142
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
the Cr of the labeled food organisms may change during the experiment. The
sedentary animals (clams, corals, tube worms) first are placed into the vessels
with nonfiltered water. When they relax after the stress caused by their transfer and become active again, this water is discharged with the aid of a syphon
and carefully replaced by the filtered water so as not to scare the animals. The
labeled food is added into the vessel when all the animals return to active
filtration. Then the experiment begins.
The duration of feeding the consumers with the labeled food must be
approximately equal to three-quarters of the estimated time of its digestion
by the given grazer species, and must be finished before the loss of the radioactive material ingested begins. For small planktonic crustaceans this incubation
time should be about lS-20min, for bivalve mollusks l-l.Sh, for fish larvae
about 40 min. When the incubation with the labeled food ends, the animals are
very gently washed out of the labeled food as described above (Sect. 3.S.2).
The main precaution during this procedure is not to disturb the animals, to
prevent their losing the ingested food. The washed animals are separated into
two equal groups, if the Re values are to be estimated directly. One group of
animals is placed in this case into the vessel with unlabeled food. The members
of the other group are first counted. Then they are rapidly transferred into the
scintillation vial (with the aid of a pipette if they are small), the excess water
from it is removed, and they are radioassayed as described above (see Sect.
3.S.2). If they are large, they (or their excized flesh) are placed into the beaker
with alkaline solution for their hydrolysis (see Sect. 3.4.2). If the direct estimation of Re is absent in the experimental protocol, all animals, after being
washed, are placed into the vessel with the unlabeled food. The water for this
incubation must be first filtered through the membrane filter. Its pH should
be adjusted to ~8.S and some S-lOmgl- 1 (wet weight) of unlabeled food must
be added to it. The volume of water for this second (assimilation) phase of the
experiment has to be taken as the minimum necessary for normal survival of
animals placed there for 1.S to 2h, for example: 100 daphnia in SOml or 3
average sized (3-4cm) clams in lSO-200mi. The duration of their incubation
in this water must be at least two times more than the estimated digestion time
to be sure that no remains of the previously ingested labeled food is present
in their guts. When this time span has elapsed, the animals are taken out of
the water in which they have been kept. To facilitate the operation the animals
could be fixed or narcotized. The main prerequisite in this operation is to
preserve quantitatively the whole volume of this water. The animals are then
subjected to radioassay, as described above for the estimation of Ra values
(see Sects. 3.S.2, 3.S.3).
The remaining water is used to estimate the radioactivity of 14C02 respired
by animals incubated in it (re, see Sect. 3.S.3), and the radioactivity of feces
(R f ). The procedure is as follows. First the pH of water is adjusted again to 8.S.
Then the whole water volume is filtered through the O.4S-llm pore size and
20-mm diameter membrane filters. The filter is placed into the scintillation
vials and radioassayed after their dissolution with the ethylene glycol (O.S ml),
Radioisotopic Methods for the Study of Nutrition in Aquatic Animals
the Cr of the labeled food organisms may change during the experiment. The
sedentary animals (clams, corals, tube worms) first are placed into the vessels
with nonfiltered water. When they relax after the stress caused by their transfer and become active again, this water is discharged with the aid of a syphon
and carefully replaced by the filtered water so as not to scare the animals. The
labeled food is added into the vessel when all the animals return to active
filtration. Then the experiment begins.
The duration of feeding the consumers with the labeled food must be
approximately equal to three-quarters of the estimated time of its digestion
by the given grazer species, and must be finished before the loss of the radioactive material ingested begins. For small planktonic crustaceans this incubation
time should be about lS-20min, for bivalve mollusks l-l.Sh, for fish larvae
about 40 min. When the incubation with the labeled food ends, the animals are
very gently washed out of the labeled food as described above (Sect. 3.S.2).
The main precaution during this procedure is not to disturb the animals, to
prevent their losing the ingested food. The washed animals are separated into
two equal groups, if the Re values are to be estimated directly. One group of
animals is placed in this case into the vessel with unlabeled food. The members
of the other group are first counted. Then they are rapidly transferred into the
scintillation vial (with the aid of a pipette if they are small), the excess water
from it is removed, and they are radioassayed as described above (see Sect.
3.S.2). If they are large, they (or their excized flesh) are placed into the beaker
with alkaline solution for their hydrolysis (see Sect. 3.4.2). If the direct estimation of Re is absent in the experimental protocol, all animals, after being
washed, are placed into the vessel with the unlabeled food. The water for this
incubation must be first filtered through the membrane filter. Its pH should
be adjusted to ~8.S and some S-lOmgl- 1 (wet weight) of unlabeled food must
be added to it. The volume of water for this second (assimilation) phase of the
experiment has to be taken as the minimum necessary for normal survival of
animals placed there for 1.S to 2h, for example: 100 daphnia in SOml or 3
average sized (3-4cm) clams in lSO-200mi. The duration of their incubation
in this water must be at least two times more than the estimated digestion time
to be sure that no remains of the previously ingested labeled food is present
in their guts. When this time span has elapsed, the animals are taken out of
the water in which they have been kept. To facilitate the operation the animals
could be fixed or narcotized. The main prerequisite in this operation is to
preserve quantitatively the whole volume of this water. The animals are then
subjected to radioassay, as described above for the estimation of Ra values
(see Sects. 3.S.2, 3.S.3).
The remaining water is used to estimate the radioactivity of 14C02 respired
by animals incubated in it (re, see Sect. 3.S.3), and the radioactivity of feces
(R f ). The procedure is as follows. First the pH of water is adjusted again to 8.S.
Then the whole water volume is filtered through the O.4S-llm pore size and
20-mm diameter membrane filters. The filter is placed into the scintillation
vials and radioassayed after their dissolution with the ethylene glycol (O.S ml),