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International Journal of Marine Science 2014, Vol.4, No.17: 160-165
http://ijms.sophiapublisher.com
161
The rates of phytoplankton growth and loss as result of
microzooplankton grazing were determined using
dilution procedure (Landry and Hasset, 1982). The
major advantage of this method is that it assesses the
rate of total phytoplankton growth along with the rate
of microzooplankton grazing on the phytoplankton.
Samples of seawater (12~15 L) were taken from the sea
surface (~ 0.5 m depth) early in the morning using
Niskin bottle and
gently poured through a 200 µm
Nitex mesh.
To have filtrate, а water volume of 6–8 L
was sieved through a glass fiber filter (Whatman GF/F;
low pressure (<0.1 atm) to avoid the breakage of the
phytoplankton cells and thus to minimize their
intrusion into the filtrate. The native sample was then
diluted with the filtrate freshly obtained by a factor of
dilution of 1.0, 0.80, 0.60, 0.40 and 0.20 in duplicates.
The factor 1.0 means an undiluted sample, while the
factor of 0.20 means the sample was diluted five times
with filtrate. The prepared solutions were then placed
into polycarbonate bottles of 1 L volume, which were
prewashed with 10% HCl and then with distilled water.
The bottles were incubated for 24 hours on board,
opened for the solar impact, and cooled down to 20
℃
by running pumped surface water of the same
temperature. As the experiment ended, the water was
filtered through Whatman GF/F filters. After filtration
filters were placed in 90% acetone (5 mL) and
chlorophyll was extracted for 24 h at 4 ºC in the dark
(Protocols JGOFS, 1994). T
he acetone extracts were
centrifuged, and their fluorescence determined before
and after acidification
in a fluorometer (excitation 440
to 480 nm, emission > 665 nm), which was calibrated
with pure chlorophyll-
a
(Sigma Chemical Co). The
precision of these measurements was high, with a
relative standard deviation of 5%.
The phytoplankton growth rate was calculated under
the daily increase of Chl
a
in the experimental bottles.
The initial concentration of Chl
a
was determined only
for the undiluted samples, while, for the diluted
samples it was recalculated according to the dilution
factor (DF). The observed daily phytoplankton growth
rate for each of the 5 dilution treatments (µ
DF
) was
calculated as:
µ
DF
= ln(Chl
a
final
/ Chl
a
initial
) (1)
The linear regression equations were calculated to
estimate the interrelations between the observed
phytoplankton growth rate (µ
DF
) and the dilution
factor (DF) as:
µ
DF
= –g·DF + µ (2)
where µ is true phytoplankton growth rate (d
-1
) and g
– the microzooplankton grazing rate (d
-1
).
For determination of phytoplankton biovolume and
species composition, 3~4 L samples of sea water were
concentrated under the nucleopore membranes (1 μm
pore size; the product of the Institute of Nuclear
Researches, Dubna, Russia) in the inverse filtering
plexiglass funnel, (Sorokin et al., 1975). After the
samples were condensed to 50 ml, they were fixed with
neutralized 1% formaldehyde (final concentration in
the sample) and immediately processed. The numbers
and dimensions of microalgae were measured in a 0.1
ml drop in 3~5 replications under the light microscope
ZEISS Primo Star (x400). The precision of these
measurements was with a relative standard deviation of
25%. Phytoplankton biovolume was calculated from
cell counts and dimension measurements assuming
simple geometric shapes. The abundance and biomass
of microzooplankton were not determined.
Nitrate, ammonium, phosphate and silicon contents
were measured using the previously described
technique (Stelmakh et al., 2013).
Mathematical treatment of all data involved using
Microsoft Office Excel 2007 and Sigma Plot 2001
software for Windows.
2 Results
By the end of May 2013, the sea surface has warmed
to about 20° C, and diurnal photosynthetic active
radiation (PAR) was 44 E/m
2
·d on the average. The
depth of the upper mixed seawater layer (UML) varied
from 4 to 19 m, being about 10 m on the average.
Nitrate content was relatively low (0.10 – 0.30
mmol/m
3
) both in the shallow- and deep-water areas
of the sea (Table 1).
Nitrogen was present as ammonium, varying from
0.50 to 1.90 mmol/m
3
. The content of phosphates was
as large as 0.20 – 0.40 mmol/m
3
, therefore N:P ratio
averaged for the entire seawater area of the
investigation was considerably lesser than Redfield
ratio (5.1 (±1.8) vs. 16:1, correspondingly).