Measurements of Feeding, Food Assimilability, and Respiration
147
solution +0.1 ml of water is added into one (blank) test tube, or 0.1 ml of
solution of some amino acid, which contains a known amount of organic
carbon between 30 to 50 /lg C is added into the second (calibration sample)
test tube. After cooling, the contents of all three test tubes are quantitatively
transferred into little (50ml) flasks and diluted to 10ml. Then O.lml 5% K1
solution is added into them, and they are closed with glass stoppers. Five
minutes later, the 12 formed is titrated with 0.01 N thiosulfate. The amount of
organic carbon corresponding to 1 ml of thiosulfate solution first is calculated
after titration results of calibration samples. Then it is possible to calculate the
content of organic carbon (W, /lgCml- l ) in the DOM solution thus analyzed.
The value of radioactivity (r, cpmml- I ) of the same concentrated solution is
determined as follows: 0.1 ml of this solution is diluted in 50 ml of water with
5-lOmg of the carrier added to it (the same but unlabeled hydrolyzate or yeast
extract). Then exact volumes 0.3 to 0.5 ml of this dilution are poured into the
scintillation vials and radio assayed in the presence of unlabeled animal material to avoid quench corrections, as described above (see Sect. 3.4.1). The
radioactivity value thus measured is calculated per 1 ml of original DOM solution (r, cpmml-l). The needed Cr value will be equal to the ratio of Wlr.
Having estimated the Cr value in the concentrated solution of labeled
DOM the latter is diluted with the distilled water or with 3.5% NaCI solution
(for application in seawater) to obtain the final content of organic carbon 30
to 50/lgCml- 1 taking into account that some 0.1-0.2ml of this working solution of labeled DOM per 100ml of water will be added into the experimental
vessels. The solution thus diluted is then heated to 95°C, cooled, and filtered
through 0.1-0.2-llm pore size membrane filter. Then it is distributed into
1-2-ml ampules. The ampUles are sealed and sterilized by boiling three times
for a half an hour at I-day intervals.
2. Preparation of water and animals and the experimental procedure
Water has to be filtered through a 0.45-llm pore size membrane or through
GF filters to avoid the particles and most of bacteria. Just before the experiment start, this water must be heated to 70°C and then rapidly cooled. The
animals selected for the experiments must be previously kept for several hours
in water prefiltered through 0.45-llm pore size membrane filter to eliminate
bacteria. Then they are washed, using the special device (Fig. 3.2F) if they are
small, and placed into the experimental vessels previously rinsed with hot
water and filled with water prepared as described above. The volume of water
should be sufficient for normal functioning of the animals: ca. 500ml per 100
daphnia or 31 per 2 clams 3 cm long. Then the portion of the labeled DOM
working solution is added. Its size is calculated in accordance with the volume
of water in the experimental vessel in order to attain a concentration of 14C_
DOM of approximately 50-100/lgCI- I . The duration of the experiment should
be between 1 to 3 h. At the beginning of incubation and during its course, 510-ml aliquots are removed from the experimental vessel at I-h intervals for
147
solution +0.1 ml of water is added into one (blank) test tube, or 0.1 ml of
solution of some amino acid, which contains a known amount of organic
carbon between 30 to 50 /lg C is added into the second (calibration sample)
test tube. After cooling, the contents of all three test tubes are quantitatively
transferred into little (50ml) flasks and diluted to 10ml. Then O.lml 5% K1
solution is added into them, and they are closed with glass stoppers. Five
minutes later, the 12 formed is titrated with 0.01 N thiosulfate. The amount of
organic carbon corresponding to 1 ml of thiosulfate solution first is calculated
after titration results of calibration samples. Then it is possible to calculate the
content of organic carbon (W, /lgCml- l ) in the DOM solution thus analyzed.
The value of radioactivity (r, cpmml- I ) of the same concentrated solution is
determined as follows: 0.1 ml of this solution is diluted in 50 ml of water with
5-lOmg of the carrier added to it (the same but unlabeled hydrolyzate or yeast
extract). Then exact volumes 0.3 to 0.5 ml of this dilution are poured into the
scintillation vials and radio assayed in the presence of unlabeled animal material to avoid quench corrections, as described above (see Sect. 3.4.1). The
radioactivity value thus measured is calculated per 1 ml of original DOM solution (r, cpmml-l). The needed Cr value will be equal to the ratio of Wlr.
Having estimated the Cr value in the concentrated solution of labeled
DOM the latter is diluted with the distilled water or with 3.5% NaCI solution
(for application in seawater) to obtain the final content of organic carbon 30
to 50/lgCml- 1 taking into account that some 0.1-0.2ml of this working solution of labeled DOM per 100ml of water will be added into the experimental
vessels. The solution thus diluted is then heated to 95°C, cooled, and filtered
through 0.1-0.2-llm pore size membrane filter. Then it is distributed into
1-2-ml ampules. The ampUles are sealed and sterilized by boiling three times
for a half an hour at I-day intervals.
2. Preparation of water and animals and the experimental procedure
Water has to be filtered through a 0.45-llm pore size membrane or through
GF filters to avoid the particles and most of bacteria. Just before the experiment start, this water must be heated to 70°C and then rapidly cooled. The
animals selected for the experiments must be previously kept for several hours
in water prefiltered through 0.45-llm pore size membrane filter to eliminate
bacteria. Then they are washed, using the special device (Fig. 3.2F) if they are
small, and placed into the experimental vessels previously rinsed with hot
water and filled with water prepared as described above. The volume of water
should be sufficient for normal functioning of the animals: ca. 500ml per 100
daphnia or 31 per 2 clams 3 cm long. Then the portion of the labeled DOM
working solution is added. Its size is calculated in accordance with the volume
of water in the experimental vessel in order to attain a concentration of 14C_
DOM of approximately 50-100/lgCI- I . The duration of the experiment should
be between 1 to 3 h. At the beginning of incubation and during its course, 510-ml aliquots are removed from the experimental vessel at I-h intervals for