WATER POLLUTION CONTROL RESEARCH SERIES
16010 EHR 08/71
  Nutrient  Sources for
Algae and  Their Control
  U.S. ENVIRONMENTAL PROTECTION AGENCY

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          WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of pollution
in our Nation's waters.  They provide a central source of
information on the research, development and demonstration
activities in the Environmental Protection Agency, through
inhouse research and grants and contracts with Federal,
State, and local agencies, research institutions, and
industrial organizations.

Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications Branch
(Water), Research Information Division, R&M, Environmental
Protection Agency, Washington, B.C. 20^60.

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         NUTRIENT SOURCES FOR ALGAE

              AM) THEIR CONTROL
Dr0 George P. Fitzgerald,  Principal Investigator
          Water Resources Center
          University of Wisconsin
         Madison, Wisconsin  53706
                  for the
       Environmental Protection Agency
              Project #16010 EHR
                  August 1971

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For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.00
                             ii

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                    ABSTRACT
Critical evaluations are presented of bioassays
for nutrient availability.  The biological avail-
ability of any required algal nutrient in a sample
of water can be determined by growth experiments
requiring 2 weeks incubation.  In addition,
relatively short-term tests can be carried out
measuring changes in certain enzymatic activities
or chemical fractions which have been shown to
reflect meaningful nutritional changes.  The lat-
ter types of tests have been useful in evaluating
the nutritional status of in situ algae.  The
selection of the type of bioassays for particular
purposes can be made from the data presented as
to what information can be obtained, the length
of time required, and the range of sensitivity
of the bioassays.  Examples are presented of
ecologically important questions which have been
answered by the different bioassays.
                      ill

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                       CONTENTS

                                                     Page
SUMMARY AND CONCLUSIONS - - r, - - ---- « -- -       1


NUTRIENT SOURCES FOR ALGAE  -------„-_-       3
   Introduction --^---------,-----       3
   Materials and Methods  ----------~-       5
      Analysis of Nutrient Content by Growth  - -       5
      Extractive and Enzymatic Analyses for p - -       9
      Rate of Ammonia Absorption for N
        Nutrition ----------------     n
      Nitrogen Fixation Rates Related to Sources
        of N and P  ----- - - -- -- ----     12
   Experimental -----------------     16
      Factors Affecting Bioassays by Growth - - -     16
   References --------------.---^     24


AEROBIC LAKE MUDS FOR THE REMOVAL
OF PHOSPHORUS FROM LAKE WATERS  - -- ------     25
   Introduction -----------------     25
   Methods and Materials  ------------     26
   Results  -------------------     26
      Availability of Phosphorus in Lake Muds
        to Algae  --__---_-----r---     26
      The Sorption of Phosphorus by Lake Muds - -     30
   Discussion ------------------     33
   References ------------------     35


EVALUATIONS OF THE AVAILABILITY OF SOURCES
OF NITROGEN AND PHOSPHORUS FOR ALGAE  ------     39
   Abstract --,-----------------     39
   Introduction ------------------     39
   Materials and Methods  ------------     41
   Results  -__----,--_---------     41
      Techniques for Evaluation of Nutrient
        Sources -----------------     41
      Solubility as a Nutritional Factor  - - - -     47
      Factors in the Sharing of Nutrients
        between Plants  -------------     54
   Discussion ------r------------     59
   References -------r-----------     60

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                       CONTENTS
ACETYLENE REDUCTION ASSAY FOR DETERMINATION
OF PHOSPHORUS AVAILABILITY IN WISCONSIN LAKES --     63
   Abstract -------------------     63
   Introduction -----------------     63
   Results  -------------------     64
      Experiments Using a Defined Medium  - - - -     64
      Field Trials  ---------------     70
   Discussion ------------------     73
   References ------------------     75
                           vi

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                        FIGURES
EVALUATIONS OF THE AVAILABILITY OF SOURCES
OF NITROGEN AND PHOSPHORUS FOR ALGAE

  1  The Effectiveness of Different Phosphorus
     Sources for the Growth of Selenastrum by
     the Exposure and Subculture Technique  - - -     46

  2  The Effect of Different Nitrogen Sources
     on the Ammonia Absorption Rate of
     Selenastrum  ,--__,-----.--_----     49

ACETYLENE REDUCTION ASSAY FOR DETERMINATION
OF PHOSPHORUS AVAILABILITY IN WISCONSIN LAKES

  1  Response of P-Starved A. flos-aquae to
     Various Levels of Available Phosphorus - - -     68

  2  Effect of Time on the Response to Phosphorus
     of Phosphorus-Deficient Anabaena Cultures  -     69
                          vii

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                        TABLES

                                                     Page
NUTRIENT SOURCES FOR ALGAE

  1  Minimum Detectable Concentrations  (mg/L)
     of Nutrients by Growth of Selenastrum
     When Algal Growth Is Measured by
     Different Techniques ------------      8

  2  The Growth of Selected Algae Taken from PAAP
     Medium and Placed in PAAP Media Containing
     Different Levels of N, P, Mg, S, and Fe  - -     18

  3  The Effect of Different Initial Cell Densities
     on the Response of Algae from PAAP Medium
     to Relatively Low Concentrations of N, P,
     Mg, S, and Fe in PAAP Medium --------     19

  4  The Response of Selenastrum and Microcystis
     Precultured in PAAP Medium to Low Levels
     of N, P, Mg, S, and Fe in Gorham's Medium  -     20

  5  The Response of Algae Precultured in Allen's
     Medium to Low Levels of N, P, Mg, S, and Fe
     in PAAP Medium ---------------     22

  6  The Effect of Different Initial Cell Densities
     on the Response of Selenastrum from Allen's
     Medium to Low Concentrations of N, P, Mg, S,
     and Fe in PAAP Medium  ----------r-     23

AEROBIC LAKE MUDS FOR THE REMOVAL
OF PHOSPHORUS FROM LAKE WATERS

  1  Lake Muds as Sources of Available Phosphorus
     for Selenastrum capricornutum (PAAP) - - - -     27

  2  Lake Muds as Sources of Available Phosphorus
     for Cladophora sp. -------------     29

  3  Rate of Sorption of Phosphorus by Different
     Aerobic Lake Muds  -------------     31

  4  Rate of Sorption of Phosphorus by Treated
     Bottom Mud of Lake Mendota under Aerobic
     Conditions -----------------     32

  5  Rate of Sorption of Phosphorus by Bottom Mud
     from Lake Mendota under Aerobic Conditions -     34
                          Vlll

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                        TABLES

                                                     Pag<
EVALUATIONS OF THE AVAILABILITY OF SOURCES
OF NITROGEN AND PHOSPHORUS FOR ALGAE

  1  Utilization of Nitrogen Sources by
     Selenastrum  ----___---,------     42

  2  Utilization of Nitrogen Sources by Selenastrum
     as Measured by Growth Attained by 1/2 mg
     Samples Placed in 25 ml PAAP  (-N) Medium after
     Different Exposure Times ----------     45

  3  The Effect of Solubility of Phosphorus on
     the Growth of Selenastrum  ---------     43

  4  Hair as a Source of Available Nitrogen
     for Selenastrum  ,--__----_-_---     52

  5  Live versus Killed Algae as Sources of
     Available Nitrogen for Selenastrum in
     Gorham's  (-N) Medium ------------     55

  6  Live versus Killed Algae as Sources of
     Available Phosphorus for Selenastrum
     in PAAP (-P) Medium  ------------     55
  7  Comparative Tests of Field Plants as Sources
     of Available Nitrogen or Phosphorus for
     Selenastrum , Microcystis / or Anabaena  - - -     53

ACETYLENE REDUCTION ASSAY FOR DETERMINATION
OF PHOSPHORUS AVAILABILITY IN WISCONSIN LAKES

  1  Response in Acetylene Reduction of Nutrient-
     Starved Anabaena f los-aquae to Various
     Nutrients  -----------------     55

  2  Acetylene Reduction by Phosphorus-Starved
     and Phosphorus-Sufficient Anabaena f los-
     aquae When Incubated in Phosphorus-
     Containing Medium  -------------     55

  3  Response of Natural Populations of Anabaena
     and of a Phosphorus-Starved Laboratory
     Culture of A. f los-aquae When Incubated in
     the Surface Waters of Lake Mendota, with or
     without a Phosphorus Supplement  ------     66
                          IX

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                   TABLES
Response in Acetylene Reduction of
Phosphorus-Starved A. flos-aquae Cultures
to Various Levels of Available Phosphorus  -     67

Response in Acetylene Reduction of Phosphorus-
Starved A. flos-aquae When Incubated in the
Surface Waters of Various Lakes  ------     71

Response of Phosphorus-Starved A. flos-aquae
When Placed in the Surface Waters of Lake
Mendota Collected at Various Times
of the Day -----------------     71

Acetylene Reduction by Phosphorus-Starved
A. flos-aquae When Incubated in Untreated
Waters and in Phosphorus-Supplemented Waters
Taken from Various Depths of Lake Mendota  -     72

Acetylene Reduction by Phosphorus-Deficient
A. flos-aquae Incubated in Lake Mendota Surface
Waters Taken near a Drainage Inlet -----     72
                      x

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                SUMMARY AND CONCLUSIONS

A critical evaluation of bioassays for nutrient availability
was made in order to define the conditions and limits under
which each method can give meaningful results.  The biolog-
ical availability of algal nutrients in a sample of water
and the response to changes in the growth- limiting nutrient
were measured by the following tests:

     The growth attained by selected algae was measured
     in spiked and untreated samples (as much as 2 to 3
     weeks of incubation required) .

     Available N of the sample was calculated from NH\-N
     absorption rates after incubation with N-limited
     algae  (1 or 2 days incubation required) .

     Available P of the sample was calculated from in-
     creases in extractable POit-P after incubation with
     P-limited algae  (1 or 2 days incubation required)
     and from increases in the rate of C2H2 reduction
     by P-limited Na -fixing algae  (1/2 hour incubation
     required) .

To determine if various sources of nutrient are capable
of supplying adequate amounts of N or P for in situ algae
or aquatic weeds the following information was used:
     N-limited algae or  aquatic weeds will  absorb
     in the dark at rates greater  than  15 ug NH^-N/10 mg/
     hour.

     N2~fixing algae grown  in environments  with  surplus
     available fixed N  (NHit+NOs) will have  relatively
     few heterocysts and low Nz fixation or CaHz reduc-
     tion rates .

     P-limited algae or  aquatic weeds will  have  less
     than 0.08 mg extractable POif-P/100 mg  plant material
     and have relatively high alkaline  phosphatase
     activities .

     P-limited Na -fixing algae will  respond to incubation
     with added POit-P by having increased rates  of
     C2H2 reduction.

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Thex following is a summary of limnological facts recently
corroborated by bioassay analyses:

     The nutritional status of certain species of algae can
     vary from lake to lake, or even from different areas
     or depths in the same lake, on the same sampling date;
     subsurface samples of planktonic algae have been shown
     to have surplus N or P at times when the same species
     in surface waters were N or P limited.

     Lake Mendota algae contain surplus N and P in spring,
     can become N or P limited  (at the same time or inde-
     pendently) during summer, and again have surplus N and
     P after the fall overturn.  This pattern may represent
     the changes that take place in similar eutrophic lakes
     with spring and fall overturns.

     Rain can be a significant source of available N to
     algae in surface waters in the Madison lakes.  Less
     dramatic increases in available P were also associated
     with certain rains.

     Filamentous green algae, such as Cladophora sp., and
     aquatic weeds that have been in an environment contain-
     ing surplus N for a week or more are usually visibly
     coated with epiphytes  (have a brown appearance).

     Under certain laboratory conditions, solubility was
     shown not to be a limiting factor in the nutrition of
     algae because the equilibrium between soluble and in-
     soluble nutrients allowed algae to obtain sufficient
     amounts of nutrients which were present in an "insolu-
     ble" form.

     Factors other than insolubility prevent the N or P of
     certain samples of aerobic lake muds from being readily
     available for the growth of algae; P-limited Spirogyra
     sp. have been found growing through layers of muds
     with 0.1% total P content.

     The nutrients of live algae and aquatic weeds are not
     effectively available to other plants even when nutrient-
     limited plants are mixed with plants containing surplus
     nutrients.  However, when plants containing surplus
     nutrients are killed their nutrients become available
     for nutrient-limited plants.  This points out a distinct
     disadvantage of chemical treatment as a means of con-
     trolling undesirable growths of aquatic weeds because
     weeds killed by chemical treatment are likely to release
     much of their nutrients to the lake water in forms avail-
     able for the growth of algae.

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              NUTRIENT SOURCES FOR ALGAE

                     INTRODUCTION

Preliminary evaluations of water pollution control projects
for the control of obnoxious growths of algae will depend
upon the relative sources of nutrients:  those which can be
controlled versus those which cannot.  Through the use of
bioassay techniques developed in this laboratory for nutri-
tional studies of problem-causing algae it is proposed to
study the sources of nutrients available to algae and eval-
uate possible means of limiting these available sources.
The factors that control the absorption and utilization of
nitrogen, phosphorus, and possible other nutrients will be
studied by the use of techniques developed for determining
if algae have surplus quantities of certain nutrients or are
limited by their supply of these nutrients.  The main ques-
tions to be investigated will be:  1) the sources of nitro-
gen and phosphorus which can serve as available nutrients
for algae; 2) the factors affecting the utilization of
various nutrients under the conditions existing in surface
versus bottom water of lakes; 3) whether the solubility of
nitrogen, phosphorus, and iron compounds in the bottom of
lakes is a factor in the nutrition of algae (precipitated
versus soluble nutrients); 4) assuming that phosphorus is
an important nutrient for algae in lakes fertilized by
sewages or treated sewages, if the phosphorus of detergents
were replaced by organic chemicals such as nitrilotriacetate
or E.D.T.A., what effect might this have on the practical
supply of phosphorus from sewages; and 5) what effect would
such a replacement have on the availability of other nutri-
ents and on the copper sulphate treatments used for the
control of algae in lakes and reservoirs.

Bioassays can be used to evaluate the amount of a particular
nutrient or nutrients available to algae or aquatic weeds
in a water sample or to assess the nutritional status of
in situ plants.  Bioassays for any required plant nutrient
can be carried out by growth experiments in the laboratory
using selected species of algae.  In addition, relatively
short-term tests can be carried out by measuring changes in
certain enzymatic activities or chemical fractions which
have been shown to reflect meaningful nutritional changes.
An evaluation of the N or P nutritional status of in situ
algae or aquatic weeds at any particular time can be made
by measuring the NEU-N absorption rates in the dark, rela-
tive amounts of POi,-P extracted and alkaline phosphatase
activities, or N2-fixation rates by blue-green algae.  Bio-
assays of water samples demonstrate the level of available
nutrients whereas bioassays with in situ plants demonstrate

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whether the environment has supplied nutrients to only a
limited extent or if there were surplus quantities avail-
able.  The latter tests also will indicate transitory changes
in nutrient sources that might have taken place between
sampling dates, such as the effects of slugs of nutrients
which might not be detected without continuous monitoring.

Through the use of the bioassays developed and evaluated
by laboratory studies several practical evaluations of in
situ nutrient sources have been made.  The results of these
tests have suggested methods that could be used to control
certain forms of eutrophication of natural waters or the
inadvisability of further pursuit of certain approaches to
eutrophication control.

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                MATERIALS AND METHODS

Analysis of Nutrient Content by Growth

The value of measuring the growth of algae in water samples
is that differentiation can be made between the total nutri-
ent content of water samples as obtained by chemical analyses
and the nutrients that are available to support the growth
of algae under certain circumstances.  The concentration of
any nutrient required for the growth of algae can be deter-
mined by measuring the growth attained by selected algal
species in dilutions of the water sample or after suitable
spikes of other nutrients are added.  Standard techniques
for this type of bioassay are being developed  (Maloney,
1970).  Growth experiments often require two or three weeks
of incubation, but preliminary results can sometimes be
detected after only one or two days.  Since long incubation
periods may allow an original source of a potential nutrient
to degrade to an available form  (such as a polyphosphate
compound degrading to orthophosphate), it is sometimes de-
sirable to expose the algae for relatively short periods of
time to the potential nutrient and then transfer them to
another media to grow on the absorbed nutrient  (Fitzgerald,
1970a).

Some factors that can influence the results of nutritional
tests  are the type, source, and amount of algae used and
the nutrients carried over when the algae are added to the
test media.  The algae to be used must be readily avail-
able and must respond to the nutrients of interest.  In
addition, the selected species should represent problem-
causing species since not all algae may respond alike to
the same environment.  There is frequent controversy as
to whether one should use the natural flora of a water
sample for nutrient bioassays or a standardized culture
that may not be related to a particular flora but which
has been shown to respond similarly to various nutrients.
As pointed out, the alga to be used must be available when
you want to test the water; if the tests are to be run on
samples taken at spring overturn of a lake, the in situ
algae  will probably not be the same as those that cause
problems in mid-summer.  If the samples must be collected
and stored until a convenient time is found for the bio-
assays, there is a good chance the original flora will no
longer be present.  It has been pointed out by  several
workers that the algal species composition of  in situ tests
frequently changed when samples were confined  for a few
days time, such as the replacement of phytoplankton by

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epiphytic types of algae that had become attached to the
walls of the container.  Therefore, except for certain
specific studies, the use of selected and tested species
of algae is preferred over the use of in situ algae for
growth bioassays.

Since algae are known to be able to concentrate certain
nutrients in excess of their present needs when they are
grown in media with surplus nutrients, this factor must be
taken into account in selecting culture media and the amount
of algae used.  The effect the media can have on nutrition
tests was demonstrated by Gerloff and Skoog (1954).  They
showed that the planktonic blue-green alga, Microcystis
aeruginosa (Wis 1036), cultured in a medium with low N
(6.8 mg N/L)  would not grow when transferred to a medium
lacking N, but Microcystis from a medium with surplus N
for maximum growth  (27 mg N/L) was able to increase twofold
in a medium lacking N.  They also showed that Microcystis
from a medium with excessive P (1.8 mg P/L) could increase
fourfold when transferred to a medium lacking P.  Using the
green algae, Chlorella pyrenoidosa (Wis 2005)  and
Selenastrum capricornutum (PAAP), and the blue-green algae,
Microcystis and Anabaena flos aquae (Ind 1444) , a series of
30 experiments have shown that there was no significant
further growth in media lacking N or P with algal inocula-
tion levels of from 10,000 to 1 million cells/ml from the
relatively dilute PAAP medium (4,7 mg N/L and 0.2 mg P/L).
Thus, if algae are cultured in relatively dilute media, the
amount of growth in subsequent media will be dependent upon
the N or P of the latter media regardless of inoculum size.
However, if algae were precultured in more concentrated
media, such as Allen's  (1952) (178 mg N/L and 45 mg P/L) or
Gorham's  (1958)  (82 mg N/L and 7 mg P/L), Selenastrum could
increase two- to threefold in N-free media and increase
fourfold in P-free media.  The growth that occurs in N-free
or P-free media is due to surplus nutrients (luxury con-
sumption) inside the algal cells.  Therefore, the amount
of algae added to test media from more concentrated media
should be low enough so the excess nutrients in the cells
would be insignificant compared to the nutrients in the
test media.  Statistical data have shown that the lowest
concentration of algae that could be readily measured by
cell counts using a haemocytometer was 100,000 cells/ml
(100 cells, 1 20, in 175 microscopic fields), so inocula-
tions at levels less than 100,000 cells/ml should be used
when cells from relatively concentrated media are used.  The
carry-over of extracellular nutrients from the preculture
medium can be minimized by washing cells in media lacking
the nutrients of interest, such as by centrifuging and re-
suspending in distilled water containing 50 mg/L of NaHCOs.

                          6

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The sensitivity of growth tests carried out with these pre-
cautions can be evaluated by the minimum detectable concen-
trations of the nutrients of interest.  By washing the green
alga, Selenastrum, from any nutrient medium and inoculating
to a concentration of 50,000 cells/ml, concentrations of
various nutrients as low as those presented in Table 1 will
consistently yield increases in the measurements used.  By
using more sensitive measurements than those indicated,
lower concentrations of nutrients could be detected.

In order to evaluate the reproducibility of data on the
relationship between algal growth measurements and nutrient
levels in natural waters, the amount of growth attained by
Selenastrum was measured by 3 to 5 tests in each of 8 samples
of water from 3 lakes in the Madison area which were pre-
served by autoclaving.  Concentrations of available N in
5 separate tests of a Lake Mendota surface water sample,
collected and preserved on August 17 and assayed during
December, 1970, were 0.15, 0.17, 0.20, 0.20, and 0.25 mg N/L.
This degree of reproducibility is typical for bioassays.  The
level of available N in 6 surface water samples collected in
mid-summer of 1970 was about 0.2 mg N/L, whereas a hypo-
limnion sample contained 1.2 mg N/L, and a surface sample
collected in December contained 0.5 mg N/L.  These values
correlate well with levels of NH^+NOj obtained by chemical
analyses of similar samples.

In general, growth of algae in water samples or with poten-
tial nutrient sources will be correlated with the growth
attained by the same inoculum under conditions of known
nutrient content:  such as growth in 0, Ix, 2x, 4x, 8x mg/L.
Growth in the absence of the nutrient of interest will in-
dicate growth attainable with the nutrients contributed by
the algae and the techniques of culturing and handling.
The concentration of nutrients used to establish standard
curves will be dependent upon the particular nutrient of
interest, but should represent as wide a range of nutrients
as will have a significant influence on algal growth.  The
growth of algae in test samples, or dilutions of test sam-
ples if concentrated sources of nutrients are suspected,
and growth attained with known concentrations of nutrients
is used to estimate the amount of nutrients available in
the test sample under the conditions of the test.

The availability of different forms of nutrients can also
be evaluated by growth experiments.  Certain relatively
insoluble sources of nutrients, such as iron-phosphorus
compounds and teeth for P, hair for N, iron pyrites for
iron, and marble for carbon, were found to be readily used
by algae, whereas other sources, such as the N or P of

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Table 1.  Minimum Detectable Concentrations (mg/L)
          of Nutrients by Growth of Selenastrum When
          Algal Growth Is Measured by Different Techniques
Nutrient
N
P
Mg
S
Fe
Absorbance
(1 cm, 750 my)
0.2
0.02
0.02
0.02
0.002
Cell Counts
( Haemocy tome ter )
0.1
0.01
0.01
0.01
0.001
Fluorometry
(Chlorophyll a)
0.1
0.01
0.01
0.01
0.001

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aerobic lake muds or the N or P contained in other live
plants, were found to be relatively unavailable.

Evaluations of the availability of sources of nutrients
also can be carried out using rooted aquatic weeds, such as
Lemna minor, a common "duckweed."  Increases in frond num-
bers or dry weight can be readily followed during incubation
periods of 2 to 3 weeks or after exposure tests of a few
hours and then subculture to N- or P-free media for growth
on sorbed nutrients.  Concentrations as low as 0.25 mg N/L
or 0.05 mg P/L will result in at least twofold increases
in growth over control  (-N or -P) cultures.  Because these
plants have roots,they can be used to differentiate between
nutrient sources available to algae and those that are avail-
able to rooted aquatic weeds.


Extractive and Enzymatic Analyses for P

An extractive procedure can be used to differentiate between
algae whi'ch have surplus or stored P and those that are
P-limited.  Algae and aquatic weeds containing adequate P
will release more than 0.08 mg PO^-P/100 mg  (dry weight) of
plant material when extracted in a boiling water bath for
1 hr.  Therefore, this extractive procedure can be used to
measure the P-nutritional status of algae or aquatic weeds
and to follow the effects of environmental changes that
might influence the P-nutrition of plants  (Fitzgerald and
Nelson, 1966).

The POi,-P in algal extracts was analyzed by the stannous
chloride method  (Am. Public Health Assoc., 1965).  All analy-
ses are reported on a dry weight basis.  Dry weight of
planktonic algae was measured on filtered samples dried in
tared vessels.  Measurements of the dry weight of plants
that could not be sampled by aliquot, such as Cladophora or
Myriophyllum, were made with the actual samples used by
filtering and drying them after the PO^-P extraction or
enzymatic tests were completed.  Dry weights of samples
after extraction with boiling water are not equal to the
total dry weight of the original sample but are accurate
enough for comparative purposes.

The extraction procedure for surplus phosphorus involved
placing 10-80 mg of washed  [Gorham's minus P(-P) medium]
plant material into 40 ml of Gorham's medium  (minus P
source; pH 7), extracting in a boiling water bath for
60 min, centrifuging or otherwise removing plant material,
and analyzing the supernatant liquid for orthophosphate.
Alternative methods using 5 min direct boiling or auto-

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claving techniques have not given as complete an extraction
as 1 hr in boiling water baths but could be of comparative
use.  Any extraction method dependent on killing the plant
tissue and allowing the POit-P to leach to the supernatant
liquid would probably be sufficient as long as results could
be correlated (Fitzgerald and Faust, 1967).  The PCH-P in
the extracts was calculated as mg P/100 mg  (dry wt) plant
material.

Alkaline phosphatase activity was determined by suspending
1-20 mg of washed plant material in 32 ml of Gorham's
(minus P) medium, adding 4 ml of buffer solution (1 M Tris,
0.01 M MgCl, adjusted to pH 8.5 with acetic acid) and 4 ml
of p-nitrophenylphosphate solution (30 mg/100 ml), and in-
cubating at 35-37C.  The relative activity was measured
after 0.25-2 hr by centrifuging 10-ml samples with 10 mg
of orthophosphate-P in 0.5 ml (to stop further enzyme activ-
ity) and measuring the optical density of the clear super-
natant liquid at 395 mu.  An alternative measurement of
alkaline phosphatase activity uses commercial enzymatic
tablets, one brand of which (Phosphatabs-Alkaline Quanti-
tative, Warner-Chilcott Labs, Morris Plains, N.J.)  uses
phenolphthalein phosphate and necessary cofactors.  Data
so obtained are comparable to results with p-nitrophenyl-
phosphate as substrate.  Activity was recorded as units of
enzyme/ing (dry wt) of plant material in 40 ml.  One unit
of alkaline phosphatase is defined as the amount of enzyme
liberating 1 mymole of nitrophenol/hr under the prescribed
conditions.

The use of the extractive procedure in laboratory studies
of the availability of P sources has indicated that 10 mg
of either the green alga, Cladophora sp, or leaves of the
aquatic weed, Myriophyllum sp, could detect P concentra-
tions as low as 0.04 mg P/L.  By using P-limited Cladophora
sp from Lake Wingra it was shown that 1% or less of the P
of various lake muds was available to the alga when tested
under aerobic conditions in the laboratory  (Fitzgerald,
1970b).

It has been found that plants that are P-limited will have
25 times as much alkaline phosphatase activity as plants
grown with surplus P.  Analysis of alkaline phosphatase
activity can thus be used to confirm that plants with low
extractable PCu-P levels are alive, but P-limited.  The
two procedures have been used together to detect long-term
nutritional changes, such as seasonal changes in the avail-
ability of P in lake waters, or recent additions of avail-
able P.  Plants that have only recently been exposed to
increased available P supplies have higher extractable
                            10

-------
  t-P but also have relatively high alkaline phosphatase
activities because the alkaline phosphomonoesterase content
is only lowered by dilution by growth of the cell under
adequate P conditions.  During the period of July 22 to 31,
1968, the Cladophora sp along the shore of Lake Mendota
appeared to be P-limited in that very little PCK-P could
be extracted  (0.03 to 0.07 mg/100 mg algae) and relatively
high alkaline phosphatase activities were recorded (about
1,000 units/mg algae).  There was a sudden increase in ex-
tractable PCU-P from the Cladophora during the period of
August 5 to 7, 1968 (probably associated with the 1.4 inches
of rain occurring then), the values going to 0.12 mg PO^-P/
100 mg on August 6, but the algae still had 1,500 units of
alkaline phosphatase/mg.  With growth under surplus P con-
ditions (probably associated with a lack of competition
because there were few phytoplankton present; Secchi depth
of 2.5 meters), by August 29 the Cladophora had 0.17 mg of
extractable POij-P/100 mg, but only 90 units of alkaline
phosphatase/mg.

Verification of these tests can be accomplished by the con-
trasting results obtained by the POit-P extraction method
and alkaline phosphatase measurements with in situ algae,
except in cases where recent sources of P were absorbed by
the plant material.  In addition, plants that appear to be
P-limited can be kept for several days in media containing
relatively high levels of P, such as Gorham's culture medium.
If these plants then absorb  P and had been able to absorb P
if it had been available in their former environment, they
will respond by increases in extractable POi^-P after incuba-
tion in an environment with known available P.  Plants that
appear to have surplus P can be incubated for a week or more
in culture media lacking only P to show that if less P had
been available in their former environment ^they would have
lower extractable PO^-P values and higher alkaline phos-
phatase activities.
Rate of Ammonia Absorption for N Nutrition

Plants that are limited by the supply of available N are
able to absorb ammonia (NH^-N) in the dark 4 to 5 times more
rapidly than plants with adequate or surplus N (Fitzgerald,
1968).  Thus, the effect of changes in the environmental
supply of N to in situ algae or aquatic weeds can be fol-
lowed as well as an evaluation made of different sources
of N in laboratory experiments by NH^-N absorption measure-
ments .

Comparative rates of NH^-N absorption in the dark were used
                           11

-------
to differentitate between plant material from cultures con-
taining surplus nitrogen and those whose growth was limited
by the available nitrogen.  From 5  to 20 mg  (dry wt) of
plant material was washed in nitrogen-free medium, placed
in 10 to 30 ml of Gorham's medium  (minus N), and 0.1 mg
NHit-N was added.  After 1-hr incubation at 25 ± 2C in the
dark, the NHi*-N content of the supernatant liquid was com-
pared to controls without plant material.  NHi*-N was ana-
lyzed by direct Nesslerization (APHA, 1965).  Results were
calculated as yg N absorbed/dO mg dry wt x hr) .  If the
plant material settled during incubation, occasional mixing
will be required to prevent local depletion of NHit-N and
erroneous rate results.  Without such a depletion, the rates
of NHit-N absorption by samples with fourfold differences in
weight will be equivalent on a dry weight basis.

The use of the rate of NHif-N absorption tests for evaluating
the concentration of available nitrogen in water samples has
not been investigated in detail.  Therefore, the minimum de-
tectable levels of available N by this technique are not
known.

Verification of results of this test can be accomplished by
incubating plants in relatively high levels of available N
or in media lacking only N and recording the changes caused
by such treatments.  If the plants had been healthy and
capable of change in their former environments, they would
respond after the incubation treatments.

An interesting correlation has been made between the N nutri-
tion of filamentous algae or aquatic weeds and the growth
of epiphytic algae.  Observations in the field and in con-
trolled laboratory tests have indicated that plants growing
for a week or more in the presence of surplus N usually
become coated  with epiphytic algae.  Therefore, the presence
of a dense coating of epiphytes on algae can be used as an
indication that the algae have had surplus N available in
their immediate past history  (Fitzgerald, 1969) .


Nitrogen Fixation Rates Related to Sources of N and P

It has recently been shown that the capacity of blue-green
algae and other plants to fix Na could be followed by measur-
ing the rate of acetylene (C2Hz) reduction by the same
nitrogenase enzymes used to fix Na  (Stewart et al, 1967) .
The ease with which the rate of reduction of CaH2 to ethylene
(CaHit) can be measured with gas chromatography has made this
measurement a useful tool in limnology.
                            12

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The general assay procedure used was to place about 1 mg
(actual dry weight of samples used determined on aliquot
samples) of algae in a serum bottle for the direct assay
of the C2H2 reduction activity or in a volume of test water
as a pre-incubation for the absorption of available P before
the C2H2 reduction assay.  The P absorption incubation period
can be as short as 30 minutes.  The algal suspension is then
concentrated by centrifugation to a volume of 1 ml for the
C2H2 reduction assay.  The C2H2 reduction measurements were
performed in 7-ml-capacity serum bottles fitted with serum
stoppers.  A 1.0-ml aliquot of test alga was added to each
bottle and 1.4 ml of C2H2  (purified grade obtained from
Matheson Co) was added to the bottle without removing the
air.  The gas pressure within the bottle then was restored
to atmospheric by pricking the serum stopper with a hypo-
dermic needle.  This simplified procedure was adopted because
experiments showed that it was unnecessary to remove N2 from
the gas phase if sufficient C2H2 was added.  Acetylene assays
were run for 30 min.  The experiments were terminated when
required by the injection of 0.2 ml of 5N
The gas phase was analyzed  for ethylene by gas chromatography
using a Varian-Aerograph model 600 D gas  chromatograph fitted
with a hydrogen  flame  ionization detector and a 9 ft long
column of Porapak R.   The instrument was  run at room tempera-
ture and high purity nitrogen gas at a flow rate of approxi-
mately 25 ml/min served as  carrier gas.

When the availability  of P  in test samples is to be measured
using P-limited  algae, all  test samles which are being com-
pared should be  set up at the same time.  For each test
water sample  (a) there should also be available a control
sample (b)  in which the algal response in phosphorus-free
medium is noted, and a second control  (c) comprising the
test water  sample plus 0.025 mg/liter of  phosphorus.  The
value for  (b) serves to show that the bioassay organism is
phosphorus  starved while the value for  (c) shows its ability
to respond  in the test water when phosphorus is available.
The relative response  for each test water thus, will be:
 (c - b)/(a  - b) .  By comparing the values for this ratio,
the relative abundance of phosphorus in each test sample can
be measured.

The quantitative determination of the amount of available
phosphorus  in a  particular  water is more  complicated, and
it is suggested  that the following series should be set up
for such a  measurement:   (1) P-free medium,  (2) P-free
medium + 0.100 mg P/liter,  (3) P-free medium + 0.050 mg
P/liter,  (4) P-free medium  + 0.025 mg P/liter,  (5) P-free
medium + 0.010 mg P/liter,  (6) test water sample,  (7) test
water sample + 0.025 mg P/liter.


                            13

-------
Tests (l)-(5) show the response of the alga to available phos-
phorus in the absence of inhibitory or competitive reactions
and can be used to prepare a standard curve which can be used
for all tests performed with the same batch of assay organism.
The value for test (7) minus test (1) gives the response to
0.025 mg/liter of phosphorus plus the amount of phosphorus in
the water sample.  The value for test (6) minus test (1)
gives the response to available phosphorus in the test water
only.  Thus the response to 0.025 mg P/liter alone and to
the available phosphorus in the test water is obtained.  These
values are then transferred onto the standard curve to give
the quantity of available phosphorus in the test water.  Thus,
the response of the algae to a series of standards and to two
samples of the test water, one with and the other without
added phosphorus, is all that is required to determine the
level of available phosphorus in a particular water.  The
same standard curve can be used for all waters tested using
the same batch of assay organism.  We always have noted a
response when phosphorus was added to phosphorus-deficient
culture medium.  This indicates that a failure to detect
acetylene reduction in test water samples with added phos-
phorus is because of some inhibitory factor in the water.
In the presence of total inhibitors the test would not be
satisfactory.

Anabaena flos-aquae is a good assay organism, but any fast-
growing N2-fixing blue-green alga presumably could be used.
The exact culture medium used is also immaterial as long as
it is free of combined nitrogen and the bioassay organism
is phosphorus starved and metabolically active at the start
of the assay.

Small volumes (25 ml) of test water may be used, but larger
volumes are more satisfactory.  A volume of 70-200 ml of
water is recommended with 6.0 mg dry weight of the assay
organism.  The volume and amount of algae should be constant
in any one test series.

The sensitivity of C2H2 reduction assays is such that re-
producible measurements can be obtained with 2 to 4
Gloeotrichia colonies per sample.  The actual amount of
algae used in field work is usually based on the dry weight
analyses of samples of concentrated plankton or the total N
analyses of the samples used after the C2H2 reduction assay.
When P-limited Anabaena is used to assay for available P in
water samples, as little as 0.01 mg P/L will cause a 100%
increase in the C2H2 reduction activity of the test alga.

The C2H2 reduction assay for nitrogen fixation capacity of
field or laboratory algae can be correlated with the rela-
                           14

-------
tive numbers of heterocysts  (clear cells in which the
nitrogenase enzymes seem to be located) and vegetative
cells.  Algae grown with adequate fixed N  (NHi* or NOa)
cannot fix Na nor do they have heterocysts.  When N-fixing
algae are used to assay for available P, a positive response
to added available P serves to indicate the test algae were
P-limited, but further evidence can be obtained by measuring
the amount of extractable POif-P of the algae.
                            15

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                     EXPERIMENTAL

Factors Affecting Bioassays by Growth

Bioassays for any plant nutrient can be carried out by growth
experiments in the laboratory using selected species of
algae.  Studies have been made of the influence on the results
of nutritional bioassays of some of the factors that can be
readily controlled, such as inoculum size, control culture
medium (source of standard curves), or source of algae (pre-
culture medium).

A theoretical approach can be used to determine the effect
of inoculum size on results of nutrition tests.  By using
the minimum biomass or equivalent measurement as a basis,
the amount of cells to give a significant increase in the
measurement can be calculated.  Thus, in the case of cell
counts using a haemocytometer, in order to have an accuracy
of 20%, the lowest reproducible cell count would be about
100,000 cells/ml  (100 cells, + 20,  in 175 microscopic
fields).  This concentration of cells is too dilute to
readily measure by absorbance or dry weight, but could
readily be measured by fluorometry or with electronic par-
ticle counters.  The general relationship between the vari-
ous measurements indicates that an absorbance  (1 cm, 750 my)
of 0.1 is equivalent to approximately 5,000,000 cells/ml and
50 mg, dry weight/L.  Therefore, bioassays which do not
produce at least 100,000 cells/ml at the final harvest can-
not be measured with reasonable accuracy by using the haemo-
cytometer cell count method.  Inoculum levels must be such
as to bring the number of cells up to a level at harvest time
where they can be counted.  Only then can a study be made
of the effects caused by different preculture media or a
determination made of the minimum detectable concentrations
of nutrients.  If more sensitive biomass measurements are
used, lower inoculum levels can be used.  However, if algae
of different forms, such as unicellular and filamentous
forms, are to be compared, measurements are restricted to
those methods adaptable to any type of algae.  Thus, absorb-
ance and fluorometry can be used under such conditions.

Inasmuch as the bioassays of interest are related to measure-
ments of available nutrients, studies have been made of the
effect of different factors on the response of algae to
various levels of N, P, Mg, S, and Fe.  Since both uni-
cellular algae, Selenastrum, Chlorella, and Microcystis,
and the filamentous alga, Anabaena, were employed in the
tests, the biomass measurement chosen for use was absorbance
(1 cm, 750 nm).
                           16

-------
The first series of tests were carried out to demonstrate
that the selected algae grown in the relatively dilute PAAP
medium and washed in NaHCOa solution  (50 mg/L) could be used
to detect the presence of available N, P, Mg, S, and Fe by
comparing the growths attained after 7 to 12 days incubation
in media lacking the nutrient of interest and with different
levels of the nutrient.  The results are summarized in
Table 2.

It is evident that algae from this dilute medium could be
used to detect the nutrients listed.  The differences be-
tween growths attained in the medium lacking any of the
nutrients and in the lowest concentrations of nutrients
indicate that, with more sensitive biomass measurements,
effects caused by considerably lower concentrations of some
of the nutrients could have been detected.  However, the
purpose of the experiment was to show that these algae could
be used to detect available forms of N, P, Mg, S, and Fe
when inoculated from PAAP medium at the concentrations used.

Further tests were carried out to determine if five- or
tenfold increases in the initial concentration of cells
from the relatively dilute PAAP medium would prevent the
detection of available forms of N, P, Mg, S, or Fe in PAAP
medium.  Selenastrum was tested at 10,000 and 100,000 cells
per ml, while Microcystis was tested at 50,000 and 250,000
cells per ml.  Typical results are summarized in Table 3.

These algae could be used at concentrations of 100,000 or
250,000 cells per ml to detect the differences between
PAAP media lacking N, P, Mg, S, or Fe and media with addi-
tions of low concentrations of these nutrients.

Since many practical evaluations of available nutrients
will not be run in the medium algae are grown in, such as
transferring algae from complete PAAP medium to PAAP medium
lacking some essential nutrient plus the source of nutrient
of interest, tests were carried out in which Selenastrum
and Microcystis were grown in PAAP medium and then inoculated
in Gorham's medium or Gorham's medium with no or low concen-
trations of N, P, Mg, S, or Fe.  The results of growths
attained after 7 to 12 days are summarized in Table 4.

The differences in the growths of these algae in media
lacking N, P, Mg, S, and Fe and in media to which low con-
centrations of these nutrients were added indicate that
when algae are transferred from the relatively dilute PAAP
medium to the more concentrated Gorham's medium, the avail-
ability of the nutrients listed could readily be detected.
                           17

-------
Table 2. The Growth of Selected Algae Taken from PAAP
         Medium and Placed in PAAP Media Containing
         Different Levels of N, P, Mg, S, and Fe.
         Growth Recorded as Absorbance (1 cm, 750 nm)
         after 7 to 12 Days Incubation.
Test Medium
Complete
-N
" +0.25
11 +0.5
11 + 1.
_p
" + 0.025
11 +0.05
" +0.1
-Mg
" + 0.025
11 +0.05
11 +0.1
_o
" + 0.025
" +0.05
" +0.1
-Fe
11 + 0.001
" + 0.002
" + 0.005
Growth Attained (Absorbance)
Initially
10,000 cells/ml
Selenastrum
0.18
.005
.025
.06
.10
0.0
.025
.06
.08
.03
.08
.10
.13
.02
.04
.06
.08
.02
.06
.08
.08
Chlorella
0.21
.04
.07
.09
.09
.005
.05
.08
.10
0.0
.04
.09
.09
.02
.04
.05
.07
.03
.04
.06
.08
Initially
50,000 cells/ml
Microcystis
0.19
.02
.03
.05
.08
.01
.07
.08
.20
.02
.12
.15
.14
.02
.06
.07
.10
.02
.03
.04
.06
Anabaena
0.18
.20
.24
.20
.18
.01
.04
.06
.09
.01
.02
.03
.06
.03
.04
.08
.09
.05
.07
.09
.11
                           18

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Table 3. The Effect of Different Initial Cell Densities
         on the Response of Algae from PAAP Medium to
         Relatively Low Concentrations of N, P, Mg, S,
         and Fe in PAAP Medium.  Growth Attained after
         7-12 Days Measured as Absorbance (1 cm, 750 nm).
Test Medium
Complete
-N
11 +0.5
-P
" +0.05
-Mg
" +0.05
-S
" +0.05
-Pe
11 + 0.005
Growth Attained as Absorbance
Selenastrum
10,000
cells/ml
0.13
.005
.04
0. 0
.06
.03
.10
.02
.06
.01
.04
100,000
cells/ml
0.13
.015
.05
.01
.07
.04
.11
.02
.05
.02
.05
Microcystis
50,000
cells/ml
0.19
.015
.05
.01
.08
.015
.15
.02
.06
.015
.06
250,000
cells/ml
0.27
.03
.06
.05
.22
.04
.12
.02
.10
.06
.10
                           19

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Table 4. The Response of Selenastrum and Microcystis
         Precultured in PAAP Medium to Low Levels of
         N, P, Mg, S, and Fe in Gorham's Medium.
         Growth Attained Measured after 7 to 12 Days
         as Absorbance (1 cm, 750 nm).
Test Medium
                 Growth Attained as Absorbance
  Selenastrum
10,000 cells/ml
  Microcystis
50,000 cells/ml
Complete

-N
 "  +0.5

-P
 "  +0-05

-Mg
 "  +0.05

-S
 11  + 0.05

-Fe
 11  + 0.005
    0.38

     .005
     .03

    0. 0
     .04

     .10
     .16

     .10
     .15

     .04
     .08
    0.66

     .01
     .04

     -015
     .06

     .01
     .12

     .14
     .21

     .015
     .27
                       20

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Some concern has been expressed by various authors for the
effects on nutritional bioassays of nutrients carried over
as surplus stored nutrients inside algae when algae from
relatively concentrated media are used as the preculture
medium.  The four algae, Selenastrum, Chlorella, Microcystis,
and Anabaena, were cultured in the relatively concentrated
Allen's medium  (178 mg N/L and 45 mg P/L) and inoculated into
PAAP media lacking N, P, Mg, S, or Fe and with low concentra-
tions of these nutrients in order to determine if such a
source of algae would negate the use of the algae for nutri-
tional bioassays.  The results with inoculations of 10,000
and 50,000 cells/ml are summarized in Table 5.

When such low initial concentrations of cells are used to
detect available sources of N, P, Mg, S, or Fe, algae from
the relatively concentrated Allen's medium can be success-
fully used.. Even at an inoculation level of 50,000 cells/ml
Microcystis and Anabaena could detect the addition of the
nutrients tested.

It was of interest, therefore, to determine the effect in-
creased inoculation densities of algae from the relatively
concentrated Allen's medium would have on nutritional bio-
assays.  Selenastrum was cultured in Allen's medium and
added to various levels of N, P, Mg, S, or Fe in PAAP
medium at initial concentrations of 10,000, 100,000 and
800,000 cells/ml.  The results of a series of tests are
summarized in Table 6.

There was a logical response to the addition of all nutri-
ents tested when an initial concentration of 10,000 cells/ml
was used.  Inoculation levels giving 100,000 cells also
could detect the addition of N, P, Mg, and S, but the growth
in medium lacking Fe was nearly as much as when Fe was added
in the case presented.  When inoculation levels of Selenastrum
from Allen's medium were increased to 800,000 cells/ml, there
was a measurable increase in the amount of growth attained
in all nutrient solutions lacking one nutrient when compared
with the growths at lower cell densities.  The Selenastrum,
however, did increase over 100% with additions of N and Mg,
but not to additions of P, S, and Fe.  Furthermore, tests
carried out with initial cell densities of one million or
more/ml have indicated that all algae tested (Selenastrum,
Chlorella and Microcystis) could increase in biomass two-
fold in media lacking N and fourfold in media lacking P when
algae were precultured in media containing surplus quantities
of N or P.  Therefore, limited amounts of growth of algae
can take place with surplus nutrients stored in algal cells
from relatively concentrated culture media, but by using
inoculation levels of 100,000 cells/ml or less the amounts
of nutrients carried over will not prevent the algae from


                           21

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Table 5. The Response of Algae Precultured in Allen's
         Medium to Low Levels of N, P, Mg, S, and Fe in
         PAAP Medium.  Growth Attained after 7 to 12 Days
         Measured as Absorbance (1 cm, 750 nm).
Test Medium
Complete
-N
" +0.5
-P
11 +0.05
-Mg
11 +0.05
-S
" +0.05
-Fe
" + 0.005
Growth Attained as Absorbance
10,000 Cells/ml
Selenastrum

0.14
.01
.04
.01
.08
.03
.10
.02
.05
-03
.10
Chlorella

0.13
.015
.07
.005
.08
.04
.07
.02
.10
.02
.05
50,000 Cells/ml
Microcystis
0.26
.015
.04
.02
.16
.05
.17
.015
.08
.02
.17
Anabaena

0.19
-
.02
.07
.01
.06
.04
.08
.09
.14
                           22

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Table 6. The Effect of Different Initial Cell Densities
         on the Response of Selenastrum from Allen's Medium
         to Low Concentrations of N, P, Mg, S, and Fe in
         PAAP Medium.  Growth Attained after 7 to 12 Days
         Measured as Absorbance (1 cm, 750 nm).
Test Medium
Complete
-N
" +0.25
" +0.5
" +1.0
-P
" + 0.025
" + 0.05
" +0.10
-Mg
" + 0.025
11 +0.05
" +0.10
-S
" + 0.025
" +0.05
11 +0.1
-Fe
" + 0.001
" + 0.002
" + 0.005
Growth Attained as Absorbance
No. Cells
10,000/ml
0.14
.01
.02
.04
.08
.01
.05
.08
.11
.03
.08
.10
.14
.02
.04
.05
.08
.03
.05
.04
.05
No. Cells
100,000/ml
0.17
.02
.04
.06
.09
.07
.10
.11
.14
.04
.08
.12
.16
.02
.04
.06
.09
.06
.06
.06
.07
No. Cells
800,000/ml
0.20
.04
.06
.08
.10
.10
.12
.12
.14
.10
.13
.16
.20
.13
.13
.15
.15
.12
.11
.15
.12
                             23

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responding to the addition of essential nutrients to media
lacking that nutrient.  By using lower initial cell densities
and relatively sensitive biomass measurements, responses to
relatively lower concentrations of nutrients could be de-
tected.
                      REFERENCES

American Public Health Association.  1965.  Standard Methods
for the Examination of Water and Wastewater.  12 ed.  Amer.
Public Health Assoc., New York.  769 p.

Fitzgerald, G. P.  1970a.  Evaluations of the Availability
of Sources of Nitrogen and Phosphorus for Algae.  J. Phycol.,
£:239-247.

	.  197Ob.  Aerobic Lake Muds for the Removal of
Phosphorus from Lake Waters.  Limnol. Oceanogr., 15:550-555.
_ .  1969.  Some Factors in the Competition or
Antagonism among Bacteria, Algae, and Aquatic Weeds.
J. Phycol., 5_:341-349.

_ .  1968.  Detection of Limiting or Surplus Nitrogen
in Algae and Aquatic Weeds.  J. Phycol., 4^:121-126.

         , and S. L. Faust.  1967.  Effect of Water Sample
Preservation Methods on the Release of Phosphorus from Algae.
Limnol. Oceanogr., 12:332-334.

_ , and T. C. Nelson.  1966.  Extractive and Enzymatic
Analyses for Limiting or Surplus Phosphorus in Algae.
J. Phycol., 2^:32-3.7.

Gerloff, G. C., and F. Skoog.  1954.  Cell Contents of
Nitrogen and Phosphorus as a Measure of Their Availability
for Growth of Microcystis aeruginosa.  Ecology, 35; 348-353.

Maloney, T. E. (ed.).  1970.  Provisional Algal Assay
Procedure.  Jt. Industry/Gov' t Task Force on Eutrophication,
New York, N. Y.  62 p.
                            24

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           AEROBIC LAKE MUDS FOR THE REMOVAL

             OF PHOSPHORUS FROM LAKE WATERS


                       ABSTRACT

Phosphorus-limited Selenastrum and Cladophora sp. have been
shown to respond by growth or extractable POit-P to as little
as 0.02 mg PCK-P.  However, these same species did not re-
spond when exposed for a period of 1 or 2 weeks to as much
as 2 mg of phosphorus in lake muds under aerobic conditions.
Studies of the rate of sorption of phosphorus by lake muds
under aerobic conditions from two lakes and three depths in
one lake indicated that as little as 0.4 g of mud could sorb
about 0.05 mg PCX-P in less than 30 minutes.  Due to these
findings, it is suggested that the sorption of phosphorus
by lake muds under aerobic conditions can be used to remove
phosphorus from lake water and to demonstrate the importance
of the control of continuous sources of phosphorus to lakes.


                     INTRODUCTION

Bioasssays with Cladophora sp. and other algae have indi-
cated these algae did become phosphorus-limited or nitrogen-
limited in Lake Mendota, Monona Bay, and Lake Wingra of
Madison, Wisconsin during the summers of 1968 (Fitzgerald
and Lee, 1970) and 1969 despite the fact the algae were
located close to muds which contained both phosphorus and
nitrogen compounds.  Therefore, the availability of phos-
phorus from lake muds for the nutrition of algae was of
interest.  Mortimer  (1941) produced evidence that phosphorus
is adsorbed strongly on ferric hydroxide or ferric hydroxide
organic complexes in the oxidized surface mud layers of lake
bottoms as long as oxygen is present in the overlying water.
Barter (1968) and others have studied the adsorption of
phosphorus by lake sediments, and Lee  (1969) has recently
reviewed this subject.  Without trying to study the various
forms of phosphorus that may be present in lake muds, tests
have been carried out to determine if the phosphorus of
lake muds is readily available to algae under aerobic con-
ditions.  When preliminary tests indicated that the phos-
phorus was not available to algae in short periods of time,
further studies on the use of lake muds to remove phos-
phorus from solutions were carried out with the objective
of evaluating this as a means of removing nutrients from
lake waters.
                           25

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                 METHODS AND MATERIALS

The PAAP (Bartsch, 1969) strain of Selenastrum capricornutum
was cultured in modified Gorham's medium  (Hughes et al.,
1958) which contained 0.2 mg P/L so the resulting culture
was phosphorus(P)-limited.  Growth of the algae was measured
by absorbance (1 cm, 750 my).  Absorbance of 0.1 is equiva-
lent to 2-6 million cells/ml, depending on their size,  and
about 50 mg/L dry weight.  Cladophora sp. from Lake Wingra,
Madison, Wisconsin, had been collected and tested for
surplus-P or P-limited conditions by the one-hour boiling -
water extraction method of Fitzgerald and Nelson (1966).
The samples used were P-limited because only 0.03 mg PCK-P
were extracted from 100 mg algae.

Mud samples were collected at mud-water surfaces from
Madison, Wisconsin lakes by hand or by use of an Ekman
dredge.  Lake Wingra and Lake Mendota (3-meter depth) sam-
ples were collected from weed beds.  The sample from the
18-meter depth of Lake Mendota was from the hypolimnion and
was anaerobic when collected.  Dried mud samples were heated
at 110°C for 1 hour or more and ashed samples were heated
at 600°C for 1 hour.  The approximate dry weights of 25 ml
mud samples used in most experiments are presented in
Table 3.

The POit-P analyses were by the molybdate-stannous chloride
method  (APHA, 1965), and the approximate total P analyses
were by the acid-persulfate method of Gales et al. (1968).
                        RESULTS

Availability of Phosphorus in Lake Muds to Algae

In order to determine if Selenastrum could use the phosphorus
(P) of lake muds for immediate growth, P-limited Selenastrum
cells were added to 150 ml of Gorham's  (-P) medium
(100,000 cells/ml) in aerated tubes (pH maintained at
7.5-8.1 by addition of 0.5% C02 in air).  Either POi+-P or
lake muds as sources of P were placed in dialysis tubes in
the cultures, and the growth of algae after 7 and 12 days
was measured by absorbance.  Controls with zero or 0.030 mg
PCK-P were used to test the response of the inoculum to the
test conditions.  Five experiments were carried out.  The
results of a typical experiment are presented in Table 1
which shows the averages of growths attained in duplicate
cultures.
                           26

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Table 1.  Lake Muds as Sources of Available Phosphorus for
          Selenastrum capricornutum  (PAAP).  Basal Medium,
          Gorham's  (-P) Medium; 150 ml/Tube; pH Maintained
          at 7.5-8.1 by Continuous Aeration with 0.5% C02
          in Air; Initial Cell Density, 100,000 Cells/ml;
          Fresh Mud Samples in Dialysis Tubes in Cultures.
Added Phosphorus Source
None
PCH-P
L. Wingra Mud (1/2 m) A
L. Wingra Mud (1/2 m) A
+ 0.03 mg PO^-P on Day 7
L. Wingra Mud (1/2 m) B
L. Mendota Mud (5 m) A
L. Mendota Mud (5 m) A
+ 0.03 mg PO^-P on Day 7
L. Mendota Mud (5 m) B
L. Mendota Mud (18 m) A
L. Mendota Mud (18 m) A
+ 0.03 mg POij-P on Day 7
L. Mendota Mud (18 m) B
Original
Total P
(mg P/Tube)
0.00
0.03
0.09
-
0.16
0.22
-
0.44
0.68
-
1.4
Ave . Growth
of Algae
as Absorbance
(1 cm 750 my)
7 Days
.005
.17
.005
-
.005
.005
-
.005
.025
-
.040
12 Days
.005
.18
-
-040
.005
-
.090
.005
-
.070
.030
                              27

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The results indicate that little or no growth was supported
by the muds/ whereas the algae exposed to 0.03 mg PO^-P
nearly reached their peak yield in 7 days.  Since negative
results with the mud samples might indicate toxicity under
these conditions, PCH-P  (0.03 mg) was added to 3 of the cul-
tures containing muds which had been incubated for 7 days.
The results after an additional incubation period of 5 days
indicated that the algae-mud cultures which had been given
additional PCK-P were able to grow, so this ruled out
toxicity as the reason for lack of growth in the presence
of the muds.  The mud sample from the 18-meter depth in
Lake Mendota provided some P for Selenastrum.  The amount
of growth attained in these samples(absorbances of 0.025
and 0.04) is equivalent to the growth to be expected from
less than 0.01 mg PCH-P.  Since the two mud samples con-
tained 0.68 and 1.4 mg total P, less than 1% of the total
P of these samples was available to the algae tested under
these conditions.  Similar tests also indicated that lake
muds did not provide available nitrogen to Selenastrum and
other algal species under equivalent conditions.

Since tests with Selenastrum might be prejudiced by the use
of laboratory algae and by using dialysis tubes for the mud
so that intimate contact with the muds was not possible,
additional tests were carried out using P-limited Cladophora
sp. from Lake Wingra.  With the use of 250 ml Erlenmeyer
flasks as containers, the P-source being tested and approxi-
mately 10 mg of Cladophora were added to 150 ml Gorham's (-P)
medium.  In all mud cultures, the Cladophora were partially
embedded in the mud.  After an incubation period of 2 days
in the culture room, the Cladophora were removed, washed,
and extracted by the boiling water method.  The average
amounts of POij-P and total P extracted from triplicate cul-
tures are presented in Table 2.

The data indicate that the Cladophora were initially P-
limited but that they were able to obtain measurable amounts
of available P from even the lowest level of POif-P added
(0.02 mg P).  The amount of PCH-P extracted from algae of
cultures with increasing PCU-P levels increased proportion-
ately to the added P up to 0.04 mg P.  A level of 0.08 mg P
appears to have been surplus for the amount of algae present
(10 mg).  The lack of any increase of extractable P in the
algae in the presence of the mud from Lake Wingra and the
3-meter depth of Lake Mendota indicates that none of the
0.8 and 1.2 mg of total P present was available to the
Cladophora.  The mud sample from the 18-meter depth of
Lake Mendota provided P to the Cladophora equivalent to
0.02 mg PCH-P.  Therefore, only 1% of the total P of this
sample was available.
                           28

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Table 2.  Lake Muds as Sources of Available Phosphorus for
          Cladophora sp.  Basal Medium, Gorham's  (-P) Medium;
          150 ml/250 ml Erlenmeyer Flasks; Approx. 10 mg
          P-Limited Cladophora  (0.03 mg PCH-P Extracted/
          100 mg Algae) from L. Wingra; Cladophora Partially
          Embedded in Mud Samples at Bottom of Flask.

Phosphorus Source
None
POif-P
PCK-P
PCK-P
L. Wingra Mud (1/2 m)
L.Mendota Mud (3 m)
L.Mendota Mud (18 m)

Phosphorus
Added
(mg Total P/
Culture)
0.00
0.02
0.04
0.08
0.8
1.2
2.2
Ave . Phosphorus
Extracted from
Cladophora
after 2 Days
(ma P/10 ma Alaae)
PCH-P
0.0032
0.0086
0.018
0.021
0.0033
0.0030
0.0090
Total P
0.006
0.018
0.024
0.028
0.008
0.009
0.018
                            29

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The Sorption of Phosphorus by Lake Muds

Since the above tests and bioassays with Cladophora sp. and
other algae from the Madison lakes indicated that lake muds
under aerobic conditions did not provide algae with adequate
available phosphorus, tests of the sorption of PO^-P by lake
muds were carried out.  In the first series of experiments
different amounts of lake muds were added to 150 ml of
Gorham's  (-P) medium in aerated tubes and the amounts of
POit-P in the supernatants were analyzed at different times.
Initial tests indicated that less than 15 yg PO^-P/150 ml
were released during a period of several days.  The amounts
of POi,-P remaining in the supernatants after the addition
of 300 lag PO^-P were then tested.  The results from a
typical experiment are presented in Table 3 which shows
average percentages of P sorbed by the lake muds.

It is evident that lake muds from aerobic areas of the lakes
as well as muds from anaerobic depths would sorb PCH-P from
solutions under these aerobic conditions.  A good propor-
tion of the POij-P sorption took place in the first 10 minutes
under these conditions, but when similar tests were carried
out with the muds encased in dialysis tubes, at least
24 hours were required for 50% or more sorption.  Thus, it
becomes apparent why PCH-P added to Selenastrum cultures is
not competitively sorbed by lake muds in dialysis tubes in
the cultures since the sorption reaction through dialysis
tubing is so slow and Selenastrum can effectively absorb
maximal quantities of phosphorus within 1 or 2 hours of
exposure  (Fitzgerald, 1970).

As various studies have mentioned different sorption re-
actions, the sorption of P by lake muds that had been dried
or ashed was compared with the sorption by untreated sam-
ples.  The averages of the percentage of P added to 150 ml
of Gorham's  (-P) medium which was sorbed by treated mud
from the 18-meter depth of Lake Mendota are presented in
Table 4.

The amount of PO^-P added and the treatments given the mud
from Lake Mendota appear to have an effect on the sorption
of P.  The percentage of P sorbed by 2.8 g of mud decreased
as the amount of PO^-P added increased, but even 60% of
1,200 yg of PO^-P could be sorbed from solution by fresh
samples within 10 minutes.  The effect of drying on the
sorption reaction was to cut the rate in half.  However,
the rate of sorption by ashed samples was very high.  With
samples from Lake Wingra and other depths of Lake Mendota,
ashed muds were also found to sorb significant amounts of P
in less than 2 minutes.  Therefore, the sorption of P by
                           30

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Table 3.  Rate of Sorption of Phosphorus by Different
          Aerobic Lake Muds.  Gorham's  (-P) Medium;
          150 ml/Aerated Tube; pH 7-8;  Supernatants of
          Mud-Containing Tubes Without  Added P Contained
          Less Than 15 ug POij-P/150 ml  at Any Time;
          300 ug PCH-P Added to 150 ml  Medium Plus Mud
          and PO^-P Remaining in Supernatant Analyzed
          after Different Times.
Mud Source
L. Wingra (1/2 m)
L. Mendota (3 m)
L. Mendota (5 m)
L. Mendota (18 m)
Mud Added
(g[Dry Weight]/
150 ml)
17
22
13
3
Ave
Phos-
10 rain
30
40
20
70
. Percentage
phorus Sorbed
60 min
65
65
25
80
180 min
80
75
30
85
                           31

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Table 4.  Rate of Sorption of Phosphorus by Treated Bottom
          Mud (18 meters)  of Lake Mendota under Aerobic
          Conditions.  Gorham's (-P)  Medium; 150 ml/Aerated
          Tubes; pH 7-8; Supernatants of Tubes of Mud
          without Added P Contained Less Than 30 yg PO^-P
          at Any Time; 300 yg or More PCK-P Added and PO%-P
          Remaining in Supernatant Analyzed after Different
          Times.
Mud
Treatment
Fresh
Fresh
Fresh
Dried
Ashed
Mud Added
Dry Weight
(g/150 ml)
2.8
2.8
2.8
2.8
2.3
Phosphorus
Added
(Ug/150 ml)
300
600
1200
300
300
Ave. Percentage
Phosphorus Sorbed
10 min
75
70
60
35
95
60 min
90
80
70
50
95
                           32

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lake muds is not related to the organic material present
(0.5 g of volatile solids per  2.8 g dry solids).

The tests reported thus far were carried out with relatively
large amounts of lake muds added to 150 ml of Gorham's  (-P)
medium.  Further tests with lesser quantities of muds and
in other media were also carried out.  Gorham's  (-P) medium
contains over 700 mg dissolved solids/liter, but PAAP (-P)
medium has less than 200 mg/liter.  Therefore, natural
waters and the more dilute PAAP  (-P) medium were used.
Different amounts of mud from  the 18-meter depth of Lake
Mendota were added to the test media which contained 1 mg
PCu-P/liter and the POt-P removed from the solution analyzed,
The results of a typical experiment with PAAP  (-P) medium
showing the average percentage of POtt-P sorbed after 30 and
60 minutes are presented in Table 5.

The data indicate that the amount of POit-P sorbed from any
volume increases as the amount of mud added increases.  With
an increase in the volume of the water in which a quantity
of mud is suspended, the percentage of PC^-P sorbed de-
creases.  Similar results were also obtained when different
lake waters were used.  One of the lake waters tested had
90 yg PO^-P/liter present, and this amount was reduced to
less than 20 yg/liter by the muds within 30 minutes, so the
POit-P of lake water appears to be sorbed in the same manner
as added PCU-P.  Therefore, these quantitative results would
be of value in predicting the  amount of mud which would have
to be used to sorb particular  quantities of P from lake
waters.
                      DISCUSSION

The early onset of phosphorus-limitation of the Cladophora
sp. along the southern  shore of Lake Mendota  (mid-June in
1968  [Fitzgerald and Lee,  1970] and early June in 1969) has
indicated that the phosphorus  supplies coming from winter
degradation, spring runoff, and lake overturn" do not remain
available in the surface waters of a lake for the nutrition
of algae restricted to  these waters.  The manner in which
available phosphorus is lost from solution is, therefore,
of considerable interest in studies of eutrophication.
POit-P has been shown to be lost from sewage plant effluents
in oxidation ponds during  relatively high pH conditions
associated with the growth of  algae, but the solubilization
of the phosphorus when  pH values decreased towards pH  8
indicated that the original loss from  solution must have
been by precipitation  (Fitzgerald, 1961).  The loss of
phosphorus added to fish ponds reported by Hepher (1,965)
                              33

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Table 5.  Rate of Sorption of Phosphorus by Bottom (18 meter)
          Mud from Lake Mendota under Aerobic Conditions.
          PAAP Medium (-P);  pH 7-8;  Aerobic Mud Samples
          Added to 1 mg P/L.
Sample
Volume (ml)
50
50
50
100
100
100
250
250
250
Mud Added
(grams [dry wt] )
0.1
0.2
0.4
0.2
0.4
0.8
0.2
0.4
0.8
PCK-P Added
(yg)
50
50
50
100
100
100
250
250
250
Ave . Percentage
Phosphorus Sorbed
30 min
30
70
80
30
60
80
20
30
50
60 min
50
70
90
50
70
90
30
40
60
                           34

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also seemed to be due to precipitation  as tricalcium phos-
phate.  However, studies on  the  availability of phosphorus
which, had been precipitated  by the  addition of relatively
high concentrations of calcium and  iron or only iron to
algal culture media indicated that  such precipitated phos-
phorus was nearly as readily available  for the growth of
several species of phosphorus-limited algae as soluble
POit-P (Fitzgerald, 1970) .  As a  matter  of fact, when teeth
were used as a source of "insoluble" phosphorus at the sug-
gestion of Lee  (G. Fred Lee, 1969,  personal communication)
it was found that dog and  shark  teeth and even petrified
shark teeth supported the  growth of several species of
phosphorus-limited algae at  about the same rate as soluble
POit-P (Fitzgerald, 1970) .  Therefore, the adsorption re-
actions suggested by Mortimer  (1941) and others must be
of more importance in the  loss in availability of phosphorus
in lake waters than mere insolubility since the data pre-
sented here indicate that  the phosphorus of lake muds is
not immediately available  to phosphorus-limited algae under
aerobic conditions.

Since phosphorus is so readily lost from lake waters early
in the spring, the importance of winter degradation, spring
runoff, and spring turnover  as sources  of phosphorus to
algal ecology is questionable.   Therefore, the data pre-
sented here and by others  (Barter,  1968) showing that lake
muds sorb phosphorus under aerobic  conditions could be of
immense ecological importance.   If  muds could be used to
sorb the phosphorus of surface waters or the epilimnion
after stratification takes place but before anaerobic con-
ditions develop in the hypolimnion, the muds could effec-
tively strip the epilimnion  of phosphorus and return it
to the bottom or to the hypolimnion.  There would be less
phosphorus in surface waters, therefore, to support ob-
noxious algal growths.   It should be pointed out that
studies by Lee  (G. F. Lee, 1970, personal communication)
have shown that very significant amounts of phosphorus are
released from aerobic lake muds  when tests are carried out
for periods exceeding one  month  and this release is more
rapid under anaerobic conditions.   Thus, information on the
relative importance of continuous sources of phosphorus to
surface waters, such as contamination from waste waters,
the regeneration from bottom muds,  or decay of plants, is
of prime importance.

It should be emphasized that if  the available phosphorus of
surface waters were to be  stripped  by the use of bottom
muds, the growth of algae  might  be  limited, but such actions
would probably have no effect on the growth of rooted aquatic
weeds.  Lake muds have been  shown by Martin et al.  (1969)
                            35

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to be main sources of nutrients for the aquatic weed,
Najas sp.  In the absence of competition with algae  (Easier
and Jones, 1949; Fitzgerald, 1969) the growth of aquatic
weeds would probably be stimulated beyond normally expected
amounts.  Therefore, the physical manicuring of weed beds
in much the same manner as used to control the growth of
grass in parks would be necessary to allow the most benefit
to be gained from aquatic environments.
                      REFERENCES

Amer. Pub. Hlth. Assoc.  1965.  Standard Methods for the
Examination of Water and Wastewater.  12 ed., APHA, Inc.,
1790 Broadway, New York, N. Y.  10019.  769 p.

Bartsch, A. .F.  (Chrm.) .  1969.  Provisional Algal Assay
Procedure.  Joint Ind.-Gov't Task Force on Eutrophication,
P.O. Box 3011, Grand Central Sta., New York, N. Y. 10017.
62 p.

Fitzgerald, G. P.  1961.  Stripping Effluents by Biological
Means.  Algae and Metropolitan Wastes Seminar, U.S.P.H.S.
Tech. Report W61-3, p.  136-139.

Fitzgerald, G. P.  1969.  Some Factors in the Competition
or Antagonism among Bacteria, Algae, and Aquatic Weeds.
J. Phycol., 5^:341-349.

Fitzgerald, G. P.  1970.  Evaluations of the Availability
of Sources of Nitrogen  and Phosphorus for Algae.
J. Phycol., 6_: 239-247.

Fitzgerald, G. P. and T. C. Nelson.  1966.  Extractive and
Enzymatic Analyses for  Limiting or Surplus Phosphorus in
Algae.  J. Phycol., 2^:32-37.

Gales, M. E., Jr., E. C. Julian,  and R. C. Kroner.  1966.
Method for Quantitative Determination of Total Phosphorus
in Water.  J. Amer. Water Works Assoc., 58:1363-1368.

Harter, R. D.  1968.  Adsorption  of Phosphorus by Lake
Sediments.  Soil Sci. Amer. Proc., 32:514-518.

Easier, A. D., and E. Jones.  1949.  Demonstration of the
Antagonistic Action of  Large Aquatic Plants on Algae and
Rotifers.  Ecology, 30:359-364.

Hepher, B.  1965.  The  Effect of  Impoundments on Chemical
and Textural Changes in Fishpond's Bottom Soils.  Bamidgehs
Bull. Fish Culture in Israel, 17:71-80.
                            36

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Hughes, E. 0., P. R. Gorham, and A. Zehnder.  1958.
Toxicity of a Unialgal Culture of Microcystis aeruginosa.
Can.J. Microbiol., 4:225-236.
Lee, G. F.  1970.  Factors Affecting the Exchange of
Materials between Lake Water and Sediments.  Eutrophication
Information Program, Univ. Wisconsin, Madison.  Literature
Review No. 1.   50 p.

Martin, J. B.,  Jr., B. N. Bradford, and H. G. Kennedy.
1969.  Factors  Affecting  the Growth of Najas in Pickwick
Reservoir.  National Fertilizer Development Center, T.V.A.,
Muscle Shoals,  Ala.  47 p.

Mortimer, C.  H.  1941.  The Exchange of Dissolved Substances
between Mud and Water  in  Lakes.  J. Ecol., 29:280-329.
                               37

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      EVALUATIONS OF THE AVAILABILITY OF SOURCES

         OF NITROGEN AND PHOSPHORUS FOR ALGAE

                       ABSTRACT

Techniques are compared for the evaluation of nutrient
sources in which the potential nutrients are in contact
with algae over their entire  culture period versus rela-
tively short-term exposure tests.  The availability of
relatively insoluble nutrients:   iron-phosphorus compounds
and teeth for phosphorus, hair for nitrogen, iron pyrites
for iron, and marble for carbon,  indicates that in many
cases the equilibrium between soluble and insoluble forms
allows algae to successfully  compete with insoluble forms
for nutrients.  Apparently, factors other than mere insolu-
bility prevent algae from obtaining available nitrogen or
phosphorus from phosphate rock or lake muds.  The facts,
(1) that live algae and aquatic weeds do not share their
adequate or surplus nutrients with nutrient-limited algae
and (2) that lake muds do not provide readily available
nitrogen or phosphorus, indicate  that once lake waters are
stripped of available nutrients by plant production, further
plant production will depend  upon nutrients from continuous
sources of nutrients, such as wastewater effluents.
                     INTRODUCTION

In order to determine if a potential source of an algal
nutrient is actually available for the growth of algae one
usually adds different concentrations of the source to
nutrient-limited cultures of algae and compares the growth
attained with cultures lacking the nutrient and with the
growth of cultures having standard amounts of known avail-
able sources of the nutrient.  One of the disadvantages of
this procedure is that algae grow so slowly that the poten-
tial source of nutrient must be present in the medium for
a week or more before one can judge whether it-was an avail-
able source of nutrient under those conditions.  During this
time of incubation one cannot be sure that the original
source has not been degraded to a more available source of
nutrient by the conditions of the test, such as the degrada-
tion of condensed phosphates to orthophosphate either in the
presence or absence of bacteria in lake waters or culture
media. (-3J  Therefore, when possible, it would be desirable
to use nutrient-availability tests which would rely on
relatively short exposure times to a potential source of
nutrient under conditions which can be readily controlled,
                           39

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such as pH, light, or temperature, and which represent some
of the factors that might be involved under field exposures
to the sources of the nutrients.

Relatively short-term exposure periods to potential sources
of nutrients under controlled environmental conditions can
be followed by removing aliquots of algae after different
exposure times and either (1) subculturing them to measure
the amount of growth attained after further incubation or
(2) measuring an enzymatic change related to the absorption
of a nutrient.  The type of test to be used will be limited
by known reactions with established nutrients.  We have
demonstrated several different techniques for the evaluation
of the availability of nitrogen from sources and a short-
term test, using the subculture technique, for evaluating
sources of phosphorus.

Besides the physical factors, such as pH, light, and the
chemical composition of a potential nutrient source, the
solubility of a compound is frequently considered to be of
importance in nutrition.  In order to evaluate some nutrient
sources that potentially might be affected by solubility,
we have demonstrated with relatively insoluble nitrogen and
phosphorus compounds that in some cases the equilibrium
between soluble and insoluble states of some compounds allows
adequate nutrients to be available to algae under the usual
growth conditions.  However, not all potential nutrients,
such as the nitrogen and phosphorus of lake muds, are readily
available to algae in short-term experiments.

Several workers *•l 2J have demonstrated how certain aquatic
animals can provide nitrogen or phosphorus for use by algae.
However, little work has demonstrated nutrient-sharing
between algae or between algae and aquatic plants of higher
forms.  In contrast, there are many articles dealing with
tracer studies of nutrient exchange.  Therefore, studies
were made of the availability of the nitrogen and phosphorus
of one alga or aquatic weed to another.  In other words,
conditions under which plants share their nutrients were
studied.

These studies are presented in order to demonstrate tech-
niques available for such investigations and to provide
information on some of the factors affecting the avail-
ability of certain potential nutrient sources.  It is hoped
that further evaluations will be stimulated from the in-
formation presented here.
                           40

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                 MATERIALS AND METHODS

The green alga, Selenastrum  capricornutum  (PAAP;  2), was used
as the main test organism.   This  alga was  cultured in modi-
fications either of PAAP medium^2-1 or of Gorham's  medium.^11-1
The techniques used to determine  whether algae were phos-
phorus-limited19-1 or nitrogen-limited(-5J have been described
previously.  The measurements of  growth of cultures were
usually done by light absorbance  measurements  (1  cm, 750 mu) .
An absorbance of 0.1 is equivalent to 2 to 6 million cells
per ml, depending upon cell  size,  and to about 50 mg/L dry
weight.  The field collections of algae, aquatic  weeds, and
lake muds came from Lakes Mendota or Wingra of the Madison,
Wisconsin area.

Analyses for NH3-N were by direct Nesslerization,1-1-1 for
PO/t-P by the molybdate-stannous chloride method,*-1'' and
for approximate total phosphorus  by the acid-persulfate
method of Gales et aJU *>10J   Analyses of the concentration
of soluble POij-P from various "insoluble"  phosphorus sources
were carried out at pH 7.5 after  filtration through glass-
pad filters  (Reeve Angel, #934 AH).  The percentage of ortho
POij-P in the commercial phosphorus sources used was analyzed:
sodium tripolyphosphate  (1.4% ortho POit-P) , sodium pyro-
phosphate  (2.5% ortho POit-PO, and sodium phosphite  (0.36%
ortho POit-P) .
                        RESULTS

Techniques for Evaluation of Nutrient  Sources

A common procedure for  evaluating  a  nutrient source  is to
add nutrient-limited  algae  to  different  concentrations of
the source, using suitable  standards as  guides.  The growth
attained after incubation is then  measured.  This method
was used to evaluate  different sources of nitrogen for
Selenastrum.  Selenastrum from PAAP  medium were placed in
PAAP  (-N) medium for  2  additional  days to ensure that cells
were N-starved.  These  algae then  were added to 150  ml of
medium in aerated  (air  + 0.5%  CO2) tubes at pH 7 to  give
an initial concentration of 100,000  cells/ml.  The growths
as absorbances after  different incubation times with
ammonia, nitrate, nitrite and  glutamic acid in a typical
experiment are presented in Table  1.

The data indicate that  the  rate  of utilization of ammonia
was slightly less than  for  the other sources  (i.e.,  data
for 5 and 6 days), but  the  pH  of the cultures with 10 mg
NH3-N/L was lower  (pH 6.3 versus 6.8)  after incubation for
                             41

-------
Table 1.  Utilization of Nitrogen Sources by Selenastrum,
          PAAP (-N) Medium, 150 ml/Tube, pH 7.



N Source
None

Ammonia


Nitrate


Nitrite


Glutamic Acid




mg N/L
0
5

10
5

10
5

10
5

10
Growth of Algae
as Absorbance of Cultures
(1 cm, 750 my)
5 Days
0.015
.06

.06
.11

.15
.13

.14
.06

.15
6 Days
0.015
.13

.15
.22

.36
.26

.36
.12

.26
11 Days
0.015
.28

.53
.30

.55
.36

.58
.19

.38
                           42

-------
6 days.  After 11 days of culture  the  growths  in ammonia,
nitrate and nitrite were essentially the  same  at each nitro-
gen level, but the growth with  glutamic acid-nitrogen was
about 65% that of the other nitrogen sources.  Because the
degradation or conversion of  the nitrogen sources before
utilization could not be ruled  out, and because pH was not
consistent, these results were  not considered  to be very
reliable.

The physical conditions of tests can be more readily con-
trolled when short-term exposures  of nutrient-limited algae
to potential nutrient sources are  used.   In order to demon-
strate this technique, phosphorus-limited or nitrogen-limited
algae were exposed to different nutrients under controlled
conditions and the subsequent growth of subcultures was used
to evaluate the relative availability  of  the nutrients.
Phosphorus sources were evaluated  by using Selenastrum grown
in PAAP medium plus at least  2  days in PAAP (-P) medium so
they were phosphorus-limited  at the time  of use.  The algae
were placed in aerated tubes  with  different levels of phos-
phorus  (0, 0.08, and 0.32 mg  P/150 ml), and after different
exposure times, duplicate 0.5 mg  (dry  weight)  samples were
removed, washed and placed in 25 ml of sterile PAAP(-P)
medium for incubation.  Growth  attained in the subcultures
for a typical experiment evaluating ortho PCH-P, tripoly-
phosphate, pyrophosphate, and phosphite are presented in
Figure 1 as the averages of duplicate  absorbances attained
after 25 days.

It was found that Selenastrum would not absorb and utilize
phosphite-phosphorus in either  light or dark exposures of
up to 26 hours; little or no  growth occurred in subcultures,
and there was no loss in phosphite concentration in the
exposure tubes.  This contrasted to no measurable phosphorus
remaining in the supernatants of the other phosphorus
sources.  Ortho PCH-P and pyrophosphate appeared to be
equally effective as sources  of phosphorus.  The apparent
increased availability of pyro  is  felt to be insignificant.
The rate of utilization of tripolyphosphate was less than
that of POit-P and pyrophosphate; the growth attained in
subcultures after 1/2- to 1-hour exposures was about 50%
that of PCK-P and pyrophosphate subcultures.   Growth of sub-
cultures was essentially the  same  after 4- to  26-hour
exposures.  The results of tests in which the  exposure tubes
were held in darkness during  the sampling periods were simi-
lar to the results obtained when tubes were held in the
light  (600 ft.C).  Exposure tests  in the  light at pH 7
(6.8-7.3) and pH 9  (8.8-9.3)  gave  equivalent results:  PCH-P
was absorbed more rapidly than  tripolyphosphate, but with
                            43

-------
Figure 1.  The Effectiveness  of  Different Phosphorus Sources
          for the Growth of  Selenastrum by the Exposure and
          Subculture Technique.  Averages of Duplicate
          Cultures.   PAAP (-P)  Medium, pH 7-7.5, 400 ft.
          Candles.
  0.5
                                       O-Control  (-p)

                                       x -Ortho

                                       A-Tri Poly

                                       D-Pyro

                                       •-Phosphite

                                        	0.08mgP/l20ml.

                                        	0.32mgP/l20ml.
     00.5
           EXPOSURE  TIME
              (Hours)
                           44

-------
longer exposures the  trlpolyphc-sphate was  as  good  a phos-
phorus source as POi,-P.

When the exposure and subculture  technique was  used with
nitrogen-limited Selenastrum,  it  was found (Table  2)  that
the subsequent growth of  0.5 rag samples  from  algae exposed
to ammonia, nitrate,  nitrite and  urea  (3.2 mg N/120 ml) in
600 ft.C light resulted in  light  absorbances  after incuba-
tion for 3 weeks of 0.06  for control  (-N)  samples  and 0.16
to 0.22 for exposures of  24 hours to the nitrogen  sources.
The rates of utilization, as measured by the  amount of growth
attained by the 0.5 mg samples taken at  increasing exposure
times, were so close  to each other for the different  nitro-
gen sources that this method could not detect any  differences
in availability of these  nitrogen sources.

The distinct disadvantage of the  techniques demonstrated thus
far has been that results are  not apparent until after a 2-
to 3-week incubation  period of the algae.  Results could be
determined more rapidly if  changes produced in  the algae
were followed.  Such  tests  could  not replace  the ultimate
test of the growth of algae with  absorbed  nutrients,  but
could be used for rapid evaluations under  specific environ-
mental conditions.  One such test is to  follow  the absorp-
tion of phosphorus by measuring changes  in the  amount of
extractable phosphorus from algae after  exposure to dif-
ferent concentrations of  phosphorus sources,  as described
in a later section.   Another short-term  technique  is  to
measure the effects of sources of nitrogen on the  rate of
NHs-N absorption in the dark by nitrogen-limited algae.
In a typical series of tests,  nitrogen-limited  Selenastrum
were added to test media  (24 mg algae/80 ml Gorham's  (-N)
medium so that 3 mg samples could be removed  after differ-
ent exposure times and then washed, and  the amount of NHa-N
absorbed in the dark  over 30 minutes measured.  A  summary
of typical results of at  least 3  tests with 0 and  7.5  mg
ammonia-, nitrate-, or nitrite-nitrogen  in the  light
(600 ft.C) at pH 7 versus pH 9 is presented in  Figure 2.

In all tests the algae from control exposure  tubes lacking
any fixed nitrogen  (-N) had NH3-N absorption  rates of
15-20 yg NH3-N/3 mg algae/30 minutes which indicated  they
were nitrogen-limited.tsj   The effect of added  ammonia was
detected within 30 minutes, but detection  of  added nitrate-
and nitrite-nitrogen  required  longer exposure times.   Ammonia
was much more effective as  a nitrogen source  by this  tech-
nique at pH 9 than at pH  7, whereas nitrate-  and nitrite-
nitrogen required less time to affect the  NH3-N absorption
rate at pH 7 than at  pH 9 with Selenastrum.   Similar  results
were obtained if the  exposures to the nitrogen  sources were
                            45

-------
Table 2. Utilization of Nitrogen Sources by
         Selenastrum as Measured by Growth
         Attained by 1/2 mg Samples Placed
         in 25 ml PAAP (-N) Medium after Dif-
         ferent Exposure Times.  400 ft. C.,
         pH 7, 3 Weeks Incubation.  Exposure:
         3.2 mg N/16 mg Algae/120 ml PAAP (-N).
N Source
None
Ammonia
Nitrate
Nitrite
Urea
Effect of Exposure Time on the
Average Growth Attained
(Absorbance, 1 cm, 750 my)
1 Hour
0.06
.08
.08
.09
.08
6 Hours
0.06
.10
.08
.11
.11
24 Hours
0.06
.21
.16
.16
.22
                       46

-------
  Figure 2. The Effect of Different Nitrogen Sources on the
            Ammonia Absorption Rate of Selenastrum.   Averages
            of Duplicate  Cultures.   Gorhain^" (-N) "Medium,
            pH 7-7,5  versus  pH 8.8-9.3,  600 ft Candles.
z o
0.5
H E
CQ
<
   a
   o>
O  ro
  X
UJ 2
                            PH7-7.5
                                 Control  (-N)
        20-
                            pH8.8-9.3

                                   Control
                                         Nitrate -N
                                         —•—.
                                         "-*•-^
                                    Nitrite-N
                                                  40
                            HOURS
                             47

-------
carried out in light or in darkness.  This technique would
appear to be suitable, therefore, for comparative tests with
potential nitrogen sources and with relatively short exposure
times necessary.


Solubility as a Nutritional Factor

Solubility should be added to the physical factors, pH, light,
and chemical structure, which might affect the availability
of a nutrient.  Solubility is not a simple factor related
only to whether algae can compete with the equilibrium be-
tween a soluble and insoluble form of a nutrient, but con-
sideration of it is complicated by the fact that if a nutri-
ent is insoluble it probably will tend to settle out of sus-
pension and away from the algae.  Therefore, only speculation
can be made of the effect of solubility in a lake environment
until more knowledge is gained as to the location and manner
of absorption of nutrients by algae.  However, tests can be
made of the availability of soluble versus insoluble nutrients
in algal cultures, and a few such examples are presented.

The solubility of phosphorus in PAAP medium (minus EDTA) can
be manipulated by the addition of FeCla:  0.2 mg P/L is 100%
soluble in PAAP; 50% soluble in PAAP + 2.5 mg Fe/L; and 15%
soluble in PAAP + 5 mg Fe/L.  The relative rate of growth of
Selenastrum in 0.06 to 0.4 mg P/L was measured when the phos-
phorus was soluble or partially insoluble and when the phos-
phorus was in the algal growth medium or separated from the
algae by semipermeable membranes.

In the simplest experiment the rate of growth of Selenastrum
(100,000 cells/ml initially) was followed in the three media
mentioned above, such that the phosphorus was soluble, 50%
soluble, and 15% soluble.  These tests were carried out in
150 ml of medium in tubes aerated with 0.5% COz in air in
order to maintain pH 7-7.5.  The growth of Selenastrum in
levels of 0.06 to 0.4 mg P/L is presented as light absorb-
ances in Table 3.

The data indicate that despite a range of solubility of the
available phosphorus from 100% to 15%, the rate of growth
was the same between 4 and 8 days at all levels of phos-
phorus , and the maximum growth attained was independent of
the media at phosphorus levels of 0.27 and 0-4 mg/L.

Since the above results might be prejudiced by the fact that
the algae were in intimate contact with the insoluble form
of phosphorus so that one could not separate the effect of
                            48

-------
Table 3.  The Effect of Solubility of Phosphorus on the
          Growth of Selenastrum.  (150 ml, pH 7-7.5)




Medium

PAAP


PAAP +

2.5 mg Fe/L


PAAP +
5 mg Fe/L




% P
Soluble1

100




-J U


1 C
lb





mg P/L
0.067
0.13
0.27
0.40
0.067
0.13

0.27
0.40
0.067
0.13
0.27
0.40
Ave . Growth
as Absorbances
(1 cm, 750 my)
4
Days
0.07
.09
.08
.10
.02
.06

.06
.07
.00
.01
.03
.04
6
Days
0.10
.16
.19
.23
,03
.13

.25
.31
.01
.02
.19
.33
7
Days
0.11
.18
.22
.27
.04
.14

.32
.40
.01
.05
.27
.41

8
Days
0.11
.19
.24
.31
.05
.15

.33
.44
.02
.08
.31
.45
 Solubility at 0.2 mg P/L Level
                           49

-------
algal surface enzymes on the insoluble phosphorus from the
effects of competition of the algae with the equilibrium
between soluble and insoluble forms of phosphorus, a series
of experiments were carried, out in which the phosphorus was
placed in dialysis tubing or on the opposite side of a
membrane filter (Millipore, 0.45 u) from the Selenas trum.
When 0.05, 0.10 and 0.20 mg P were placed in about 30 ml
of PAAP medium inside dialysis tubing and the tubing placed
in 750 ml of PAAP medium in one-liter Erlenmeyer flasks,
the growth of Selenastrum (initially 100,000 cells/ml) in
the culture flask attained absorbances of 0.13, 0.15 and
0.27 after 5 days.  When the basal medium in the tube and
culture was PAAP + 5 mg Fe/L (solubility of 0.2 mg P/L = 15%),
the same levels of phosphorus resulted in absorbances of
0.03, 0.15, and 0.32 after 5 days.  Similarly, the rate of
growth of Selenastrum in the medium in which the phosphorus
was only 15% soluble was approximately the same as in the
PAAP medium when the phosphorus was placed on the opposite
side of a membrane filter from the algae (500 ml Bellco
spinner flask-#3008, 0.45 y Millipore filter).

We infer from these data that the solubility of phosphorus
in culture media in which the reaction of iron and phos-
phorus may result in different solubilities of the phosphorus
is not a limiting factor for the growth of algae.  The
chemical equilibrium between insoluble and soluble phosphorus
under these conditions is apparently such that phosphorus is
available for algal growth; the rate of solubility of phos-
phorus from the iron-phosphorus complex is faster than the
rate of growth of the alga, Selenastrum.  Similar tests using
phosphorus which had been precipitated with relatively high
levels of both calcium and iron at pH 9-9.5 also indicated
that insoluble phosphorus was readily available for the
growth of Chlorella pyrenoidosa (Wis. 2005).

Lee (G. Fred Lee, 1969, personal communication) suggested
that forms of insoluble nutrients from nature be tested for
availability of nutrients.  Suggestions included:  teeth
and rocks for phosphorus, hair for nitrogen, and iron pyrites
for iron.  Accordingly, we have tested various types of teeth
as sources of phosphorus for Selenas trum.  A dog tooth
(poodle, puppy, Sherry's Silver Dusay, a 70 mg canine tooth),
a shark tooth (unidentified, 2.9 g) and 3 petrified shark
teeth (unidentified, 1.7 g total), when placed in PAAP (-P)
medium inoculated with 100,000 Selenastrum cells/ml, or
separated from the algae by dialysis tubing, supported the
rate of growth and ultimate growth attained by at least
0.2 mg ortho POi»-P/L.  Repetition of these tests gave the
same results each time, so it was concluded that washing
                            50

-------
the teeth, or repetitive growth of algae did not exhaust the
supply of phosphorus supplied by the teeth.

Hair was used to demonstrate "insoluble" N sources.  The
results from one test in which various sources of well
rinsed hair were added directly to Gorham's (-N) cultures
of N-limited Selenastrum  (100,000 cells/ml) are presented
in Table 4.

It was found that the rate of growth of the Selenastrum
appeared to be faster in the control culture with 5 mg
N03-N/L than with the various hairs, but hair from a cat
and two dogs appeared to support good growths.  The human
hair used in this test had been cut about 2 years before
the test, so an additional test with fresh hair was also
carried out, but the results with as much as 100 mg (dry
weight) indicated human hair to be a relatively poor
nitrogen source.  Since such negative results might in-
dicate a toxicity reaction, NOa-N (5 mg/L) was added to
a culture containing 20 mg of human hair in which the
Selenastrum had not grown appreciatively over a 7-day
period.  Within 24 hours the absorbance of the culture had
increased from 0.01 to 0.05 and the culture had become
green.

During the course of experiments on the availability of
sources of iron, two series of tests were carried out with
natural crystals of iron pyrites which were assumed to be
very insoluble because they had resisted solution under
natural conditions in the river in which they were found.
It was found that although control PAAP (-Fe, +EDTA) cul-
tures attained barely measurable growth, Selenastrum were
able to use the iron pyrites as a readily available source
of iron, and the resulting growth was equivalent to that
supported by the Fed3 usually used.

Similar tests of carbon sources for Selenastrum and the
bloom-forming blue-green alga, Microcystis aeruginosa
(Wis. 1036), have indicated that under conditions in which
carbon was a limiting nutrient in cultures in PAAP or
Gorham's medium (flasks 1/2-1/3 filled with medium and
closed with rubber stoppers) the carbon from increasing
concentrations of either calcium carbonate or marble,
despite their being relatively insoluble sources of carbon,
could support increasing amounts of growth of these algae.

As an approach to more ecologically important sources of
algal nutrients, samples of Florida phosphate rock  (Nat.
Bur. Std. #120 a)  (0.9% of phosphorus as soluble. P in PAAP
medium) were added to Selenastrum cultures (100,000 cells/ml,
                            51

-------
Table 4. Hair as a Source of Available Nitrogen for
         Selenastrum.  Gorham's Medium (-N), pH 1,
         150 ml/Tube.

N Source
None
N03-N
Human (Brown)
ii ii
ii ii
Cat (Persian)
H ii
ii H
Dog (Irish Wolfhound)
H H ii
H ii H
" (Poodle)

Dry Weight
(mg/Tube)
0
0.75
10
20
40
10
20
40
10
20
40
20
Growth of Selenastrum
as Absorbance
(1 cm, 750 my)
5 Days
0.02
.31
.02
.02
.04
.04
.07
.15
.04
.08
.14
,06
9 Days
0.02
.42
.03
.02
.03
.08
.16
.36
.06
.13
.22
.10
                             52

-------
150 ml PAAP [-P]).  This, rock was either dispersed in the
cultures or separated from the algae by dialysis tubing so
as to provide as much as 5 mg of total P/L for the cultures.
It was found that only a slight growth of Selenastrum was
supported by the rock source of phosphorus; up to 5.0 mg
total P/L supported as much growth as 0.05 mg ortho PCK-P/L.
Ultrasonic treatment of the rock suspension did not increase
the availability of the phosphorus.  The addition of 3 pet-
rified shark teeth to one rock culture which had supported
little growth for 1 week resulted in the tripling of the
amount of algae after 4 days of further incubation.  It was
concluded that only a fraction of the total phosphorus of
this rock was available for Selenastrum, and this conclusion
is in contrast to the results with relatively insoluble
sources of phosphorus tested before.  In other words, some
factor other than simple insolubility was inhibiting the
availability of the phosphorus of the Florida phosphate
rock.

In experiments^6-' evaluating the immediate availability of
phosphorus of lake muds under aerobic conditions for the
growth of algae in short-term experiments, it was found that
whereas phosphorus-limited Selenastrum nearly reached their
peak yield in 7 days with 0.03 mg PCU-P/150 ml cultures,
cultures containing as much as 2 mg of total phosphorus in
lake muds supported little or no growth when muds from
2 lakes and from 3 depths of one of the lakes were tested.
Since negative results might indicate toxic conditions in
these experiments, additions of PCH-P (0.03 mg) were made
after 7 days of incubation to 3 of the cultures containing
muds.  The results of an additional incubation period of
5 days indicated that the algae-mud cultures were able to
grow with the additional PCH-P, so this ruled out toxicity
as a reason for lack of growth in the presence of muds.
Additional tests with field collections of phosphorus-limited
Cladophora sp. also indicated this alga could not obtain
increased extractable phosphorus compounds from 48-hour ex-
posures to various lake muds, but could with added PO^-P.
When lake muds were tested for available nitrogen it was
repeatedly found that as much as 8 mg of lake-mud nitrogen
did not support measurable growth of Selenastrum, but that
0.8 mg NOs-N supported good growth  (absorbance of 0.3).

It is apparent, therefore, that while certain relatively
insoluble forms of nutrients may be readily available for
the nutrition of algae, certain natural forms of phosphorus
or nitrogen, such as phosphate rock and lake muds, were not
readily available sources of nutrients.
                            53

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Factors in the Sharing of Nutrients Between Plants

Observations and results of field tests^ have suggested
that algae do not effectively share their nutrients with
one another.  It is well known that under certain circum-
stances the nitrogen from N2-fixing algae can be found in
other plants,1'13'1 and it is generally assumed that algae
could be the base of organic matter production in recently
denuded areas.  However, the question of whether one viable
alga or plant will effectively support the growth of another
within short-time periods under natural conditions has had
only negative answers thus far. *-8-'  Therefore, laboratory
tests have been carried out to determine what factors in-
fluence the sharing of nutrients of one algal species with
another.

A series of tests were carried out in which the potential
sources of nitrogen or phosphorus were either placed in the
cultures or separated from the cultures by dialysis tubes,
and the growth of nitrogen-limited or phosphorus-limited
Selenastrum (100,000 cells/ml) under these conditions was
compared.  Preliminary results indicated that live algae
did not share their nutrients.  Therefore, more detailed
tests were carried out in which live algae and algae gently
killed by freeze-thawing were compared as to the availability
of their nitrogen or phosphorus.  The freeze-thawing technique
was shown to kill 99% or more of Selenastrum tested.  The
amounts of algae added as nutrient sources were calculated
as dry weights taken from equivalent amounts of algae.  The
results of the tests are presented in Tables 5 and 6 as
absorbances of cultures and summarized as the calculated
average absorbance per 10 mg of added algae.

The data of Table 5 indicate very little growth of nitrogen-
limited Selenastrum was supported by live algae, but killed
algae which were not nitrogen-limited supported good growths
of this alga.   Very little growth was attained in cultures
which depended upon nitrogen from samples either from the
nitrogen-limited Cladophora sp. from Lake Mendota or from
the nitrogen-limited Selenastrum from a Gorham's medium
containing only 7.5 mg N/L.

The data of Table 6 indicate that, as in the case with nitro-
gen-limited Selenastrum, phosphorus-limited Selenastrum are
not provided available phosphorus by live algae.  Similar
evidence of this nature was noted when it was found that a
phosphorus-limited Cladophora sp. (0.07 mg PCH-P extracted/
100 mg algae)  grew at the bottom  (3-meter depth) of Lake
Mendota where it was intimately mixed with the aquatic weed,
                           54

-------
Table 5.  Live versus Killed Algae as Sources of Available Nitrogen for
          Selenastrum in Gorham's (-N) Medium (150 ml/Tube, pH 7).
N Source
None
N03-N (5 mg N/L)
N03-N (10 mg N/L)
Live Algae:
N-limited Cladophora sp.1
Surplus-N Cladophora sp.2
N0-Fixers Anabaena sp. +
Aphanizomenon sp.
Bloom Microcystis sp.
Anabaena flos-aquae
(Ind. 1444)
Surplus-N Selenastrum
PAAP
Killed Algae (freeze-thaw)
N-limited Cladophora sp.1
Surplus-N Cladophora sp.2
Surplus-N Cladophora sp.3
N0-fixer Anabaena sp.
Bloom Microcystis sp.
Anabaena flos-aquae
(Ind. 1444)
N-limited Selenastrum
Surplus-N Selenastrum

Source
of Alga

L. Mendota
6/17/69
Monona Bay
6/17/69
L. Mendota
6/17/69
L . Monona
7/23/69
Gorham's(-N)
Culture
Gorham's
Culture
L. Mendota
6/17/69
Monona Bay
6/17/69
L. Mendota
7/25/69
L. Mendota
6/19/69
L . Monona
7/23/69
Gorham's(-N)
Culture
Gorham's
(7.5 mg N/L)
Gorham ' s
(82 mg N/L)

Approx .
Algal
Dry Weight
(mg/Tube)
—
10
40
10
40
15
44
19
57
10
40
10
40
10
40
20
40
20
40
10
20
19
57
10
40
10
40
10
40
Growth of Selenstrum
after 7-8 Days
Absorbance
(1 cm, 750 my)
0.02
0.32
0.60
0.03
0.02
0.02
0.02
0.06
0.16
0.02
0.02
0.02
0.03
0.02
0.04
0.02
0.05
0.14
0.15
0.12
0.44
0.31
0.37
0.23
0.54
0.08
0.16
0.01
0.01
0.12
0.58
Av.
Absorbance
per 10 mg
Algae

.010
.008
.036
.005
.010
.012
.015
.050
.060
0.23
0.10
.050
.004
0.15
1 N-limited Cladophora sp:
2 Surplus-N Cladophora sp
3 Surplus-N Cladophora sp
                            20 pg NH3-N absorbed/10 mg/hr.
                            (Monona Bay):  5 lag NH3-N absorbed/10 mg/hr.
                            (Lake Mendota):  5 pg NH3-N absorbed/10 mg/hr.
                                    55

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Table 6.  Live versus Killed  Algae as Sources of Available Phosphorus
          for Selenastrum in  PAAP  (-P)  Medium (150 ml/Tube, pH 7).




P-Source
(Dialysis Tube)
None
PO^-P (0.03 mg P/Tube)
Live Algae
P-limited Cladophora sp.1
(Monona Bay)
P-surplus Cladophora sp.2
(L. Mendota)
P-surplus Anabaena sp.(90%)3
(L. Mendota)
P-surplus Microcystis sp.lf
(L . Monona)
Killed Algae (freeze-thaw)
P-limited Cladophora sp.1
(Monona Bay)
P-surplus Cladophora sp.2
(L. Mendota
P-surplus Anabaena sp . 3
(L. Mendota)
P-limited Aphanizomenon sp.5
(Monona Bay)
P-surplus Microcystis sp.1*
(L. Monona)


Approx .
Algal
Dry Weight
(mg/Tube)
—
—

10
40
10
40
10
20
19
57

10
20
5
10
10
20
8
23
19
57
Growth of Selenastrum
after 6-9 Days


Absorbance
(1 cm, 750 my)
0.01
0.20

0.02
0.02
0.02
0.02
0.07
0.14
0.01
0.01

0.18
0.24
0.32
0.50
0.43
0.70
0.05
0.14
0.24
0.75
Av.
Absorbance
per 10 mg
Algae



0.008

.008

.07

.003


.14

.55

.37

.06

.13

1 P-limited Cladophora  sp.
2 P-surplus Cladophora  sp.
3 P-surplus Anabaena  sp.
  P-surplus Microcystis sp.
  •*• «-f «•*• £• -I.M-M **J. w ^ \s*-^j *-r *~ -^w  M1T       ^
-------
Myriop'hyllum s,p.f which, contained  surplus  phosphorus
(Q.33 nig PCK-P extracted/lQQ mg  tip  leaves)  at  the  time of
sampling (9/4/69).

The amount of growth  attained by phosphorus-limited Selenas-
trum in the presence  of killed algae was found  to be de-
pendent upon whether  the  phosphorus  source (algae)  had been
itself phosphorus^limited or contained  surplus  phosphorus:
5 mg of Cla'dophora with surplus  phosphorus supported more
growth of Seleriastrum than 20 mg of  phosphorus-limited
Cladophora; and  10 mg of  Ahabaena  sp. with surplus  phos-
phorus supported 3 times  as much growth of Selenastrum as
23 mg of phosphorus-limited Aph anizomenon  sp.

The data on the  sharing of nutrients by algae presented
thus far have only concerned the use of the green alga,
Selenastrum.  In order to demonstrate that similar  results
could be obtained with other algae,  comparative tests were
also carried out with the bloom-forming blue-green  alga,
Microcystis aeruginosa (Wis. 1036),  and the N2-fixing blue-
green alga, Anabaena  flos-^aquae  (Ind. 1444) .  Typical results
are summarized in Table 7 as the averages  of absorbances
attained after 14 days of culture  when  different sources
of nitrogen and  phosphorus were  evaluated.

As in previous studies, live plants  provided neither nitro-
gen nor phosphorus to the test algae.   It  is of interest
to point out that samples of phosphorus-limited Cladophora,
even when killed, did not support  the growth of the test
algae, but the other  plants with surplus nutrients  did
share available  nutrients when they  were killed.

Under the conditions  of these tests  the minimal reproducible
growth of test algae  that was detectable by the light ab-
sorbance measurements used would be  representative  of the
growth attained  with  about 0.008 mg  P or 0.06 to 0.2 mg N
per culture.  It is evident from the data  presented that
neither live algae nor live aquatic  weeds  did provide such
quantities of available nutrients.   Live algae  or weeds may
have provided lesser  quantities  of available nutrients which
could not be detected by  such crude  techniques  as used here.
The facts of importance are that algae  or  weeds from the
field or from cultures with surplus  nitrogen or phosphorus
do not share their nutrients unless  they are killed.  There-
fore, once nitrogen or phosphorus  are absorbed  by plant
materials, it must be assumed that they are not readily
available in significant  amounts until  the death of the
plant.
                             57

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en
oo
             Table 7.   Comparative Tests of Field Plants as  Sources  of  Available Nitrogen or
                       Phosphorus for Selenastrum, Microcystis,  or Anabaena.



Medium
(75 ml/125 Erl.)
Gorham's (-P)
ii

ii
ii

ii

K
ii
Gorham's (-N)
ti

ii
ti


Nutrient Source
and Amount
(mg/culture)
None
POif-P - 0.015 mg
Surplus-P Cladophora1- 20 mg
Live
Killed5
P-Limited Cladophora3- 20 mg
Killed
Myriophyllum Leaves'*- 20 mg
Live
Killed
None
N03-N - 0.38 mg
Surplus-N Cladophora2- 20 mg
Live
Killed
Av. Growth Attained after 14 Days
(Absorbances, 1 cm, 750 mu)
Selenastrum
capricornutum
(PAAP)
0.01
.14

.01
.38

.01

.01
.10
.01
.24

.02
.23
Microcystis
aeruginosa
CWis. 1036)
0.02
.10

.01
.08

—

.03
.15
.00
.06

.01
.13
Anabaena
flos-aquae
(Ind. 1444)
0.02
.07

.01
.24

.02

.01
.12
—
—

—
—
             1  Surplus-P  Cladophora  sp.  (0.30 mg  PO^-P  extracted/100 mg  algae)  -  Lake Mendota 10/7/69
             2  Surplus-N  Cladophora  sp.  (9  yg NH3N utilized/10 mg/hour)  -  Lake  Mendota 10/7/69
             3  P-Limited  Cladophora  sp.  (0.02 mg  PO^-P  extracted/100 mg) - Lake Wingra 10/7/69
             4  Myriophyllum sp.  (0.05  mg  P04-P  extracted/100 mg  leaves)  -  Lake  Wingra 10/7/69
             5  Killed  - plants killed  by  freeze-thaw (2 cycles)

-------
As Ixas been pointed out,  tests  of  the  availability of
nutrient sources when  they  are  in  contact with  algae over
the entire period required  for  the growth of the  algae
(1-2 weeks) do not differentiate between the availability
of the original source of the nutrient and  its  degradation
products.  Therefore,  for critical analyses of  the immediate
availability of nutrient  sources,  tests with short-exposure
periods are preferable.   Such tests are required, of course,
if one wants to evaluate  nutrient  sources for algae held
in the dark.

The results presented  here  on the  effects of pH and light
versus darkness have indicated  that algae can absorb nitro-
gen and phosphorus compounds under all conditions tested.
The significance of these results  is that pH (in  a range
of pH 7-9) and darkness do  not  affect  the ability of the
algae tested to absorb nitrogen and phosphorus.  Therefore,
nutrients could be absorbed by  the algae in areas of lakes
below the penetration  of  light.  The algae  then could rise
or be carried to photic zones where subsequent  growth could
take place.  The sources  of nutrients  available to the algae
in these subsurface areas are still questionable, but the
results with solubility as  a factor indicate that simple
solubility may not be  the only  reason  why lake  muds and
natural rocks did not  serve as  sources of nutrients when
tested in short-term experiments.   It  must  be emphasized
that the negative results with  the tests of lake muds and
rocks as nutrient sources reported here were carried out
over fairly short periods of time  (1-2 weeks).  Although
the laboratory results reported substantiate field tests
as to the lack of available nitrogen and phosphorus from
lake muds for algae in situ, it should be emphasized that
studies by Lee  (personal  communication, 1970) have shown
that very significant  amounts of phosphorus are released
from aerobic lake muds when tests  are  carried out for
periods exceeding 1 month.  This release is even more rapid
under anaerobic conditions.  Therefore, the ecological
significance of the contribution of phosphorus  from lake
muds can not be judged properly until  more  studies are
carried out.

An interesting point when discussing solubility as a factor
in the availability of a  compound  is the similarity seen
in nutrition and toxicity tests.   It has been shown with
studies of the toxicity of  dichlone (2,3-dichloronaphtho-
quinone) ^ that 1 mg/L of  the  chemical does not prevent
the growth of Chlorella under standard conditions, but
                            59

-------
that 1.5 mg/L will accomplish this.  Yet, the solubility
of dichlone is only 0.1 mg/L, so the amounts that are in-
sufficient and sufficient for algistatic action are both
in excess of the limit of solubility.  Also, it has been
shown that insoluble forms of copper were as effective as
algistats and algicides in algal cultures when compared
with soluble forms of copper.^7J  However, when copper is
used as an algicide in field applications, sources of copper
which remain soluble in the waters in which they are used
are much more effective than the usual application of copper
sulfate which readily precipitates under field conditions
(R. M. Stern, 1970, personal communication).  Therefore,
in both toxicity and nutrition, although laboratory studies
may show that insoluble sources of compounds are available
to algae, under practical field conditions insoluble sources
may be lost from the environment of the algae and not be
effective.

The fact that live algae and aquatic weeds do not share
their adequate or surplus nutrients with nutrient-limited
algae, even when the same strains are used, as in the tests
with Selenastrum as the source of nitrogen or phosphorus
for nutrient-limited Selenastrum, has considerable eco-
logical importance.  It can be concluded that once nitrogen
and phosphorus are tied up in living plant material those
nutrients are not available for the nutrition of other
plants until the original plants die.  Therefore, once
lake waters are stripped of available nutrients by plant
production, further plant production will depend upon
nutrients from continuous sources of nutrients, such as
wastewater effluents, the regeneration of nutrients from
muds, or the decay of the original flora.  Knowledge of
the relative rate of supply from each source of nutrients
could be of great value in evaluating the influence of any
potential nutrient source to a lake environment.
                      REFERENCES

 1.  APHA.  1965.  Standard Methods for the Examination
     of Water and Wastewater.  12th ed.  APHA, N. Y.

 2.  Bartsch, A. F. (Chairman).  1969.  Provisional Algal
     Assay Procedure.  Joint Ind./Gov't Task Force on
     Eutrophication, P. 0. Box 3011, Grand Central Sta.,
     N. Y.  10017.

 3.  Clesceri, N. L. and G. F. Lee.  1965.  Hydrolysis of
     Condensed Phosphates - I: Non-Sterile Environment.
     Int. J. Air Water Pollution, 9:723-742.
                           60

-------
 4.   Fitzgerald,  G.  P.   I960.   Loss of Algicidal Chemicals
     in Swimming Pools.   Appl.  Microbiol.,  £: 269-274.

 5.   Fitzgerald,  G.  P.   1968.   Detection of Limiting or
     Surplus Nitrogen in Algae and Aquatic  Weeds.  J.
     Phycol., 4_: 121-126.

 6.   Fitzgerald,  G.  P.   1970.   Aerobic Lake Muds for the
     Removal of Phosphorus from Lake Waters.  Limnol.
     Oceanogr. 15:550-555.

 7.   Fitzgerald,  G.  P.  and S.  L. Faust.  1963.  Factors
     Affecting the Algicidal and Algistatic Properties
     of Copper.  Appl.  Microbiol., 11:345-351.

 8.   Fitzgerald,  G.  P.  and G.  F. Lee.  1970.  Use of Tests
     for Limiting or Surplus Nutrients to Evaluate Sources
     of Nitrogen arid Phosphorus for Algae and Aquatic
     Weeds.   (Submitted for publication).

 9.   Fitzgerald,  G.  P.  and T.  C. Nelson.  1966.   Extractive
     and Enzymatic Analyses for Limiting or Surplus Phos-
     phorus in Algae.  J. Phycol., 2_: 32-37.

10.   Gales, M. E., Jr., E. C.  Julian, and R. C.  Kroner.
     1966.  Method for Quantitative Determination of Total
     Phosphorus in Water.  J.  Amer. Water Works  Assoc.f
     58:1363-1368.

11.   Hughes, E. 0., P.  R. Gorham, and A. Zehnder.  1958.
     Toxicity of a Unialgal Culture of Microcystis
     aeruginosa.  Can.  J. Microbiol., £: 225-236.

12.   Pomeroy, L. R., H. M. Mathews, and H.  S. Min.  1963.
     Excretion of Phosphate and Soluble Organic  Phosphorus
     Compounds by Zooplankton.  Lironol. Oceanogr., 8^50-55.

13.   Stewart, W. D. P.   1967.   Transfer of  Biologically
     Fixed Nitrogen in a Sand Dune Slack Region.  Nature,
     214:603-604.
                           61

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    ACETYLENE REDUCTION ASSAY FOR DETERMINATION OF

      PHOSPHORUS AVAILABILITY IN WISCONSIN LAKES
          W. D. P. Stewart, G. P. Fitzgerald,
                   and R. H. Burris
                       ABSTRACT

Phosphorus-starved cells of Anabaena flos-aquae rapidly in-
crease their capacity to reduce acetylene to ethylene when
they receive phosphorus.  This response may be used as a
bioassay for detecting available phosphorus in aquatic eco-
systems.  The sensitivity of the method compares favorably
with conventional methods for measuring dissolved ortho-
phosphate, and has the additional advantage that it measures
available phosphorus.  Studies on Wisconsin lakes show that
available phosphorus generally is present, that the concen-
trations are higher at the lower depths than at the surface,
and that there may be diurnal variations in the available
phosphorus content of surface waters.  Important sources of
available phosphorus in  Lake Mendota are the waters below
the thermocline and the input from storm sewers.
                     INTRODUCTION

Eutrophication of aquatic ecosystems has become intensified
in many areas within recent years and it is considered gen-
erally that nitrogen and phosphorus are the most important
nutrients that contribute to eutrophication. (-1~"f<1  However,
Stewart, Fitzgerald, and Burris1-5' J found, using the
acetylene reduction technique^7'B) in lakes, that when
metabolically active heterocystous blue-green algae were
present they were invariably capable of fixing N2.  This
infers that even if it were possible to eliminate completely
the input of combined nitrogen  into lakes, blooms of nitrogen-
fixing algae would still occur  if phosphorus was available.
Thus it is important to know the quantities of available
phosphorus in, and being added  to, lakes, and to date there
has been no completely satisfactory method1-9 '* OJ whereby
this may be determined.

This paper reports a modification of the acetylene reduction
technique which may allow rapid and sensitive determinations
of the available phosphorus in  aquatic ecosystems.  The bio-
assay is based on the following rationale:  if a culture of
a nitrogen-fixing blue-green alga is grown on molecular
                            63

-------
nitrogen under conditions of phosphorus starvation, the or-
ganism should respond to the addition of available phosphorus
with increased growth and metabolism.  Because the organism
fixes N2, the enhancement of metabolism when phosphorus is
supplied should be reflected in an increased capacity for
the reduction of acetylene to ethylene.

                        RESULTS

Experiments Using a Defined Medium

Experiments were conducted to determine the response of blue-
green algae to various nutrients.  The algae were grown in
Gorham's complete nitrogen-free medium and were transferred
after washing into a medium in which one of phosphorus, iron,
magnesium, or sulfur was omitted.  When the algae showed nu-
trient deficiency they were returned to complete Gorham's
nitrogen-free medium.  The data in Table 1 show that within
2.0 hr nutrient-depleted algae responded to phosphorus but
not to the other elements.  A longer incubation may have elic-
ited a response to the other nutrients, but a long incubation
period defeats the object of a rapid bioassay technique.
Nevertheless, bioassays for other elements based on the acety-
lene reduction assay may be reasonable, when it is appreciated
that existing bioassay methods^9'10-* require 10-14 days.

The response to phosphorus depends upon the test alga being
starved  initially as is shown in Table 2.  Natural popula-
tions of algae could not be used satisfactorily as assay
material either (Table 3), because sometimes a response was
obtained and sometimes it was not.  This irregularity prob-
ably reflects the ability of algae to store phosphorus which
can be utilized later in phosphorus-deficient waters.

The data in Table 4 show that phosphorus-starved algae respond
to as little as 3.0 yg phosphorus/liter, but that high con-
centrations saturate the system.  Addition of the same amount
of phosphorus may induce different enhancements in acetylene
reduction in different experiments and this usually reflects
different degrees of phosphorus starvation in the test algae
used.  The type of response shown in Figure 1 is character-
istic for phosphorus levels in the range 0-0.25 mg/liter.

The time course of the response  (Fig. 2) shows a lag phase of
almost 15 min followed by a 15-min period of rapid response.
After 30 min there was little further increase in the rate of
acetylene reduction.  The positive value obtained over the con-
trol at 0 time presumably resulted from phosphorus uptake during
the period in which the algae were being separated from the phos-
phorus-containing medium by centrifugation immediately after
the incubation period and prior to measurement of acetylene
                            64

-------
Table 1. Response in Acetylene Reduction of
         Nutrient-Starved Anabaena flos-aguae
         to Various Nutrients.
Deficient
 Nutrient
    Test
   Medium
nmol
1 . 0 ml/mi n
Sulfur


Iron


Magnesium

Phosphorus
- sulfur
+ sulfur

- iron
+ iron

- magnesium
+ magnesium
- phosphorus
+ phosphorus
   0.057
   0.064

   0.228
   0.212

   0.465
   0.452

   0.027
   0.349
Incubation period prior to the 30 min acetylene
reduction assay was 2 hr; the volume of test
water used was 70 ml, the nutrient concentra-
tions in complete medium were (mg/liter): S,10;
P, 7; Mg, 1; Fe, 0.05.  Each value is the mean
of triplicate determinations.
Table 2. Acetylene Reduction by Phosphorus-
         Starved and Phosphorus-Sufficient
         Anabaena flos-aquae When Incubated
         in Phosphorus-Containing Medium.
Pre treatment
+ phosphorus
- phosphorus
Subsequent
Treatment
- phosphorus
+ phosphorus
- phosphorus
+ phosphorus
nmol CaHit/
1 . 0 ml/min
0.406
0.392
0.030
0.349
Pretreatment period was 4 days; the subsequent
incubation period prior to the 30 min CzHa
reduction assay was 2 hr; the volume of solu-
tion used was 70 ml.  Each value is the mean
of triplicate determinations.
                    65

-------
Table 3.  Response of Natural Populations of Anabaeha
          and of a Phosphorus-Starved Laboratory
          Culture of A. flos-aquae when Incubated in
          the Surface~Waters of Lake Mendota, with or
          without a Phosphorus Supplement.
Date
8/21/1969
9/3/1969
9/3/1969
Alga Used
Natural Bloom of
Anabaena
Natural Bloom of
Anabaena
P-Starved
Laboratory Culture
of
Anabaena flos -aquae

Treatment
(mg P/Liter)
Untreated
+0.085
+0.143
Untreated
+0.085
+0.143
Untreated
+0.085
+0.143
nmol CaHit/
1.0 ml/mi n
0.23
1.94
2.01
0.42
0.40
0.41
0.02
0.23
0.20
Period of incubation prior to the CaHa reduction assay
was 2 hr; volume of water used was 35 ml.   In the ex-
periments of Sept. 3rd the natural populations of
Anabaena and of the P-starved A.  flos-aquae were incu-
bated in aliquots of the same test water.   Date of
collection of water samples is given in column 1.
                          66

-------
Table 4. Response in Acetylene Reduction of
         Phosphorus-Starved A. flos-aquae
         Cultures to Various Levels of
         Available Phosphorus.
Phosphorus
Added
(mg/Liter)
nmol CzHi*/
1 . 0 ml/min
Increase Over
P-Deficient
Cultures
(%)
Experiment 1
0.000
5.700
1.430
0.360
0.087
0.014
0.010
0.118
0.119
0.125
0.119
0.048
-
1080
1090
1150
1090
380
Experiment 2
0.000
0.010
0.005
0.003
0.001
0.036
0.116
0.066
0.042
0.036
-
222
83
17
0
Expt.  1:  Incubation period prior to the
30 min C2H2 reduction assay was 60 min;
the volume of the test water used was 25 ml,

Expt.  2:  Incubation period was 60 min and
the volume of test water used was 500 ml.

Each value is the mean of triplicate deter-
minations .
                      67

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Figure 1.  Response of P^starved A. flos^aquae to Various
           Levels of Available P."
 c
 •H
 g


 O
  •

 H
 ffi  n  7
 CM  U ' *•
 u

 en
 
-------
Figure 2.  Effect of Time on the Response to Phosphorus
           of Phosphorus-Deficient Anabaena Cultures
   0.4
                         Time (min)


The incubation period prior  to the 30 min C2H2 reduction
assay was in the presence of 0.02 mg P/liter for the time
indicated on the figure; the volume of test solution used
was 70 ml.  Each value is the mean of triplicate deter-
minations .
                             69

-------
reduction.  A 30-min incubation in the test water before
the start of the acetylene reduction assay is probably suf-
ficient enough to provide reliable results.
Field Trials

The data in Table 5 show that phosphorus-starved Anabaena
responded to incubation in the surface waters of Lake
Mendota, Lake Wingra, and Lake Monona, indicating that each
contained available phosphorus with the highest levels
occurring in Lake Mendota.  Table 6 shows that available
phosphorus is highest in Lake Mendota in the morning and
decreases during the day.  Thus, available phosphorus levels
may build up in lakes at night and decrease during the day
when vigorous algal metabolism occurs as a result of photo-
synthesis.  Others^13'1'*'' have also reported a more rapid
uptake of phosphorus in the light than in the dark.  Moire
experiments are required to determine whether the varia-
tions which we detected are characteristic of the surface
waters of Lake Mendota.

Variations in the levels of available phosphorus at various
depths of Lake Mendota were examined.  Table 7 shows that
the assay organism responded markedly when placed in un-
treated waters from all depths with the greatest response
being obtained in waters from 15 m and 18 m; the lake was
18.5 m deep at the sampling site studied and the thermocline
was located near 10-12 m.  When phosphorus was added to
these waters, there was a marked improvement in the. nutrient
quality of the upper waters.  In contrast there was less
response to added phosphorus in the waters from 18 m and
particularly from 15 m.  These results suggest that phos-
phorus was limiting particularly in the surface waters and
that at the lower depths, although more available phosphorus
was present, there was also present an inhibitory factor of
some type.

A practical application of the bioassay is illustrated with
data obtained on waters of University Bay Creek, which dis-
charges storm sewer drainage from urban areas of Madison
into Lake Mendota.  Table 8 shows that the phosphorus-
limited test algae responded markedly when placed in these
waters.  The response persisted in lake water near the inlet
of the creek, but the response decreased as the water was
diluted in the main part of the lake.  It is apparent that
the runoff from urban areas may drastically influence the
nutrient balance of Lake Mendota.
                            70

-------
Table 5.  Response in Acetylene Reduction of Phosphorus-
          Starved A. flog-aquae when Incubated in the
          Surface Waters of Various Lakes.
Sample
Lake Mendota
Lake Monona
Lake Wingra
Treatment
Untreated
+ Phosphorus
Untreated
+ Phosphorus
Untreated
+ Phosphorus
nmol C2!U/
1.0 ml/min
0.12
0.16
0.04
0.11
0.08
0.33
Increase on
Adding P
33
175
313
Period of incubation prior to the C2H2 reduction assay was
60 min; volume of water used was 70 ml; samples collected
on Aug. 6, 1969; phosphorus was added as 1.0 mg K2HPOit/liter,
Each value is the mean of triplicate determinations.
           Table 6. Response of Phosphorus-Starved
                    A.  flos-aquae when Placed in
                    the Surface Waters of Lake
                    Mendota Collected at Various
                    Times of the Day.
Sampling Time
7 a.m.
12 noon
4 p.m.
8 p.m.
nmol CaHU/
1 . 0 ml/min
0.07
0.02
0.01
0.01
Incubation period prior to the 30 min C2H2 reduction assay
was 60 min; volume of test water used was 200 ml.  Samples
collected on Sept. 9, 1969.  Each value is the mean of
triplicate   determinations.  The times given are central
standard time.
                           71

-------
Table 7. Acetylene Reduction by Phosphorus-Starved
         A, flos-aauae when Incubated in Untreated
         Waters and in Phosphorus-Supplemented
         Waters Taken from Various Depths of Lake
         Mendota.
Depth
(m)
0
2
8
10
15
18
nmol CaHit/l.O ml/min
Untreated
Lake Water
0.04
0.07
0.06
0.06
0.16
0.16
Lake Water +
0.01 nag P/Liter
0.43
0.38
0.38
0.34
0.15
0.23
Period of incubation prior to the 30 min C2H2 reduc-
tion assay was 75 min; volume of lake water used was
200 ml; value for the control medium -P was 0.02 nmol
CaHU/1.0 ml/min.  Samples collected on Sept. 12, 1969.
Each value is the mean of triplicate determinations.
Table 8. Acetylene Reduction by Phosphorus-Deficient
         A_. flos-aquae Incubated in Lake Mendota
         Surface Waters Taken near a Drainage Inlet.
        Test Water
nmol
1.0 ml/min
University Bay Creek
University Bay at Inlet of Creek
25 m Offshore from Inlet
250 m Offshore from Inlet
1.000 m Offshore from Inlet
   0.42

   0.42

   0.14

   0.06

   0.04
University Bay Creek water was sampled at a point just
before it entered the lake.  The period of incubation
prior to the CzEte reduction assay was 60 min; volume
of lake water used was 200 ml; samples collected on
Sept. 10, 1969; value for the control medium -P was
0.03 nmol CaEU/l.O ml/min.  Each value is the mean
of triplicate determinations.
                         72

-------
                      DISCUSSION

Two main methods of measuring phosphorus in aquatic eco-
systems are currently employed.  The most common is to
determine chemically the levels of orthophosphate.  This
method takes no account, however, of other important types
of phosphorus which may be present.  For example. Galloway
and Krauss1-15-1 showed that inorganic polyphosphates could
be utilized by Chlorella at comparable rates to orthophos-
phate, and substances such as glucose-6-PO^ may be utilized
by marine algae. l-16J  The other method is to measure the
extractable phosphorus present in algae and to correlate
this with the amount of alkaline phosphatase activity of
the algae. U2J  The latter method, though useful, has the
disadvantage that  the algae rapidly assimilate and store
phosphorus when it is available; thus, the phosphorus con-
tent of an alga is no real indication of the available
phosphorus present in a particular water at a particular
time.

The present results, although preliminary, indicate that a
bioassay method based on the acetylene reduction technique
may be practical and have advantages over previously avail-
able methods.  The method which we suggest is basically
that presented in  the Methods section, but the following
additional points  are relevant.

     Anabaena flos-aquae is a good assay organism, but any
     fast-growing  N2-fixing blue-green alga presumably
     could be used.  The exact culture medium used is also
     immaterial as long as it is free of combined nitrogen
     and the bioassay organism is phosphorus starved and
     metabolically active at the start of the assay.

     Small volumes (25 ml) of test water may be used, but
     larger volumes are more satisfactory.  A volume of
     70-200 ml of  water is recommended with 6.0 mg dry
     weight of the assay organism.  The volume and amount
     of algae should be constant in any one test series.

     The data in Figure 2 suggest that an incubation period
     of 30 min with constant shaking is adequate.  The
     algal suspension is then concentrated by centrifuga-
     tion to a volume of 3.0 ml and three 1.0-ml aliquots
     are then incubated under acetylene for 30 min.

     All test samples which are being compared should be set
     up at the same time.  For each test water sample
      (a) there should also be available a control sample
                            73

-------
     (b)  in which, the algal response in phosphorus-free
     medium is noted, and a second control (c) comprising
     the test water sample plus 0.025 mg/liter of phosphorus.
     The value for (b)  serves to show that the bioassay
     organism is phosphorus starved while the value for
     (c)  shows its ability to respond in the test water when
     phosphorus is available.  The relative response for
     each test water thus will be: (c - b)/(a - b).  By
     comparing the values for this ratio, the relative
     abundance of phosphorus in each test sample can be
     measured.

The quantitative determination of the amount of available
phosphorus in a particular water is more complicated, and
it is suggested that the following series should be set up
for such a measurement:  (1) P-free medium, (2)  P-free
medium + 0.100 mg P/liter,  (3) P-free medium + 0.050 mg P/
liter,  (4) P-free medium + 0.025 mg P/liter,  (5) P-free
medium + 0.010 mg P/liter,  (6) test water sample + 0.025 mg P/
liter.

Tests (l)-(5) show the response of the alga to available
phosphorus in the absence of inhibitory or competitive re-
actions and can be used to prepare a standard curve which
can be used for all tests performed with the same batch of
assay organism.  The value for test (7) minus test (1)  gives
the response to 0.025 mg/liter of phosphorus plus the amount
of phosphorus in the water sample.  The value for test (6)
minus test (1) gives the response to available phosphorus
in the test water only.  Thus, the response to 0.025 mg P/
liter alone and to the available phosphorus in the test
water is obtained.  These values are then transferred onto
the standard curve to give the quantity of available phos-
phorus in the test water.  Thus, the response of the algae
to a series of standards and to two samples of the test water,
one with and the other without added phosphorus, is all that
is required to determine the level of available phosphorus
in a particular water.  The same standard curve can be used
for all waters tested using the same batch of assay organism.
We always have noted a response when phosphorus was added
to phosphorus-deficient culture medium.  This indicates
that a failure to detect acetylene reduction in test water
samples with added phosphorus is because of some inhibitory
factor in the water.  In the presence of total inhibitors
the test would not be satisfactory.

The overall sensitivity of the phosphorus bioassay is good.
For example, the stannous chloride method recommended by
the American Public Health Association (1965)  *-17-1 detects
orthophosphate-phosphorus levels as low as 10 yg/liter,
                           74

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whereas the acetylene bioassay technique is probably more
sensitive and detects available, rather than total, phos-
phorus.  Also, biological nitrogen fixation and phosphorus
availability can be studied with similar equipment.

       v
                      REFERENCES

 1.  Fogg, G. E.  1965.  Algal Cultures and Phytoplankton
     Ecology.  Athlone Press of the University of London,
     London.  126 p.

 2.  Lund, J. W. G.  1965.  Ecology of Fresh Water Phyto-
     plankton.  Biol. Rev., 40:231-293.

 3.  Stewart, W. D. P.   1968.  Nitrogen Input into Aquatic
     Ecosystems, p. 53-72.  In:  Algae, Man and the Environ-
     ment , D. F. Jackson (ed.), Syracuse University Press,
     Syracuse.

 4.  Kuhl, A.  1968.  Phosphate Metabolism of Green Algae,
     p. 37-52.  In:  Algae, Man and the Environment,
     D. F. Jackson  (ed.), Syracuse University Press,
     Syracuse.

 5.  Stewart, W. D. P.,  G. P. Fitzgerald, and R. H. Burris.
     1967.  In situ Studies on N2 Fixation Using the
     Acetylene Reduction Technique.  Proc. National Academy
     of Sciences, 58:2071-2078.

 6.  Stewart, W. D. P.,  G. P. Fitzgerald, and R. H. Burris.
     1968.  Acetylene Reduction of Nitrogen-Fixing Blue-
     Green Algae.  Arch. Mikrobiol., 62:336-348.

 7.  Dilworth, M. J.  1966.  Acetylene Reduction by Nitrogen-
     Fixing Clostridium  pasteurienum.  Biochim. Biophys.
     Acta, 127:285-294.

 8.  Schollhorn, R., and R. H. Burris.  1966.  Study of
     Intermediates in Nitrogen Fixation.  Fed. Proc.,
     25:710.

 9.  Skulberg, 0. M.  1966.  Algal Cultures as a Means to
     Assess the Fertilizing Influence of Pollution.  Proc.
     3rd  Internat'l  Conference on Water Pollution Research,
     Section I, No. 6, p. 1-15.

10.  Bartsch, A. F.  (Chrm.).  1969.  Provisional Algal Assay
     Procedure.  Joint Ind.-Gov't Task Force on Eutrophica-
     tion, P.O. Box 3011, Grand Central Sta., New York,
     N. Y.  10017.  62 p.
                            75

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11.  Hughes, E.  0., P.  R. Gorham, and A. Zehnder.  1958.
     Toxicity of a Uni.algal Culture of Microcystis
     aeruginosa.  Can.  J. Microbiol., 4j225-236.

12.  Fitzgerald, G. P., and T.  C. Nelson.  1966.  Extractive
     and Enzymatic Analyses for Limiting or Surplus Phos-
     phorus in Algae.  J. Phycol., 2_:32-37.

13.  Kuhl, A.  1962.  Zur Physiologic der Speicherung kon-
     densierter anorganischer Phosphate in Chlorella.
     Vortr. Botan. hrsg. Deutsch. Botan. Ges^(N.F.),
     !L:157-166.

14.  Kylin, A.  1964.  An Outpump Balancing Phosphate-
     Dependent Sodium Uptake in Scenedesmus.  Biochem.
     Biophys. Res. Comm., 16;479-500.

15.  Galloway, R. A., and R. W. Krauss.  1963.  Utilization
     of P Sources by Chlorella, p. 569-575.  In: Studies
     of Microalgae and Photosynthetic Bacteria,  Japanese
     Soc. Plant Physiol., Tokyo.

16.  Keunzler, E. J.  1965.  Glucose-6-Phosphate Utilization
     by Marine Algae.  J. Phycol., 1^:156-164.

17.  American Public Health Association.  1965.   Standard
     Methods for the Examination of Water and Wastewater.
     Amer. Public Health Assoc., New York.  769  p.
                           76

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        ACKNOWLEDGMENT
This research was supported by
Grant 16010 EHR from the Federal
Environmental Protection Agency.
The aid of Mrs. S. L. Faust with
the analytical analyses is grate-
fully acknowledged.

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    Accession Number
                        Subject Field & Group
                                         SELECTED WATER RESOURCES ABSTRACTS
                                              INPUT TRANSACTION  FORM
    Organization

          University of Wisconsin, Madison, Water Resources Center
    Title
          NUTRIENT SOURCES  FOR ALGAE AND THEIR CONTROL,
 10
    Author(s)
    Fitzgerald, George P,
                           16
Project Designation

   EPA Project 16010 EHR 08/71
                           2] Note
 22
    Citation
 23
Descriptors (Starred First)
 *Algae,  *Analytical  techniques, *Bioassay,  *Cycling nutrients,
 *Environmental effects,  *Essential nutrients,  *Eutrophication,
 *Limnology, *Nitrogen  compounds, *Nutrient  requirements,  *Phos-
 phorus compounds, Aquatic algae, Aquatic microbiology,  Aquatic
 weeds, Balance of nature, Nitrogen fixation, Nutrients, Water
 pollution effects
 25
    Identifiers (Starred First)
 27
Abstract
 Critical evaluations  are presented of bioassays  for nutrient
 availability.  The biological availability of  any  required algal
 nutrient in a sample  of water can be determined  by growth experi-
 ments requiring 2 weeks incubation.  In addition,  relatively
 short-term tests can  be carried out measuring  changes in certain
 enzymatic activities  or chemical fractions which have been shown
 to  reflect meaningful nutritional changes.  The  latter types of
 tests have been useful in evaluating the nutritional status of
 in  situ algae.  The selection of the type of bioassays for par-
 ticular" purposes can  be made from the data presented as to what
 information can be obtained, the length of time  required, and the
 range of sensitivity  of the bioassays.  Examples are presented
 of  ecologically important questions which have been answered by
 the different bioassays.
Abstractor
                          Institution
 WR:t02 (REV JULY 1969)
 WRSIC

*U. S. GOVERNMENT PRINTING OFFICE: 1972 — lf84-<>82/22
                                    SEND TO- WATER RESOURCES SCIENTIFIC INFORMATION CENTER
                                          U.S. DEPARTMENT OF THE INTERIOR
                                          WASHINGTON, D. C. 20240


                                                                * GPO: 1969-359-339

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