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                                          903979002
[    U.S. ENVIRONMENTAL  PROTECTION AGENCY
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     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

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                             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.

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                          TABLE OF CONTENTS




                                                                 Page




  I.   Introduction 	    1




 II.   Conclusions 	    6



III.   Experimental 	    7




 IV.   Discussion of Results 	   18




  V.   Recoirmendations 	   30




 VI.   References 	   31

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                               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

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                                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

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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-

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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"











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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

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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-

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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.

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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-

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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

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 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.

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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-

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    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-

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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-

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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

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    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-

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    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-

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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:

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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 .

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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-

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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

-------











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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)

-------