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Chapter 8

Nitrogen
Assoc. Prof. Nguyen Phuoc Dan

General consideration
The compound of nitrogen are of great importance in water resource, in the atmosphere and in the life
processes of all plants and animals.
The chemistry of nitrogen is complex because of the several oxidation states that nitrogen can assume and
the fact that changes in oxidation state can be brought about by living organism.
Nitrogen can exist in 07 oxidation states and essentially all are of environment interest:
-3 0 +1 +2 +3 +4 +5
NH3 – N2 – N2O – NO – N2O3 – NO2 – N2O5

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General consideration
Nitrate serves to fertilize plant life and is converted to
protein (organic nitrogen)
NO3- + CO2 + green plant + sunlight → protein

Atmospheric nitrogen enters the nitrogen cycle is

though conversion to protein by nitrogen-fixing
bacteria
N2 + nitrogen-fixing bacteria → protein

Ammonia and ammonium compounds are applied to

soil to supply plants with ammonia for further
production of proteins.
NH3 + CO2 + green plant + sunlight → protein
The urine contains urea resulting from the metabolic
Nitrogen cycle
breakdown of proteins. Urea is hydrolyzed rather
rapidly by the enzyme urease to ammonium carbonate.
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General consideration
The feces of animal contain significant amounts of unassimilated
protein matter (organic nitrogen). Its protein matter are
converted to ammonia by the action of heterotrophic bacteria,
under aerobic or anaerobic conditions
Protein (organic-N) + bacteria → NH3
The ammonia released by bacterial action on urea and proteins
may be used by plants directly to produce plant protein.
if it is released in excess of plant requirement, the excess is
oxidized by the autotrophic nitrifying bacteria.
One group (nitrosomonas) covert ammonia under aerobic
conditions to nitrite and produce energy from the oxidation:
nitrosomonas
2NH3 + 3O2 2NO2- + 2H+ + 2H2O

The nitrite is oxidized by another group of nitrifying bacteria

(nitrobacter) to nitrate:
nitrobacter 4
2NO2- + O2 2NO3-

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General consideration
The nitrate formed may serve as fertilizer for plants.
Nitrate produced in excess of the needs of plant life is carried away in water percolating through the soil because
the soil does not have the ability to hold nitrate.
Under anaerobic conditions nitrate and nitrite are both reduced by denitrifying bacteria (denitrification) to N2 gas.
Some denitrifying bacteria produce N2O that releaves from soil or water to enter the atmosphere.
Denitrification causes serious loss of fertilizing matter in soils when anaerobic conditions develop.
Nitrogen content in natural water always higher than domestic or industrial wastewater.
In domestic wastewater, the nitrogen exists inorganic (65%) and organic (35%) type.
The nitrate can be suitable for plants as nutrient. If the plants are not absorb NO3- and NH4+ completely so they are
dissolved into rain water and then discharged to the river by surface run off and to groundwater by infiltration.
In wastewater treatment, the nitrate removal is required to prevent from growth of algae and aquatic plants.

Atmospheric Concerns with Nitrogen Species

The three major environmental problems associated with
nitrogen species in the atmosphere: (i) photochemical
smog, (ii) global warming, and (iii)stratospheric ozone
depletion
Photochemical smog is a smog type produced when UV
light from the sun reacts with NOx in the atmosphere, and
leads to produce high O3 concentrations and organic
chemicals → eye irritation, reduced air visibility, crop
damage.
It is visible as a brown haze, and is most prominent during
the morning and afternoon, especially in densely
populated, warm cities.
The automobile in the dense urban area is a primary
producer of two of the ingredients of photochemical
smog, partially oxidized organic matter and NOx.

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Atmospheric Concerns with Nitrogen Species

Atmospheric Concerns with Nitrogen Species

Besides CO2, the gaseous nitrogen oxides also exhibit a greenhouse effect.
Stratospheric ozone plays a key role in protecting life on earth trom the harmful effects of excessive ultraviolet
radiation
N2O and NO2 are both converted to NO in the atmosphere, and NO reaching the stratospheres reacts with
ozone to result in its depletion.

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Aquatic Concerns with Nitrogen Species

Most of the nitrogen is originally present in the form of organic
(protein) nitrogen and ammonia.
The organic-N is converted to NH4+, and later on, if aerobic
conditions are present, oxidation of NH4+ to NO2- and NO3-.
Waters that contained mostly organic and ammonia nitrogen
were considered as the polluted water.
Drinking waters with high nitrate content often caused
methemoglobinemia in infants,
The QCVN 01-1 2018/BYT has set a maximum contaminant level
requiring that the NO3--N ≤ 2 mg/L and the NO2--N ≤ 0.05 mg/L in
public water supplies

Aquatic Concerns with Nitrogen Species

Nitrite can interact with amines chemically (when chlorinating for disinfection) or enzymatically to form nitrosamines,
which are strong carcinogens.
The formation of N-nitrosodimethylamine (NDMA) by these processes during wastewater treatment and has become
an issue recently in wastewater reuse projects and contaminated groundwater supplies.
Ammonia reacts with chlorine to form chloramines, which are sloweracting disinfectants than free chlorine
→ Ammonia is added to drinking water supplies when a disinfection residual in water mains is desired as chloramines
do not decompose as rapidly as chlorine

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Ammonia
+
Ammonia in water exists under 02 types: NH3 and NH4 depend on pH of environment.
pH is low: presented in cation NH4+ .
pH is high: presented in gas.
+
The NH3/NH4 ratio depends on temperature

pH
Temperature
6,0 6,5 7,0 7,5 8,0 8,5 9,0 9,5
1 0,009 0,028 0,09 0,28 0,89 2,77 8,25 22,1
10 0,019 0,059 0,19 0,59 1,83 5,56 15,7 37,1
20 0,040 0,125 0,40 1,24 3,82 11,2 28,4 55,7
30 0,081 0,254 0,80 2,48 7,46 20,3 44,6 71,8

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Ammonia
Ammonia presented in surface water or groundwater, is derived from the organic matter decomposition by
anaerobic microorganisms.
Ammonia that is found in raw water used for water supply in case of it is contaminated by the waste-stream.
In water network, ammomia use the chloramine compound which it increases the bactericidal time of residual
chlorine when transports it in pipeline.
In anaerobic conditions, ammonia can be formed from nitrate base on the denitrification of bacteria.
The NH3 content in domestic wastewater, industrial food wastewater and anothers can be very high. NH3 content in
groudwater is stable.
The NH3 content in surface water is change, depend on hydrological regime of water source. In the winter, the NH3
content is always higher than summer because oxidation process to nitrate which takes place at the low
temperature.

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Nitrite - nitrate
Nitrite is unstable compound. The denifitrication process in anaerobic condition transfer TNOx to nitrogen gas.
Beside, nitrite is derived from wastewater of the electrochemical industrial process.
In equilibrium status, nitrite concentration in water very low, it in surface water always lower 0,02 mg/l.
Nitrate is inorganic compound of nitrogen which it has the highest valence. It’s sustainable in water environment
and has the major source follow as:
The nitrogen compounds oxidation process in soil.
The washing surface process or runoff from fields to rivers and lakes carry a large amount of fertilizer that plants
could not absorb.
The domestic or chemical, food industrial wastewater, etc. contain a large amount of nitrogen compounds.…
In surface water, nitrate often trace type but in groundwater, nitrate content sometimes very high.

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Nitrite - nitrate
Nitrite is intermediate production of oxidizing ammonia or nitrogen ammonia process in aerobic
conditions base on Nitrosomonas bacteria. After that, the nitrite is oxidized to nitrate base on
Nitrobacter bacteria. + − −
NH 4 ⎯⎯
K n
→ NO2 ⎯⎯
K
m
→ NO3

With: Kn, Km is nitrification rate constant

The equations of nitrite and nitrate reactions:
NH 4 + + 1,5O2 ⎯⎯⎯⎯⎯
Nitrosomonas
→ NO2 − + H 2O + 2 H +
NO2 − + 0,5O2 ⎯⎯⎯⎯
Nitrobacter
→ NO3−
NH 4 + + 2, 0O2 → NO3− + H 2O + 2 H +

The nitrification need 4,57g O2 for 1g N-NH4. Nitrosomonas and Nitrobacter is anaerobic bacteria which
they are suitable for temperature from 20-30oC.
In no residual oxygen conditions but it’s has organic carbon, some bacteria will eliminate nitrate or nitrite
to create oxygen for oxidizing organic process.
NO3 − + 4 H + + 5Chöõu cô → 5CO2 + 2 N 2 + 2 H 2O 14

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Organic nitrogen - Total nitrogen

All nitrogen present in organic compounds may be considered organic nitrogen. it includes amino acid, amines,
amides, imides, nitro derivatives and some other compounds.
Most of the organic nitrogen that occurs in the wastewater is in the form of proteins or their degradation
products: polypeptides and amino acids.
Kjeldahl nitrogen is organic nitrogen and ammonia nitrogen in the sample was determined after transfer them to
inorganic types.

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Application of nitrogen data

At the present time, data concerning the nitrogen compounds that exist in drinking water supplies
are used largely in connection with disinfection practice.
The amount of ammonia nitrogen present in a water determines to a great extent the chlorine
needed to obtain free chlorine residuals in breakpoint chlorination and determines to some extent
the ratio of monochloramine to dichchloramine when combined chlorine residuals are involved.
By controlling nitrification when not required, aerobic-treatment costs can be kept at a minimum.
When nitrogen oxidation is required, the undersatnding nitrogen changes through nitrification and
denitrification is essential for process control.
When wastewater sludges are sold for their fertilizing value, the nitrogen content of the sludges is a
major factor in determining their value for such purpose.
The productivity of natural waters in terms of algal growths is related to the fertilizing matter that
gains entrance to them. Nitrogen in its various forms is a major consideration.

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Methods of analysis
Nitrogen exists in 04 forms that are of interest in water sources.
Nitrate → N-NO3-
Nitrite → N-NO2-
Ammonia → N-NH3
Organic nitrogen

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Ammonia analysis
Nessler method
According to a classical colorimetric method with sample was filtered and decanted by the
appropriate pretreatment methods used zinc sulfate and sodium hydroxide
This method can measure the amount of ammonia by treating with Nessler reagent that is
highly alkaline solution of potassium iodine, mercury. This substances combine with
ammonia in alkaline environment to create colloidal dispersion with golden-brown color,
this color intensity express directly in original ammonia content. This reaction can be
described by the equation:

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Ammonia
Nessler method (cont.)
analysis
In order to avoid obstacles by impurities in the sample can react with a reagent,
ammonia should be separated by sample distillation method. The colorimetric will
be performed on distilled solution.
The major obstacles of method:
Ca2+ content excess 250 mg/l, ammonia is lower than actual. To avoid this need to adjust
pH before distilled sample, phosphate buffer solution is used to precipitate calcium as
calcium phosphate.
Some acyclic amines, cyclic, organic chloramine, acetone, aldehydes, alcohol and other
organic compounds can cause many obstacles. These substances can create to yellow or
green color or becomes cloudy when adding Nessler reagent into distilled solution.
Sulfur compounds create precipitation with Nessler reagent, can remove sulfur by
carbonate lead.
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Ammonia
Phenate method analysis
It involves the addition of an alkaline phenol solution together with hypochlorite and a
manganous salt.
The manganese catalyzes the reaction involving phenol, hypochlorite and ammonia to
produce indophenol which has an intense blue color.
This method has obstacles to similar as Nessler method.

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Ammonia
Distillation method analysis
The phenate method is subject to serious error from extranous color and turbidity.
This method is used to separate the ammonia from interfering substances and
measurement of ammonia nitrogen can then be made in a number of ways,
including the phenate method.
At pH levels above 8, the equilibrium is displaced far enough to the right so that
ammonia is liberated as a gas along with the steam produced when a sample is
boiled

NH 4 + ⎯⎯ → NH 3  + H +
A borate buffer is added to maintain the pH in a range of 9,5 in order to displace the
equilibrium far to the right. Higher pH levels aren’t recommended because of the
danger of some ammonia being released from organic sources at the temperature of
boiling water. 21

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Ammonia analysis
Distillation method
Experience has shown that essentially all the free ammonia will be expelled from
solutions whose pH is maintained at 9,5 by the time 200ml of water has been
distilled when samples of 500 – 1000ml are used.
The distillate is collected by passage of the vapor through a condenser and then
into an acid solution contained in a flask.
The acid solution converts the free ammonia in the vapor to ammonium ion,
which can’t volatilize from the condensate solution. It used depends upon the
analysis that follows.
For water samples that contain small amounts of ammonia nitrogen, the usual
procedure is to measure the amount of nitrogen in the distillate by the phenate
method.
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Ammonia analysis
Distillation method
Many analysts find it convenient to distill an amount that exceeds 200ml to avoid close attention at the end of the
distillation. The calculation can be made, provided that the volume of distillate is known:

VD 1000
mg / l N − NH 3 = N
VDN s
With:

VD The volume of distillate (mL)

VDN The volume of distillate actually analyzed (mL)
N The N-NH3 content found in the analyzed portion of the distillate (mg)

s The volume sample used for distillation (mL)

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Ammonia analysis
Volumetric analysis
When samples contain more than 2mg/l of ammonia nitrogen, as is the case with domestic and many industrial
wastes, the concentration can be determined by titration with a standard solution of sulfuric acid after distillation
and absorbtion of ammonia in boric acid as previously described.
Boric acid (excellent buffer) combines with ammonia in the distillate to form ammonium and borate ion:

→ NH 4 + + H 2 BO3−
NH 3 + H 3 BO3 ⎯⎯
This causes the pH to increase somewhat as was dicussed under buffer action, but the pH is held in a favorable
range for absorbtion of ammonia by the use of an excess of boric acid.
The ammonia may then be measured by back titration with a strong acid such as sulfuric acid.

H 2 BO3− + H + ⎯⎯
→ H 3 BO3

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Ammonia analysis
Volumetric analysis
When the pH of the boric acid solution has been decreased to its original value, an amount of strong acid
equivalent to the ammonia has been added.
The titration is most easily conducted by potentionmetric methods which eliminates the need for internal
indicators.
Many water chemists prefer to use a N/14 or 0.0714N, solution of sulfuric acid for the measurement of
ammonia nitrogen.
Since each milliliter of 0.0714N acid is equivalent to 1mg of nitrogen, its use eliminates the need for the 0.28
factor required when 0.02N acid is used

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Nitrite analysis
Nitrite nitrogen seldom appears in concentrations > 1mg/l, even in waste-treatment-plant effluents. Its
concentration in surface waters and groundwaters is normally much below 0,1 mg/l.
Nitrite analysis: The colormetric method and ion chromatography.
The colormetric method: a modification of the Griess-Ilosvay diazotization method is used. This employs
the use of 02 organic reagents: sulfanilamide and N-(1 napththyl)-ethylenediamine dihydrochloride.

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Nitrate analysis
Screening by UV Spectrophotometry
Nitrate ions in water absorb UV radiation with a wavelenght of 220 nm. For this reason optical methods of
analysis can be used to measure nitrate.
The method is quite sensitive but requires a spectrophotometer that can be operated in the UV range.
Any materials that absorb 220 nm radiation will interfere in this analysis. That includes nitrite, hexavalent
chronium and many different organic compounds.
Ion chromatographic and capillary ion electrophoresis methods
The ion chromatographic method is useful for analysis with nitrate concentrations greater than about 0,2 mg/l.
In this way many of the normal interferrences in nitrate analysis can be avoided.
The method also permits the simultaneous analysis for other anions in the sample.
The disadvantage of this method is the cost for purchase and maintance of the instrument.

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Nitrate analysis
Nitrate electrode method:
The nitrate electrode is a liquid membrane electrode can detect the presence of nitrate nitrogn down
to concentrations of about 1mg/l.
The advantage is that once calibrated, analysis for nitrate is rapid.
The electrode is readily adapted to continous monitoring and to automatic process control.
Disadvantage are that several common ions such as chloride and bicarbonate cause interference and
the electrode isn’t sensitive with the lower nitrate concentrations frequently encountered.

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Nitrate analysis
Cadimium reduction method
The cadimium reduction method offers a highly snesitive procedure for nitrate
analysis.
A filtered sample with added NH4Cl-EDTA solution is passed through a specially
prepared column containing amalgamated cadimium granules.
pH =8,5
NO3− + H 2O + Cd ⎯⎯⎯→ NO2 − + 2OH − + Cd 2+
Nitrate is quantitative reduced by the cadimium to nitrite. The nitrite produced is
determined by the diazotization method previously described.
Nitrite originally present in the sample is also measured, this procedure in effect
measures the sum of nitrate plus nitrate nitrogen.

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Nitrate analysis
To determine the nitrate concentration, a separate analysis of nitrate alone is required and this value is
subtracted from the results of the cadimium reduction procedure. Because of the sensivity of the diazotization
method for nitrite, nitrate concentration as low as 0,01 mg/l can be detected.
Samples with nitrate nitrogen concentrations greater than 1 mg/l can also be measured if the samples are
diluted prior to passage through the cadimium column.
A standared nitrate solution should be passed through the column on occasion to establish that quantitative
conversion of nitrate to nitrite is being obtained.
If not, then the column should be reactivated or the sample flow rate should be adjusted to obtain quantitative
conversion.

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Nitrate analysis
Disadvantage of method:
The suspended matter prevent the flow of water sample in the column and limit the contact area between
the sample and Cd particles. Thereby reducing the reduction efficiency.
→ For the high turbidity water samples need to filter through the glass with a holes diameter of 0,45 micron
prior to sample analysis.
The iron, copper or other metal ions with a concentration which above a few mg/L will reduce the
reduction efficiency of Cd.
→ To add EDTA solution in the samples to remove the effects of ions.
Grease can cover the surface of Cd particles.
→ Can remove it by extracting samples with organic solvents.Coù
Strong oxidizing substance such as residual chlorine can impact to reduce the performance of Cd column
reduction.
→ To check the amount of residual chlorine in the sample and remove by adding Na2S2O3 solution.

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Nitrate analysis
Brucin-Sulfanilic method
Principals
In strongly acidic environment, the reaction between nitrate and brucin create sulfur compounds with
yellow color is applied to determine the amount of nitrate by colorimetric method.
However, not color also follow Beer-Lambert law, experimental research proven the color intensity depend
on the heat from the reaction.
To control the temperature, acid sulfuric concentration and reaction time will be calculated previously so
that all developments are always occur in the best conditions and less affected by the surrounding
environment.

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Nitrate analysis
Brucin-Sulfanilic method
Disadvantage of method:
All strong oxidizing matters and the reducing agents in the sample may interfere with this method
→ Orthotolidin be used to cover the oxidant.
The amount of residual chlorine higher than 5 mg/L will affect the results of analysis.
→ To reduce residual chlorine by residual arsenic sodium.
Fe2+ and Fe3+ as well as other metals with the valence is 4 when its content is less than 1 mg/L can be
considered to impact insignificantly.
The transformation nitrite can be prevented previously by acid sulfanilic.

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Organic nitrogen - Kjeldahl method

Most organic compounds containing nitrogen are derivatives of ammonia and this the nitrogen has an oxidation
state of -3. destruction of the organic portion of the molecule by oxidation frees the nitrogen as ammonia.
The Kjeldahl method employing sulfuric acid as the oxidizing agent is standard procedure.
In the reaction, carbon is oxidized to carbon dioxide, while the sulfate ion is reduced to sulfur dioxide.
The amino group is released as ammonia but, of course, can’t escape from the acid environment and is held as
an ammonium salt.


CH 3CHNH 2 COOH + 7H 2SO 4 ⎯⎯ → 3CO 2 + 6SO 2 + 8H 2O + NH 4 HSO 4

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Organic nitrogen - Kjeldahl method

The oxidation proceeds rapidly at temperatures slightly above the boiling
point of sulfuric acid (340oC)
The boiling point of the acid is increased to about 360-370oC to enhance
the rate of oxidation by addition of sodium or potassium sulfate.
The complete digestion of organic matter is essential if all organic
nitrogen is to be released as ammonia.

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Organic nitrogen - Kjeldahl method

This can best be done by listing the changes that samples undergo during digestion:
1. Excess water is expelled, leaving concentrated sulfuric acid to attack the organic matter
2. Copious white fumes form in the flask at the time sulfuric acid reached its boiling point
3. The mixture turns black, owing to the dehydrating action of the sulfuric acid on the organic matter.
4. Oxidation of carbon occur. Boiling during this period is characterized by extremely small bubble
formation due to release of carbon-dioxide and sulfur-dioxide.
5. Complete destruction of organic matter is indicated by a clearing of the sample to a “water-clear”
solution.
6. Digestion should be continued for at least 20 min after the samples appear clear to ensure complete
destruction of all organic matter
Once the organic nitrogen has been released as ammonia nitrogen, it may measured in a manner similar to
that described in the discussion of ammonia nitrogen. The excess sulfuric acid must be neutralized and the pH
of the sample adjusted to about 11.

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Question
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ASSIGNMENT
1. Would you expect to find the highest concentration of each of the following in raw
domestic wastewater or in the effluent from an aerobic biological waste treatment
plant? Why?
(a) Organic-N; (b) NH3-N; (c) NO2—N; (d) NO3—N

2. What biological processes might be used to reduce the total nitrogen content of a
wastewater?

3. What limit is placed on the nitrogen content of drinking water and why?

4. What is NOx and what environmental problems are associated with it?

5. What are the oxidation states of nitrogen, and what environmental problem or
problems may be associated with each?
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ASSIGNMENT
6. How many organic nitrogen concentration in a wastewater be determined?

7. What methods are available for analysis of nitrate nitrogen?

8. Determine the total nitrogen concentration in mg/L of a water sample containing

0.25 mM ammonia, 0.02 mM nitrite, 0.75 mM nitrate, and 1.3 mg/L organic nitrogen
9. Determine the total nitrogen concentration in mg/L of a water sample containing 1.3
mg/L ammonia, 0.25 mg/L nitrite, 15.1 mg/L nitrate, and 0.9 mg/L organic nitrogen.

10. The total nitrogen concentration of a water sample is found to be 11.2 mg/L. How
might the organic nitrogen content be determined without chemical analysis for the
organic nitrogen content of the sample? What is the pros and cons of your method
over over direct chemical analysis for organic nitrogen?
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ASSIGNMENT

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Environmental significance
Nitrogen is seen to be an essential component of all living things, excessive concentrations of certain nitrogen
species in some compartments of the environment can lead to significant environment problems.
The 03 major environmental problems associated with nitrogen species in the atmosphere are photochemical
smog, global warming and stratospheric ozone depletion.
Photochemical smog results when partially oxidized organic matter, NOx and sunlight come together under certain
meteorological conditions.

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Environmental significance
Indicator of sanitary quality
It has long been known that polluted waters will purify
themselves, provided that they’re allowed to age for
sufficient periods of time.

Nitrogen present origially in water in the form of

organic nitrogen (protein) and ammonia. As time
progresses, the organic nitrogen is gradually converted
to ammonia nitrogen, and later on, if aerobic conditions
are present, oxidation of ammonia to nitrite and nitrate
occurs.

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Environmental significance

Nutritional and related problems

All biological processes employed for wastewater treatment are dependent upon
reproduction of the organism employed.
In planning waste treatment facilities it becomes important to know whether the
waste contains sufficient nitrogen for the organisms. If not, any deficiency must
be supllied from outside sources.
Nitrogen is one of the fertilizing elements essential to the growth of algae.
Nitrogen analysis are an important means of gaining information on this problem.

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Environmental significance
Oxidation in rivers and estuaries
The autotrophic conversion of ammonia to nitrite and nitrate requires oxygen and so the discharge
of ammonia nitrogen and its subsequent oxidation can seriously reduce the DO levels inriver and
estuaries.
Nitrogen analyses are important in assesing the possible significance of the problem and in the
operation of treatment processes designed to reduce ammonia discharge.

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Environmental significance
Control of biological treatment processes
Determinations of nitrogen are often made to control
degree of purification produced in biological treatment.
Ammonia and organic nitrogen are first biologically
converted to nitrite and nitrate by aerobic treatment
In some states, ammonia nitrogen limitations have
been imposed because of suspected toxic effects upon
fish life. It’s well known that un-ionized ammonia is
toxic but that the ammonium ion is not. Since the
realtionship between the 02 is pH dependent, a
discussion is in order.

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