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Journal of Honors Lab Investigations 1(1): 19 - 21

Ammonia, Nitrite and Nitrate Nitrogen in the effluent of Columbia Waste Water.
J an Weaver

Abstract: The EPA has identified nitrogen enrichment as a leading cause of water quality degradation. One of the
important contributors to nitrogen enrichment is incompletely treated sewage from municipal waste water treatment
plants. I tested the effluent from the Columbia Waste Water Treatment Plant (WWTP) to see if it would meet the
EPAs Ambient Water Quality Criteria Recommendations for ecoregion 11 (central and eastern forested uplands)
of 0.3 ppm nitrogen. I also wanted to see if the relative proportions of reactive nitrogen species - ammonia, nitrite
and nitrate, were consistent with their place in the nitrogen cycle; in other words, for nitrogen coming directly from
organic sources, I expected this order of relative concentrations: ammonia >nitrite >nitrate. I used tests for the
three species that rely on comparing color development to standard charts. The combined values for all three kinds
of nitrogen were 17. 1 ppm, nearly 60 times the recommended level for ambient water quality. Clearly, it is a good
thing that the effluent is eventually piped to the Missouri River where it can be diluted. The order of concentration
was ammonia - 13.6 ppm >nitrate - 2.5 ppm >nitrite - 1.0 ppm, so nitrate was higher than nitrite, which was
unexpected. The higher levels of nitrate may be due to the fact that it was easier for the aerobic bacteria which
convert nitrite to nitrate to find suitable conditions, than it is for the anaerobic bacteria which convert nitrate to
atmospheric nitrogen.

Introduction
In the National Water Quality Inventory report to Congress in 1996, nitrogen was identified as one of the
leading causes of degradation of water quality in U.S. lakes, rivers and estuaries. Nitrogen has been implicated in
the development of a dead zone in the Gulf of Mexico and in the population explosion of the diatom Pfiesteria
which was identified as a major cause of fish-kills off the North Carolina coast in the 1990s (Cantilli 1998).
Smil (1997) points out that human activities have added 150 metric tons of reactive nitrogen a year to the
biosphere, almost the equivalent of all the nitrogen fixed by bacterial activity. This reactive nitrogen, N that has
been oxidized to the NOx form or reduced to NH
3
, can play a powerful role in shaping ecosystems. The sources of
this reactive nitrogen include combustion of fossil fuels, runoff of fertilizers from fields and sewage from human
and animal sources. It is the addition of reactive nitrogen from human sewage that I deal with in this paper.
The Columbia Waste Water Treatment Plant (WWTP) uses a complete-mix activated sludge process which
maintains naturally occurring living microorganisms at high population levels. These microorganisms consume
dissolved suspended organic material as a source of food. In the process, they promote clumping of biological
masses that settle to the bottom of the treatment cell as sludge. The sludge is then disposed of by injection into farm
fields. The effluent, the liquid portion of the sewage from which 90-95% of the pollutants have been removed, is
pumped to a series of constructed wetlands where planted cattails use up some of the remaining pollutants
(mainly reactive nitrogen). This polished effluent is piped to Missouri Department of Conservation wetlands at
Eagle Bluffs, and from there to the Missouri River. (City of Columbia, 2001)
The majority of the reactive nitrogen in waste water will be ammonia (NH
3
), but through nitrification
(actually chemical oxidation) by Nitrosomonas and then Nitrobacter, the ammonia is oxidized to nitrite (NO
2
-) and
then nitrate (NO
3
). In the presence of denitrifying bacteria, found only in anaerobic soils or sediments, the nitrates
can be reduced back to atmospheric N
2
, which is not reactive and does not contribute to water quality degradation.
I propose to test the effluent from the WWTP for ammonia, nitrite and nitrate-nitrogen to see how much
total nitrogen is present in the sewage after treatment. I will compare this to recommended levels of nitrogen of 0.3
mg/l (=ppm) in the EPAs Ambient Water Quality Criteria Recommendations (Grubbs 2000). I will also compare
the relative amounts of the different species of nitrogen. My expectation is that ammonia-nitrogen, which is the
form found in organic matter like feces and urine, will predominate. The next highest concentration of nitrogen
should be in the nitrite form, since this is the next step in the nitrogen cycle. The nitrate-nitrogen should be in the
lowest concentrations.

Methods
I made arrangements with the Columbia Waste Water Treatment Plant to collect a sample of the plant effluent
as it left the plant property to go to the constructed wetlands. I collected approximately 200 ml on October 12,
2001 at 4 pm. There had been a heavy storm in the Columbia area and storm water had infiltrated the waste water
system, significantly increasing the flow through the plant. In situations like this, the excess water is diverted to a
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separate 1 treatment tank. After allowing solids to settle and skimming the surface, this waste water is combined
with waste water that has received secondary treatment and released from the plant.
To test ammonia-nitrogen I used a test kit from Tetra Test designed to measure NH
3
/NH
4
+in fish tanks.
After rinsing the 10 ml test tube with the water being tested, I used a pipette to transfer 5 ml of sample water to the
test tube. Then I added 14 drops of reagent #1 to the tube (making sure to hold the reagent bottle upside down),
sealed it, and shook it gently. Finally, following the same procedure I added 7 drops from reagent bottle #2, shook
the solution, and then 7 drops from reagent bottle #3, shaking again to mix. After allowing 20 minutes for color to
develop, I matched the color in the tube to the color on the chart to estimate ammonia content.
If the color development was too intense to read off the chart, I diluted the sample of waste water by a
standard percentage, performed the test again, and corrected for the dilution factor.
To test nitrite and nitrate-nitrogen, I used test strips from Carolina Biological Supply. I placed
approximately 50 ml of the effluent in a small beaker and dipped a test strip into the water, making sure it was
wetted by the effluent, for 2 seconds. I removed the strip, shook off the water and waited 1 minute before reading
the strip. To read the strip, I compared the color development to a color chart which reported nitrite and nitrate-
nitrogen in ppm. If the color development was deeper than the scale allowed, or if it fell between colors, I diluted
the sample and redid the test, multiplying the estimated ppm by the dilution factor to get a corrected value. If the
color fell between two colors on the chart, I interpolated the value.
All the test kits measured nitrogen in mg/l, this is equivalent to ppm or parts per million and all results were
reported as ppm.

Results
The ammonia-nitrogen from WWTP was quite high and I had to dilute the sample by a factor of 4 to get a
readable result. With a 4x dilution, the reading was slightly darker than the color for 3 mg/l (or 3 ppm).
Interpolating on the color chart, I estimated the value was about 3.5 ppm. Multiplied by the dilution factor of 4,
that gave a value of 14 ppm ammonia-nitrogen for the WWTP sample (Figure 1.).
For nitrite-nitrogen, the color development was very close to 1.0 ppm. To double check the estimate, I
diluted the sample to 1/5 and got a reading of 0.2 ppm. This reading, multiplied by the dilution factor of 5, gives
the same result as the undiluted effluent, 1.0 ppm.
For nitrate-nitrogen, the color development was close to 3.0 ppm. However, when I diluted the sample to
1/5 and repeated the test, I got a reading of 0.5 ppm, which when multiplied by the dilution factor, gives a result of
2.5 ppm.

Ammonia Nitrite Nitrate
0
5
10
15
20
Nitrogen Species







P
a
r
t
s

P
e
r

M
i
l
l
i
o
n

i
n

W
W
T
P

W
a
t
e
r

S
a
m
p
l
e
s

Figure 1. PPM of Ammonia, Nitrite and Nitrate in water from the Columbia WWTP.

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Discussion
When the three reactive forms of nitrogen are added together, ammonia-14 ppm +nitrite-1.0 ppm +nitrate-
2.5 ppm, I get a total nitrogen of 17.5 ppm. This is much higher than the EPAs recommended ambient water
quality criteria of 0.3 ppm, in fact nearly 60 times higher. Clearly the effluent from Columbia's WWTP could not
be directly released into the environment without being diluted by 60 times as much water. Since the plant treats an
average of 16 million gallons of sewage a day, that means the effluent would have to be discharged into a river with
a flow of 960 million gallons a day. The local creek, where effluent was discharged up until the construction of the
wetlands, is clearly incapable of handling that load.
I had expected the reactive nitrogen to be in the order ammonia >nitrite >nitrate, however, there was more
nitrate in the sample than nitrite. Since nitrogen has to go through the nitrite form to become nitrate, it suggests that
the transformation from nitrite to nitrate happens more rapidly than the transformation of nitrate to atmospheric
nitrogen. This result makes sense since the oxidation of nitrite to nitrate requires less restrictive conditions than the
anaerobic soils required by denitrifying bacteria.
It would be interesting to test water samples from the other parts of the wastewater treatment
system, the constructed wetlands, the Eagle Bluffs wetlands and the Missouri River, to see if the total
nitrogen concentration goes down and if the relative amounts of the different species of reactive nitrogen
change*.

Citations
City of Columbia. Columbia Regional Waste Water Treatment Plant Homepage. Anonymous. update: Feb
21, 2001. access:
Oct 17, 2001. http://www.ci.columbia.mo.us/dept/pubw/sewer_utility/Wwtppg_4.htm
EPA, Office of Water. National Strategy for the Development of Regional Nutrient Criteria. Cantelli, B.
upate: Oct 8, 1998. access: Oct 17, 2001. http://www.epa.gov/OST/standards/nutsi.html
EPA, Office of Water. Grubbs, G. H. 2001. Ambient Water Quality Criteria Recommendations for
Ecoregion 11:
Central and Eastern Forested Uplands. Grubbs, G. H. update Dec 2000. access: Oct 17, 2001.
http://www.epa.gov/waterscience/criteria/nutrient/ecoregions/rivers/rivers_11.pdf
Smil, V. 1997. Cycles of Life: Civilization and the Biosphere. Scientific American Library.