Document 6530222

NOTES
AND
COMMENT
CHANGES IN INORGANIC PHOSPHATECONCENTRATION
OCCURRING DURING SEAWATER SAMPLE STORAGE'
The measurement of dissolved inorganic
phosphate is an integral part of many
oceanographic research programs, but it is
only practical to perform the analyses at
sea on larger research vessels. The storage
of samples for later analysis is often required.
Unfortunately,
if samples are
stored without treatment, marked changes
in the level of inorganic phosphate occur.
These changes may be increases arising
from the bacterial or enzymatic decomposition of organic phosphorus or decreases
from utilization
by growing bacteria and
plankton or by adsorption on detritus or
sample bottle walls, or both. The problem
of sample storage for phosphate determinations is therefore of importance.
Various solutions to this problem have
been recommended (for example, Ibanez
Gomez 1933; Harvey 1948; Heron 1962).
Currently, the two most commonly used
techniques
utilize
polyethylene
storage
bottles, with chloroform
treatment
for
short-term
storage (Harvey
1955) and
quick-freezing for long-term storage (Collier and Marvin 1953). However, the effectiveness of even these methods has been
questioned (Murphy and Riley 1956; Hassenteufel, Jagitsch, and Koczy 1963; Jones
1963 ) .
In the initial stages of a survey of estuarine waters of Ecuador, it was noted that
preservation by quick-freezing was providing erratic results, and it was hypothesized
that significant changes were taking place
during the freezing and thawing processes.
Experiments were conducted to establish
a method of preservation suitable for samples of waters characterized by high phosphate levels, dense bacterial and plankton
population, and heavy particulate organic
l This research was partially
supported by the
Inter-American
Tropical Tuna Commission, Scripps
Institution
of Oceanography,
La Jolla, California.
detrital loads. The experiments were designed to test whether the addition of
chloroform to the sample immediately after
collecting and before freezing: a) would
stabilize the samples between thawing and
analysis, b ) would act as a safety factor
should the freezing be delayed or should
the samples accidentally thaw before analysis, and c) would not significantly increase
the concentration of inorganic phosphate.
EXPERIMENTAL
TECHNIQUE
The seawater used for all experiments
was collected from the Ester0 Salado of the
Golfo de Guayaquil, Ecuador. The water
characteristics of the samples at the time of
collection were: 24.4-28.2C, 27%32.9a/co
S, and 4.12-6.58 pg-at. PO,-P/liter.
Samples
were transported (within 20 min) to the
laboratory in 5-gal ( 19-liter ) polyethylene
carboys and transferred to a lo-gal (38liter) Pyrex carboy where they were kept
gently agitated by a magnetic stirrer. The
drawing
of loo-ml subsamples for the
various experiments was started within 45
min of collection. Subsamples were usually
placed in 4-0~ ( 112 ml) polyethylene screwtop bottles and, depending upon experimental design, were either quick-frozen in
an ethyl alcohol-Dry Ice bath or maintained
at room temperature. The frozen samples
were stored at -5 to -lOC until thawed for
analysis. Subsamples incubated at room
temperatures were exposed to ambient temperatures between 18.2 and 21.3C. Chloroform was added to the subsamples with a
volumetric syringe and Whatman No. 1
filter paper was used in those cases where
experimental design required filtration.
All
analyses were conducted using a Beckman
model DU spectrophotometer following the
method presented by Strickland and Parsons ( 1960). Statistical tests for significance were made at the ;p = 0.05 level.
325
326
NOTES
AND
COMMENT
Seven experiments were conducted and
845 samples were analyzed. Depending
upon the expected variability, three to five
samples made up a subset. The complete
data, including statistical treatment and a
detailed description of each experiment,
are available from the author. Only the
most pertinent results are discussed here.
Seawater is saturated with chloroform at
approximately 0.7% v/v. The addition of
this quantity of chloroform effectively inhibits changes in the phosphate concentration of unfrozen seawater samples for up to
6 hr, even under the extreme conditions
represented by the Gulf of Guayaquil
samples (Fig. 1). This experiment was
based on 125 subsamples, with the first
subset receiving no treatment, the second
a 0.4% v/v treatment, etc. The level of
phosphate within the carboy (represented
by the “zero time” values) also decreased
with time similar to the subsamples receiving no treatment.
A sample of seawater may be likened to
a loosely organized but actively metabolizing system in which bacterial and planktonic growth, death, and regeneration and
various extracellular enzymatic and absorptive processes bring about a series of
simultaneous increases and decreases in the
phosphate content of the seawater samples.
The complex cycle of changes that may
develop from the interactions of system
components is well illustrated by one of
the experiments where both frozen and unfrozen samples showed a decrease for the
first 3 hr, followed by a significant
increase before entering the characteristic
longer-term decrease in phosphate concentration ( Fig. 2). The analyses of samples
representing the unfrozen and frozen subsets were 25 hr out of phase, indicating that
the cycle of change was not an artifact introduced by the experimental technique.
It further indicates that the cycle of change
is characteristic of a particular system and
does not change even when the system is
frozen, thereby delaying the development
of the cycle for more than 24 hr.
FIG. 2. A comparison
between the variation in
phosphate concentrations
of unfrozen and frozen/
thawed seawater samples receiving no chloroform
treatment.
FIG. 3. A comparison
between the variation in
phosphate concentration
of unfrozen and frozen/
thawed
a prefreezing
__ ^ seawater samples receiving
chloroform
treatment,
c 6CtiC :
90I
FIG. 1. A comparison
between the variation in
uhosohate concentration
of unfrozen seawater samples deceiving chloroform
treatments
(in % v/v).
The initial concentrations
varied from 4.41 to 5.28
pug-at/liter.
RESULTS
AND
DISCUSSION
NOTES
TABLE
1.
AND
327
COMMENT
A comparison of the inorganic phosphate concentration
water treated with 0.770 v/v chloroform.
Values
of unfroxen and frozen/thawed
in pg-at. P04-P/liter
Unfrozen
Frozen
-CHCL,
Time
interval
Initial
+ 35
+ 70
+205
+405
+14.5
+ 27
+ 48
+ 72
+ 96
min
min
min
min
hr
hr
hr
hr
hr
+CHCL,
-CHCL,
SD
Mean
SD
Mean
SD
Mean
5.174
5.265
5.194
5.104
5.046
5.098
5.043
3.032
3.168
2.791
0.118
0.059
0.090
0.040
0.025
0.071
0.159
0.599
0.459
0.625
5.229
5.276
5.288
5.192
5.238
5.124
5.009
4.774
5.239
5.376
0.024
0.063
0.027
0.048
0.088
0.068
0.182
0.304
0.196
0.115
5.147
5.005
5.009
4.882
4.921
4.672
3.480
2.668
2.051
2.068
0.032
0.134
0.091
0.140
0.045
0.024
0.245
0.242
0.925
0.179
5.198
5.194
5.167
5.167
5.083
5.236
5.344
5.299
5.269
5.242
Total
number
of samples:
Subsamples
per treatment
As a working hypothesis, it can be assumed that the two predominant system
components are the increase on phosphate
concentration resulting from the enzymatic
decomposition of organic material and the
decrease resulting from phosphate utilization by a developing bacterial population,
These experiments suggest that the enzymatic process often predominates during
the initial 3-hr period (Table 1 ), and thereafter an exponentially developing bacterial
population commences to utilize phosphorus
in excess of that released by the enzymatic
process, resulting in a net decrease in phosphate concentration.
Theoretically,
it would be desirable to
remove the particulate
organic material
that acts as substrate for many of the
processes, either by filtering or centrifuging
the samples, a concept supported by the
work of Heron ( 1962). In a practical
sense the time delay introduced by such
set:
min
SD
0.071
0.043
0.075
0.080
0.122
0.095
0.195
0.056
0.107
0.053
160
4
sample treatment could allow a larger
change to take place than if the entire
system were quickly deactivated by, for
example,
quick-freezing.
Unfortunately,
freezing can increase the rate at which
the phosphate
concentrations
decrease
( Fig. 2). Such an increase in net change
could result from the disruption of organic
material in the sample, thereby providing
increased substrata for bacterial growth or
adsorptive processes or both. However,
since the decrease can be minimized by
the chloroform treatment ( Fig. 3) it seems
reasonable to assign it to the bacterial component.
When samples were treated with 1.2%
v/v chloroform, a significant difference was
often, but not always, noted between the
filtered and unfiltered samples; this indicates that leaching can take place under
certain circumstances (Fig. 1 and Table 2).
Of additional interest, the data suggest that
values, of inorganic phosphate concentrations
in filtered
1.2yS v/v chloroform
treatment.
Values in pg-at./liter
Time
+15
Treatment
Unfiltered
Filtered
Difference
Standard error of
the difference
+CHCL,
Mean
TABLE 2. A comparison, with initial
unfiltered
seawater, receiving
sea-
+45
min
+90
and
interval
min
+180
min
+24
hr
-0.009
+0.012
0.021
-0.012
-0.074
0.062
+0.011
-0.074
0.085
+0.046
-0.044
0.090
+O.lOS
-0.091
0.199
0.012
0.019
0.021
0.028
0.016
328
NOTES
ANDCOMMENT
samples treated with chloroform appreciably in excess of saturation exhibit a much
higher variability
( coefficient of variation
at 1.2% v/v = 0.344) than samples receiving chloroform treatment at or near saturation (coefficient of variation at 0.7% v/v =
0.283), thus emphasizing the need for care
during the addition of chloroform for treatment of phosphate samples.
Perhaps the most important aspect of
these experiments is to emphasize that the
rate of change of phosphate level with time
is not constant but depends upon the biological and physical characteristics of the
sample. It can be expected to vary from
region to region, from season to season,
and it is doubtful whether there is any one
sample treatment that will fulfill the requirements of all experimental designs.
CONCLUSIONS
a. The low levels of chloroform occasionally recommended to stabilize samples
(as low as 0.05% v/v) are often insufficient
to achieve stabilization in highly productive regions.
The optimum
chloroform
treatment is slightly less (0.7% v/v) than
that required to saturate the sample.
b. If the samples are quick-frozen without chloroform treatment, it is imperative
that they be analyzed immediately upon
thawing. The process of freezing and thawing can bring about a marked increase in
the rate of change of inorganic phosphate
level.
c. The addition of chloroform at levels
near saturation (O.&OS% v/v) does not
significantly increase the level of inorganic
phosphate.
d. The rate of change of inorganic phosphate in quick-frozen samples treated with
AN ICE
RECORD
OF
Wind-induced waves are reflected in the
University of Wisconsin Laboratory of Limnology boat slip to produce a standing
wave that crests approximately 3 m from
the boat slip door which acts as a reflector.
Ice is built up along the side of the slip by
chloroform is significantly less than that in
frozen or unfrozen samples without chloroform.
e. The addition of chloroform to samples
before freezing tends to stabilize the samples during the time between thawing and
analvsis.
,
MALVERNGILMARTIN
Hopkins Marine Station,
Stanford University,
Pacific Grove, California
93950.
REFERENCES
COLLIER, A. W., AND K. T. MARVIN.
1953. Stabilization
of the phosphate ratio of sea-water
by freezing.
Bull. U.S. Bur. Fisheries,
79:
71-76.
1948. The estimation
of phosHARVEY, H. W.
phate and total phosphorus in sea waters. J.
Marine Biol. Assoc. U.K., 27: 337-359.
HARVEY, J. W.
1955. The chemistry and fertility of sea waters.
Cambridge,
London, England. 224 p.
HASSENTEUFEL, W., R. JAGITSCH, AND F. F. KOCZY.
1963. Impregnation
of glass surface against
traces.
Limnol.
sorption
of
phosphate
Oceanog., 8: 152-156.
HERON, J. 1962. Determination
of phosphate in
water after storage in polyethylene.
Limnol.
Oceanog., 7: 316-321.
IBANEZ GOMEZ, 0.
1933. Note on the effect of
salts in the determination
of phosphates
in
sea-water
by Deniges method.
J. Conseil,
Conseil Perm. Intern.
Exploration
Mer, 8:
326-329.
JONES, P. G. W.
1963. The effect of chloroform
on the soluble inorganic phosphate content of
unfiltered sea-water. J. Conseil, Conseil Perm.
Intern.
Exploration
Mer, 28: 3-7.
MURPHY, J., AND J. P. RILEY.
1956. The storage
of sea-water samples for the determination
of
dissolved inorganic phosphates.
Anal. Chim.
Acta, 14: 818-819.
STRICKLAND, J. D. H., AND T. R. PARSONS. 1960.
A manual of sea water analysis.
Bull. Fisheries Res. Board Can. 125. 185 p.
STANDING
WAVES
splashing water and is undercut by wave
action. The ice, therefore, serves as a record of wave height.
The photographs were taken at about
1200 CST on 4 December 1964. The maximum diameter of pancake ice shown in