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EXERCISE 5
MEASURING CARBON DIOXIDE PRODUCTION IN AQUATIC ANIMALS

Introduction
Every energy-consuming process in the body liberates heat so that heat production of an animal
reflects its metabolic rate. The relationship is consistent so that a traditional definition of metabolic
rate is rate of heat production. Heat output from an animal may be measured directly (direct
calorimetry) or indirectly (indirect calorimetry). The latter method may be based on either
respiratory gas exchange or material balance.

By respiratory gas exchange, it means that the amount of oxygen used up and the amount of
carbon dioxide produced in metabolism have their calorific or heat equivalents. By material
balance, it means that the measurement is based on chemical energy content of all materials
entering and leaving the body. By the first indirect method, for example, the aerobic oxidation of
carbohydrates with the production of one milliliter of carbon dioxide produces heat equivalent to
5.05 gram-calories.

Carbon dioxide production was commonly used several decades ago to measure metabolic rate.
But, since it has been found that the conversion factor between CO 2 production and heat
production would vary strongly among the different foodstuff, oxygen consumption is preferred
nowadays. The method can only become accurate enough if the particular foodstuff being
catabolized in the cells is specified. Despite this limitation, though, the measurement of carbon
dioxide when particularly applied to aquatic animals has its distinct advantages which are 1) the
high solubility of the gas in water and 2) the facility of measurement by a simple titrometric method.

You will be determining the metabolic rate of an aquatic animal like a clam, a tadpole, or a fish.
These animals may give the idea that they are not producing heat because they are cool to the
touch. Like all other animals, they are producing heat: but since their rates of heat production are
low and their insulation inadequate, they cannot be warmed appreciably by the heat they
produced. They are examples of “cold-blooded” animals or poikilotherms (poikilos means
“variegated” or “manifold”) or ectotherms because their body temperature can vary depending on
external thermal conditions. The thermal influence on metabolism can best be seen in the
poikilotherms. In general, their resting metabolic rate raises appreciably and exponentially with
the body temperature.

Objectives:

1. To estimate and compare the rate CO2 small aquatic animals by titrometric method
2. To relate CO2 production with metabolic rate in aquatic animals

Compiled and edited from: Experiments in Animal Physiology of Glorina N. Pocsidio, Ph.D. and
Biol-123 Gen. Animal Physiology Lab Manual of Julissah Evangelio, M.Sc.
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Materials Needed

Aquatic animals of your choice (e.g snails, small fish, tadpole)

Two bottles (about 150 mL capacity)
500 mL dechlorinated tap water
Phenolphthalein Rubber or cork stopper
0.04% NaOH 2 5-mL measuring pippettes
Thermometer Medicine dropper
Triple beam balance

Procedure

1. Draw about 500 mL dechlorinated tap water and add 5 drops of phenolphthalein. If the
water turns pink, carefully exhaled air through it until it just turns clear.

2. Fill the 150 mL bottles (labeled A and B) with the clear water. Place in bottle A an aquatic
animal that will fit in the bottle comfortably. Or if the organism that you selected is small
(such as an aquatic snail or tadpoles) collect 10 or more organisms, determine their total
weight and place them in bottle A. Bottle B will serve as control and should not contain
any animal.

3. Note the time at the beginning of the test. After 30 minutes, remove the animals from bottle
A. Determine the CO2 content of the water in each of the bottles using the following
method:

a. Important: write down the level of the NaOH solution before starting the titration!
Slowly add 0.04% NaOH using a measuring pipette. Swirl the water as you add the
NaOH solution. Add enough 0.04% NaOH until the water turns pink and stays pink for
one minute after swirling.

b. Record the volume (in mL) of NaOH solution used in the titration. Multiply the number
of mL of 0.04% NaOH solution used by 10 since each mL of 0.04% NaOH solution
combines with 10 µM of CO2.

c. Repeat the experiment on similar animal/s at 10 0C below and above room

temperature. The activity of the animals during each measurement should be observed
and recorded.

4. Weigh the animal(s) after all experiments are done.

5. After weighing the animal/s, compute the respiration rate for each temperature used using
the following equation:

µM CO2 of A − µM CO2 of B
𝑅𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 (µM CO2)/g/hr) =
weight (g) × time (hr)

Compiled and edited from: Experiments in Animal Physiology of Glorina N. Pocsidio, Ph.D. and
Biol-123 Gen. Animal Physiology Lab Manual of Julissah Evangelio, M.Sc.
 3

For example: If bottle A had 32 µM CO2 and bottle B had 11 µM CO2, and the animals
weighed a total of 7.3 g, while the recorded time of the experiment was 35 minutes, then:

32− 11
𝑅𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 = 7.3 × 0.58 = 4.9 µM CO2/g/hr

If larger animals are to be used in the experiment, obtain a pair of bottles of equal size big
enough to hold them. Measure the volume of the bottles and follow procedures as given
above but with this modification: determine the µM CO2 content of a 100 mL sample from
each bottle and use this information to compute for the µM CO2 content of the entire water
sample.

6. Data summarized in table or plot in graph (You can choose which is can best present your
data/observation). I wanted to see how you will be able to arrange your data and interpret
your results)

7. Answer the guide questions and use them to further discuss the results obtain.

Guide Questions

1. How does respiration rate vary with temperature, activity, size of the animal? Discuss also
in terms of the relationship between metabolic rate and body mass.

2. In the aerobic catabolism of carbohydrates, lipids and proteins, corresponding heat

production per mL of CO2 produced in gram calories are 5.05, 6.67, and 5.57 respectively.
What is the metabolic rate of the animals in terms of heat production?

3. What do you mean by weight-dependent, surface-dependent, or intermediate metabolic

rate? How may this be determined from a graph of CO2 production rate vs weight?

4. What could be the possible sources or error in the experiment?

Compiled and edited from: Experiments in Animal Physiology of Glorina N. Pocsidio, Ph.D. and
Biol-123 Gen. Animal Physiology Lab Manual of Julissah Evangelio, M.Sc.