118
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
the number of animal specimens. The wet chromic oxidation procedure cannot
give 100% combustion of organic material. Usually its efficiency is around
90%. The above value of 600flgC should therefore be controlled empirically
by combustion aliquots of an amino acid or peptone solution with the known
content of organic carbon.
3.4.3 Determination of Radioactivity of Respired CO2 and Feces
In practical use of the 14C method, it is often necessary to measure the losses
of radiocarbon by labeled organisms (food or consumers' losses) as respired
CO2 (rc). These losses are measured via radioessay of the bicarbonate and CO2
carbon in the water in which these organisms were incubated. These measurement are made either by CO2 distillation or by its direct precipitation as
BaC03• Direct radioassay of the water in which the labeled animals have been
kept is hardly applicable in this case, one cause being low sensitivity. SubsampIes of 2-4ml of water, which could be radio assayed with the use of special
water-accepting scintillation cocktails are too small for correct estimation of
the comparatively low specific radioactivity of respired 14C02. The second
sause is the possible presence in the same water sample of radioactive liquid
excretions of animals (see below). To determine the values of rc by distillation
of 14C02 from the acidified water, an exact subsample of 50 or 100mi containing the exposed animals is taken from the aquarium, and transferred into
the flask of the apparatus (Fig. 2.3); 5m15% hyamine or phenethylamine solution in methanol is added into the absorber. The sample in the flask is acidified
with 5 ml of 20% H 2S04 added to the flask via the funnel. After establishing
the flow of COrfree air, the sample is boiled for 2-3 min. About lOmin later,
when the distillation of the CO2 thus formed is completed, the alkaline solution is discharged from the absorber directly into the scintillation vial, and its
radioactivity (r;) is measured after the addition of 15 ml scintillation cocktail.
The value of rc can then be calculated as follows: rc = (r; Va K)/(V; N) flg
Csp.-t, if Va is the total volume of water in the aquarium, ml; V; the volume
of water taken into the flask for the 14C02 distillation, ml; N the number of
animals kept in the aquarium, and K the mean counting efficiency of liquid
scintillation samples with the organic material of consumers' bodies.
A second, simpler way to estimate rc is direct precipitation ofTC02 carbon
as BaC03 and its radioassay by liquid scintillation counting. This method is
applicable mostly for work with freshwater hydrobionts because, in seawater,
too large BaC03 + BaS04 precipitates form after addition of BaCI2. In the first
case, up to 50ml of water can be thus treated, in the second only 3-5 ml.
The procedure for TC02 precipitation is as follows. A sample of freshwater
(25-40ml) is taken from the aquarium. Having been rendered alkaline
with 1 ml of 0.2 N KOH solution, it is filtered through a membrane filter
(0.2-0.4flm). Five ml 2N KOH and 3ml of Baetz solution, are added to the
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
the number of animal specimens. The wet chromic oxidation procedure cannot
give 100% combustion of organic material. Usually its efficiency is around
90%. The above value of 600flgC should therefore be controlled empirically
by combustion aliquots of an amino acid or peptone solution with the known
content of organic carbon.
3.4.3 Determination of Radioactivity of Respired CO2 and Feces
In practical use of the 14C method, it is often necessary to measure the losses
of radiocarbon by labeled organisms (food or consumers' losses) as respired
CO2 (rc). These losses are measured via radioessay of the bicarbonate and CO2
carbon in the water in which these organisms were incubated. These measurement are made either by CO2 distillation or by its direct precipitation as
BaC03• Direct radioassay of the water in which the labeled animals have been
kept is hardly applicable in this case, one cause being low sensitivity. SubsampIes of 2-4ml of water, which could be radio assayed with the use of special
water-accepting scintillation cocktails are too small for correct estimation of
the comparatively low specific radioactivity of respired 14C02. The second
sause is the possible presence in the same water sample of radioactive liquid
excretions of animals (see below). To determine the values of rc by distillation
of 14C02 from the acidified water, an exact subsample of 50 or 100mi containing the exposed animals is taken from the aquarium, and transferred into
the flask of the apparatus (Fig. 2.3); 5m15% hyamine or phenethylamine solution in methanol is added into the absorber. The sample in the flask is acidified
with 5 ml of 20% H 2S04 added to the flask via the funnel. After establishing
the flow of COrfree air, the sample is boiled for 2-3 min. About lOmin later,
when the distillation of the CO2 thus formed is completed, the alkaline solution is discharged from the absorber directly into the scintillation vial, and its
radioactivity (r;) is measured after the addition of 15 ml scintillation cocktail.
The value of rc can then be calculated as follows: rc = (r; Va K)/(V; N) flg
Csp.-t, if Va is the total volume of water in the aquarium, ml; V; the volume
of water taken into the flask for the 14C02 distillation, ml; N the number of
animals kept in the aquarium, and K the mean counting efficiency of liquid
scintillation samples with the organic material of consumers' bodies.
A second, simpler way to estimate rc is direct precipitation ofTC02 carbon
as BaC03 and its radioassay by liquid scintillation counting. This method is
applicable mostly for work with freshwater hydrobionts because, in seawater,
too large BaC03 + BaS04 precipitates form after addition of BaCI2. In the first
case, up to 50ml of water can be thus treated, in the second only 3-5 ml.
The procedure for TC02 precipitation is as follows. A sample of freshwater
(25-40ml) is taken from the aquarium. Having been rendered alkaline
with 1 ml of 0.2 N KOH solution, it is filtered through a membrane filter
(0.2-0.4flm). Five ml 2N KOH and 3ml of Baetz solution, are added to the