
26.9
26.0
27.1
26.6
26.9
28.0 0 6.3 13.2
27.2
26.7
27.6
27.5
27.9
27.9
27.7
27.0
27.1
Day 2
A Ave.
Glucose Glucose Ave. D.O,
mg/1 ave. mg/1 pH mg/1
7.3 20.8 5.9 6.9
6.9
7.1
8.7
6.8
8.5 21.9 5.7 6.9
6.7
6.7
5.3
9.4
Day 2
9.9 16.5 6.0 5.4
10.9
10.2
12.2
9.5
10.5
10.1
10.2
9.8
10.2
11 .0 15.5 5.3 6.4
12.0
13.4
10.4
9.9
9.4
n .0
10.9
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Table 4. Rate of glucose respiration during inhibition of nitrification
r»
u Day 0
TCMP
M Inhibited
Sample
r»
Uninhibited
'"* Sample
M Day 2
'"* TCMP
MI Inhibited
Sample
Uninhibited
IJ Sample
" Day 4
TCMP
,., Inhibited
. Sample
Uninhibited
' * Sample
i *
* *
.1 Ave.
Glucose Glucose Ave. D.O.
mg/1 ave. mg/1 pH mg/1
23.6 0 6.7 15.7
26.2
26.7
27.1 0 6.8 15.6
27.6
25.6
9.0 16.5 6.1 7.3
9.0
10.4 16.2 6.0 7.3
10.8
10.5
3.0 22.3 5.9 5.7
3.3
4.3 22.4 5.9 6.0
4.6
4.4
Day 1
i Ave.
Glucose Glucose Ave. D.O.
mg/1 ave. mg/1 oH mg/1
12.8 12.3 6.2 9.2
13.6
15.0 12.6 6.1 9.0
14.3
13.2
Day 3
5.4 20.2 6.1 6.8
5.2
6.8 19.4 5.9 6.8
7.7
7.6
it
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Figure 2. Effect of the inhibitor on the rate of glucose respirati
ion
a»
E
O
u
O
Control •
TCMP O
Days of Incubation
-------
Assays on TCMP treated samples consistently gave lower glucose
values than the control samples. Bottles which were assayed
*j immediately after preparation demonstrated this same pattern and this
-------
Fourteen identical biiodate standards were also analyzed using
the starch end point in the Hinkler determination. The average
difference in the titrant required for inhibited and uninhibited bottles
was 0.03 ml, which indicated that ~CMP did not affect the starch end
point determination.
Potomac River Study
With the completion of the preliminary experimentation using
synthetic samples, the use of TCMP in the determination of nitrogenous
oxygen demand was tested using environmental samples. Potomac River
samples were assayed for NOD during the summer of 1977. Nitrogen analyses
were limited to TKN. The river historically3 had a pattern of rapid
biological activity and long term incubation was expected to yield
essentially complete nitrification. The potential MOD was calculated
from the TKN originally in the sample as: (TKN) x 4.57 = potential NOD.
This compared favorably with the NOD measured using the nitrification
inhibitor with an average difference of 0.9 mg/1. The results are
compiled in Table 5. It should he emphasized that the potential MOO
estimate from the TKN may not occur. However, the coefficient of
linear correlation (r=0.88) suggested that after 20 days of incubation
nitrification was generally complete and that the method utilizing
TCMP gave reasonable NOD results.
Lehigh River Study
The inhibitor TCMP was also employed in an intensive nitrification
study undertaken on the Lehigh River during fall 1977. The study
included the determination of nitrogen states and dissolved oxygen
depletion of unaltered and inhibited samples at several times during
a long term incubation interval. The data are presented in Figure 3
and Tables 5 and 7 and reflect the difrerent sample types and
oreoarations involved:
-------
Table 5. Comparison of the potential NOD and the actual MOD
measured using TCMP (mg/1)
Potomac River Samoles
L*
U
n>
ILJ
f »
If
fm
L«
»•»
i*
1*
'•-
l«
Un
I •*
t*
1"*
C.
I'*
L,
r*
E
L
r*
u
r
i.
L*
C
c
NOD2Q
(TCMP)
2.2
2.3
4.4
5.2
n.o
n.i
4.0
3.6
3.0
2.6
1.4
1.5
2.6
5.3
5.6
6.8
5.5
3.8
2.4
3.6
LA
1.4
7.3
4.8
5.0
Potential
NOD
(4.57)(TKN)
3.4
3.2
3.8
9.4
11.4
10.1
6.2
4.9
3.9
2.8
2.1
1.7
2.7
4.5
5.5
5.9
4.1
3.3
2.8
2.3
2.00
1.5
6.7
5.8
5.9
NOD?0
(TCMP)
3.3
4.4
4.0
3.8
1.8
3.0
2.7
4.0
4.4 .
3.4
4.1
3.5
6.6
6.8
4.2
1.6
1.2
7.1
4.7
5.1
4.9
4.3
5.2
4.9
5.6
Potential
MOD
(4.57)(TKN)
4.9
4.0
3.4
3.1
2.5
2.2
2.2
4.1
6.3
5.3
5.0
5.1
5.8
6.1
3.7
2.2
1 .8
8.0
5.4
5.8
5.0
4.4
5.6
5.5
3.7
Potential
NOD2Q NOD
(TCMP) (4.57)(TKNJ
2.0 2.1
2.2 1.9
4.5 4.8
8.9 6.5
11.0 8.4
—
3.6 3.3
3.0 2.1
2.5 1.3
3.0 1.8
linear correlation
coefficient = 0.88
with n = 58
-------
1. unaltered samples - river stations
2. seeded samples - industrial effluents
3. seeded and diluted samples - sewage treatment plants
and industrial effluents
The average difference between the two NOD methods for river samples,
with an oxygen demand of less than 10 mg/1, was 0.4 mg/1 (n=128 and
s.d.=0.349). The seeded effluent samples had an average MOD difference
of 0.5 mg/1 (n=42 and s.d.=0.463). The increased error and variability
of the results reflects the added measurements of the seeded blank
made for both nitrogen conversions and oxygen depletion determinations.
The average NOD difference for seeded and diluted effluent samples was
5.7 mg/1 (n=36 and s.d.= 7.83), which represented an average error
of 10% for the NOD. The NOD error for diluted samples was amplified
by the dilution factors of 15 and 30 necessary for the BOD analysis.
A paired t-test of the nitrogenous oxygen demand over the combined
206 paired data sets established at the 95S confidence level (t=.75)
that there was no significant difference 1n the results of the two
NOD methods.
Station 031, an industrial effluent sample from a steel plant
slag leachate was unique in that the outfall had an average 30020.3]
of 753 mg/1 and an average initial TKN of 359 mg/1 on the three days
it was sampled. However, nitrate and nitrite were not formed after 31
days of incubation. The sample was analyzed for phenol and cyanide
and was found to contain 35.9 mg/1 total phenol and 50 mg/1 cyanide.
This suggested that the outfall was toxic to nitrifying bacteria,
but not to the heterotropnic species present.
-------
fcj
Figure 3. NOD of Lehigh River samples calculated
from nitrogen analyses and measured using
the inhibitor, TCMP
(U
*-o
_o
» 3
• ~O
U
f'1
IL4
i*
40
30
20
6-
5
4-
3-
2-
'•^.THEORETICAL
RELATION
x fwo sampl«s with
identical results
8
Observed NOD (inhibitor) mg/l
10 20 30 40
in
-------
Table 6. NOD of seeded Lehlgh industrial effluent samples determined by analysis of nitrogen
conversions and by measurement with TCMP
Days of N02~Nf N02~Nj
Ddte-Sfa. Incubation
10/05 005
111 as t furnace
006
Blast furnace
cool ing
007
Illast furnace
cool ing
008
Illast furnace
cool ing
010
Meat t real merit
cool Ing
012
Scale pit
014
Saw house
cool ing
10/06 005
IHast furnace
6
12
29
6
12
29
6
12
29
6
12
29
6
12
29
6
12
29
6
12
29
6
12
31
mg/1
.082
.710
0
.182
.100
0
.178
.075
0
.132
0
0
.149
.057
0
.082
.294
0
.113
.143
0
.177
.764
0
mg/1
.048
.048
.048
.061
.061
.061
.045
.045
.045
.045
.045
.045
.050
.050
.050
0
0
0
.041
.041
.041
.054
.054
.054
AN02-N
mg/1
.034
.662
- .048
.121
.039
- .061
.133
.030
- .045
.087
- .045
- .045
.099
.007
- .050
.082
.294
0
.072
.102
- .041
.123
.710
- .054
N03-Nf
ing/1
1.538
1 .690
3.04
1.648
2.040
2.19
1 .762
2.065
2.23
2.068
2.240
2.340
1.661
1.793
1.980
1.598
1.646
1.940
1.607
1.737
1.880
1.613
2.176
2.940
N03-Ni
mg/1
1 .576
1.576
1.576
1.673
1.673
1.673
1.749
1.749
1.749
2.039
2.039
2.039
1.684
.684
.684
.584
.584
.584
1.593
1.593
1.593
1 .656
1.656
1.656
AN03-N
mg/1
- .038
.114
1.464
- .025
.367
.517
.013
.316
.481
.029
.201
.301
- .023
.109
.296
.014
.062
.356
.014
.144
.287
- .043
.520
1.284
BOD
mg/1
2.4
8.0
9.8
2.3
4.3
5.8
2.2
4.4
5.7
2.1
3.8
5.4
1.8
3.6
5.5
1.6
3.6
6.1
1.9
3.3
5.9
3.6
8.2
11.2
3.43
(AN02-N
+AN03-N)
mg/1
- .01372
2.66168
4.85688
.32928
1.39258
1.56408
.50078
1.18678
1 .49548
.39788
.53508
.87808
. 26068
.39788
.84378
.32928
1.22108
1.22108
.29498
.84378
.84378
.27
4.22
4.22
1.14
(AN03-N)
mg/1
- .04332
.12996
1.66896
- .0285
.41838
.58938
.01482
.36024
.54834
.03306
.22914
.34314
- .02622
.12426
.33744
.01596
.07068
.40584
.01596
.16416
.32718
- .05
.59
1.46
NOD*
calc.
mg/1
0
2.8
6.5
.3
1.8
2.2
.5
1.5
2.0
.4
.8
1.2
.2
.5
1.2
.3
1.3
1.6
.3
1.0
1.2
.2
4.8
5.7
NOD
TCMP
mg/1
.7
4.8
5.7
.7
1.6
2.2
.7
1.6
2.0
.3
1.3
1 .8
.9
1 .4
2.3
.5
1.5
2.8
.4
.6
1.7
1.0
4.8
6.1
-------
r -3 c 3 S3 c ? r ~a r-a n r a ' n r 1 n E~a t ? r i r "5 c a c i c a
Table 6. (con't) NOD of seeded Lehigh industrial effluent samples determined by analysis of
nitrogen conversions and by measurement with TCMI1
3.43
(AN02-N 1.14 MOD* NOD
Hays of N02-Nf N02-Ni AN02-N N03-Nf N03-Nj AN03-N BOH MN03-M) (AM03-M) calc. TCMI1
Date-Sta. Incubation mg/1 mg/1 mg/1 mg/1 mg/1 mg/1 mg/1 mg/1 mg/1 mg/1 inrj/1
10/06 006 6 .285 .065 .220 1.715 1.775 - .060 3.2 .55 - .07 .5 1 .0
Blast furnace 12 .045 .065 - .020 2.115 1.775 .340 4.8 1.10 .40 1.5 1.3
cooling 31 0 .065 - .065 2.160 1.775 .385 6.4 1.10 .40 1.5 ?.2
007 6 .224 .044 .180 1.796 1.756 .040 3.6 .75 .05 .8 1.5
Blast furnace 12 0 .044 - .044 2.120 1.756 .364 5.0 1.10 .41 1.5 1.3
cooling 31 0 .044 - .044 2.100 1.756 .344 6.6 1.03 .39 1.4 2.4
008 6 .153 .054 .099 2.067 2.026 .041 3.5 .43 .05 .5 .4
Blast furnace 12 0 .054 - .054 2.190 2.026 .164 5.3 .40 .19 .6 0.6
cooling 31 0 .054 - .054 2.190 2.026 .164 7.5 .40 .19 .6 2.0
010 6 .206 .058 .148 1.724 1.782 - .058 3.4 .30 - .07 .2 .9
Heat treatment 12 0 .058 - .058 1.970 1.782 .188 4.9 .50 .20 .7 0.8
cooling 31 0 .058 - .058 1.970 1.782 .188 7.0 .50 .20 .7 2.0
012 6 .217 0 .217 1.683 1.660 .023 3.1 .80 .03 .8 .8
Scale pit 12 000 1.970 1.660 .310 5.4 1.06 .35 1.4 1.4
31 000 2.000 1.660 .340 8.0 1.17 .40 1.6 2.8
014 6 .172 .047 .125 1.678 1.703 - .030 3.4 .33 - .03 .3 .5
Saw house 12 0 .047 - .047 2.000 1.703 .297 5.1 .86 .34 1.2 1.5
cooling 31 0 .047 - .047 1.980 1.703 .277 7.0 .79 .32 1.1 1.6
*NOD = 3.43 (AN02 + AN03) + 1.14 (AN03)
where A=final - initial
-------
Table 7. NOD of seeded and diluted SIP and industrial effluent samples determined by analysis
of nitrogen conversions and by measurement with TCMP
Ddte-Sta. 1
1 0/04
Al 1 en town
STP
015
Coke works
031
Sldg
ledchate
liethlehem
SIT
10/05
All en town
STP
015
Coke works
031
Sidy
1 fdclld te
Bethlehem
STP
Days of
Incubation
6
12
29
6
12
29
6
12
20
6
12
29
6
12
29
6
12
29
6
12
29
6
12
29
N02-Nf
mg/1
8.4
13.74
0
9.93
55.53
0
0
0
0
12.72
21.33
0
1.395
11.94
0
1.125
26.4
14.835
0
0
0
1.515
17.7
0
N02-Nj
mg/1
.27
.27
.27
.45
.45
.45
.12
.12
.12
.33
.33
.33
.045
.045
.045
.195
.195
.195
0
0
0
.045
.045
.045
ANOo-N
mg/1
8.13
13.47
- .27
9.48
55.08
- .45
- .12
- .12
- .12
12.39
21.0
- .33
1.35
11.895
- .045
.93
26.205
14.64
0
0
0
1.47
17.655
- .04
N03-Nf
mg/1
4.89
7.26
21 .0
2.10
2.67
60.6
0
0
0
2.49
4.77
26.1
3.015
4.05
15.99
.825
2.19
13.665
0
0
0
.330
2.79
20.85
N03-Nj
mg/1
4.44
4.44
4.44
1.41
1.41
1.41
0
0
0
0
0
0
2.355
2.355
2.355
.825
.825
.825
0
0
0
.330
.330
.330
AN03-N BOD
mg/1
.45
2.82
16.56
.69
1.26
59.19
0
0
0
2.49
4.77
26.1
.66
+1.695
13.635
0
1.365
12.84
0
0
0
0
2.46
20.52
mg/1
54
93
120
57
204
264
241.5
417.0
1203.0
99
120
189
21
64.5
90
4.5
64.5
103.5
123
244.5
576
36
105
129
3.43
(AN02-N
+AN03-N)
mg/1
29.4294
55.8747
55.8747
34.8831
193.2462
201.4782
- .4116
- .4116
- .4116
51.0384
88.3911
88.3911
6.8943
46.6137
46.6137
3.1899
94.5651
94.2564
0
0
0
5.0421
68.9945
70.2464
1.14
(AN03-N)
mg/1
.513
3.2148
18.8784
.7866
1.4364
67.4766
0
0
0
2.8386
5.4378
29.754
.7524
1.9323
15.5439
0
1.5561
14.6376
0
0
0
0
2.8044
23.3928
NOD*
calc.
mg/1
29.9
59.1
74.8
35.7
194.7
269.0
0
0
0
53.9
93.8
118.1
7.6
48.5
62
3.2
96.1
108.9
0
0
0
5.0
71.8
93.6
NOD
TCMP
mg/1
33
63
75
46.5
189
249
0
0
0
60
102
115
10.5
48
52
3.0
64.5
103.5
0
0
0
19.5
78
81.0
-------
Days of
Date-Sta. Incubation
10/06 6
Allentown 12
STP 31
015
Coke works
031
Slag
leachate
Bethlehem
STP
6
12
31
6
12
31
6
12
31
lf d/^d ifi
analysis of nitrogen conversions and
102-Nf
mg/1
7.77
3.375
0
N02-Nj
mg/1
.03
.03
.03
AN02-N
mg/1
7.74
3.345
- .03
N03-Nf
mg/1
3.405
12.195
15.57
N03-Nj
mg/1
2.52
2.52
2.52
7.515
24.135
0
0
0
0
7.8
10.335
0
.285
.285
.285
0
0
0
0
0
0
7.23
23.85
- .285
0
0
0
7.8
10.335
0
1.185
2.685
27.285
0
0
0
.57
9.585
19.92
.810
.810
.810
0
0
0
.135
.135
.135
" i E~ "2 £"1 £2 1
uStriai "ffmonc samples a
by measurement with TfMP
AN03-N
mg/1
.885
9.675
11.05
.375
1.875
26.475
0
0
0
BOD
mg/1
47.0
79.5
106.5
25.5
88.5
121.5
121.5
256.5
510.0
3.43
(ANO?-N
+AN03-N)
mg/1
29.58
44.66
44.66
26.09
88.24
90.81
0
0
0
etlrmlned 3!>y
.435
9.45
19.79
64.5
117.0
159.0
3.43
(ANO?-N
+AN03-N)
mg/1
29.58
44.66
44.66
1.14
(ANOa-N)
mg/1
1.01
11.03
14.88
MOD*
calc .
IIHJ/I
30. G
05. 7
b9.5
NOD
TCMP
mg/1
32.0
55.5
60 . 0
28.25
67.86
67.88
.4
2.14
30.18
0
0
0
.50
10.77
22.56
26.5
90.4
121.0
0
0
0
28.3
78.6
90.4
25.5
81.8
115.5
0
0
0
39.0
81 .0
99.0
*NOf) = 3.43 (AN02 + AN03) 4- 1.14 (AN03)
where A= final - initial
*LA = laboratory accident
-------
Conclusions
The results of this study on synthetic, river, sewage treatment
plant and industrial effluent samples suggested that:
1) TCMP was an effective inhibitor to nitrification.
The inhibitor stopped the nitrification of ammonia by
inhibiting the formation of nitrite.
2) TCMP did not inhibit the conversion of nitrite to nitrate.
3) TCMP did not inhibit the respiration of glucose.
4) TCMP did not significantly contribute to the C30D even
after 31 days of incubation at 2Q°C.
5) The determination of MOD using the difference in oxygen
depletion in inhibited and uninhibited 603 bottles was auick
and easy. This method did not involve the expensive equipment,
nor time associated with the chemical analysis of nitrogen
states to determine the NOD.
6) The inhibitor did not interfere •vith the determination of
oxygen by the azide modified Winkler or electrode methods.
7) The inhibitor method yielded reliably accurate MOD determinations
References
1. Standard Methods for the Examination of 'later and 'Jastawatsr,
14th ed., APHA, 1975.
2. Srinath, E.G., Raymond, L.C., Loehr, M. and ?ra:
-------
** 4. Stensel, H.D., McDowell, C.S. and Ritter, E.D., "An Automated
" Biological "litrifi cation Toxicity Test," J.H.a.C.F. . 48, 10,
p. 2348-2350, (October 1976).
5. Breed, R.S., Murrv E.G.D., and Hitchens, A.P., Beraev's Manual of
Ul "—
Determinative Bacteriology, 6th ed., The Williams and Wilkens.
L« 6. Painter, H.A., "Microbial Transformations of Inorganic Nitrogen,"
n» Prog. Hat. Tech. vol. 8, Mos.4/5, pp. 3-29 Pergamcn Press, 1977.
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CE 755A6 prepared for the Office of Water Research and Techno!ocy.
Lm
3. Wezernak, C.T. and Gannon, J.J., "Evaluation of Nitrification in
r
^ Streams," J_._ Sanitary Engineering Div., Proc. of American Soc.
r» of Civil Engineers, p. 883-395, (Oct. 1968).
^ 9. Wezernak, C.T. and Gannon, J.J., "Oxygen-Nitrogen Relationships in
'* Autotrophic Nitrification," Applied Microbiology, 15, p. 1211-1215,
I.
(Sept. 1967).
, 10. Montomgery, H.A.C. and Borne, B.J., "The Inhibition of Nitrification
in the BOO test," J. Proc. Inst. Sew. Purif., p. 357-368, 1966.
L 11. Young, J.C., "Chemical Methods for Nitrification Control,"
p» 24th Industrial Waste Conference, Part J_I_ Purdue University,
U p. 1090-1102, 1967.
^" 12. Van Kessel, J.F., "Factors Affecting the Denitrification Rate
in Two Water-Sediment Systems," Water Research, 11, p. 259-267,
r
u (July 1976).
_ 13. Goring, C.A., "Control of Nitrification by 2-Chloro-6 (Trichloromethyl)
I- Pyridine," Soil Science, 93, p. 211-218, (Jan. 1962).
r
u»
r
L
-------
14. Mullison, W.R. and Norn's, M.G., "A Review of Toxicological , Residual
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and Food Chemistry, 12, p. 207-209, (May-June 1964).
16. Young, J.C., "Chemical Methods for Nitrification Control," J.VLP.C.F.,
45, 4, p. 637-646, (April 1973).
17. Laskowski, O.A., Q'Melia, E.C., Griffith, J.D. at al, "Effect of
2-Chloro-6-(Trichloromethyl) Pyridine and irs Hydrolysis Product
6-Chloro-?icolinic Acid on Soil Microorganisms," J. o_f Env. Qual ity, 4:
p. 412-417, (July-Sept. 1975).
18. Chemistry Laboratory Manual-Bottom Sediments, compiled by Great
Lakes Region Committee on Analytical Methods, E.P.A. Dec. 1969.
19. Methods For Chemical Analysis of Hater and Hastes. E.D.A., 1974.
20. Strickland, J.D.H., and Parsons, T.R., A Practical Handbook gf_
Seawater Analysis, Queen's Printer, Ottawa, 1968, p. 173-174.
21. Bundy, L.G., "Control of Nitrogen Transformations," Ph.D.
Dissertation, Iowa State University, 1973.
22. Quastel, J.H., and Scholefield, "Biochemistry of Nitrification in
Soil," Bact. Rev., 15, 1951, p. 1-53, 1951.
23. Tandon, S.P., "Effect of Organic Substances on Nitrita Formation
by 'litrosomonas," Symp. 3iol . Hung., 11 , p. 233-223, 1 972 .
------- |