r
L.
903979002
[ U.S. ENVIRONMENTAL PROTECTION AGENCY
r
r
c
r
u
C
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L
C
MIDDLE ATLANTIC REGION-III 6th and Walnut Streets, Philadelphia, Pennsylvania 19106
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EPA 903/9-79-002
ALGAL NUTRIENT STUDIES OF THE
POTOMAC ESTUARY
(Summer 1977)
Annapolis Field Office
Region III
Environmental Protection Agency
Joseph Lee Slayton
E. R. Trovato
-------
DISCLAIMER
The mention of trade names or commercial products in this report
is for illustration purposes and does not constitute endorsement or
recommendation by the U. S. Environmental Protection Agency.
-------
TABLE OF CONTENTS
Page
I. Introduction 1
II. Conclusions 6
III. Experimental 7
IV. Discussion of Results 18
V. Recoirmendations 30
VI. References 31
-------
TABLES
Page
1. Station Locations 3
2. Algal Growth/Assay Media 10
3. Summary of Assay/Analysis Results 19
4. Ammonium Uptake Rates/Nitrogen Distribution 24
5. N2 Fixat ion/Acetylene Reduct ion 26
6. Filtered vs Centrifuged Methods 29
-------
FIGURES
Page
1. Map of Study Area 2
2. Sample Preparation Flow Chart 8
3. Standard Curve for Alkaline Phosphatase Activity 13
4-7. Chlorophyll a vs HMI 20-23
-------
I. Introduction
During the summer of 1977 an intensive survey of the middle reach
of the Potomac River (Figure 1, Table 1) was undertaken by the A.F.O.
As part of this work the nutrient requirements of the phytoplankton
present were studied using the following laboratory tests: NH.-N
uptake; alkaline phosphatase enzyme activity; extractable surplus
orthophoshate; tissue analysis for carbon, nitrogen and phosphorus
content; and nitrogen fixation by acetylene reduction. These bio-
assays were conducted in the Potomac from Gunston Cove to Possum
Point during August and September 1977.
The ammonium uptake test was designed to assess the bio-avail-
ability of nitrogen to algae. Algae are spiked with ammonia and if
a rapid rate of absorption of nitrogen with time is observed this
signifies that nitrogen is limiting potential algal growth.
Algae have the ability to store phosphorus when it is encountered
P
in amounts beyond the immediate biological need. Previous studies
have determined that this stored phosphorus is easily extracted and
is thought to be stored as orthophosphate; polyphosphate chains and/
or as very labile organic compounds which breakdown to orthophosphate
with heat (100°C). Algae containing significant luxury phosphate are
not limited in their growth by phosphorus.
When ambient bio-available phosphorus is depleted in the water
column, algae may activate the production of alkaline phosphatase
enzyme. This enzyme cleaves phosphate from the stored luxury phosphate
chains/compounds. The presence of significant alkaline phosphate enzyme
is indicative of algae limited in their potential growth by phosphorus
-1-
-------
-------
Station Number
P-8
P-4
1
1-A
2
3
4
5
5-A
6
7
8
8-A
9
10
10-B
11
12
13
14
15
15-A
16
Station Number
S-l
S-2
S-3
S-4
S-5
S-6
S-7
S-8
Table 1
Station Name
Chain Bridge
windy Run
Key Bridge
Memorial Bridge
14th Street Bridge
Hains Point
Bellevue
Woodrow Wilson Bridge
Rosier Bluff
Broad Creek
Ft. Washington
Dogue Creek
Gunston Cove
Chapman Point
Indian Head
Deep Point
Possum Point
Sandy Point
Smith Point
Maryland Point
Nanjemoy Creek
Mathias Point
Rt. 301 Bridge
Treatment Plant Name
Piscataway STP
Arlington STP
Blue Plains STP
Alexandria STP
Westgate STP
Hunting Creek STP
Dogue Creek STP
Pohick Creek STP
RMI
0.0
1.9
3.4
4.9
5.9
7.6
10.0
12.1
13.6
15.2
18.4
22.3
24.3
26.9
30.6
34.0
38.0
42.5
45.8
52.4
58.6
62.8
67.4
Buoy Reference
C "1"
FLR-231 Bell
C "87"
N "86"
FL "77"
FL "67"
R "64"
FL "59"
N "54"
R "44"
N "40"
N "30"
G "21"
N "10"
C "3"
-------
and forced to draw upon reserve phosphate to meet their nutrient re-
quirements. If phosphorus was depleted to a critical level in the
estuary, measured concentrations of luxury phosphate would be expected
to decrease and the activity of alkaline phosphatase would be expected
to increase. Studies^ have found that these changes are not immediate
and a lag time occurs before the biological changes, related to phos-
phorus deficiency, are expressed.
Several species of algae, notably blue-green algae, have the ability
to meet their nitrogen requirement by reducing free nitrogen (N2) from
the air and incorporating it into cellular organic compounds. Algae
grown in an environment containing adequate fixed nitrogen (NH^ or NO^)
do not fix N2 without a preliminary starvation period during which the
nitrogenase enzymes can develop.^ The triple bonds of N2 are extremely
stable and breakage of these bonds involved in nitrogen reduction dic-
tates that fixation requires considerable energy input. Cells capable
of fixing nitrogen will use NH^ or NOJ preferentially because less
energy is required.^^
The nitrogenase enzyme complex is comprised of two major protein
components, Fe-protein and Mo-Fe-protein, each composed of several
subunits. Nitrogen is reduced by the enzyme complex to ammonia as
electrons flow from a reducing agent to the Fe-protein, then to the
Mo-Fe-protein and finally to nitrogen. The ammonia formed in these
processes is subsequently employed in amino acids, which are the
building blocks of protein. The nitrogen fixing activity of algae
is often restricted to specialized cells termed heterocysts. These
-------
are enlarged, clear (reduced pigmentation) cells, which apparently do
not produce 02 since oxygen is thought to deactivate nitrogenase.10
It has been found^ that nitrogenase can reduce a variety of
multiple bond substances in addition to molecular nitrogen. These
include N02, NJ, RCN, RNC, and RCCH. Acetylene is reduced by this
system to ethylene which is not further affected. Algae actively
fixing nitrogen will produce ethylene when incubated with acetylene.
Bulk elemental analysis of the phytoplankton standing crop gave
an indication of the carbon, nitrogen and phosphorus bound in algal
cells. This information when ratioed to chlorophyll a. gives a means
of predicting algal C, N, and P from the more easily measured
chlorophyll a concentration. To increase the comparability of these
elemental analyses to cells of different sizes, the cell concentrations
of C, N, and P were also reported on a dry weight basis. A problem
with the comparability of elemental analysis is the varying amount of
sheath material observed with different algal species. This problem
makes it difficult to establish a reliable relationship between elemen-
tal composition ratios measured and the nutrient status of the algae
being studied.
-5-
-------
II. Conclusions
A. The average composition of the phytoplankton present in the
study area was (mg/ug);
Org C/ = 0.028; PO,/ = 0.002; TKN-N/ = 0.007
/chlor a /chlor a /chlor a
The predominate phytoplankton species present during the study
period was the blue-green algae Oscillatoria spp.
B. No significant alkaline phosphatase activity was detected
during this study and together with the average luxury phosphate
of 0.45 mg PO^/100 mg algae (dry) suggested that phosphorus was
not limiting growth.
C. No significant nitrogen fixation was detected during the
study period.
D. Ammonium uptake rate varied markedly with station location
and a negative correlation, r = -.80 (n = 4), was determined
for ammonia absorption rate vs (N02 + NO-j)-N concentration. The
absorption rate increased from 0.0 ug NH^-N/10 mg algae/hr. at a
(N02 + NO.)-N concentration of 0.352 mg/1 at Chapman Point to
7.5 ug NH.-N/10 mg algae/hr. when the nitrate + nitrite-nitrogen
4-
concentration became less than 0.04 mg/1 at Possum Point. This
indicated that the reach from Chapman Point to Possum Point was
becoming nitrogen limited.
E. Approximately 50$ of the algal TKN-N was refractory to the
Technicon Autoanalyzer (phenolate/helix method) without preliminary
manual digestion.
F. Elemental analysis data for phosphorus was obtained by Millipore
filtration and by centrifugation. The results obtained were not
significantly different.
-------
III. Experimental
A. Chlorophyll a was determined on an untreated portion of the
sample via a 90$ acetone extraction of a Millipore filtrate from
100 ml of the sample.5
B. Sample preparation procedures (Figure 2) required the exist-
ence of a significant bloom (>50 ug/1 chlorophyll aj. so that
errors due to "non-agal particulate material" would be minimized
and so that sufficient algae could be concentrated to run the
necessary tests. The sample preparation procedures involved:
1. Centrifuge algal sample in 50 ml aliquots (8) at 3K HPM
for 5 minutes. The sample was stored at 4°C during this
procedure.
2. Collect 10 ml of supernatant as a blank from each
centrifuge tube in a 125 ml Srlenmyer flask stored on ice.
Discard all but a few drops of the remaining liquid in the
tubes.
3. Resuspend pellets in >.50 ml of river v/ater blank (super-
natant). The volume of the sample centrifuged and the volume
to which the resultant algal pellet was diluted was recorded.
Microscopic examination revealed that no apparent morphological
damage was suffered by the predominant phytoplankton species
present.
C. Elemental Analyses
1. TKN-N (NH_ plus organic nitrogen): 5 ml of algal
suspension was diluted to 25 ml in a volumetric flask using
Super Q - Milli Ro deioniaed water. A blank was run using
-7-
-------
Figure 2
Sample Premration
Sample (stored on ice)
1-4 liters
Centrifuged (3K RPM - 5 minutes)
(stored on ice)
Algal Pellet
Supernatant Discarded
(except for 100-500 ml)
Resuspens ion of Pellet with clear filtrate
\
Algal Suspension
m>
Appropriate subsamp\es and dilutions
N
"N2 Fixation
Elemental
Analysis
TKN-N TOC TP
Alkaline
Phosahatase
Luxury
Phosphate
Absorption
-------
5 ml of supernatant river water diluted to 25 ml in Super Q -
Milli Ro deionized water.
These samples were then manually digested: 10 ml aliquot
of each was placed in reflux tubes and 8.0 ml of
digestion solution was added. The tubes were placed over
flame until boiling and reflux stopped. The contents of
the tubes were washed with deionized water and brought to
50 ml using a graduated cylinder.
The resultant digests were analyzed using the Technicon
Autoanalyzer phenolate method.
2. TOG: 5 ml of algal suspension was diluted to 25 ml in
a volumetric flask using Super Q deionized water. A blank
was run using 5 ml of supernatant river v/ater diluted to 25
ml in Super Q deionized water. The TC and 1C were then
determined on a Beckman 915 TOO analyzer.
3. Total Phosphate: 25 ml of sample and blank were prepared
as above by dilution of 5 ml of sample to 25 ml with deionized
water. The sample and blank were placed in aluminum foil
covered pyrex test tubes to which ammonium persulfate and
sulfuric acid were added and autoclaved at 15 psi for 30
minutes. The digests were then analyzed for total phosphate
by the Technicon automated ascorbic acid reduction method.
-------
D. Table 2
Growth Media1 used in laboratory studies:
Gorham ' s
Complete
Solution
me/1
K2HP04
NaN03
MgS04-2H20
CaCl2.2H20
Na2Si03.9H20
Na2CO^
Ferric Citrate
Citric Acid
(Na2)E.D.T.A.
39.0
496.0
75.0
36.0
53.0
20.0
6.0
6.0
1.0
Gorham 's
(Minus P)
Solution
me/1
0.0
496.0
75.0
36.0
58.0
20.0
6.0
6.0
1.0
Gorham ' s
(Minus N)
Solution
me/1
39.0
0.0
75.0
36.0
58.0
20.0
6.0
6.0
1.0
Volume of Stock
ml per liter cone, stock
1 ml
10 ml
1 ml
1 ml
10 ml
1 ml
10 ml
1 ml
1 ml
19.5g/500 ml
24.8g/500 ml
37.5g/500 ml
18.0g/500 ml
2.9g/500 ml
10.0g/500 ml
0.3g/500 ml
3.0g/500 ml
l.Og/500 ml
E. Luxury Phosphate^
1. Spin down two sets of 5 ml aliquots of algal suspension
at 3K RPM for 5 minutes and discard supernatant.
2. Lightly wash pellet with 10 ml of Gorham's (P-minus)
solution adjusted to pH 7 with acetic acid.
3. Pour off liquid and wash cells with Gorham's (P-minus)
pH 7 solution into an Erlenmyer flask to a total volume of 40 ml,
4. Cover with aluminum foil and place one flask into
boiling water for 60 minutes.
5. The other set is immediately centrifuged and the super-
natant analyzed for PO^.
6. After one hour repeat step #5 for the first extracted set.
-1C-
-------
7. Calculate the net (by difference) extracted PO^/
100 rag algae (dry weight).
Definition: Extracted algae that give less than 0.03 mg PO//
100 rag algae (dry weight) are considered to be
phosphorus limited.
F. Alkaline Phosphatase Activity2
1. Centrifuge 5 ml of algal suspension and discard supernatant.
2. Wash pellet with 10ml of Gorham's (P-minus) adjusted to
pH 9.0 with acetic acid.
3. Wash cells into Erlenmyer flask with 32ml of Gorham's
(P-minus) pH 9.0 solution.
4. Add 4 ml of 1M THIS solution which is also 0.01 M MgCl2
and adjust pK to 8.5 with acetic acid.
5. Add 4 ml of p-nitrophenyl phosphate solution (30 mg/100 ml).
6. Incubate glass stoppered flask with mixing for 15 to 20
minutes at 35-37°C.
7. Stop when color is within standard curve by adding 0.5 ml
of orthophosphate (20 mg PO^/ml) stock solution.
8. Filter material through .45 u Millipore membrane filter and
analyze liquid.
9. Read absorbance at 395 nm in 2 cm cells with 2.0 nm slit.
10. Run standard curve of nitrophenol, (color is pH dependent)
with:
32ml Gorham's (P-minus) adjusted to pH 9.
4ml of the Tris Buffer.
4^1 of standard solution.
-11-
-------
11. Standard curve concentrations (after reagent addition):
0; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0xlO~5M p-nitrophenol.
a. Preparation of standard solutions:
(1) Prepare a stock of p-nitrophenol of 1.3911g/l
(2) 20 ml of this solution was diluted to 200 ml
with deionized water to generate a working stock.
(3) 5; 10; 15; 20; 25; and 30 ml of the working
solution is diluted to 100 ml with deionized water
to generate: 0.5; 1.0; 1.5; 2.0; 2.5; 3.0xKT4M
solutions .
(4) When 4 ml of these solutions is diluted to 40 ml
total with reagent, the standard curve at the 10~^M
level is generated.
b. Characteristic Calibration Curve (Figure 3)
mg
Concentration
0.0
0.5 x 10"5M
1.0 x 10~5M
1.5 x 10'5M
2.0 x 10~5M
2.5 x 10~5M
3.0 x 10~5M
Absorbane
0.000
0.193
0.362
0.536
0.733
0.902
1.076
p-nitronhenol
0.00
0.70
1.39
2.08
2.78
3.45
4.17
-12-
-------
I i I i i i f j 11
, ; \ i ; ! 4 I f ; l i f i ; \
Figure 3
STANDARD CURVE FOR ALKAKINE FHOSFHATASE ENZYME ACTIVITY
1.000
.900 -
.800 --
.700 - -
.600 - -
CO
8 .500--
t»
B
o
CD
.400 --
.300
.200 --
.100 --
0
I
n , , , ^ , , , , 2f-~i -' > ft ' r- r
mg p-nitrophenol
-------
12. Determine mu moles of nitrophenol liberated/hr. per
milligram of algae (dry weight).
Definition:
a. 1 unit of enzyme activity is equivalent to 1.0 mu
mole of nitrophenol per hour per mg dry weight.
b. xv 1000 enzyme units/mg algae/hour represent algae
considered phosphorus limited.
c. This test is generally a confirming test since
changes in enzyme activity per changes in nutrient level
are slow to occur.
A check standard of bacterial alkaline phosphatase
(12 units/mg from the Worthington Biochemical Corporation)
was run as a positive control check with each batch of samples
analyzed.
G. Ammonia Absorption Rate
1. Centrifuge 2 sets of 5 ml aliquots of algal suspension
at 3K RPM for 5 minutes. Discard the supernatant.
2. Pre-wash pellets with 10 ml of Gorham's N-minus, adjusted
to pH 8.0 with acetic acid and discard liquid.
3. Wash pellets into a flask with 30 ml of Gorham's N-minus
adjusted to pH 8.0.
4. Spike both sets with 0.5 mg NH^C1-N/1.
5. Centrifuge the first set immediately and analyze super-
natant for NH^-N.
6. Incubate the other flask in the dark at 63°C with occa-
sional mixing for one hour.
-14-
-------
7. Centrifuge and assay supernatant for NH^-N.
Threshold Limit; Nitrogen-starved algal cells were found to
assimilate NH^-N 4 to 5 times more rapidly than normal cells
under optimum nitrogen conditions. The limit cited is that
algae are considered nitrogen limited if they absorb more than
+ 7
15 ug NH,-N/10 mg dry algae per hour . This threshold rate,
however, was observed to vary from species to species. The
comparison of NHt-N assimilation rates measured for algae from
different locations in the Potomac River study area, associated
with different in situ nitrogen concentrations was thought to be
more meaningful. A drastic rate of increase (--4 or 5 times)
from one location to another was taken to indicate changes in the
availability of nitrogen for assimilation purposes and suggested
that nitrogen was limiting growth.
H. N2 Fixation3
1. Sample preparation:
a. Concentrate 2 liters of sample (770906-16, 17, 19)
for algae as described previously and bring to 25 ml
total volume with river water supernatant. (Blank)
b. Add 10 ml of each concentrate to two 40 ml septum
vials.
2. Seal the vials with an injectionable septum (air tight
pharmaceutical type).
3. Inject 1.5 ml of acetylene (C2H2) into each vial using a
5 ml disposable syringe.
4. Immediately inject 0.2 ml of 5N HgSO^ into one set to act
as a control blank.
-15-
-------
5. Shake all flasks and vent by pricking with a hypodermic
needle.
6. Incubate in a 'rater bath in direct sunlight for 1 1/2
hours at 29°C (~ambient surface water temperature).
7. The reduction reaction was stopped by the injection of
0.2 ml of 5N H2SO^. The samples were stored at 4°C until
gas chromatographic analysis.
8. The G.C. and experimental conditions were as follows:
a. Column temperature: 50°C
b. Flow 25 ml/minute of Helium
c. Column: pcrapack N, 30-100 mesh, 6 ft. with 0.2 mm I.D.
d. Retention time:
Ethylene: 1.75 minute
Acetylene: 3.55 minute
e. Room temperature: 24°C; 30.13" Hg barometric pressure.
9. Chlorophyll a. concentration was determined as described
previously, and using the measured TKN-N/chlorophyll a. ratio
(0.007) the mg of algal TKN-N was determined and the results
were reported as ng acetylene reduction per mg algal TKN-N.
10. The volume of the vials (60.0 ml) was determined using
the weight of water at room temperature.
11. The procedure for diluting the stock ethylene was crude
but the reduction test was run more as a qualitative assay to
detect significant nitrogen fixation rather than a strictly
quantitative rate determination. The dilution and spikes
were as follows:
-------
a. Stock ethylene preparation (rr.w. 28.04 gm/mole)
(l) Ethylene was assumed an ideal gas or PV = nRT.
R = 0.082 1 atm K"1 mol"1
T = 24°C or 297.14 K
P = 30.13" Hg x 2.54 cm/in x jj^M = 1.0070 AHA
V = 60 ml bottle or 0.060 1
PY (1.007) (0.060)
n = OT = {0!082) (297) = °'Q°2^ m°leS °f gES
in stock
(2) Inject 0.5 ml directly into 60 ml gas tight vial:
.00248 moles
ml = Q0021 moles or <57g
oO ml
b. Dilute 5/60 by volume using a gas tight syringe and
gas tight bottle:
.00248 moles x -^ = .X0207 moles in dilution
(1) Inject 0.5 ml directly:
.0002(77 moles x 0.5 ml = .00000173 mole or .049 mg
60 ml
(2) Inject 1.0 ml directly:
. 000207 moles x 1.0 ml = .0000034 mole or .095 mg
60 ml
c. Dilute 1/60 by volume using a gas tight syringe and
gas tight bottle:
.00243 moles x _1 = .0000413 moles in dilution
60 ml
Inject 0.5 ml directly:
.0000413 x 0.5 ml = .00000034 mole or .0095 mg
60
The area of the G.C. peaks for these standards was used
to determine the concentration in the unknown samples .
-------
IV. Discussion of Results
The elemental analyses and special bioassay results are compiled
in Table 3. The location and chlorophyll a distribution of the stations
sampled for this study are given in Figures 4-7. It should be empha-
sized that these results are based on the overall phytoplankton standing
crop. The alkaline phosphatase activity (>1000 enzyme units/mg algae/
hour)2 indicative of phosphorus starved algal cells was not encountered
in any of the study samples. The average luxury phosphate measured,
0.45 mg PO//100 mg algae (dry), was in excess of the established thres-
hold level for phosphorus limitation of 0.03 mg PO^/100 mg algae (dry).2
Little difference was observed in luxury phosphate measured at the up-
stream and downstream stations. The inorganic phosphate concentration
increased in the bloom area with an average of 0.214 mg/1 PO^ measured
over the study stations. The alkaline phosphatase, luxury phosphate,
and ambient inorganic phosphorus data indicated that adequate phos-
phorus was present for maximum growth during the study period.
The inorganic nitrogen source for algal growth was limited to
(N02 + NO.O-N in the bloom area, Table 4. This was a result of the
rapid nitrification of the ammonia entering the river upstream of the
study area. The distribution of measured ammonium uptake rates
relative to (N02 + NO^)-N measured in the Potomac are also included
in Table 4. Though this data is sparse, a significant increase in
uptake rate occurred with (N02 + NO^J-N depletion on the August 29
analysis between Chapman Point (0.0 ug NH^/IO mg algae/hour) and
Possum Point (7.5 ug NH^/10 mg algae/hour). This data (n = 4) was
used to generate the correlation coefficient of -0.8. The increased
-13-
-------
Table 3
SUMMARY OF ASSAY/ANALYSIS RESULTS
Locat ion
Chapman Pt.
Indian Head
Deep Pt.
Guns ton Cove
Chapman Pt.
Indian Head
Chapman Pt.
Indian Head
Deep Pt.
Possum Pt.
Date
8-1-77
8-1-77
8-3-77
8-22-77
8-22-77
8-22-77
8-29-77
8-29-77
8-29-77
8-29-77
Average
Sta.
9
10
10-B
8 -A
9
10
9
10
10-B
11
mg/ug
.036
.028
.029
.037
.026
.037
.013
.012
.027
.034
.028
mg/mg
W
Tss
.152
.213
.211
.238
.283
.157
.162
.213
.376
,43?
.244
mg/ug
P04
.003
.002
.002
.003
.002
.003
.001
.001
.001
.002
.002
mg/mg
TSS
.014
.017
.015
.018
.018
.012
.015
.022
.016
.020
.017
Sample
TP Pi
.503
.472
.4»9
.764
.751
.736
.799
.759
.850
,846
,o94
.161
.157
.157
..227
.238
.259
.176
.205
.275
.282
.214
mg/ug
TKN-N
Chi. a
.013
.008
.009
.008
.006
.008
.003
.003
.004
.005
.007
mg/mg
TKN-N
TSS
.055
.060
.065
.052
.060
.035
.038
.058
.052
.050
.053
1
Luxury
Phosphate
.37
-
-
.25
.38
.15
.51
.67
.53
.73
.45
2
Alkaline
Phosphatase
ND
-
-
ND
ND
ND
ND
ND
ND
ND
<57 E.U.
3
0.3
-
-
0.3
0.2
0.3
0.0
0.4
4.2
'7,5
ug/1
Chi.
60.
66.
76.
306
264
283.
261
300
294
199,
a
0
0
5
5
5
1. Luxury Phosphate: mg PO//100 ing Algae (dry)
2. Alkaline Phosphatase: 1 E.U. = 1.0 mu Moles Nitrophenol/mg algae/hour
3. NH^-N Absorption: ug NH/-N/10 mg Algae/hour
-------
Figure 4
ppb Chlorophyll a
-------
Figure 5
. -t-
-------
Figure 6
+ r*
o:-
o
CM
-
CM
O
m
ppb Chlorophyll a.
+ O
-22-
-------
.+. tt. .+....+....+.... -K ...
j:o i . i i i t i i ,
+....+....+....-*-....+..
j i ! i i i i I
..+.... +.,. . , +.
i t i i i
+. . . + .
- Sta. 13 Smith Point
24S +
210 +
175 +
tJ
a'
p
H
O
O
If
"^
M
H
140
105
- Sta. 11 Possum
Point
ep Hoint
Sta.
9
Chapmai Point
A Aunsion Cove tt - Sta. 12 Sandy foint
'- Sta. 10 Indian Head
Chlorophyll a
CD
0. 0 +M
+.
4.
+....+....+..
20 28
24
.+....+....+....+....+..
36 44 52
40 43
KM I
+.
64
ro
0.
SEP 6 1977
-------
Table 4
Ammonium Uptake Rates/Nitrogen Distribution
Location
Chapman Pt.
Guns ton Cove
Chapman Pt.
Indian Head
Chapman Pt.
Indian Head
Deep Pt.
Possum Pt .
Location
Guns ton Cove
Chapman Pt.
Indian Head
Deep Pt.
Possum Pt.
Sandy Pt.
Smith Pt.
Date
8-1-77
8-22-77
8-22-77
8-22-77
8-29-77
8-29-77
8-29-77
8-29-77
Date
Sta . *1
8-A
9
10 0
10-B
11
12
13
Sta. ug NH^-N/10 mg Algae/hour mg NH^-N/ug
9 0.3 0.4
8-A 0.3 0.5
9 0.2 0.2
10 0.3 0.5
9 0.0 0.0
10 0.4 8.0
10-B 4.2 12.2
11 7.5 23.7
8-1-77 8-22-77 8
Chi. a/hour
x 10"5
x 10"5
x 10"5
x 10"5
x 10"5
x 10"5
x 10~5
;-29-77
MH^f (N02 + N03)-N NH+f (N02 + N03)-N NH+'j1 (N02 + N03)-N
.928 0.3 .131
.710 0.2 .130 0.0
.3 .495 0.3 .089 0.4
.378 ND L.2
.122 ND 7.5
**ND .126
ND .317
.352
.110
ND
ND
ND
ND
Note: The NH^-N concentration was less than 0.02 irg/1 over these dates and
stations except for Sta. 13 on 8-22-77 which had an NH^-N concentration
of 0.052 mg/1.
*NHtt = ug NH^-N/IO mg Algae/hour
^ 4
**ND = not detectable = <0.04 mg (N02 + NCO-N/1
-------
rate of ammonium absorption (>7.5x) corresponded to a decrease in
inorganic nitrogen from 0.352 mg/1 (N02 + NOo)-N at Chapman Point to
less than 0.04 mg (N02 + NO^-N/l at Possum Point. The rate of NH^-N
absorption by algae and aquatic weeds in the dark has been shown to be
4-5 times greater for plants which are N-limited as compared to plants
n
with sufficient available nitrogen. This indicated that this reach of
the Potomac, Chapman Point to Possum Point, was becoming nitrogen
limited .
As the (N02 + NO^)-N source was depleted it was of interest
whether nitrogen fixation might be occurring. Nitrogen fixation had
been observed in a marine species of Oscillatoria.1*-1 but fresh water
species are considered non-N2 fixers,11 In general algae may utilize
N2; NH^; or (N02 + N03)-N as their nitrogen source if the proper enzyme
systems are present and activated. The use of Np and (N02 + NO^)-N
require additional reactions and energy. Algae capable of utilizing
these sources of nitrogen preferentially select ammonia, since it
involves the most efficient source of cellular nitrogen.^'7 The
nitrogen fixation data is compiled in Table 5 and indicates that no
significant acetylene reduction (<.073 n moles C2H,/mg N/hour) was
measured although ambient inorganic nitrogen became non-detectable
between Deep Point and Sandy Point on September 6 when the acetylene
fixation procedure was carried out. Values of 126-230 n moles C2H,/mg
N/hour have been reported as indicative of high efficiencies of
acetylene reduction,11 and rates of 30-60 n moles C2H^/mg N/hour are
considered significant.
As a check on the laboratory procedures involved in centrifugation
-------
Table 5
FIXATION/ACETYLENE REDUCTION
Eth.ylene Measurements
Location
Date
Vol. Injected
Sta. cc
Acetylene Srandard
Air Blank
Deep Pt.
Possum Pt.
Sandy Pt.
Smith Pt.
Standards :
Ethylene
Ethylene
9-6-77
9-6-77
9-6-77
9-6-77
5/60
5/60
10-B
10-B Blk
11
11 Blk
12
12 Blk
13
13 Blk
0.2
0.5
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
0.5
Net Area
Ethylene Area (Blk)
637
(ND Level)
1541 0
2158
2103 289
1817
2225 302
1923
2950 709
2241
4,150,447
3.781.067
x 3,965,757
Ethylene
(mp)
0
ND
ND
8.7 10"6 mg
.049 mg
.049 mg
Acetylene Area
10, 129, 733
10, 894, 579
12, 074, 981
11, 794, 779
11, 672, 681
10, 765, 493
11, 584, 374
11, 032, 906
10, 681, 130
Agal TKN-Nitropen (Ethylene Experiment)
Location
Indian Head
Deep Pt.
Date Sta.
9-6-77 10
9_6-77 10-B
Volume
of Sample
Concentrated
-
2 liters
Final
Volume of
Suspension
-
25 ml
Chi. a Cone.
Volume TKN-N/ in Orginal
Incubated Chi. a Sample ug/1
_
10 ml .007 180
mg Ambient Inorganic
Algal Nitrogen (mg/1)
TKN-N (NOg + NO-})-N NH^-N
-
1.008
.725 ND (-, .04)
.172 ND
-------
Co co ha o
S P O ro
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c+
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till
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z >
r? 5 cr
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-------
and sample concentration, dilution, etc., the elemental analysis results
were compared to a second method (filter method). The results are com-
piled in Table 6. The columns designated "total" represent analytical
results (C, N, and P) on the unaltered samples. The column labeled
"filtered" represents the elemental analyses of the filtrate after
filtration through 0.45 u Millipore filters. The algae were held on
the filter and the differences of filtered and unfiltered results were
taken as the algal material. A paired t-test of the phosphorus results
revealed that there was no significant difference at the 95% confidence
level and 9 degrees of freedom between the results of the two methods
with t = 1.195. The nitrogen data (Table 6) was consistently lower
for the filtered experiments. The TKN-N for the filtered data did
not incorporate the preliminary manual digestion used in the centrifuge
procedure. The results suggest that 50$ of the algal nitrogen was
refractory to the TKN-N Technicon Autcanalyzer without preliminary
manual digestion. A paired t-test of filtered TKN-N data (corrected
for recovery) and centrifuged data established that there was no
significant difference at the 95$ confidence level and 9 degrees of
freedom with t = 0.958. The good comparison between these experimental
approaches suggest that the analytical procedures were accurate and
precise. The basic assumption inherent in both was that the primary
suspended material was algae. This assumption was not tested but algae
assays and analyses were limited to the peak-bloom area where the
assumption would be most reasonable.
-28-
-------
Table 6
FILTERED vs CENTRIFUGED METHODS
Total Filtered
ore POz/Chl. a
Location
Chapman Pt.
Indian Head
Deep Pt.
Date Sta.
8-1-77 9
8-1-77 10
8-1-77 10-B
Gunston Cove 8-22-77 8-A
Chapman Pt.
Indian Head
Chapman Pt .
Indian Head
Deep Pt.
Possum Pt.
8-22-77 9
8-22-77 10
8-29-77 9
8-29-77 10
8-29-77 10-B
8-29-77 11
TP
.503
.472
.469
.764
.751
.736
.799
.759
.850
.846
£i
mg P04
.161
.157
.157
.162
.238
.259
.176
.205
.275
.282
IE
/I
.204
.212
.210
.240
.243
.270
.256
.250
.279
.366
Pi Chi. a
.102
.114
.120
.227
.170
.207
.122
.136
.290
.310
ug/1
60.0
66.0
76.5
306
264
283.5
261.0
300.0
294
199.5
x
Filter Centrifuge
.004
.003
.003
.002
.002
.002
.002
.001
.002
.002
= .002
v
t
r
»
= 9
003
002
002
003
002
003
001
001
001
002
002
= 1.152
= .50
Location
Chapman Pt.
Indian Head
Deep Pt.
Gunston Cove
Chapman Pt .
Indian Head
Champan Pt.
Indian Head
Deep Pt.
Possum Pt.
Date Sta.
8_1_77 9
8-1-77 10
8-1-77 10-B
8-22-77 8-A. 1
8-22-77 9 1
8-22-77 10 1
8-29-77 9 1
8-29-77 10 1
3-29-77 10-B 1
3-29-77 11
Total
TKN NH-
*4
.685 ND
.651 ND
.600 ND
.439 ND
.254 ND
.227 ND
.378 ND
.328 ND
.111 ND
.885 ND
Filtered
2_ TKN
5 N/l
.338
.313
.288
.344
.362
.353
.526
.426
.342
.334
NH3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Chi. a
ug/1
60.0
66.0
76.5
306
264
283.5
261.0
300.0
294
199.5
x
N
ors/Chl. a
Filter
Filter Centrifuge
.006
.005
.004
.004
.003
.003
.003
.003
.003
.003
= .004
v
t
r
*
*
= 9.00
008
013
009
008
006
008
003
003
004
005
007
«
x2
012
010
008
008
006
006
006
006
006
006
007
= .958
= .67
-------
V. Rec ommendat ions
A. It is recommended that future work with algal bioassays be
split into two areas of concern. Algae from the peak bloom area
(highest chlorophyll a concentration) should be employed in the
elemental analysis work. This will ensure adequate phytoplankton
necessary for the required analyses. Limiting nutrient analyses
should be stressed in areas downstream from the peak bloom, where
algae are encountering less productive conditions.
B. It is recommended that future ^-fixation work involve con-
centration and incubation of phytoplankton in situ. In addition
to providing the natural setting for incubation, larger quantities
of algae should be obtained to insure that the TKN-N determinations
are in the optimal range of the test. The practice of reporting
acetylene reduction in terms of total Kjeldahl nitrogen limits
the test to some degree by the lack of sensitivity of the TKN-N
analysis relative to the gas chromatographic determination of
ethylene.
-------
VI. References
1. O'Shaughnessey, J. C., McDonnell, Archie J., "Criteria for
Estimating Limiting Nutrients in Natural Streams". Ins t. for
Research on Land and Water. Pennsylvania State University,
Res. Pub. No. 75.
2. Fitzgerald, G. P. and Nelson, T. C., "Extractive and
Enzymatic Analysis for Limiting or Surplus Phosphorus in Algae",
Journal of Phycology. Vol, 2, 1966, pp. 32-37
3. Williams, L. R., "Heteroinhibition as a Factor in Anabaena
f'Ips-aquae Waterbloom Production", Proceedings of Biostimulation
Nutrient Assessment Workshop. EPA - Corvallis, October 1973.
4. Fitzgerald, G. P., "Bioassay Analysis of Nutrient Availability",
Nutrients in Natural Waters. John Wiley and Sons, Inc., 1972.
5. Strickland, J. D. H., and Parsons, T. R., "A Manual of Sea
Water Analysis", Bulletin 125, Fisheries Research Board of Canada.
Ottowa, I960, p. 185.
6. Environmental Protection Agency, Methods for Chemical Analysis
of Water and Wastes. 1974, p. 182.
7. Fitzgerald, G. P., "Detection of Limiting or Surplus Nitrogen
in Algae and Aquatic Weeds", Journal of Phycology. Vol. 4, 1968,
pp. 121-126.
8. Stewart, W. D., Maque, T., Fitzgerald, G. P., and Burris, R. H.,
"Nitrogenase Activity in Wisconsin Lakes of Differing Degrees of
Euthrophication", New Phvtol.. (1971), 70, pp. 497-509.
9. Slayton, J. L., Trovato, E. R., "Carbonaceous and Nitrogenous
Demand Studies of the Potomac Estuary", Annapolis Field Office,
EPA, 1979.
10. Carpenter, E. J., "Marine Oscillatoria (trichodesmium):
Explanation for Aerobic Nitrogen Fixation Without Heterocysts",
Science 191, March 1976, pp. 1278-1280.
11. Mague, T. H. and Burris, R. H., "Acetylene Reduction as an
Indicator of Biological Nitrogen Fixation in the Great Lakes",
Limnology and Oceanography.
12. Skinner, K. J., "Nitrogen Fixation", Chemical and Engineering
News. Oct. 4, 1976, pp. 23-35.
13. (Author unknown) "Reduction Point to N2 Fixation Mechanisms:
Nitrogen Fixing Enzymes Catalyze the Reduction of Acetylene, Azide,
Cyanides, Methyl isocyanide and nitrous oxide", Chemical and
Engineering Newsf Jan. 30, 1967, p. 32.
-31-
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA 903/9-79-002
4. TITLE AND SUBTITLE
ALGAL NUTRIENT STUDIES OF THE POTQI/AC ESTUABY
7. AUTHOR(S)
J. L. Slayton
and S. ?.. Trovato
9. PERFORMING ORGANIZATION NAME AND ADDRESS 1
Annapolis Field Office, Region III
U.S. Environmental Protection Agency
Annapolis Science Center
Annapolis , Maryland 21401
12. SPONSORING AGENCY NAME AND ADDRESS
Sane
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
Summer 19'7"7
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
13. TYPE OF REPORT AND PERIOD COVERED
In House; Final
14. SPONSORING AGENCY CODE
3PA/9C3/00-
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The nutrient requirements of the phytoplankton of the Potomac Estuary vere
studied during the summer of 1977 employing the following laboratory tests :
NHJ-N uptake, alkaline phosphatase enzyme activity; extractable surplus
orthophosphate; tissue analysis for carbon, nitrogen and phosphorus content;
and nitrogen fixation by acetylene reduction. The results indicated that
the bloom of Oscillatoria v/as limited by nitrosen and that adeauate -hos-ohoris
?;as present.
17. KEY WORDS AND DOCUMENT ANALYSIS
3. DESCRIPTORS b.lDENTIFI
Algae - - Luxury
Nutrients Ammon
Mitroj
Alkal
TT}_OT3'
13. DISTRIBUTION STATEMENT 19. SECUH
TTMpT A<
rl^Liabi TO ^UBLiC 20. SECUR
UMCU
ERS/OPEN ENDED TERMS c. COSATI Field/Group
/ phosphorus
Lum uptake
jen fixation
ine phosphatase
rfcal analysis
TY CLASS IThis Report) 21 NO. OF PAGES
3SIFIED OR
TY CLASS (Thispage) 22. PRltE
\SSIFIED
EPA Form 2220-1 (9-73)
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