DETERMINATION OF PARTICULATE ORGANIC NITROGEN

DETERMINATION OF PARTICULATE ORGANIC NITROGEN

175
NOTES AND COMMENT
In the second, the patterns of distribution
of a dye solution passing outwardly through
a sampling head are not necessarily similar
to the pattern of inwardly moving water.
Finally, although some data on dye movement into the slit were obtained by greatly
accelerating the flow rate, we are not convinced that these patterns are identical to
those occurring under normal conditions.
Our conclusion on the sampling pattern
of the instrument can be only tentative.
We feel that the pattern either resembles
a torus or a toroid with an expanded horizontal dimension.
This opinion is compatible with that of Schooley ( 1967) who
has made elaborate studies of wake collapse in stratified fluids. Assuming a torus,
our calculations indicate that this sampler
iS many times more efficient than typical
open-ended, vertical samplers as long as
the volumes sampled are comparable.
However, if volumes in excess of a few
liters are desired from horizontal layers of
water, it is essential to consider the increase of the cross-sectional diameter of the
toroidal sampling pattern. In such cases,
sampling heads of larger diameter or horizontal movement of the sampling head
during operation may be necessary to avoid
contamination from layers other than the
one desired. Probably a certain amount
of horizontal oscillation often occurs during
sampling operations, and this action may
improve the effectiveness of obtaining a
sample from a narrow horizontal layer by
collecting from more than one torus or
toroid.
We are grateful to E. Gruner, P. Kenny,
and members of the Aquatic Tutorial,
Center for the Biology of Natural Systems
for suggestions and support in this work.
BRUCE C. PARKER
GEORGE LEEPER
WILLIAM HURNI
Department of Botany,
Washington Uniumsity,
St, Louis, Missouri
63130.
REFERENCES
JoERIs L . s . 1964.
A horizontal
sampler for
collection of water samples near the bottom.
Limnol. Oceanog., 9 : 595-598.
LUND, J. W. G. 1954. The seasonal cycle of
the plankton diatom, Melosira it&a
(Ehr)
Kutz subsp. subartica o. Mull.
J. Ecol., 42:
151-179.
SCHOOLEY, A. H.
1967.
Wake collapse in a
stratified fluid.
Science, 157 : 421-423.
DETERMINATION OF PARTICULATE ORGANIC NITROGENS
Most studies on the distribution
and
composition of the particulate matter in
ocean water report the organic carbon content but not the organic nitrogen content.
One reason for this is that the carbon content can be determined on samples of 1.0
liter or less by the method described by
Menzel and Vaccaro ( 1964)) whereas
methods for determination of the nitrogen
content generally require 4.0 liters of
water. These methods for nitrogen determination include 1) the Dumas combustion
( Menzel and Ryther 1964)) 2) oxidation
of organic nitrogen to nitrate which is
then determined calorimetrically
( Eppley,
Holmes, and Paasche 1967)) and 3) a
Kjeldahl digestion followed by determina’ This research was supported by U.S. Atomic
Energy Commission
Contract
No. AT ( 1 l-l ) -34,
Project 108. UCSD P108-71.
tion of ammonia by the Nessler reagent
( Strickland and Parsons 1965 ) .
The basic calorimetric reaction whereby
ammonia is determined by complexing with
ninhydrin and hydrindantin was developed
by Moore and Stein (1954) and modified
by Jacobs ( 1962). Dal Pont and Newell
(1963) applied this reaction to determination of organic nitrogen in the particulate
fraction from ocean samples; their method
involved collection of the particulate fraction by adsorption onto an aluminum
hydroxide cake that was then digested and
analyzed for nitrogen. By using glass-fiber
filters and by modifications in the digestion procedure and subsequent calorimetric
determination
of the ammonia with ninhydrin-hydrindantin,
the volume of sample
required has been reduced by a factor of
five; the speed and simplicity of the determination have also been increased as com-
176
NOTES AND COMMENT
1.6
I-
O
IO
ug
20
30
40
50
NITROGEN
FIG. 1. Optical density of reference solutions
of ammonium sulfate after reaction with ninhydrin and hydrindantin.
Line A, undiluted samples
containing 0 to 10 ,ug of nitrogen; line B, samples
containing
10 to 50 ,ug of nitrogen, diluted 1 to
10 after color development.
See text for details.
pared with that described by Dal Pont and
Newell. The sensitivity of this reaction is
such that 1.0 liter of deep ocean water
is sufficient for a reliable determination
of the nitrogen content of the particulate
fraction. This method, which has been in
use during the past two years for routine
nitrogen determinations,
is described in
detail below.
METHOD
Reagents
1. Digestion mixture. Dissolve 0.2 g of
selenium dioxide in 200 ml of distilled
water. Add 110 ml of concentrated sulfuric
acid, and take to 1.0 liter with distilled
water.
2. Anti-bumping granules. Hengar gran-
ules are broken into small pieces in a
mortar and pestle and then cleaned in hot
sulfuric acid-dichromate
cleaning solution
for 2 hr. The granules are rinsed three
times with distilled water and dried at 60C.
3. Ninhydrin-hydrindantin
reagent. Dissolve 0.5 g of ninhydrin
and 0.075 g of
hydrindantin dihydrate in 20 ml of ethylene
glycol monomethyl ether. Add 5 ml of
acetate buffer and mix. This solution is
dark red and should be used as soon as
possible after mixing as it deteriorates
within a few hours.
4. Acetate buffer. Dissolve 136 g of
sodium acetate trihydrate in 100 ml of distilled water. Add 25 ml of glacial acetic
acid and dilute to 250 ml with distilled
water.
5. Sodium hydroxide solution. Dissolve
40 g of NaOH in 1,000 ml of distilled water.
6. Ammonium sulfate standard. Dissolve
1.18 g of ( NH4 ),SOh in distilled water and
take to 500 ml. One ml of this solution
contains 500 pg of N. Store at -20C.
7. Phenolphthalein
indicator.
Dissolve
0.2 g of phenolphthalein
in 50 ml of 80%
ethanol.
8. 50% ethanol.
Filtration of sample
Sample volumes generally range from
100 ml for rich coastal water to 1.0 liter
for all samples below 100 m. Filter the
sample at one-third atmospheric pressure
through a 25mm glass-fiber filter (Whatman GF/C ) that has previously
been
heated at 450C for 2-3 hr to eliminate all
organic material in the filter. Using clean
forceps, place the filter in an acid-washed
Pyrex test tube ( 18 x 150 mm) that has
been marked at the 7.0-ml level. The
filter is not rolled or folded, but placed
against the wall of the tube, with the filtered material toward the inside of the
tube. Carefully push the filter down the
test tube so that the bottom of the filter
touches the bottom of the tube. Place
parafilm over the top of the tube and store
at -20C until analysis. With any series of
samples, include at least one blank. Blanks
are prepared as above, but in lieu of a
sample, filter 10 to 20 ml of seawater that
177
NOTES AND COMMENT
has previously been filtered
HA Millipore filter.
through
an
Standards
Make serial dilutions of the ammonium
sulfate solution and pipette LO-ml aliquots
into clean test tubes ( 18 x 150 mm). The
concentrations usually used for the reference curve are 0, 2, 5, 10, 20, and 50 pg
of N per tube.
Digestion
Add 1.0 ml of the sulfuric acid digestion mixture and one piece of a boiling
granule to each test tube (samples and
standards)
and heat on an electrical
Kjeldahl digestion rack for about 2 hr. The
heating receptacles of the digestion rack
are modified to receive the ends of the
test tubes by cutting elliptical openings in
squares of stainless steel sheet and clipping
these in place over the heating elements.
The tubes are placed so that the filters
are toward the underside of the tube, which
ensures that the refluxing acid will saturate
the filter. During the latter part of the
digesion period, the acid is refluxing down
the walls of the test tube for about 3 cm.
Color formation
Add 3.0 ml of distilled water and one
drop of phenolphthalein
indicator to each
tube. After cooling the tubes in an ice
bath, add sufficient sodium hydroxide solution (3.5 to 4.0 ml) to make the contents
of each tube slightly alkaline. To avoid
adding too much base, the contents of each
tube are held on the vortex mixer while
sodium hydroxide solution is added with a
pipette until a permanent pink color is
obtained. Add distilled water if necessary
so that the total volume in the tube is
7.0 ml. Cool the tubes in an ice bath and
centrifuge at 1,500 X g for 5 min. Draw off
3.0 ml of the supernatant liquid and transfer to a clean test tube ( 18 x 150 mm).
Add 2.0 ml of the ninhydrin-hydrindantin
color reagent, mix, and heat at 1OOC in a
boiling water bath for 20 min. Remove
and cool in a water bath at room temperature.
Measurement of optical density
In dilute solution the ninhydrin-ammonia-hydrindantin
complex is stable at room
temperature for at least several hours. In
samples containing 20-50 pg of N, there
is a tendency for precipitation
of the colored complex. To eliminate this difficulty
and also to reduce the optical density
values, all samples and standards that look
darker than the 10 pg of N standard are
diluted shortly after removal from the hot
water bath. To dilute, add 1.0 ml of the
colored solution to 9.0 ml of 50% ethanol
in a clean test tube. If any dilutions are
made, also dilute one reagent blank. The
optical density of the blanks, samples, and
standards are then measured against distilled water in a l-cm cell at 570 mp.
When the optical density of the blanks
and standards is plotted against nitrogen
concentration, the relationship is a straight
line throughout the range of O-50 pg of N
(see Fig. 1). The amount of nitrogen in
any sample is then obtained by reference
to the standard curve as shown in Fig. 1.
Sensitivity and precision of method
As shown in Fig. 1, the range over
which nitrogen can be reliably determined
by this procedure is 0.5-50 pg of N. Ten
replicate samples (2 ml) of a culture of
the green marine flagellate Dunaliellu tertiolecta gave a mean of 4.9 pg of N, with
a standard deviation of 0.24 pg.
APPLICABILITY
OF METHOD
The method as described above has been
used here during the past two years for
the determination
of organic nitrogen in
the particulate matter from ocean samples
( Holm-Hansen,
Strickland, and Williams
1966). It offers several important advantages over the previously used method of
coating a Millipore filter with a layer of
MgC03 and determination of nitrogen by
the Nessler reagent (Strickland and Parsons
1965). Not only is it more sensitive, but
the sampling procedure on board ship is
far simpler as no rinsing and centrifugation
steps are required. Even though the effective pore size of the glass-fiber filter is
apparently larger than the MgCOs-coated
178
NOTES AND COMMENT
Millipore filter, the amount of particulate
matter retained by these filters is essentially identical, at least in regard to organic
nitrogen content. To test this, replicate
samples of ocean water from 10 and 200 m
were filtered through both types of filters,
and the particulate matter digested and
analyzed for organic nitrogen by the ninhydrin-hydrindantin
method. The nitrogen
values for the Millipore filter samples were
9.6 and 7.0 pg of N per liter at 10 and
200 m, respectively, while the corresponding values for the glass filters were 9.9
and 7.0 pg of N per liter.
This method using glass filters and the
ninhydrin-hydrindantin
color reaction is
also ideally suited for the determination of
nitrogen content of laboratory cultures of
phytoplankton.
The simplicity of the sampling procedure combined with the sensitivity of the calorimetric
determination
permit many determinations
to be made
during a short time interval with a small
volume of algal suspension. The results
of one such study dealing with the chemical composition of NUV&X!U pelliculosa
during silicon starvation synchrony have
been described by Coombs et al. ( 1967).
OSMUND HOLM-HANSEN
Institute of Marine Resources,
University 0f Calif orniu,
La Jo&z 92038.
REFERENCES
COOMBS, J., W. M. DARLEY, 0. HOLM-HANSEN,
AND B. E. VOLCANI.
1967. Chemical composition of Navicula pelliculosa during silicon
starvation
synchrony.
Plant Physiol.,
42:
1601-1606.
DAL PONT, G., AND B. NEWELL.
1963. Suspended organic matter in the Tasman Sea.
Australian
J. Marine Freshwater
Res., 14:
155-165.
EPPLEY, R. W., R. W. HOLMES, AND E. PAASCHE.
1967. Periodicity
in cell division and physiological behavior of Ditylum
brightwellii,
a
marine planktonic
diatom during growth in
light-dark
cycles.
Arch.
Mikrobiol.,
56 :
305-323.
HOLM-HANSEN,
O., J. D. H. STRICKLAND, AND
P. M. WILLIAALS.
1966. A detailed analysis of biologically
important
susbtances in a
profile
off
Southern
California.
Limnol.
Oceanog., 11: 548-561.
JACOBS, S. 1962. The quantitative
determination of nitrogen by a further modification
of
the indanetrione
hydrate
method.
Analyst,
87: 53-57.
MENZEL, D. W., AND J. H. RYTHER.
1964.
The composition
of particulate
organic matter in the western North Atlantic.
Limnol.
Oceanog., 9 : 179-186.
-,
AND R. F. VACCARO. 1964. The measurement of dissolved organic and particulate
carbon in seawater.
Limnol.
Oceanog., 9:
138-142.
MOORE, S., AND W. H. STEIN. 1954. A modified ninhydrin
reagent for the photometric
determination
of amino acids and related
compounds.
J. Biol. Chem., 211: 907-913.
STRICKLAND, J. D. H., AND T. R. PARSONS. 1965.
A manual of sea water analysis.
Bull. Fisheries Res. Board Can. 125, 2nd ed. 203 p.
THE EFFECT OF TEMPERATURE, LIGHT INTENSITY, AND PHOTOPERIOD
ON COCCOLITH
In a recent paper Watabe and Wilbur
( 1966) presented graphs showing the dependence of coccolith formation in Coccolithus huxleyi (Lohmann)
Kamptner on
temperature. They used two slightly differing methods for estimating the extent
to which coccoliths were formed in their
cultures. Essentially, both procedures de1 This investigation
was
Atomic Energy Commission
34, Project 108.
supported
Contract
by
U.S.
AT( 11-l )-
FORR/IATION~
pended on measuring the relative proportions of naked and coccolith-forming
cells
that grew up at different temperatures,
starting from identical inocula. The inocula
contained a mixture of naked and coccolith-forming
cells.
The percentage
of coccolith-forming
cells was found, by either method, to be
2-3 times greater between 18 and 24C,
which is the temperature range for optimal
growth, than at either 7, 12, or 27C. However, Watabe and Wilbur expressed doubt