LABORATORY: NITROGEN EXCRETION (REVISED NOV 1, 2012)
PURPOSE: The main aim of this lab is to study excretion of nitrogenous waste in
aquatic and amphibian animals. Animals excrete nitrogenous waste primarily in the
forms of ammonia (NH3 and NH4+), urea, and uric acid. Most animals have the ability
to produce each of these products, however in most cases one type of waste
dominates. For example, most of the nitrogenous waste of humans is urea (making us
ureotelic animals), although we also produce some ammonia and uric acid. Most
animals in a given taxon have the same excretory strategy, although there are
exceptions within most taxa. The nitrogenous waste strategy can also be altered in
response to the environment. For example, toadfish can switch from ammoniotelism to
ureotelism under some conditions. The rate of N excretion in an animal can change
with metabolic rate, and other physiological and environmental factors. In this lab, you
will determine the rate of nitrogen excretion in animals living in aquatic environment –
teleost fish and aquatic to terrestrial environment - toad.
Questions: What are the primary N-excretion sites (organ or tissues) in
teleost fish? What are the physiological and adaptive advantages of
water-breathing animals to excrete ammonia as the major N-waste? Why
might you expect an embryonic fish to produce urea? Why do you think there
is a change in metabolic waste from ammonia to urea in the stages of
development in amphibians? Urea is also used in hibernation. Can you give 3
possible functions of urea that might help the toad to survive hibernation?
Animal Procedures
In this lab, each pair of students will be measuring ammonia and urea excretion in
goldfish and toad over a period of 1hr.
Goldfish and Toad
Each pair of students will use one goldfish (Carassius auratus) and one toad (Bufo
marinus ).
Questions: What are the biochemical and physiological consequences of a
strategy involving each of the three main excretory products?
Experimental Procedure: Ensure that you have 2 experimental tanks (glass jars)
equilibrated to room temperature. Begin the experiment by gently transferring the test
fish and toad to their respective jar partially filled with water. Use the 5ml plastic syringe
to remove your first 2ml water sample, which will reflect the starting level of nitrogen in
the water. Transfer the water sample to a microcentrifuge tube then close the tube. You
will repeat the sampling at 20min, 40min and 60min.
Throughout the experiment, monitor the fish for movements of the opercula; ventilation
rate (opercular movements per min) is an index of metabolic rate in these fish.
You will be expressing the rate of ammonia and urea production by animal in terms of
moles N per min per g of fish (1 molecule of ammonia has 1 N, urea has 2 N). Before
returning your animal to the proper holding tank, it must be weighed. What is the best
way to weigh fish without subjecting them to stress from air-exposure? You will also
need to know the total volume of water in the experimental chamber.
Table 1. Goldfish weights and volumes
Animal
Mass (g)
Volume of water in the
chamber (ml)
Goldfish
Toad
Ammonia and urea concentrations
You will be measuring both ammonia and urea in both toad and goldfish.
Caution: These chemicals are very corrosive, so handle them with extreme care.
Ammonia
Ammonia is produced in the breakdown of amino acids, as well as other nitrogenous
compounds. Most enzymatic reactions generate ammonium (NH4+) rather than
ammonia (NH3), however once these chemicals are in solution, they quickly equilibrate
according to the following reaction.
NH3 + H2O NH4+ + OHIn many cases, the term “ammonia” is used to describe the combination of NH 4+ and
NH3 without discriminating between the two forms. Conversely, ammonium refers
specifically to NH4+. The ammonia assay is a chemical reaction that leads to the
formation of a coloured product. You will each be measuring ammonia production from
your goldfish or toad.
Preparing ammonia standards
Your assay will compare a series of unknowns (your samples) to solutions of known
ammonia concentration (standards). To prepare these standards, set up a dilution
series using a 100 µM ammonia stock solution. Your goal is to make up 1 ml of
solutions of 0, 20, 40, 60, 80, 100 µM ammonium chloride (NH4Cl) in microcentrifuge
tubes. The table below is provided so that you can plan your dilutions in advance.
Table 1. Ammonia standard curve.
[Ammonia]
µmol/l
Volume
of 100 µM
NH4Cl
Volume of
H2O
0
µmol/l
20
µmol/l
40
µmol/l
60
µmol/l
80
µmol/l
100
µmol/l
0 µl
1000 µl
1000 µl
0 µl
Premade reagents
Ammonium chloride standard: 100 M in water
The assay requires four reagents that are prepared and provided by the instructor.
Reagent 1: Sodium hypochlorite (AKA bleach, 6% solution)
Reagent 2: Alkaline citrate (35%), (35g sodium citrate, 4g NaOH in 100ml H2O).
Reagent 3: Salicylate (40% sodium salicylate)
Reagent 4: Sodium nitroprusside (0.02g/100ml). It is light sensitive and must be kept in
fridge.
Reagent 5: 1ml Reagent 1 plus 5 ml Reagent 2 in a 15ml Falcon tube.
Spectrophotometric Assay for Ammonia
You will perform your ammonia assays in a 96 well spectrophotometer plate, with
standards and samples arranged as below.
A
B
C
D
E
F
G
H
1
0
20
40
60
80
100
2
0
20
40
60
80
100
standard
3
0
20
40
60
80
4
0
20
40
60
5
0
20
40
60
6
0
20
40
60
7
0
20
40
60
8
9
10
11
12
100
Goldfish
Toad
Dispense the following solutions into the 96 well plate as follows.
1. Use a repeat pipette to transfer 200l of standard or unknown sample into the well.
2. Using a repeat pipette, dispense 25l of Reagent 3. Set the P200 pipette to 150l
and mix the contents of each well by drawing the solution up and down 3x.
3. Use the repeat pipette to dispense 25l of Reagent 4. Mix the contents of each well
as above.
4. Use the repeat pipette to dispense 25µl of Reagent 5 into each well. Mix the
contents again and then incubate the plate at room temperature in the dark for 1hr.
5. Read the samples on the SpectraMax plate reading spectrophotometer @ 650mm.
Open the file called “Ammonium Measurements”. It has the appropriate settings to
read your samples, arranged as above. Ask your TA if you are curious about the
various settings.
6. Click on the “Read” icon near the top of the program. If you have loaded your
samples properly, the program will create a standard curve (from your standards)
and calculate the concentrations of ammonia in your unknowns.
7. To verify that your numbers are meaningful, first open the “Standard Curve” tab to
verify that your standard curve is linear. Do all the points lie near the curve? Are
any of the standards (or samples) bad duplicates? Ask your TA for advice on
what to do with problem values.
Urea
Urea is produced in many animals through the ornithine-urea cycle. Once produced, it
can be released immediately across gills or skin, or be collected by the kidney for
excretion or storage in a urinary bladder. The urea assay is a chemical reaction that
leads to the formation of a coloured product.
Preparing urea standards
Constitute standard concentration series from urea stock (100umol urea/l or
2mmol urea N/l). Label 2 sets of 6 glass tubes for standard solution according to
Table 4. For 1ml of urea standard solution in each tube, you will need to dispense
into the labeled glass tube in following manner (P1000 pipettes) and mix well:
Table 2. Urea standard curve.
[Ammonia]
µmol/l
Volume
of 100 µM
urea
Volume of
H2O
0
µmol/l
20
µmol/l
40
µmol/l
60
µmol/l
80
µmol/l
100
µmol/l
0 µl
1000 µl
1000 µl
0 µl
Premade Reagents
Urea standard: 100 M in water
Reagents 6 and 7 are pre-made by the instructor and stored in the fridge.
Reagent 6: 100ml 85% H3PO4, 300ml H2SO4, 600ml H20, 1 g FeCl3
Reagent 7: 500mg diacetyl monoxime, 10mg thiosemicarbizide dissolved in 20ml H2O.
You will make Reagent 8 just before you need it for the assay.
Reagent 8: Combine 5ml Reagent 6 and 2.5ml Reagent 7 in a graduated cylinder.
Cover the top with parafilm and mix. Be extremely careful with this solution. Do not spill
the reagent or inhale the fumes.
Spectrophotometric Assay for Urea
Label 2 sets of glass tubes for each sampling time point for each experimental
animal according to the tables below.
1
2
3
4
5
6
7
8
9
10
11
12
A
0
0
0
0
0
0
0
B
20
20
20
20
20
20
20
C
40
40
40
40
40
40
40
D
60
60
60
60
60
60
60
E
80
80
80
F
100 100 100
Goldfish
Toad
G
standard
H
1. Dispense 0.6ml of standard or unknown water sample into the labelled
microcentrifuge tubes.
2. Dispense 0.3ml Reagent 8 into each tube, cap and shake to mix it well.
3. Place these tubes in a floating rack in a boiling water bath. Leave them there in
the dark for 10min.
4. Cool the tubes in a cold water bucket and vortex the tubes.
5. Transfer 250ul of sample and standard to the designated wells in the 96 well
plate and read the endpoint absorbance at 525 nm.
Calculations for Ammonia and Urea
The output from the spectrophotometer will tell you what concentration of ammonia (and
urea) was in your samples in (M, or moles per liter of water in the tank). You will need
to translate this information into a rate of ammonia (or urea) production per g animal
(mol per min per g). There are several ways to reach this goal, but try this approach if
you prefer.
Step 1. Translate the concentration (M) to an amount (moles) of ammonia (or urea)
produced by the animal at any given time. Apart from the spectrophotometric output,
what do you need?
Step 2. Using Excel, create a plot that relates production of ammonia and urea at each
time. Which is the determinant axis and which is the indeterminant axis? The slope of
the line gives you the total ammonia excretion rate for ammonia (or urea) for that
animal (moles per min).
Step 3. Correct this rate of ammonia or urea production per animal weight to reflect
differences in body mass. This gives you a mass-specific rate of ammonia (or urea) N
production per g animal.
Questions: Were the rates of ammonia and urea production linear over time in
fish and toad? Why? Do you expect the composition of N waste excretion
different between fish and toad? Why?
References
Principles of Animal Physiology (2008) CD Moyes and PM Schulte. Information on
nitrogenous waste excretion can be found in Chapter 10
Wright P.M. Nitrogen excretion: three end products, many physiological roles. J. exp.
Biol. 198:273-281