collected of Sptrogyra sp which had grown through layers of
mud (0.09% P) settled from the dredging operation in Murphy's
Creek. The total P of the muddy water from which the algae
were collected and the extractable P0O,-P of the algae were
measured and are summarized in Table 13 as averages of
triplicates.

Table 13. COMPARISONS OF THE TOTAL PHOSPHORUS CONTENT
OF MUDDY WATERS WITH THE EXTRACTABLE PO,-P OF IN SITU ALGAE

Total P
of water Extracted P0O,-P
Sample Source Alga (mg P/1) (mg P/100 mg algae)
1 winke winers Cladophora sp 0.30 0.027
2 wiake Wingea Cladophora sp 0.31 0.015
3 Wtabe Wings Cladophora sp 0.82 0.023
4 ee oetgre? Cladophora sp 0.19 0.039
s MEEZENC cindophone sp 00
© Riuply'e"cresk SPéreavra sp 0.26 noes
7 Dredged muds, Spirogyra sp 0.26 0.059

Murphy's Creek

There was considerable total P in the wind-stirred waters
surrounding the Cladophora samples, ranging from 0.2 to 0.8 mg
P/1l, but the extractable PO,-P of these algae indicated they
were still P-limited (algae containing more than 0.08 mg
extractable PO,-P/100 mg are considered to have adequate
available P).° The extractable PO,-P of samples of Cladophora
from Lake Wingra before and after this wind storm was about
0.02 mg P/100 mg algae. Sptrogyra sp and Zygnema sp up-
stream from the dredging area of Murphy's Creek contained

0.03 to 0.07 mg extractable PO,-P/100 mg algae, It is there-
fore evident that although there was considerable P stirred

51


Descriptions of variations in lake treatments are listed
below.

Table 34. LAKE TREATMENT WITH ALUMINUM

Concentration
Lake Chemical used Al_(mg/1)
snake liquid alum 12 mg/l
liq sodium aluminate in 80% total lake volume
Long liquid alum 14 mg/1
liq sodium aluminate in surface 1 ft
: se 7.3 mg/l
Pickerel liquid alum in total lake volume
Horseshoe granular alum 18 mg/l

in surface 2 ft

Description of lakes treated with aluminum -

Snake Lake - Vilas/Oneida Counties - Snake Lake is a slightly
acid, brown-water seepage lake of potentially high fertility.
It has a_ surface area of 5 hectares and a volume of

1.1 x 105m’. The maximum depth is about 6 meters and the
length of the shoreline is 1.2 km. The bottom sediment is
muck overlying sand and peat. For 22 years the lake received
discharge from the Village of Woodruff sewage treatment plant,
resulting in the loss of the natural fish population and ex-
cessive nutrient enrichment. This nutrient source was
eliminated in 1964, but the lake remained highly eutrophic
even though nutrient influx was drastically reduced. As a
means of renewal, this lake was subjected to dilutional
pumping in October 1969. (pilot test) and July-August 1970

as part of the Inland Lake Renewal Program. During the latter
pumping period approximately three lake volumes of water were
pumped from the lake. The lake was then refilled by seepage
from groundwater. Subsequent limnological evaluations showed
that although nutrient concentrations in the lake were reduced,
the levels remained sufficiently high that additional attempts
for renewal were deemed desirable. Nutrient inactivation was
attempted in May 1972, when the lake was treated with alum
and sodium aluminate.

Horseshoe Lake - Manitowoc County - Horseshoe Lake is an

alkaline, clear-water lake of medium to high fertility. It
has a surface area of 9 hectares and a volume of about

115


Growth (Dry Weight, mg/1)

1,500 |—-

1,250

1,000

750
500
250
0 1! L 1 |
0 0.01 0.02 0.03 0.04

Iron Concentration (mg Fe/1)

Fig. 4. Growth of Selenastrum
in Different Concentrations of Iron
Gorham's Medium Fe-EDTA 25 Days
Mean and 95% Confidence Limits

17


9FT
Biologically Available Phosphorus (mg/L)

—
i
i
— O Epilimnion
[—— A Hypolimnion
1.0E-—
lb
-0leE—
A A
001 ; rv A _ A a a T T
Jan. 36 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 42. Biologically Available Phosphorus - Long Lake, 1972


Tet
Biologically Available Phosphorus (mg/L)

10

© Epilimnion

A Hypolimnion

T T T T T T T T T T T T
30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 37. Biologically Available Phosphorus - Snake Lake, South Basin, 1973


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technolcgy
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
‘on the effects of pollution on humans, plant and
animal species, and materials.- Problems are
assessed for their long- and short-term
influences. investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments. ,

EPA REVIEW NOTICE

This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency,
nor does mention of trade names or commercial products con-
stitute endorsement or recommendation for use.


The medium was used (autoclaved) in triple strength for the
stock cultures, and (made up either without phosphorus or
without nitrogen) in normal strength for the reference
cultures. There was no autoclaving or filtering of the
reference medium.

Assay procedure -

Lake waters were treated according to the following protocol:

Concentration (mg/1)
Treatment of added nutrient Replicates

Lake water control 0
" " 0.05P
0.5N
0.05P, 0.5N
+ Fe 0.05P, 0.5N, 0.03Fe
'e 0.1P, 0.03Fe
'e 2.0N, 6.03F

tt tees
Zuovygy
tet
gy Ze

A
B
c
D " "
E
F
G

WWWWWWW

Treatment A provided a measure of sample fertility and
was used as a base of comparison for the other treatments.

Treatments B and C provided the data for determination
of the limiting nutrient of the sample by comparing
this response to that of the control.

Treatment D was included to check the possibility that
both nitrogen and phosphorus were in relatively short
supply, and additions of either of the nutrients added
singly would not yield a measurable response.

Treatment E was designed to test for possible sample
toxicity. Since algal growth is usually limited by
only nitrogen, phosphorus, or possibly iron, unless
toxic, virtually all waters, no matter how infertile,
should produce measurable growth with this spike.

Treatments F and G were designed to force the sample
into a situation where either phosphorus or nitrogen
was limiting, and biologically available concentrations
were determined by comparison with the proper reference
curve.

111


weights of the fronds per culture. It was found that in the
range of 10 to 100 fronds produced per culture with different
nutritional levels, there was a direct relationship with dry
weights of the fronds. In N and P nutrition tests 3 or 4
fronds would weigh 1 mg, dry weight, whereas under limiting-
Fe conditions about 8 fronds were required to produce 1 mg.
The latter effect was very obvious since cultures limited by
Fe produced relatively small plants which suggested that
limiting-Fe conditions may restrict growth (size of plants)
with considerably less influence on multiplication.

If the inoculum for nutrition tests is set at a certain number
of fronds per culture, as compared to inoculations with algae
based on the number of cells/ml of culture, the volume of the
culture will have an influence on the results. In order to
demonstrate this factor, nutritional tests were carried out
with different N or P concentrations in 25, 50, and 100 ml
volumes, using an initial inoculum of 2 fronds per culture.
The average numbers of fronds from duplicate cultures are
summarized in Table 17.

It can be seen from the data that, with increased volume, more
fronds are produced at a constant concentration of nutrient.
The effect on nutritional bioassays would be that 0.25 mg N/1L
could be detected in 50 and 100 ml volumes, but 1 mg N/1 would
be needed to cause a twofold increase over control (-N) in

25 ml volumes. P concentrations of 0.05, 0.10, and 0.20 mg P/1
could be detected in 100, 50, and 25 ml volumes, respectively.
It is thus apparent that when using an inoculation of 2 fronds
per culture, the volume of the culture must be reported and
kept constant for each series of comparative tests.

The previous study indicates how the numbers of fronds produced
per culture can be used as an index to the amount of available
nutrient present in a culture. Since 3 weeks or more of incu-
bation are required for the maximal growth of cultures, com-
parative growth tests with different sources of nutrient

might not be reliable because of possible conversion of one
form of nutrient to another more readily available form over
such a long exposure time. Therefore, a series of tests were
carried out in which the duckweed was exposed for relatively
short periods of time to different sources of nutrients,

placed in media lacking the nutrient of interest, and grown

on the sorbed nutrient over a 3- or 4-week period. Prelimi-
nary tests indicated that comparative tests could be carried
out by placing 4 fronds in 50 ml of supplemented Gorham's medium
containing either 5 mg N/1l, 2.5 mg P/l or 0.025 mg Fe/1l, and
after different sorption times, transferring 2 fronds each to
duplicate 25 ml volumes of Gorham's medium lacking the nutrient
of interest. The fronds developed in the media lacking

60


vet
Algal Production (mg/L Dry Wt.)

i

loo

B
°
|

we
°

Ol

O Epilimnion

4 Hypolimnion

Jan.

T
30

Fig.

40.

T T T
120 150 180 210 240
Time (Days)
Algal Production - Long Lake, 1973


Table 23. THE EXTRACTABLE PO.-P
OF CLADOPHORA SP INCUBATED ONE DAY
IN DIFFERENT CONCENTRATIONS OF PHOSPHORUS
10 mg samples of Cladophora from Lake Mendota
per 500 ml Gorham's (-P) medium
One hour boiling water extraction

Phosphorus concentration Extractable PO,-P
in medium (mg P/1) (mg P/100 mg algae)
none 0.025
0.04 0.076
0.08 0.116
0.12 0.162

of phosphorus that the algae sorb from the test waters over
a l-day period, a test to determine the amount of growth
sustained by the phosphorus in the samples has been used.
By adding Selenastrum eapricornutum (AAP)*® at a final con-
centration of 1,000 cells/ml to control cultures of AAP
medium containing known levels of PO,-P and to the test
waters, a comparison of the growth attained in the respective
cultures can be made, and the concentrations of available
phosphorus in the test water can be calculated. Because of
the fact that in some lake and river samples phosphorus is
not the limiting nutrient, in order to allow the’ algae to
utilize all of the phosphorus available for growth, the
sample frequently needs to be supplemented with nitrogen
and iron. Therefore, some samples have 2 mg NO;-N/1,

0.03 mg Fe plus 0.3 mg EDTA/1, or both, added.

The sensitivity of the method used to measure the growth

attained will determine the length of incubation required
and the range of concentrations of the nutrient over which

71


Table 1.

COMPARISONS OF TOTAL PHOSPHORUS ANALYSES OF ALGAE

Percentage of phosphorus
 HzSO,-HNOa method

H2SO,-persulfate

No. 0
Alga Source samples Ave 6% Ave 62
Chlorella '
pyrenoidoea somone 8 1.18 0.091 1.17 0.042
(Wis 2005)
Cladophora Monona Bay
sp 6/25/69 8 0.378 0.016 0.346 0.024
Rhizoectontum L. Mendota
sp 9/7/71 8 0.157 0.008 0.146 0.016
%6 = standard deviation


The underlined fluorometer responses in Table 37 should be
used for comparison since they represent maximum growth since
the maximum growth occurs at a later time when larger amounts
of assimilative nutrients are available. The data show a
growth response, principally to phosphorus, in the treated
waters, with a minor increase when both nitrogen and phos-
phorus were added.

Bioassay data for the four lakes treated with aluminum are
given in Figures 20 to 49 and are summarized in Tables 38
and 39.

The data given for Horseshoe Lake are all post-treatment
assays, so no direct evidence of the effect of the treatment
is provided by the bioassays. A detailed description of the
Horseshoe effort is given by Peterson et al.' They reported
generally positive results of the treatment and identified
reduced levels of phosphorus in the hypolimnion. during the
summers of 1970-72 as one measure of the effectiveness of
aluminum treatment. Unfortunately, bioassay data for periods
of summer stratification are lacking for the most part, but
high algal production in hypolimnetic samples during 1973,
along with the occurrence of nitrogen limitation-in these
samples, may indicate a progressive return to pretreatment
conditions. Additional sampling will be required to establish
this with certainty.

Horseshoe Lake has a mean hydraulic residence time of only a
about 0.7 years, and at times receives substantial quantities
of nutrients in runoff from the watershed; both of these
factors would tend to shorten the time during which a single
application of aluminum would influence the lake. However,
even if the lake is reverting to its pretreatment condition,
as suggested by the data for the summer of 1973, four summers
have elapsed since the lake-was treated. This result gives
some insight into the permanence which might be expected from
aluminum treatment in hardwater lakes similar to Horseshoe.

Repetitive assays for the north and south basins of Snake
Lake were initiated in the fall of 1971. Although it was not
possible to obtain a full year of pretreatment data--the lake
was treated in May, 1972, it is clear that aluminum treatment
has reduced the overall fertility of Snake Lake. Comparison
of fall and spring data before and after treatment shows
approximately a tenfold reduction in biomass produced in both
hypolimmetic and epilimnetic samples. In addition, no samples
were nitrogen limited since the'time of treatment. Prior to
that time, none of the samples were phosphorus limited.

120


9eT
Algal Production (mg/L Dry Wt.)

10

1.0

al

01

100 -— N
Oxpilimnion
Akvypolimnion
On
2
c wy PN
- P
q t T T T T T T T T T T
Jan. ‘0 6 90 120 150 180 210 240 270 300 330 360

. Time (Days)
Fig. 22. Algal Production - Horseshoe Lake, 1973


(mg/L)

OLT
Biologically Available Phosphorus

10 LK
@) Epilimnion
i 4 Hypolimnion
1.0 L—
——
+01
iz
001
T T
gan. 30 60 do ido ib0 180 2to 240 abo Joo 340 360
Time (Days)
Fig. 61. Biologically Available Phosphorus - Larson Lake, 1971


only 5 ml of Gorham's (-P) medium so the duckweed floated
only about 1 cm from the mud surface. The results of the
experiment as the average number of fronds per test condi-
tion after 6 weeks incubation are graphically summarized
in Figure 12.

Little growth (5 fronds) of the duckweed took place in the
P-free Gorham's medium, but 27 fronds developed in the pres-
ence of 0.005 mg PO,-P. It is evident that no available P
was released to the overlying medium in the case where the
mud containing 2 mg of total P was overlain by 30 ml of
Gorham's (-P) medium since the duckweed in these tubes had
no more growth than those with only Gorham's (-P) medium.
There was considerable growth (27 fronds) of the duckweed
whose roots penetrated the mud layers which were presumed
to have developed an anaerobic environment during the long
incubation period (6 weeks). Observations of these cultures
for an additional 8 weeks also indicated that, evén with
additional incubation, the duckweed in cultures in which the
roots could not touch the mud layers did not grow any farther.

These tests thus indicate how tests can be carried out to
contrast the availability of P in lake muds to algae or float-
ing aquatic weeds versus that to aquatic weeds whose roots

can penetrate to anaerobic layers of mud. As one would expect
from chemical studies of the release of PO,-P to the over-
lying aerobic waters of lakes,” there is available P in lake
muds which are under anaerobic conditions. One should be
aware, however, of the fact that P0O,-P released from anaerobic
lake muds into an aerobic environment can be competitively
sorbed by muds under the aerobic environment.!

References

1. Fitzgerald, G. P. Aerobic Lake Muds for the Removal of
Phosphorus from Lake Waters. lLimnol. and Oceangr.
15:550-555, 1970.

2. Fitzgerald, G. P. Bioassay Analysis of Nutrient Avail-
ability. In: Nutrients in Natural Waters, Allen, H. E.,
and J. R. Kramer (ed.). New York, John Wiley and Sons,
1972. p. 147-170.

66


0.10

°
.

o
es}

°
.

°
an

0.04

o
.

i=]
nN

Water - Soluble PO.,-P (mg P/1)

0.30F

0.25;- F ALGAE
0.20r

0.15f

Algae - Extractable P
(mg PO,-P/100 mg Algae)

0.05 ¥ . 7 7 T
Apr May June July Aug Sept
1972

Fig. 6. Comparison of the Soluble PO.-P of Lake Kegonsa Waters
and the Extractable PO,-P of in situ Algae at the Outlet

24


0.06

0.044

0.02

WATER - Soluble P0,-P (mg P/1)

0.15P

0.10,

ALGAE - Extractable P
(mg POy-P/100 mg Algae)
—o—

0.05 4 4 1 L 1

Apr May June July Aug Sept
Fig. 18. Comparison of the Soluble P0,-P of Lake Kegonsa

Waters and the Extractable P0O,-P
of-in sttu Algae at the Outlet

95


Extractable PO,-P (mg P/100 mg Algae)

A Cladophora sp in 100 ml Gorham's Medium

@ Cladophora sp in 1,000 ml Lake Mendota

O Gloeotrichia sp Water
0.30 in 200 ml Gorham's (-P) Medium
(3 Hours)
(20 Hours)
0 ! l l n 1
0 0.1 0.2 0.3 0.4 0.5

Added POy-P (mg P/1)

Fig. 9. Sorption of POv-P by Cladophora sp
and Gloeotrichta sp in Different Volumes
and Different Incubation Times

31


The dual set of assay data--north and south basins--is in-

cluded: to depict the degree of overall variability inherent
in the sampling and algal assays. The two basins of Snake

Lake are very similar and would not be expected to respond

differently to treatment. This similarity is reflected in

the assay results, Seasonal trends are the same and abso-

lute quantities are in good agreement.

Algal production in samples from Long and Pickerel Lakes was
consistently much lower than for any of the other lakes
studied. Most samples produced less than 0.5 mg/l, both
before and after treatment. Algal production in samples from
Pickerel Lake did not exceed 0.5 mg/l during the summer fol-
lowing treatment, whereas in 1972, prior to treatment, three
hypolimetic samples produced 5 mg/l. However, except for
these three samples, the remaining pre- and post-treatment
data were very similar.

Biomass was generally limited by phosphorus in samples from
both Pickerel and Long Lakes prior to treatment, so no shift
.in the limiting nutrient was anticipated as a result of the
aluminum treatment. Post-treatment data showed this to be
the case; nitrogen was identified as limiting in only one of
97 samples from these lakes. The assay results alone are not
sufficient to document the effect of treatment on the general
fertility of these lakes.

Hypolimnetic aeration -

The technique of aerating the hypolimnetic waters of lakes
without destruction of the thermocline appears to be a viable
method of improving the recreational value of many lakes.

It has been demonstrated that aerobic conditions can be main-
tained in the bottom waters, and when the integrity of the
thermocline is retained, it is possible in some cases to
provide conditions satisfactory for cold-water fisheries.

In addition, the flux of nutrients from sediments to the
overlying waters may be reduced as a result of the induced
aerobic conditions.

The aeration units used in the study lakes consisted of three
major components: a riser stack, separator box, and return
pipes. The riser section used was approximately 0.5 m in
diameter and extended from the bottom to the lake surface.

156


Table 16. CHANGES IN COMPOSITION OF LAKE WINGRA
CLADOPHORA SP AFTER INCUBATION
WITH DIFFERENT CONCENTRATIONS OF PHOSPHORUS
10 mg Cladophora/500 ml Lake Mendota water
20 hours sorption

Cladophora
phosphorus concentration

Phosphorus concentration (mg P/100 mg algae)
added to Lake Mendota water Extractable

(mg _PO,-P/1) PO,-P Total P
0.0 0.076 0.085
0.02 0.092 0.13
0.04 0.15 0.17

, 0.08 0.22 0.30

It is evident that the total P of either type of Cladophora
increased when the algae were incubated 24 hours in the
presence of increasing levels of PO,-P in Lake Mendota water.
From the concentrations of total P of the Cladophora in the
dilutions of Crawfish River water in Lake Mendota water, it
can be calculated that 0.20 to 0.21 mg/l of available P was
present in the Crawfish River sample, Chemical analyses of
the river water showed that it contained 0.26 mg soluble
PO,y-P/1 and 0.61 mg total P/l, thus indicating that most of
the soluble PO,-P was available to the Cladophora, but there
was a lot of total P present in the muddy water that the algae
were not able to utilize under these aerobic conditions.

Similar tests of the availability of the P of aerobic lake
muds, using as a measurement the growth of the duckweed,
Lemna minor, have also indicated that the P of lake muds was
not available even when the roots of this aquatic weed were
in intimate contact with aerobic muds for as long as 8 weeks.
However, this is not a natural environment for aquatic weeds.
As has been pointed out, lake muds are layered so that they
contain areas which are anaerobic with PO,-P in the inter-
stitial waters. The advantage of duckweed over algae as a
bioassay organism is that the availability of the in situ P
of layered lake muds can be measured. The roots of aquatic
weeds can penetrate an anaerobic environment while the rest

56


OVT
Biologically Available Phosphorus (mg/L)
a

i

-01

001

OEpilimnion

Aduypolimnion

T
Jan. 30

Fig.

36.

T T T T T T T T T

60 90 120 150 180 210 240 270 300
Time (Days)

Biologically Available Phosphorus - Snake Lake, South Basin, 1972

330

360


0.16

0.12

Extracted PO,-P (mg P/100 mg Algae)

O 9/6/72
& 8/16/72

{ it i

0 0.05 0.10 0.15
PO.-P Added to Water (mg P/1)

Fig. 7. Concentration of Extracted PO,-P from Lake Wingra
Cladophora sp after One-Day Incubation with P0,-P

Added to Lake Wingra Water
(10-20 mg algae/400 ml water)

27


SECTION V

DUCKWEED (LEMNA MINOR) FOR NUTRITIONAL BIOASSAYS

Introduction

The aquatic plant duckweed (Lemna minor) is an example of a
rooted aquatic plant that is readily cultured under labora-
tory conditions. Its characteristic flotation makes it
readily harvested or transferred from medium to medium, and
simple frond counts can be used as an approximate measure
of its growth (increase in dry weight) under different en-
vironmental conditions.

Materials and Methods

The duckweed used in these tests has been cultured in this
laboratory under algae-free conditions: for several years.
Treatments of field collections of duckweed frond with
chlorine had made it free of other plants. It was maintained
in Gorham's medium, but most nutritional tests were carried
out using supplemented Gorham's medium containing 20 mg Ca/l
(double normal concentration), EDTA (7 mg/l), and minor ele-
ments of ASM medium. Preliminary tests indicated that duck-
weed transferred from Gorham's medium to medium lacking some
essential nutrient would increase from an original inoculum
of 2 fronds per 50 ml to 10 or more fronds, whereas fronds
from Gorham's medium containing growth-limiting amounts of
the nutrient of interest exhibited little subsequent growth
when transferred to media lacking that essential nutrient.
Consequently, the inoculum (usually 2 fronds/50 m1) for most
tests came from plants transferred from the supplemented
Gorham's medium to media lacking N, P, or Fe and were incubated
in the latter media for a week or two. When transferred to
fresh media lacking the nutrient of interest, these fronds
usually only increased from the original inoculum of 2 fronds
to 4 or 5 fronds. The length of incubation required to detect
differences between the numbers of fronds in different levels
of an essential nutrient was about 2 weeks and maximal growth
required 3 to 4 weeks incubation.

Results and Discussion

Most data accumulated using duckweed in nutritional bioassays
were reported as the number of fronds produced in the cultures.
Early experiments were recorded as both frond numbers and dry

59


RAIN

(Inches)

Soluble P0,-P (mg P/1)

Soluble POs-P (mg P/1)

0.04

0.02

© Lake Mendota
@ Lake Monona

OLake Waubesa
@ Lake Kegonsa

Oct Nov Dec Jan Feb

Fig. 19. Comparison of the Soluble PO,-P
of Madison Area Lakes, Fall and Winter, 1972-1973

96

Mar


6ST
Algal Production (mg/L Dry Wt.)

100

10

-O1

_ Oepilimnion
Quypolimnion
- O-------____
PN
T T T T T T T T Tv T qT T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 52. Algal Production - Mirror Lake, 1971


Table 36.

GROWTH RESPONSE OF AAP ORGANISMS
TO NUTRIENT REMOVAL AND SPIKES

Added nutrients

Fluorometric
response units

Equivalent mg/1
dry weight

in mg/l (mean and range) of organisms
AAP medium (-) P 21 (15-27) -
" " (-) P + 0.025 P 1000 (980-1000)> -
" " (-) P+ 0.05 P 2300 (2200-2600) -
" " (~) P + 0.075 P 3700 (3600-3900) -
AAP medium (-) N 9 (7-10) -
" " (-) N+ 0.25 WN 600 (580-620) -
" " (-) N+ 1.0 N 2700 (2600-2800) -
Autoclaved lake water 2800 (2400-3200)° 90.
+ 0.05 P 2400 (2400-2500) -
+ 0.5 N 4600 (4200-5200) -
+ 0.05 P+ 0.5 N 3600 (3000-4000) -
+ 0.05 P + 0.5 N+ 0.03 Fe 3800 (3700-3800) -
+ 0.1 P + 0.03 Fe 2200 (2000-2300) -
+ 0.2 N + 0.03 Fe 6000 (5700-6200) -
Supernate of treated water 22 (20-25) ° 0.70
+ 0.05 P 1500 (940-2100) -
+0.5 WN 25 (23-27) -
+ 0.05 P+ 0.5 N 1600 (1300-1800) -
+ 0.05 P + 0.5 N + 0.03 Fe 1000 (640-1100) -
+ 0.1 P + 0.03 Fe 1000 (950-1100) -
+ 0.2 N + 0.03 Fe 44 (41-49) -
Supernate + phosphate 1900 (1700-2200) ° 61.
+ 0.05 P 2000 (1700-2400) -
+0.5 N 2600 (2400-2900) -
+ 0.05 P+ 0.5 N 2700 (2300-2900) -
+ 0.05 P + 0.5 N + 0.03 Fe 2400 (1900-2800) -
+ 0.1 P+ 0.03 Fe 2000 (2100-2800) -
+ 0.2 N + 0.03 Fe 2700 (2400-3400) -

'gased on 3 determinations except as noted.
Based on 2 determinations.
°Based on 5 determinations.

118


the ultimate answer for most comparisons, the 2- to 3-week
incubations required cause one to seek comparative methods
involving less time.

Other methods that can be used to measure the rate of sorp-
tion of P by algae or aquatic weeds without waiting for the
plants to grow with the sorbed P are measurements of P lost
from the culture medium or analysis of the plants for P
sorbed. Chamberlain and Shapiro® used the loss of P from
algae culture media to compare the biologically available P
of lake waters with different chemical analyses for P. The
present study uses the rate of loss of P from different
media to compare the rates of sorption by algae and aquatic
weeds. In addition, the comparative ability of algae to
sorb P is measured by increases in the extractable P0,-P®
or total P of the algae. The main emphasis has been placed
on whether different species of aquatic plants could sorb P
to the exclusion of other species or whether the plants
tested merely share available P. The ecological implica-
tions of these results and other studies will be discussed
from the point of what might take place in the balance of
algae versus aquatic weeds in lakes which become P-limited
due to the control of P sources to the lake environment.

Materials and Methods

The algae and aquatic weeds used for most of these studies
were field collections from the Madison, Wisconsin area.
Comparative tests indicated that the rates of P sorption
measured were not influenced when different lake waters were
compared with Gorham's (-P) medium. Therefore, many of the
results reported are for lake waters with naturally high P
concentrations (sample from hypolimnion of Lake Mendota) or
lake waters supplemented with P to bring the level up to
that sometimes found to be present in hypolimnion samples
(0.4 mg P/l).

The rate of .decrease of soluble PO.-P from media caused by
algae or weeds was followed by the molybdate-stannous
chloride method.’ Approximately 100 mg (dry weight) of
plants were placed in 2 liters of media containing 0.4 mg
PO,-P/1. Duplicate 50 ml samples were removed at different
times for analyses of the soluble PO,-P remaining. At the
end of the experiment the actual dry weight of the plant
material was measured so results could be reported as mg P
sorbed/100 mg algae or weeds/hour. In this manner, many

37


Table 28. THE PHOSPHORUS OF THE ROCK AND YAHARA RIVERS:
BIOASSAYS VERSUS CHEMICAL ANALYSES

Phosphorus concentration (mg P/1)
Bioassays-available phosphorus

Chemical
inday sorption 49 gays ———analyses _
Extractable growth Soluble

Samples PO,-P Total P (AAP) PO,-P Total P
Rock River 0.14 -
Newville-above 0.11 0.090 0.25 (0.28) 0.50
Yahara River .

Rock River 0.17
Indianford-above 0.10 0.060 0.30 (0.23) 0.41
Yahara River .

Rock River 0.77
U.S. 14-below 0.56 0.65 0.86 (0.96) 1.2
Yahara River

Yahara River 0.62
Wis 59-below 0.51 0.60 1.2 (1.0) 1.2
Badfish Creek °

Yahara River 1.9
Fulton-below 1.4 0.95 1.5 (1.9) 2.6

Badfish Creek

@analyses in parentheses are of soluble POy-P
of samples for AAP growth tests

about 0.1 mg available P/1l even before the junction with the
Yahara River. The effect of the Yahara River on the phosphorus
content of the Rock River is shown by the increase in available
phosphorus to more than 0.5 mg/l. The differences in the
samples collected from the Yahara River probably reflect the
fact that the sample collected at Highway 59 was close to the
junction with the Badfish Creek and may not have been as well
mixed with the Badfish Creek water as the sample collected
farther downstream (Town of Fulton).

79


ACKNOWLEDGMENTS

Significant contributions to this research
effort were made by Mrs. Selma Faust, Mrs.
Cynthia Nadler, Mrs. Frances Johnson, the

Wisconsin Department of Natural Resources,
and the University of Wisconsin. Their

participation and support are gratefully
acknowledged.


Abstract

CONTENTS

List of Figures

List of Tables

Acknowledgments

Sections

I Summary and Conclusions

Ir Chemical and Bioassay Methods for Nutrient
Analyses

III Rates of Phosphorus Utilization by Algae and
Aquatic Weeds

Iv The Release, Sorption, and Availability to
Algae of Phosphorus from Lake Muds

Vv Duckweed (Lemna minor) for Nutritional
Bioassays

VI Correlations Between Algal Bioassays and
Chemical Analyses to Evaluate the Effects of
Wastewater Phosphorus on Receiving Waters

VII Comparative Chemical and Bioassays Analyses
of the Nutrients in the Madison Area Lakes

VIII

Lake Restoration Effects as Measured by Algal
Assay: Bottle Test ,

iii

vii

36

47

59

68

86

106


ert
Algal Production (mg/L Dry Wt.)

loo L—
Zz O Epilimnion
Zz 4 Hypolimnion
10 }—
lobe
leo PN
- °
=
-O1 v T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Time (Days)
Fig. 38. Algal Production ~ Long Lake, 1971


REFERENCES

1.

7.

American Public Health Association. Standard Methods
for the Examination of Water and Wastewater. New York,
U.S. Public Health Association, 1971. p. 518-532.

Environmental Protection Agency. Algal Assay
Procedure: Bottle Test. Washington, D.C., National
Eutrophication Research Program, August 1971. 82 p.

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

Gerloff, G. C. Evaluating Nutrient Supplies for the
Growth of Aquatic Plants in Natural Waters. In:
Eutrophication: Causes, Consequences, Correctives.
Washington, D.C., National Academy of Sciences, 1969.
Pp. 537-555.

Fitzgerald, G. P., S. L. Faust, and C. R. Nadler.
Correlations to Evaluate Effects of Wastewater Phosphorus
on Receiving Waters, Water and Sewage Works 120:48-55,
January 1973.

Fitzgerald, G. P., and G. F. Lee. Use of Tests for
Limiting or Surplus Nutrients to Evaluate Sources of
Nitrogen and Phosphorus for Algae and Aquatic Weeds.
Madison, Water Chemistry Program Report, University of
Wisconsin-Madison, 1970. 31 p.

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

Fitzgerald, G. P. Some Factors in the Competition or
Antagonism among Bacteria, Algae and Aquatic Weeds.
J. Phycol. 5:351-359, 1969.

Fitzgerald, G. P. Evaluations of the Availability of

Sources of Nitrogen and Phosphorus for Algae.
J. Phycol. 6:239-247, 1970.

34


est
Biologically Available Phosphorus (mg/L)

10-—

BR
°

I

°
fa

-001

O Epilimnion

4 Hypolimnion

Jan.

Fig. 49.

T
210

T T
240 270

90 120 150 180

Time (Days)
Biologically Available Phosphorus - Pickerel Lake, 1973

T
300

330

T
360


Table 22. AVAILABILITY OF THE NITROGEN OF AEROBIC
LAKE MUD FOR THE GROWTH OF DUCKWEED (LEMNA MINOR)

2 fronds/50 ml Gorham's medium 3 weeks incubation
Average
Source Concentration frond number per
of nitrogen (mg_N/1) triplicate culture
NO3-N 0.0 12
NO3-N . 0.25 17
NO3-N 0.5 24
NO3-N 1.0 39
NO3-N 2.0 56
NO3-N 5.0 78
Lake Wingra mud (2 m1) 13
Lake Wingra mud (5 ml) 12
take Wangee mud (2m1)+2.5 24

The previous tests had indicated that the P of a lake mud
sample was not available for the growth of duckweed when the
roots of the plant could touch the mud under aerobic condi-
tions. The difference between tests with algae and aquatic
weeds, however, lies in the fact that aquatic weeds can be
tested under conditions where their roots can penetrate an
anaerobic environment while the rest of the plant remains

in an aerobic environment, such as occurs in normal growth
in a lake.

Consequently, an experiment was carried out comparing the
availability of the P in lake muds to duckweed floating so
far above the mud surface that its roots could only absorb P
which first passed through an aerobic column of medium, with
the availability to duckweed whose roots could penetrate the
mud and presumably extend into any anaerobic environment
developed in the mud. Two fronds were placed in 20 cm cul-
ture tubes containing Gorham's (-P) medium. Duplicate
cultures contained 50 ml of Gorham's (-P) medium or this
medium plus 0.005 mg PO.~-P. To four tubes containing 25 ml
of Lake Wingra mud (2.0 mg total P/tube), 30 ml of Gorham's
(-P) medium were added so the duckweed floated 8 cm above
the surface of the mud. The second set of mud tubes contained

65


samples taken from the stream below Lake Kegonsa and the City
of Stoughton contained relatively high phosphorus concentra-
tions, these samples were diluted to 50% and 20%, respectively,
(double-distilled water with 15 mg NaHCO3;/1). Some samples
were supplemented with 2 mg NO3;-N/1, 0.03 mg Fe plus 0.3 mg
EDTA/1, or both. Growth of Selenastrum in the cultures was
followed, and the maximum growth was shown to have been reached
after 9 days incubation.

Table 25. COMPARISONS OF PHOSPHORUS ANALYSES
OF YAHARA RIVER AND BADFISH CREEK:
BIOASSAYS VERSUS CHEMICAL ANALYSES

Phosphorus concentration (mg P/1)

Bioassays : Chemical analyses
1-day sorption 9 days Soluble

Sample and extraction growth PO,y-P Total P
Yahara River below

Lake Waubesa 0.07 0.10 0.054 0.14
Yahara River below

Lake Kegonsa 0.12 0.13 0.11 - 0.23
Yahara River below

City of Stoughton 0.27 0.28 0.28 0.40

Badfish Creek below 8.0 _ 7.7 8.0

City of Oregon

The growth of Selenastrum increased with increases in PO,~P
in the control cultures, indicating that the test alga was
able to respond to different levels of phosphorus. The re-
sults of the tests in the river waters showed that growth
was increased by the addition of nitrogen or nitrogen plus
iron. The maximum level of growth in the supplemented river
samples was used to calculate the amount of available phos-
phorus in the samples, by reference to the growth attained
with known concentrations of phosphorus (second column,
Table 25).

The calculated concentrations of available phosphorus from

the Selenastrum growth test are compared to results from the
one-day bioassay and chemical analyses in Table 25. These

74


EPA -660/3-73-023
February 1974

Ecological Research Series



by results of tests of some of the tributaries to the Madison
lakes.

Lake Mendota - University Bay Creek is essentially a storm
sewer serving a portion of the west side of the City of
Madison. In addition, it receives some cooling waters from
the University of Wisconsin campus which insure a continuous
but minor flow. In 1970, samples of water from this creek
were tested and found to have spectacular amounts of avail-
able P, effects of this P were noted in the creek and at 25
and 250 meters from shore by using P-limited Anabaena and
No-fixation tests.* In 1971, chemical analyses indicated
approximately 0.1 mg P/l was present in two samples of creek
water, but tests for the availability of the P were negative
because the water samples were toxic to P-limited Anabaena.

It was at this time that biocides were being used in some

of the University of Wisconsin cooling towers. In 1972,

8 tests were carried out on University Bay Creek waters during
periods of low and high flow (rain). The soluble P0,-P ranged
from 0.025 to 0.12 mg P/1l. In 6 tests no available P was
detected by sorption tests, and in the other two tests less
than 1/3 of the soluble P0,~-P present was available. However,
when creek waters were autoclaved (6 tests) or allowed to age
in the laboratory for 8 days (6 tests), the soluble P0O,-P

of the treated creek waters was available in both sorption
tests with Cladophora and growth tests with Selenastrum,
Autoclaving these samples did not change the concentration

of soluble P0,-P so the reason for decreased toxicity is not
yet known.

The Yahara River and Token Creek are tributaries entering
Lake Mendota from the north and represent the main source of
water. Both have flows during dry periods. Upstream samples
collected from the Yahara River near the Town of Windsor
during the period of August through December, 1972 indicated
that, although there were 0.10 to 0.15 mg soluble PO,-P/1
present, there was no available P in 6 sorption tests and
less than 1/3 of the PO,-P was available in 3 tests. No
increase in availability could be detected in membrane- |
filtered samples or by the addition of EDTA (7 mg/l). Auto-
claved samples containing 0.14 mg PO,-P/l were also found

to contain no available P when tested in growth tests with
Selenastrum., Upstream samples of Token Creek water (Wis
Highway 19) were tested 6 times, and in 4 tests less than

' 30% and in 2 tests only 50% of the soluble P0,-P was avail-
able by sorption tests. In contrast, 22 tests with downstream

100


with any of these lakes. Table 42 gives the biologically
available P concentration, the limiting nutrient and the
biomass produced for each lake sample.

Horseshoe Lake inlets -

Because phosphorus levels in Horseshoe Lake seemed to be in-
creasing during the early spring months of 1972, a series of
water samples were taken to try to identify the source of this
phosphorus. From February to August of 1973, 12 samples of
inlet water were analyzed for algal growth potential (mg/l dry
weight) and biologically available phosphorus. The results

are given in Table 43.

Table 43. BIOASSAY RESULTS FOR SAMPLES
FROM INLETS TO HORSESHOE LAKE

Soluble Biologically

ortho-P Avail P Biomass
Site Date mg/l mg/1 mg/l
1/3/73 0.110 0.08 9
2/7/73 0.156 0.46 74
3/6/73 0.304 >0.2 120
Weir 4/18/73 0.086 0.10 32
5/23/73 0.104 0.02 23
6/26/73 0.182 0.19 140
7/26/73 0.151 0.07 100
1/3/73 0.079 0.06 12
3/6/73 0.320 >0.2 250
Culvert 5/23/73 0.104 0.04 31
6/26/73 0.149 0.18 110
7/26/73 0.109 0.04 66
: 2/7/73 0.044 0.06 29
Driveway 4/18/73 0.151 0.16 99

The 1973 increase in productivity of Horseshoe Lake over that
of 1971 and 1972 could be attributed to the high concentrations
of phosphorus entering the lake throughout the entire growing
season.

174


Table 39. NUTRIENT LIMITATION IN SAMPLES
FROM LAKES TREATED WITH ALUMINUM

Number of samples
which responded
to additions of:

Total

Lake Year Location N P N&P_ samples
Long 1971 epi - - 1 1
(treated 5/72) hypo - - - 0
1972 epi - 4 8 12
hypo - 5 8 13
1973 epi - “3 3 6
hypo 1 3 2 6
Pickerel 1971 epi - 3 - 4
(treated 4/73) hypo - 2 - 3
1972 epi - 10 1 12
hypo - il 1 14
1973 epi - 12 1 13
hypo - 12 1 13

123


TST
Biologically Available Phosphorus (mg/L)

10

ia
°

e

°
fa

001

= O Epilimnion
Auypolimnion
=
L
T —T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Fig. 47,

Time (Days)
Biologically Available Phosphorus - Pickerel Lake, 1971


ABSTRACT

The availability of nutrients in selected Wisconsin
lakes was measured in laboratory studies utilizing
both sorption and growth algal assays. These tests
were conducted to evaluate contributions of phos-
phorus to the Madison area lakes from septic tanks,
agricultural runoff, and urban drainage and to
measure changes in the nutritional status of six
lakes which were manipulated for water quality
improvement by nutrient inactivation or hypolimnetic
aeration. Characteristics of the assay techniques
are discussed and results are compared to chemical
determinations of plant nutrients.

ii


samples of the Yahara River water (Wis Highways 19 and 113)
indicated that the soluble PO,-P was available either as raw
samples in sorption tests or as autoclaved samples in growth
tests with Selenastrum. Thus, there appears to be a differ-
ence in the availability of compounds analyzed as soluble
PO,-P in these rivers or creeks. Downstream samples appear
to have nearly the same chemical analyses for soluble PO,-P,
but the P present is readily available to algae, in contrast
to the P of upstream samples.

Lake Monona - The main source of water to Lake Monona is the
Yahara River coming from Lake Mendota. We have tested two
tributary sources of P to the Yahara River below Lake Mendota,
the Tenney Park Lagoon and the Johnson Street storm sewer.
The lagoon is a shallow lake, and from July through mid-
August, 1972, it was filled with aquatic weeds (mainly
Myriophyllum sp). The lagoon also has a very heavy popula-
tion of ducks. In mid-August the majority of the weeds were
cut by mechanical harvesters and removed from the area.
Preliminary tests indicated that more soluble P0,-P was coming
out of the lagoon than entered, so a series of analyses were
made of the water and in situ algae in the lagoon area to
evaluate the lagoon as a source of P to the Yahara River and
Lake Monona, to detect any changes in the lagoon after the
weed removal operation, and to measure any changes after the
fall overturn of Lake Mendota. The data collected are sum-
marized in Table 31. On three different dates in early
August, it was found that the water coming from the lagoon
contained significantly higher concentrations of soluble
PO,-P and available P, and the algae at the outlet and close
below contained higher amounts of extractable PO,-P (i.e.,
data of 8/4/72). Samples collected soon after the weed
removal operation indicated that the lagoon outlet waters
still had more P than inlet waters (data of 8/21/72), but no
more than before the weeds were removed. Thus, the weed
cutting operation, which consisted of cutting, collecting,
and roller-squeezing to remove entrained surface moisture
before removal from the lagoon area, did not cause a measur-
able increase in the P content of the outlet water of the
lagoon. Tests in mid-September also indicated that surplus P
was coming from the lagoon system. It was therefore con-
cluded that the P released from the lagoon was not related
to the presence, removal process, or absence of the dense
growths of water milfoil. After the turnover of Lake
Mendota, near early October, the results of further tests

in the lagoon area indicated that the lagoon outlet contained

101


2%

Table 5. COMPARISONS OF THE EXTRACTABLE PO,-P

FROM FRESH VERSUS DRIED ALGAE

Extracted PO,-P

(mg P/100 mg algae)

Fresh algae?

Dried algae”

No. of
Algae Source samples Ave of Ave Ch
Ulothri« sp meen 12 0.67. «0.046 = 0.500.037
Spirogyra sp hake ay ieee 8 0.51 0.054 0.35 0.021
Cladophora sp tare ea 8 0.050 0.013 0.045 0.006
Rhizoclonium sp tan ee 8 0.067 0.003 0.050 . 0.008

®a1gae extracted, dried and weighed
Palgae dried, weighed and extracted
°Standard deviation


The growth of Selenastrum in known concentrations of phosphorus
and in dilutions of the river water samples was measured after
5 and 12 days by cell counts and chlorophyll a measurements
(fluorometry). From the results of the cell count data after
5 days growth, calculations were made of the available phos-~
phorus in the original river water samples and are presented
in the third column of Table 29.

Comparisons of the data from the two bioassays and chemical
analyses of soluble P0O,-P indicate that all three analyses
agree quite well. The concentrations of phosphorus present

in the different samples indicate that there is available
phosphorus in the Rock River and its two tributaries, the
Crawfish and Bark Rivers, even before the sewage effluents

of the Cities of Jefferson and Fort Atkinson are added to the
Rock River. The 0.5 mg PO,-P/l in the Rock River below Fort
Atkinson and above Lake Koshkonong can be contrasted with the
0.1 mg PO,-P/1 in the Rock River below Lake Koshkonong.
Although the lake apparently acts as a sink for the phosphorus
of the Rock River at this time of year (September, 1971), the
river below the lake still contains phosphorus in excess of
that required for growth of the algae and aquatic weeds in the
lake, in contrast to the situation in the case of the three
lakes in the Madison area, Lakes Mendota, Monona, and Wingra.

Conclusions

Although only a limited number of field samples contained
total phosphorus that did not analyze as soluble ortho PO,-P,
it is evident that soluble ortho PO,-P analyses correlate
with bioassays for available phosphorus and total phosphorus
analyses do not, indicating that either method (bioassay or
chemical ortho PO.-P analysis) could be used to measure the
phosphorus available for the growth of algae.

There was close agreement of results between the two types

of bioassay: the one-day sorption-extraction test versus the
long-term growth assay, even though the two methods differ
greatly. While the one-day sorption-extraction technique
relies on mere sorption, by a relatively large amount of algae,
of the phosphorus from the medium, the growth test requires
that nitrogen or iron-EDTA, or both, be added to some test
waters for the minute inoculum (1,000 cells/ml) of Selenastrum
to grow and utilize all of the available phosphorus. Thus,
the longer incubation times and the need to supplement certain
samples with additional nitrogen or nitrogen and iron in order
for the algae to utilize all of the available phosphorus for

81


and chemical analyses of the phosphorus leaving all five of
the Madison area lakes during the late summer of 1971.

Two tests were carried out for the available phosphorus
concentration in the lake waters by the sorption-extraction
bioassay. In one test Cladophora sp from Lake Mendota was
used, and in the other Cladophora sp from Lake Wingra was
used. Duplicate 5-10 mg samples were incubated 20 hours in
500 ml of the control and test waters. The algae were then
rinsed and extracted for PO,-P. The results of the test
with Lake Mendota Cladophora are summarized in the first
columm of Table 26 as the calculated concentrations of avail-
able phosphorus obtained from comparisons of extracts of
algae from control and test waters.

Table 26. COMPARISON OF THE PHOSPHORUS CONTENTS
OF THE OUTLETS OF MADISON AREA LAKES:
BIOASSAY VERSUS CHEMICAL

Lake T=day Sorption —-Ty-aays ore

outlet and extraction growth (AAP) ortho PO, Total
Mendota 0.011 0.014 0.01 0.05
Wingra 0.010 0.030 0.02 0.05
Monona 0.005 0.024 0.02 0.05
Waubesa 0.030 0.041 0.050 0.090
Kegonsa 0.10 o.1L 0.12 0.15

It is evident that the samples from the outlets of Lakes
Mendota, Wingra, and Monona contained barely detectable
available phosphorus. There was considerably more available
phosphorus in the sample from Lake Kegonsa than from Lake
Waubesa (0.10 mg versus 0.03 mg P/l). Similar results were
obtained when Cladophora from Lake Wingra was used.

The growth of Selenastrum after 10 days incubation in the

lake outlet samples supplemented with nitrogen and iron was
also used to calculate the concentrations of available phos-
phorus. These results are summarized in the second column

of Table 26. The value reported for the Lake Wingra sample
(0.03 mg P/1) calculated from cell count data is significantly

76


Comparative tests showed that duckweed could obtain avail-
able P from anaerobic lake muds if their roots could pene-
trate the mud but not if they were suspended out of reach
of the mud surface. This indicates how aquatic weeds can
compete with the growth of algae in environments where
surface waters are P-limited.

CORRELATIONS BETWEEN ALGAL BIOASSAYS AND CHEMICAL
ANALYSES TO EVALUATE THE EFFECTS OF WASTEWATER
PHOSPHORUS ON RECEIVING WATERS

Nutrient surveys of the Yahara and Rock Rivers found phos-
phorus in excess of that required by the existing algae

and aquatic weeds upstream from the effluents of the cities
of Stoughton, Oregon and Madison. The algal bioassays in-
dicated that soluble orthophosphate is the form of phosphorus
in river waters that can be used by algae.

Analyses of the phosphorus content of the Madison area lakes -
Chemical analyses and bioassays of 58 collections of samples
indicate that all the lakes become relatively devoid of P

by early summer, but, while Lakes Mendota, Monona, and Waubesa .
continue to have relatively low P levels until fall overturn,
Lake Kegonsa had spectacular increases in its P. content during
August and September. These were probably associated with
septic tank drainage from shoreline cottages. In contrast,
during the winter Lake Kegonsa demonstrated a tremendous
capacity to assimilate the P added from Lake Waubesa and the
smaller tributaries so that relatively little soluble P0,-P
left the lake. These data confirm results obtained in 1943-44.

Sources of phosphorus to the Madison lakes - Comparisons of
the availability of lake-water soluble PO,-P and the soluble
POy-P of creek or river waters have demonstrated that ground
waters and some storm sewer discharges contain P compounds
that are not available to algae. The availability of these

P compounds appears to increase when the waters flow through
widespreads in the river or with certain treatments in the
laboratory. Thus, the value of bioassays in conjunction with
chemical analyses for evaluating nutrient sources for lakes
has been demonstrated.


10.

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

American Public Health Association. Standard Methods
for the Examination of Water and Wastewater. New York,
U.S. Public Health Association, 1971. p. 518-532.

Gales, M. E., Jr., E. C. Julien, and R. C. Kroner.
Method for Quantitative Determination of Total Phos-
phorus in Water. Amer. Water Works Assn, 58:1363-1368,
1966.

Fitzgerald, G. P. Some Factors in the Competition or
Antagonism among Bacteria, Algae and Aquatic Weeds.
J. Phycol. 5:351-359, 1969.

Fitzgerald, G. P. Aerobic Lake Muds for the Removal

of Phosphorus from Lake Waters. Limnol, and Oceanogr.
25:550~555, 1970.

46


€9T

10 E—
= OEpilimnion
.— A Hypolimnion
i)
3
Pico b—
= CL A
on
Fa] L
u
Q
E>} =
[a
a
fe)
s
a
ao.
‘3 L f
a L ze) H
~N
z ° 9 vows J
— jo \ 7 2
Dt oN | Sf 2
q 7 No I \ x Nov
g o----- \ \
5.01 ES \ Q \ |
3 !
g iz \ sy \
a L_ \ / , /
= \ \/
/ \/
\ 7
\/ \i
2001 T T T x T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days) .
Fig. 56. Biologically Available Phosphorus - Mirror Lake, 1972


were not providing surplus P to the other lakes at a time
Lake Kegonsa was obtaining P above the concentration its
aquatic plants could utilize, This increase in P in Lake
Kegonsa is therefore believed to be due to septic tank
drainage. Confirmation of these data during 1973 will
provide a firm basis for evaluating the proposed sewerage
of the Lake Kegonsa area population and a separation of the
effects of farm runoff in this very fertile area versus the
effect of septic tank drainage. Since the levels of P in
this lake are so spectacular before the sewerage system
(0.1 mg P/1l), it should provide a very visible test of the
effect of cottage population sewerage programs on the level
of eutrophication of a lake.

Application of Algal Assay Procedures

Comparative AAP tests using Selenastrum, Microcystis, and
Anabaena have been carried out in water samples from the
outlets of the five Madison area lakes, Mendota, Monona,
Wingra, Waubesa, and Kegonsa. All the lakes were covered
with ice at the sampling times. Two samples were tested in
January, 1973 when there were high flows due to rain and
snow melts, and two samples were tested in February, 1973
during a period of subfreezing weather when there were no
surface runoffs. The maximum growth attained by the algae
added to the lake waters was measured as in vivo chloro-
phyll a using a fluorometer. The instrument used was most
sensitive to the chlorophyll of Selenastrum so, at about
equal dry weight concentrations, the relative amounts of
chlorophyll measured were 10, 1, and 1.5 for Selenastrum,
Microcystis, and Anabaena, respectively. The growth of the
algae in all lake waters which were spiked with a combina-
tion of N, P, and Fe (NAAM level) was relatively high so
algal growth attained was dependent upon: the lake tested,
Mendota usually supporting maximal growth and Wingra sup-
porting the least growth; whether the samples were collected
during high or low flows; and what spikes of N, P, Fe, or
combinations were added. The relative growths:of the algae
are presented in Table 30 as maximal fluorometry units
attained in the autoclaved water samples and in the presence
of added N and Fe.

The data show that the growth of Selenastrum was most con-
sistent in similar samples and is the alga of choice for most
growth tests using these three algae because, even with
inocula of 1,000 cells per mi compared to 50,000 cells per ml
for the other algae, the maximal growth was attained in 7

to 9 days whereas the other algae usually required 2 weeks

or more. The results of the tests with no added spikes of

88


SELECTED WATER 1 Repott No. a
RESOURCES ABSTRACTS . : eee

INPUT TRANSACTION FORM —

v

4

APPLICATIONS OF GROWIH AND SORPTION ALGAL ASSAYS

-- 8. Perforating Orgsization

“Report No.

athest : :
Fitzgerald, G. P., and Uttormark, P. D. 1. 8

Wisconsin University, Madison.

L. Dost ect rant Ne
R-801361
ad aiid aa

EPA-660/3-73-023, February 1974.

Abstract

The availability of nutrients in selected Wisconsin lakes was measured in laboratory
studies utilizing both sorption and growth algal assays. These tests were conducted
to evaluate contributions of phosphorus to the Madison area lakes from septic tanks,
agricultural runoff, and urban drainage and to measure changes in the nutritional
status of six lakes which were manipulated for water quality improvement by

nutrient inactivation or hypolimetic aeration. Characteristics of the assay
techniques are discussed and results are compared to chemical determinations |

of plant nutrients.

Vea.

ive.

We

escripto}

*Aquatie plants, *Chemical analyses, *Assay techniques, *Phosphorus sorption,

‘Nutritional bioassays, Growth rates, Algal assays, Eutrophication, Sampling,

Nutrient inactivation, Hypolimnetic aeration, Wastewater, Lake restoration,
Aerobic lake muds, Anaerobic sediments, Toxicity, Culture medium, Contact time,
Fluorometry

lee vit

*Madison area lakes, *Wisconsin lakes

COWRUG Pele & Group 05C

49, Sectiity Clask.
iy 2, Report) *
20. Securisy Class...

Send To:

WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D.C. 20240

Abotravtor P. D. Uttormark

Wisconsin Univ., Madison



Zot
Biologically Available Phosphorus (mg/L)
T

10/—

01

001

° Epilimnion

ay Hypolimnion

Jan.

30 60

Fig. 55.

T T T T T
90 120 150 180 210 240 270
Time (Days)

Biologically Available Phosphorus - Mirror Lake, 1971



Number in Tube Is Average Number
of Fronds/Tube

Control Cultures Lake Wingra Mud Cultures
(50 ml Gorham's (-P) Medium (2 mg Total P/Tube)
) A

6 27 5 27
Y

(-P) +0.005 mg PO,-P * 30 ml 5 ml

Gorham's (-P) Gorham's (-P)

Medium Medium

Fig. 12. The Availability of Lake Mud Phosphorus
for the Growth of Duckweed (Lemna minor)
2 Fronds/Tube 6 Weeks Incubation

67


°
w
°

0.05

Soluble PO,-P (mg P/1)

i} i 1
ro) 0.05 0.10 0.15
Available P (mg P/1)

Fig. 8. Relationship Between Available Phosphorus
(24-Hour Sorption Tests) and Soluble PO.-P
in Madison, Wisconsin Lake Waters
8/16/72 and 9/6/72 Cladophora sp

28


Table 42. MISCELLANEOUS BIOASSAY RESULTS

Biologi-
Soluble cally Limiting
Depth ortho-P avail P Biomas& Nutrient
Lake Date inm mg/L mg/l mg/l {P or N)
2 - 0.001 1.7 P
W/12/71 gg - 0 0.7 P
2 0 0.003 1.7 P
W/22/71 gg 0 0.002 1.5 P
2 - 0.002 1.4 P
11/30/71 7 .
Shadow 8 0.003 1.6 P
2 0 0 1.0 P
W2/2l/7l gg 0 0 112 P
2 0.032 0.009 1.0 P
2/24/72 0.036 0.007 1.2 P
2 0 0 0 P
S/11/72 4g 0 0 0 P
12/22/71 2 0.03 0.013. 2.9 P
. 1 0.009 0.018 1.8 P
Windfall N 2/2/72 5 0.06 0.012 2.3 ?
1 0 0 0.06 -
5/5/72 0 0.12 8.5 -
12/22/71 2 0.038 0.029 9.2 P
Windfall s
0.026 0.021 1.8 P
2/2/72 4 0.11 0.068 20. N
<5 0.005 0 0.4 P
White VI/73 975 91016 0 118 P
2/7/73, 1 0.023 f) 0.3 P
Horsehead 3/2/73 1 0.01 P
5/3/73 1 0.029 0.095 -
8/8/73 1 0.055 04 P

173


It is evident that the P-limited algae from Lake Wingra were
able to remove P from the medium at a more rapid rate than
algae collected from Lake Mendota. The rates of P sorbed
during this experiment were 0.057 and 0.035 mg PO,4-P/100 mg
algae/hour, respectively. Later in the summer, other samples
of Cladophora sp from Lake Mendota were found to be P-limited
(less than 0.08 mg PO,-P extracted/100 mg algae) and had

P sorption rates equal to those measured with Cladophora sp
from Lake Wingra.

A summary of the results of tests of the rates of P sorption
by different aquatic plants is presented in Table 7 as the
mg P sorbed/100 mg algae/hour. The plants are identified as
to their source and whether they were P-limited at the time
of testing. The test for whether the plants were P-limited
was the analysis of the PO,~-P extracted in one hour by a
boiling water bath treatment. Plants which contain less than

Table 7. RATES OF PHOSPHORUS SORPTION BY AQUATIC PLANTS
BY ANALYSIS OF THE MEDIUM VERSUS TIME
100 mg/2,000 ml plus 0.4 mg PO,-P/1

Sorption rate

Plant material and source (mg _P/100 mg/hour)

Cladophora sp a

P-limited (0.05% P“)-Lake Wingra 0.05

Surplus-P (0.15% P)-Lake Mendota 0.02
Rhizoclonium sp

P-limited (0.07% P)-Lake Mendota 0.07
Pithophora oedogonium

Low-P (0.1% P)-P-free medium 0.03

Surplus-P (0.4% P)-lab aquaria none
Gloeotrichia sp

Surplus-P (0.3% P)-Lake Mendota 0.05
Myriophyllum sp

Plant tips (0.2% P)-Lake Mendota 0.008

Tip leaves (0.2% P)-Lake Mendota 0.010

Basal leaves (0.04% P)-Lake Mendota 0.015

Lemna minor (duckweed)
P-limited - P-free medium 0.003

as P means mg PO,-P extracted/100 mg algae

40


biologically available nitrogen in the sample. In addition,
checks were run for possible toxicity in the sample, and a
series of reference cultures were incubated along with the
lake samples to establish the sensitivity of the inoculum
to increasing levels of N and P. It is emphasized that the
algal assay results alone are not sufficient to evaluate
the efficacy of the lake renewal techniques under study.

The purpose of this study was to evaluate the usefulness

of the standardized Algal Assay Procedure: Bottle Test under
the practical constraints of a demonstration program. A
summary of the assays conducted as part of this program is
given in Table 33.

Methods and Procedures

Sample preparation -

Upon receipt, normally on the day after collection, the
sample waters were autoclaved at 15 psi for’ 15-30 minutes,
cooled, sealed and stored at room temperature until tested,
Precipitates which form as a result of autoclaving were
redissolved by bubbling CO2 through the sample. The pH was
then adjusted to 7.5 + 0.3 by aeration.

Chemical analysis -

Complete chemical analyses of the raw samples were conducted
by the Wisconsin Department of Natural Resources. In addi-
tion, because in certain waters the phosphorus concentration
is altered upon autoclaving, each autoclaved sample was
analyzed for soluble PO.y-P by the stannous chloride method
(Standard Methods). Thus, the phosphorus concentrations
obtained from the bioassays were comparable to those obtained
from chemical analyses.

Inoculum -
Algal species ~- The bioassay organism used throughout the

study was Selenastrum capricornutum. Stock cultures were

109


aquatic weeds. Studies with algae and the rooted aquatic
weed, Lemna minor (duckweed), have indicated that neither
type of plant can utilize the P of lake muds under aerobic
‘conditions."»'° However, duckweed was able to utilize P from
lake muds that were stratified so some depths reached by the
roots of the duckweed became anaerobic and P0,-P was released
to the interstitial water of the muds. Thus, in an aquatic
environment in which the sources of P to the surface waters
are controlled so the waters are stripped of available P
during the growing.season of the algae and aquatic weeds,
there will probably be preferential conditions for the growth
of aquatic weeds through the use of P in layers of lake muds
which is unavailable to algae. From a management point of
view, the growth of aquatic weeds is preferable since they
can be more readily harvested from selected areas than can
growths of planktonic algae.

References

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

2. Fitzgerald, G. P., and G. F. Lee. Use of Tests for
Limiting or Surplus Nutrients to Evaluate Sources of
Nitrogen and Phosphorus for Algae and Aquatic Weeds.
Madison, Water Chemistry Program Report, University
of Wisconsin-Madison, 1970. 31 p.

3. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Burris.
Acetylene Reduction Assay for Determination of Phosphorus
Availability in Wisconsin Lakes. Proc. National Acad.
Sci. 66:1104-1111, 1970.

4, Fitzgerald, G. P. Bioassay Analysis of Nutrient Avail-
ability. In: Nutrients in Natural Waters, Allen, H. E.,
and J. R. Kramer (ed.). New York, John Wiley and Sons,
1972. p. 147-170.

5. Chamberlain, W., and J. Shapiro. On the Biological
Significance of Phosphate Analysis; Comparison of Standard
and New Methods with a Bioassay. Limnol. and Oceanogr.
14:921-927, 1969.

45


OfT

100—b— N
= fe. =
[— © Epilimnion 7 ~ PN
NY7 >
ft 4 Hypolimnion Oo
~ 10e—
# =
iS =
4
a —
a
“XN
o
&
1.0
6°
ol L
y
9 L
EI
Ls]
g
a ~_
4
3
D
@ ip
i
-O1 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 26. Algal Production - Snake Lake, North Basin, 1971


0.08 mg PO,-P/100 mg algae are considered to be P-limited.®
The general conclusions that can be drawn are that the rates
of P sorption by algae are fairly similar, and the rates

by aquatic plants are generally slower under the conditions
of.these tests.

It is evident that the rates of P sorption by the different
aquatic plants are affected by whether or not the plants are
P-limited. The most conspicuous effect of surplus P condi-
tions was with the green alga, Pithophora oedogonium, which
was cultured in aquaria to prevent the growth of other algae.?®
Growth under these conditions of surplus P (0.4 mg extractable
PO,-P/100 mg) caused the algae to contain a maximal amount

of P and, under the conditions of the test, they did not sorb
additional P. When these algae were cultured in P-free medium
for a few weeks, the extractable PO,-P was reduced to 0.1 mg P/
100 mg and the algae sorbed P at a rate similar to Cladophora
sp under similar conditions. Similarly, the P sorption by
basal leaves of Myriophyllum sp from either Lake Mendota or
Wingra was always more rapid than by leaves from the tips of
the plants. The P content of tip leaves is higher because
under P-limiting conditions P migrates in the plant to the
more actively growing portions of the plant.

The significance of the rates of P sorption by algae in the
range of 0.02 to 0.07 mg P/100 mg algae/hour is evident when
the chemical composition of algae is considered. For instance,
field collections of Cladophora sp contain 0.1 to 0.4 mg P/
100 mg algae, depending upon whether or not they are P-limited
at the time of collection. The measured rates of P sorption
indicate that the time required to double the P content of
P-limited Cladophora would be about 2 hours. This corresponds
to the 2 hours required for the maximal sorption of P by
Selenastrum in the exposure and subculture for growth tests
reported earlier.!

Measurements of P sorption based on decreases in the concen-
trations of soluble PO,~P in culture media are readily carried
out, but are not direct measurements of the P sorbed by the
plants, In addition, no direct evidence can be obtained by
this method of the rate of P sorption by individual species

of algae in mixtures. For the latter type of information, in-
creases in the P content of the algae or aquatic weeds rather
than mere loss from solution must be measured. The methods
for measuring the increase in sorbed P that we have used were
analysis of either the extractable P0O.-P or the total P of the
algae. Since the sorption rate of P by the blue-green alga,
Gloeotrichia sp from Lake Mendota, was known to be equal

41


OL

, Crawfish R.

Madison

Rock River

Cs ee eee ae ae we fm 1 Fort

! Atkinson

Effluent

Lake
Koshkonong

Madison Sewage

'
'
'
Mime Se oe Stoughton
—_

Badfish Cr.

o&

Yahara R. 0!

Fig. 13. General Diagram of the Madison Lakes,
Yahara River-Rock River System

Bark R.



The phosphorus content of the Rock River
and two of its tributaries -

In order to determine the source of the phosphorus found in
the Rock River above the junction with the Yahara River,
upstream samples of the Rock River and two of its tributaries
were tested with bioassays and chemical analyses. The Rock
River was sampled at County Highway B above the City of
Jefferson and at Wisconsin Highway 59 below Lake Koshkonong.
The Crawfish and Bark Rivers were sampled near their junctions
with the Rock River. Therefore, the amount of phosphorus in
the Rock River above the Cities of Jefferson and Fort Atkinson
could be compared with the phosphorus in the river below these
stations. It was also possible to test if the plankton and
aquatic weeds of Lake Koshkonong caused the lake to act as a
sink for the phosphorus of the Rock. River.

The phosphorus sorbed by P-limited Cladophora sp from Lake
Wingra was measured as extractable P0O,-P after 22 hours incu-
bation in control samples with known concentrations of phos-
phorus and in dilutions of the field samples. The results
of the tests were used to calculate the available phosphorus
concentrations in the river water samples (2nd column,

Table 29).

Table 29. THE PHOSPHORUS OF THE ROCK RIVER AND
SOME TRIBUTARIES: BIOASSAYS VERSUS CHEMICAL ANALYSES

Phosphorus concentration (mg P/1)

Bioassays Chemical analyses
1-day 5 days Soluble

Sample sorption growth PO,-P Total P
Rock River at Co B 0.28 0.18 0.23 0.37
Crawfish River 0.22 0.28 0.21 0.44
Bark River 0.65 0.57 0.55 0.92
Rock River at Wis 106 0.50 0.57 0.51 0.68
Rock River at Wis 59 0.10 0.19 0.14 0.30

80


10.

11.

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

Fitzgerald, G. P. Bioassay Analysis of Nutrient
Availability. In: Nutrients in Natural Waters,
Allen, H. E., and J. R. Kramer (ed.). New York,
John Wiley and Sons, 1972. p. 147-170.

35


Tet

Table 38.

AVERAGE ALGAL BIOMASS

PRODUCED IN SAMPLES FROM LAKES TREATED WITH ALUMINUM®
Epilimnion Hypolimnion
Dec- Mar- June- Sept- Dec- Mar- June-  Sept-
Lake Year Feb May Aug Nov Feb May Aug Nov
1971 0.60 1.8
fected 5/70 1972 4.4 = 220. 0.76 6.0 8.6 38.
1973 20. 1l. 1.4 22. 18. 57.
Snake 1971 47. 260.
(north basin), 1972 83. 8.9 0.36 #41.5 86. 50. 0.96 2.0
treated 5/72 1973 2.1 1.5 1.1 10. 2.0 1.1
Snake 1971
(south basin), 1972 90. 0.37 0.64 74. 0.41 0.55
treated 5/72 1973 1.2 0.76 1.8 2.0 1.1 1.1
Long, - 1971 0.45
trestea 5/72 1972 0.64 0.28 0.13 0.17 0.51 0.24 0.095 0.16
1973 0.38 0.11 0.36 0.22 0.16 0.41
: 1971 0.83 0.19 1.2 0.096
treated 4/73 1972 1.6 0.11 2.1 0.32 2.1 1.2 2.1 0.77
1973 0.42 0.30 0.26 0.39 0.82 0.30

9a11 values given in mg/l dry weight.


Table 32. COMPARISON OF CHEMICAL ANALYSES OF PHOSPHORUS
AND AVAILABLE PHOSPHORUS IN LAKE WATERS AND CREEKS

Chemical analyses

Available P

Soluble
Date and Total P PO,-P Sorption Growth
sample area (mg P/1) (mg_P.1) (mg _P/1) (mg_P/1)
9/25/72
L. Waubesa 0.080 0.034 0.028 -
L. Kegonsa 0.13 0.064 0.080 -
Halverson Cr 0.16 0.11 0.060 -
Door Cr 0.14 0.088 0.050 -
11/15/72
L. Mendota - 0.15 0.18 0.07
L. Monona - 0.11 0.085 0.07
L. Waubesa - 0.092 0.045 0.06
L. Kegonsa - 0.078 0.028 0.05
Halverson Cr - 0.13 0.035 0.032
Door Cr - 0.10 0. 0.026
11/27/72
L. Mendota - 0.13 0.10 -
L. Waubesa 0.066 0.034 0.030 -
Halverson Cr 0.19 0.026 0. -
Door Cr 0.20 0.056 0. -
12/18/72
L. Mendota 0.14 0.14 0.12 0.12
L. Waubesa 0.073 0.046 0.053 0.068
L. Kegonsa 0.080 0.050 0.055 0.058
Halverson Cr 0.17 0.060 0. 0.005
1/18/73
L. Mendota - 0.13 0.13 0.16
L. Monona - 0.080 0.121 0.11
L. Waubesa - 0.046 0.06 0.062
L. Kegonsa - 0.02 0.02 0.02
Halverson Cr - 0.20 0.19 0.18
Door Cr - 0.38 0.36 0.31
3/15/73°
L. Mendota 0.19 0.10 0.12 -
L. Monona 0.14 0.10 0.10 7
L. Waubesa 0.17 0.12 0.14 -
L. Kegonsa 0.15 0.044 0.05 -
Halverson Cr 0.16 0.082 0. -
Door Cr 0.27 0.14 0.12 -

ani gh flow periods

105


alone yielded a significant increase in biomass, but a sig-
nificent response was obtained by a combined addition of
these nutrients.

This procedural definition of "limiting" provides information
regarding the relative supply of one nutrient as compared

to another, but is not indicative of the abundance or scarcity
of a nutrient on an absolute scale. Thus, a shift from
phosphorus-limitation to nitrogen-limitation could result

from either an addition of phosphorus or a reduction in the
supply of nitrogen.

Results and Discussion

Nutrient inactivation/precipitation -

Nutrient inactivation by chemical means has been proposed as
one method by which excessive algal growth in lakes might be
reduced. In particular, the use of aluminum to reduce in-
lake concentrations of phosphorus appears to hold promise.

The effectiveness of aluminum as a precipitating agent for
phosphorus has been demonstrated in laboratory studies and
has been used in industrial and municipal treatment processes.
Treatment with aluminum results in the formation of hydrous
oxide and a phosphate precipitate of low solubility. The
flocculant precipitate effectively reduces the phosphorus
concentration (dissolved inorganic and particulates) by sorp-
tion, precipitation, and physical entrapment. Removal of
other nutrients (as well as color and turbidity) is also
accomplished during settling or flotation.

Bioassays were conducted on samples from four lakes which
were treated with aluminum: Horseshoe, Snake, Long, and
Pickerel Lakes. In general, chemicals were applied to the
lakes by injection through a manifold at a depth of about
30 cm behind propeller-driven water craft. This provided
good mixing and permitted in sttu floc formation.

Commercial standard ground aluminum sulfate (alum)
(AlL2(SO,) 3 * 14 H20), liquid aluminum sulfate and sodium
aluminate (NazO * Al203 ° 3 H20) were used as sources of
aluminum III. The liquids were diluted before addition;
the ground alum was added as a slurry without further dilu-
tion.

114


growth potential and, after treatment, algal growth in the
samples was stimulated by phosphorus additions, not nitrogen.
Addition of phosphorus to near original levels in treated
waters resulted in substantial recovery of growth potential,
and the reconstituted samples showed algal response to added
nitrogen.

The experiment suggested that phosphorus limitation (at least
for AAP organisms) can be induced in Snake Lake waters by
Al(III) treatment at a concentration of about 10 mg/l.

Mirror Lake water (March, 1972) - An assay technique that
would show changes in growth rates was used for Mirror Lake.
The data have implications for the Snake Lake results. A
volume of autoclaved lake water was treated with 10 mg
Al(III)/1, settled, and decanted. Reactive-P and fluoro-
metric growth response were determined on autoclaved, super-
natant, and supernatant-plus-nutrients samples as shown in
Table 37.

Table 37. ALGAL RESPONSE AND PHOSPHORUS CONTENT
OF ALUMINUM (III)-TREATED MIRROR LAKE WATERS

Fluorometric
response units (mean) Reactive P
Day 4 Day 7 Day 9 mg/l

Autoclaved lake water 6800 14000 14000 0.52
Supernatant treated water 1500 1200 890 0.08
Supernate

+ NAAM (=P) components 1300 880 510 0.08
Supernate

+ 0.44 mg/l P 3300 12000 11000 0.52
Supernate

+ phosphate and nitrate 3000 15000 = 15000 0.52
Supernate

+ 0.44 mg/l P + NAAM (-P) 6300 15000 13000 0.52

The lake water was obtained from the 10-12 m depths of Mirror
Lake in a strata which had apparently not been mixed during
fall overturn, resulting in high (0.52 mg/l) phosphorus values.

119


€rT
Algal Production (mg/L Dry Wt.)

100-—

= O Epilimnion

_—— 4 Hypolimnion

10———_

PN
-O
PN
-O1 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Time (Days)
Fig. 39, Algal Production - Long Lake, 1972


LyT
Biologically Available Phosphorus (mg/L)

10

01,

001

© Epilimnion

4 Hypolimnion

Jan.

Fig. 43,

T T T T
90 120 150 180 210 240 270

Time (Days)
Biologically Available Phosphorus - Long Lake, 1973


EPA~660/3-73-023
February 1974

APPLICATIONS OF GROWTH

AND SORPTION ALGAL ASSAYS

by

Dr. George P. Fitzgerald
~ and
Dr. Paul D. Uttormark
University of Wisconsin
Madison, Wisconsin 53706

Grant R-801361
Program Element 1BA031

Project Officer

Mr. Thomas E. Maloney
National Environmental Research Center
Corvallis, Oregon 97330

Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460

For sale by the Superintendent of Documents, U.8. Government Printing Office, Washington, D.C, 20402 - Price $2.10


Let
Algal Production (mg/L Dry Wt.)

loo b 9
N O Epilimnion
4A Hypolimnion
10k
=
1.0
leo
+01 1 1 T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 33. Algal Production - Snake Lake, South Basin, 1972


high levels of P during this period, this effect on the algae
of Lake Waubesa was more spectacular. The decrease in the P
content of the algae in late May was probably associated with
the algal blooms occurring in all the lakes (mainly green
algae and diatoms). There was another peak in the P content
of the algae of Lake Waubesa in June, again probably associated
with rains and surface runoffs. This increase in the P content
of the in sttu algae during a period of little change in the
soluble PO,-P of the water demonstrates the value of in situ
algae in detecting sources of P that are not great enough to
exhibit surplus P in the water. The large amounts of rain
during July through September after the farm fields and marshes
were green did not appear to affect the surplus P of the lake,
measured as soluble P0,-P, but the P content of the algae
indicated that both Lakes Monona and Waubesa received enough
available P during this period for the algae to have about
0.12% P at a time when the algae of Lake Mendota had less than
0.08% P. Since Mendota receives relatively little urban drain-
age compared to Lake Monona, the increased P in the algae of
Lake Monona may be due to this source. However, the soluble
PO,-P of Lake Wingra, which receives over 90% of its P from
urban drainage from the City of Madison, was always barely
detectable and the in situ algae always P-limited. The only
exceptions were when ducks congregated near the sampling
stations on Lake Wingra and relatively higher P values were
found in water and algae samples. The P of the Lake Waubesa
algae is probably due to a combination of farm runoff and
septic tank drainage from the areas around this lake that

are not yet sewered.

The soluble PO,-P of Lake Kegonsa increased after ice out and
then decreased in mid-May. The algae also exhibited a peak
in their P content during this period. The soluble P0O,-P of
Lake Kegonsa increased to about 0.04 mg P/l in early June,
but then fell to 0.02 to 0.03 mg P/1l during late June and
early July, much like the results in Lake:Mendota. Despite

a series of rains (July 12 to 19) which amounted to about

1.8 inches, the P content of Lake Kegonsa and its’ alyae did
not increase. Therefore, surface runoffs do not appear to be
a major source of P to this lake at this time There was a
spectacular increase in the soluble PO,-P of Lake Kegonsa, to
about 0.1 mg P/l, during August through September. The P
content of the algae also increased during this period. Since
the P in all of the other Madison lakes did not increase
during July and August, the increase in the P of Lake Kegonsa
is due to local factors. The lack of an increase in the
soluble POu.-P of Lake Waubesa might be used to indicate that

98


1.

References

Swingle, H. S., B. C. Gooch, and H. R. Rabanal. Phos-
phate Fertilization of Ponds. In: Proc. 7th Annual
Conference, S.E. Assoc. Game and Fish Comm. Hot Springs,
Oct. 1963. p. 213-218.

Fitzgerald, G. P. Bioassay Analysis of Nutrient Avail-
ability. In: Nutrients in Natural Waters, Allen, H. E.,
and J. R. Kramer (ed.). New York, John Wiley and Sons,
1972. p. 147-170.

Martin, J. P., Jr., B. N. Bradford, and H. G. Kennedy.
Factors Affecting the Growth of Najas sp in Pickwick
Reservoir. Muscle Shoals, National Fertilizer Development
Center, TVA. F70-ACD2. 1970. 47 p.

Lee, G. F. Eutrophication. Eutrophication Information
Program, University of Wisconsin, Madison, Wis. Occasional
Paper No. 2. Sept. 1970. 39 p.

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

Fitzgerald, G. P. Aerobic Lake Muds for the Removal

of Phosphorus from Lake Muds. lLimnol. and Oceanogr.
15:550-555, 1970.

58


P sorption by Cladophora during 15 hours incubation in Lake
Wingra waters was tested. A summary of the results as the
averages of duplicate samples is presented in Table 10.

Table 10. THE EFFECT OF LAKE WINGRA PLANKTON
ON THE SORPTION OF PHOSPHORUS BY CLADOPHORA SP
15 to 20 mg Lake Wingra Cladophora/1,000 ml Lake Wingra water
24 hours incubation

Extractable PO,-P of Cladophora
after incubation
(mg _PO.,-P/100 mg algae)

Lake water

Plankton (17 mg/1) Lake water + 0.04 mg PO,-P/1
None (centrifuged) 0.024 0.075
Live (as is) 0.026 0.033
Dead (autoclaved) 0.080 0.13

The presence of live plankton did not affect the P sorption
by the Cladophora in unsupplemented Lake Wingra water, but
caused less P to be sorbed by the Cladophora in the lake
water with an additional 0.04 mg PO,-P/l. When the plankton
was killed by autoclaving the lake water, releasing avail-
able P for sorption by the Cladophora, the extractable P0O,-P
increased from 0.024 to 0.080 mg P/100 mg algae. This dead
plankton had no effect on the further sorption of P by the
Cladophora. When 0.04 mg PO«-P was added to the autoclaved
lake water, the extractable PO,-P of Cladophora increased as
much as that of Cladophora from supplemented centrifuged lake
water, Therefore, the sorption of P by mixtures of algae
results in competition between species for the available P,
but there is no evidence that one type of algae can prevent
another type from sorbing P.

The general results of these experiments have shown that
different algae or aquatic weeds have P sorption rates which
would indicate that they probably would compete with each
other for supplies of available P, but that no plant that
was P-limited could be prevented from using a portion of the
available P. This would mean that in a lake environment the
quantity of available P in the lake water would not preferen-
tially influence the growth of different species of algae or

44


in samples from the hypolimnion. Whereas the production in
epilimnetic samples in 1973 was about twice as large as in
1972, hypolimnetic samples in 1972 yielded more than ten
times as much algae as the 1973 samples.

Nitrogen was identified as the nutrient which most frequently
limited cell production in the 1972 samples. Nitrogen limita~
tion was particularly prevalent in the hypolimnion samples

in which 10 of 12 determinations showed a significant response
to an addition of nitrogen alone. It should be noted that
these results indicate that large amounts of phosphorus were
available; nitrogen was in short supply only in relative terms.
Sufficient nitrogen was available to permit biomass production
in excess of 100 mg/l. However, in 1973, nitrogen limitation
was detected on only one occasion, and algal production and
biologically available phosphorus were much lower in the
hypolimetic samples.

Bioassay data for Larson Lake (Figures 58-63, Tables 40-41)
showed a lesser effect of aeration than was noted in Mirror
Lake, however, only about 6 months of comparable pre- and
post-treatment data were obtained, A slight increase in cell
production in both the epilimnion and hypolimnion occurred

in 1973 as compared to 1972. The shift from nitrogen-limited
to phosphorus-limited samples, noted in Mirror Lake, also
occurred in Larson Lake, but the effect was less pronounced.

Miscellaneous Studies

Drawdown and winter aeration -

Horsehead Lake - Oneida County - A program of baseline data
collection was initiated on Horsehead Lake (144 ha, 3.7m
maximum depth). This lake has experienced water quality
problems relating to both weeds and algae, and a combined
treatment of drawdown for sediment consolidation coupled with
winter aeration is contemplated for the future. Tentative
treatment dates have not been scheduled. Algal assays were
included as part of the baseline data collection program.

As shown in Table 42, a total of 12 assays were conducted
on Windfall, White, and Shadow Lakes. These were conducted
as part of short-term assessments of aquatic plant control
(Windfall), urban runoff studies (Shadow), and winter aera-
tion (White). No further work was undertaken in connection

165


ALGAE - Extractable P
(mg POy-P/100 mg Algae)

WATER - Soluble PO,-P (mg P/1)

0.30 F

0.20

0.05

Fig. 16.

Apr May June July Aug Sept
Comparison of the Soluble PO«-P of Lake Monona
Waters and the Extractable PO,-P
of in situ Algae at the Outlet

93


1. Lake water (as is) - to record relative nutrient
status of sample.

2. Lake water plus P spike (NAAM level) - to record
response to an increase in available P.

3. Lake water plus N spike (NAAM level) - to record
response to an increase in available N.

4. Lake water plus Fe spike (NAAM level) - to record
response to an increase in available Fe.

5. Lake water plus N+Fe spikes (NAAM level) - to
Measure the concentration of available P in the
sample.

6. Lake water plus P+Fe spikes (NAAM level) - to
measure the concentration of available N in the
sample.

7. Lake water plus N+P spikes (NAAM level) - to
measure the concentration of available Fe in the
sample.

8, Lake water plus N+P+Fe spikes (NAAM level) - to
determine if algal growth is limited by a toxic
condition.

Lake water samples which support relatively high concentra-
tions of algae will, of course, be more fertile than those
which support only sparse growth. If a spike of a single
nutrient causes ‘a significant increase in growth it means
that this nutrient is the nutrient that is limiting the
growth of algae in that lake water sample. Caution should
be used in interpreting responses to the addition of NAAM-Fe
since this is added in the form of Fe-EDTA, and without
further tests it cannot be determined if the response was

to Fe or EDTA (a strong chelating agent which might reduce
the toxicity of certain chemicals). As has been pointed out,
the response of algae to spikes of single nutrients may be
limited because two or more nutrients might be in relatively
short supply. Therefore, the algae may not respond to an
addition of N without also adding Fe. The additions of two
spikes at a time will provide enough nutrients so the growth
of the algae will be proportional to the concentration of
the third nutrient in the lake sample in the caSe of mid-
western waters which usually are only limited by N, P, or
Fe contents. If the growth of algae in waters spiked with

18


oT

10

BR
.
°

o
.
=

eye ry pp Pyrrmm
yeep | |

Growth (Fluorescent Units)

0.01

0.001

Fig. 1.

0

2 4 6 8 10
Age of Culture (Days)

Rate of Growth of Selenastrum in
NAAM Medium

Growth (Dry Weight - mg/l)

L 4 L rf
% 1 2 3 4 5

NO3 Concentration (mg N/1)

Fig. 2. Growth of Selenastrum in
Different Concentrations of NO3-N
in NAAM Medium
7 Days Mean and 95% Confidence Limits


During the sampling period of 1972 when the lakes were covered
with ice, April 10 to 18, the soluble P0,-P concentrations

in Lake Mendota were around 0.1 mg P/1, and the extractable
PO,-P of the im sttu algae in the lake outlet was very high,
in the range of 0.5 to 0.8% P. The concentration of soluble
PO,-P in Lake Monona was about 0.065 mg P/l and the algae had
0.2% P or higher. In contrast, the concentration of soluble
PO,-P in both Lakes Waubesa and Kegonsa was about 0.02 mg P/1,
and the algae had extractable PO,-P levels of about 0.15 to
0.17% P. The results of analyses on Lake Mendota during the
ice period of 1972-73 were about the same as in April, 1972.
There was a gradual decrease in the soluble P0O,-P of Lake
Monona from about 0.1 mg P/1 in December to 0.07 mg P/1l in
February. There was then an increase to about 0.1 mg P/l in
March. In contrast to the results of April, 1972, during the
ice period of 1972-73 the soluble P0O.,-P of Lake Waubesa re-
mained at about 0.05 to 0.07 mg P/l from December to March.
From mid-January to March, the soluble P0,-P of Lake Kegonsa
was less than 0.03 mg P/l, values very similar to those in
April, 1972. Also, the algae (Ulothrix) in the outlet of
Lake Kegonsa in March, 1973 had only about one-half the
extractable P of the Ulothrix of Lake Mendota at this time.

After the ice breakup in 1972 there was a gradual decrease in
the soluble P0,-P of Lake Mendota from about 0.1 mg P/1 to
about 0.03 mg P/l by mid-June. The extractable P0O,-P of the
algae remained relatively high until a drastic decrease to
0.1% P during June. The algae (Cladophora sp) of Lake Mendota
remained P-limited (less than 0.08% extractable P) through
September 6. They contained surplus P at September 11 and
afterwards, although the fall overturn was not detected by
chemical analyses of lake water until September 25. This
demonstrates that sources of P are detected by analyses of

in situ algae before they cause an increase in the surplus P
of the lake waters (measured as soluble PO,-P left in the
water after algae have removed what they could).

There was a similar decréase in. the soluble PO,-P of Lake
Monona, from 0.07 mg P/l to about 0.02 mg P/l by mid-June.

As in the case of Lake Mendota, the P of the algae in Lake
Monona remained relatively high, 0.2 to 0.3% P, until mid-
June when it dropped to below 0.1% P. The soluble PO,-P of
Lake Waubesa remained at about 0.02 to 0.03 mg P/1 throughout
the spring and summer of 1972. However, there was a definite
peak in the per cent P of algae in late April and early May,
perhaps associated with rains and surface runoffs at this
time. Since the algae of Lake Monona already had relatively

97


ST
Algal Production (mg/L Dry Wt.)

100k

01

Jan.

O Epilimnion

A Hypolimnion

T T T T T T
90 120 150 180 210 240
Time (Days)

Fig. 50. Algal Production - Mayflower Lake, 1973


Air was injected at the bottom of the riser which "pumped"
water to the surface and increased the dissolved oxygen
content during ascent. A separator box was attached to the
riser section at the lake surface. Bubbles were allowed

to escape to the atmosphere at the separator, and the aerated
water was then returned to the hypolimnion through flexible
pipes.

Lakes receiving hypolimetic aeration -

Mirror Lake - Waupaca County - Mirror Lake is a drainage

lake of low color and moderate fertility. It has a surface
area of 5.3 ha and a maximum depth of 13 m. What was once

an excellent trout fishery has been compromised by an anoxic
hypolimnion created by storm sewer input. The lake's basin

is well sheltered, consequently spring and/or fall mixing may
be ineffectual or even absent during some years. The renewal
approach consists of hypolimnetic aeration during summer and
winter, coupled with total mixing to extend and amplify periods
of overturn in spring and fall. The equipment was installed
and tested in the fall of 1972, and hypolimnetic aeration began
in January 1973.

Larson Lake - Lincoln County - Larson Lake is a small, brown-
water seepage lake of moderate to low fertility. It has a
surface area of 5 ha and a maximum depth of 9 m, The shore-~
line is partly bog-like in nature, but there is no appreciable
mat on the lake's surface. Available data indicate that
Larson Lake seldom, if ever, mixes thoroughly in either spring
or fall. The extensive hypolimnion is generally without
measurable dissolved oxygen. Hypolimnetic aeration began on
February 2, 1973.

Results of algal assays on samples from Mirror Lake are
plotted in Figures 52 through 57 and are summarized, by
season, in Table 40. These data provide a general measure
of changes in lake fertility, but are not sufficient to
detect short-term effects induced by the intermittent periods
of aeration.

Samples from the epilimnion of Mirror Lake consistently pro-
duced more algal biomass in 1973 than in 1972. The increase
was greatest during the period from June through August.
However, the increase in algal production in epilimnetic
samples was small in comparison to the decrease in production

157


TOT
Algal Production (mg/L Dry Wt.)

100

BH
°o

Fy

e
°

BR

01

OEpilimnion

Atypolimnion
\ PN
b----—----9
= D , P
=
T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Time (Days)
Fig. 54. Algal Production - Mirror Lake, 1973


PO,-P in quantitative tests. The algae that have been suc-
cessfully used in sorption tests were usually filamentous
types (Cladophora (+ epiphytes), Rhizoclonium, Odeogonium,
Ulothriz, Mierospora, Spirogyra, Gloeotrichia, or mixed
diatoms) because of the ease with which they could be har-
vested from sorption solutions. Test solutions were usually
harvested by pouring through plankton nets and the algae
separated into duplicate samples for analyses. Algae that
are relatively P-limited are usually used for sorption tests,
but successful tests have been carried out with algae with
initial extractable PO,-P concentrations of as little as
0.02% and as high as 0.14%. The response of two collections
of Cladophora sp from Lake Wingra (8/16/72 and 9/6/72) to
different amounts of PO,~-P added to Lake Wingra water and
incubation (10 to 20 mg algae/400 ml) with shaking or aeration
for one day is presented in Figure 7 as the average concen-
trations of extracted PO,-P from the algae.

It can be seen that there is a direct relationship between
the concentration of POs-P added to Lake Wingra water
(original PO,y-P concentration below 0.02 mg P/1) and the
extractable PO,-P of the Cladophora over the range of 0.05

to 0.15 mg P/l. Similar relationships have been found when
the total P of the algae was analyzed after incubation with
known PO,-P concentrations, the P content of the algae going
from 0.11% P in the medium lacking P to 0.32% P in the medium
containing 0.075 mg PO,-P/l. The data from control cultures
in known concentrations of P were used to calculate the avail-
able P concentrations in 22 samples of water from 5 lakes in
‘the Madison, Wisconsin area after one-day sorption tests of
untreated samples (8/16/72 and 9/6/72). The results of the
tests are presented in Figure 8 as the concentrations of
soluble P0O,-P chemically analyzed in the waters and the con-
centrations of available P calculated from the sorption tests.

These data indicate that, for these lake water samples, P
analyzed as soluble P0,-P was available P as measured by the
sorption test. A word of caution: certain types of waters
(creeks and storm sewers) have been found to have chemicals
that analyze as soluble P0O,-P but which are not all avail-
able P. As a typical example, the extractable PO,-P from
P-limited Cladophora sp from Lake Wingra after 19 hours in-
cubation in lake and creek waters is presented in Table 6
along with the original soluble P0O.,-P content and the cal-
culated available P content using results from spiked Lake
Wingra water as the controls. It is evident that the soluble
PO,-P of Lakes WaubeSa and Kegonsa as well as the added spikes
of PO,~P were 100% available to the Cladophora. In contrast,

26


LOT
Algal Production (mg/L Dry Wt.)

100

10

01

=
L—
OEpilimnion
Akypolimnion
;_ O°
ya
T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Fig. 58.

Time (Days)
Algal Production - Larson Lake, 1971


SECTION VIII
LAKE RESTORATION EFFECTS

AS MEASURED BY ALGAL ASSAY: BOTTLE TEST
Introduction

The overall objectives of this aspect of the project were:

1) to measure changes in the nutrient status of lakes brought
about by lake restoration procedures, and 2) to evaluate the
usefuiness of the Algal Assay Procedure: Bottle Test as a
predictive and interpretive tool for lake management.

This project was conducted in conjunction with the Inland

Lake Renewal and Management Demonstration Program, a coopera-
tive project involving the University of Wisconsin-Extension
and the Wisconsin Department of Natural Resources. As part

of that program, several Wisconsin lakes were manipulated

for improved water quality. Restoration techniques included
dilutional pumping, nutrient inactivation/precipitation with
aluminum (alum or sodium aluminate) and hypolimnetic aeration
without destratification. The lakes under study were moni-
tored for a wide spectrum of physical, chemical and biological
parameters. This project expanded the data collection program
by the addition of algal assay results and, at the same time,
provided data for evaluation of algal assay results against

a backdrop of diverse limnological parameters.

The experimental design of the bioassays was based solely on
maximum cell production; exponential growth rates were not
measured. Each assay was designed to yield the following
information for a particular sample: 1) the algal biomass
produced by the sample (autoclaved), 2) the nutrient which
limited cell production, 3) the quantity of biologically
available phosphorus in the sample, and 4) the quantity of

107


Cras
Biologically Available Phosphorus (mg/L)

10

1.0
c

Oxrpi limnion
Auypolimnion

Ol / \

E / \

/ \ 7
t- / \ 7
\
; oe
01
001
° T a) T T T TT T T “T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 24. Biologically Available Phosphorus ~ Horseshoe Lake, 1972


69T
Algal Production (mg/L Dry Wt.)
we
°

100.—.

01

O Epilimnion
A Hypolimnion

Jan. 30 60 90

T T T T U T UJ T

120 150 180 210 240 270 300 330
Time (Days)

Algal Production - Larson Lake, 1973

360


Table 12. SORPTION OF PHOSPHORUS BY AEROBIC LAKE MUDS
300 ml mud/3,000 ml lake water + 0.6 mg PO,-P

Percentage
Time soluble PO,-P remaining
(minutes) Monona Bay University Bay
1 74 61
5 44 58
15 28 52
20 18 30

The release of PO.-P from lake muds by a dredging operation

has also been studied. Lake muds were being dredged from

Lake Monona to a marsh near Murphy's Creek (outlet of Lake
Wingra to Lake Monona). During a period of dredging, it

was found that the supernatant waters which entered Murphy's
Creek contained 0.13 mg soluble PO,-P/l and 0.64 mg total P/1l
whereas the waters upstream contained less than 0.02 mg
PO,-P/1l. Since downstream samples of Murphy's Creek con- |
tained considerably less soluble P0O,-P than the supernatant
entering the creek, tests were made of the sorption of P0O,-P
by mud in the dredging supernatant. The soluble PO,-P concen-
tration of aerated dredging supernatant which contained fine
grey sand to silt decreased to 0.076 mg P/l after one day and
0.030 mg P/1l after two days. When dredged mud was suspended
in river water supplemented with 0.4 mg PO,-P/1, 20, 30, and
45% of the PO,-P of 75 ml of the water was sorbed in 10 minutes
by 120, 240, and 480 mg mud, respectively. Thus, it appears
that muds disturbed by this dredging operation released soluble
PO,-P to Murphy's Creek, but that the sorption of P by aerobic
muds took place.

Availability of phosphorus to in situ algae -

Since the previous studies had indicated that P could be re-
leased from disturbed lake muds, it was of interest to deter-
mine how important such sources of P were to in situ algae.

Samples of Cladophora sp were collected from 3 sites in Lake
Wingra and 2 sites in Monona Bay on days when wind storms had
caused the surface muds to be suspended and to actually coat
the algae at the time of sampling. In addition, samples were

50


alge increased from 0.06% P to 0.11% P when incubated in
water samples containing 0.05 mg PO,-P/l, whereas when incu-
bated in sewer waters containing 0.10 mg PO,y-P/1 (undiluted)
or 0.05 mg PO,-P (diluted with P-free culture medium), the
algae had extractable PO,-P concentrations of only 0.06% P.
Tests carried out (12/20/72) with autoclaved samples using
Selenastrum, when Lake Mendota contained 0.15 mg PO,-P/1 and
the sewer water contained 0.057 mg PO,-P, indicated that
available P was present in the sewer water. When P was
added to the sewer waters, the growth of Selenastrum was
significantly higher than when tested "as is" or with the
addition of N plus Fe. Thus, the lack of availability of
the storm sewer P was not due to only toxic factors.

The results of these tests indicate that: 1) the Tenney
Park Lagoon system adds available P to the Yahara River
during the summer but dilutes the level of P in the river
after fall overturn; 2) the source of the higher levels

of P in the Tenney Lagoon could not be related to the pres-
ence of weeds or a new type of cutting operation carried
out; 3) the Johnson Street storm sewer contributes compounds
to the Yahara River which analyze as soluble P0O,-P but are
not available for in sttu algae or test algae.

Lake Kegonsa - The main tributary to Lake Kegonsa is the
Yahara River coming from Lake Waubesa. Tests carried out

on this section of the river indicated that there was little
change in the chemistry of the water or availability of P
from the Lake WaubeSa outlet to the Lake Kegonsa inlet.

Two smaller tributaries, which are believed to have less
than 10% of the flow from Lake Waubesa, are Halverson and
Door Creeks. These small creeks have a continuous flow,
pass through relatively fertile farm lands, but are un-
modified by passage through widespread areas. Bioassays

for available P were carried out with these creek waters on
26 dates from August, 1972 to March, 1973. A summary of
results is presented in Table 32 as a comparison of analyses
for total P and soluble P0O,-P with available P calculated
from 1-day sorption tests or growth tests using Selenastrum,

It is evident from the data that the available P of the lake
water samples is more closely related to the soluble POy4-P

than total P, as has been pointed out previously." In contrast
to the soluble P0,-P of lake waters, the soluble POs-P of the
creek waters was sometimes not available. There appears to be
a difference in the availability of the P from surface runoffs

104


SECTION VI
CORRELATIONS BETWEEN ALGAL BIOASSAYS
-AND CHEMICAL ANALYSES TO EVALUATE THE EFFECTS

OF WASTEWATER PHOSPHORUS ON RECEIVING WATERS
Introduction

The use of algal growth tests to measure the concentration
of a limiting nutrient in a water sample is based on the
assumption that, under the conditions of the test, the growth
attained by the algae will be directly dependent upon the
concentration of the limiting nutrient. Of the nutrients
required for plant growth in an aquatic environment, the con-
sensus of ecology-oriented scientists!,? is that phosphorus
is most able to be controlled to a degree which will allow
regulation of its fertilizing effect. In order to get a
perspective of the relative amounts of .phosphorus available
for algal growth contributed by the Madison, Wisconsin area
lakes and cities to the Yahara and Rock Rivers, a series of
bioassays and chemical analyses of river samples has been
carried out.

It was assumed that during late summer the growth of algae
in the Madison area lakes would cause them to act as sinks
for available phosphorus in the Yahara River system. If

the rivers below the lake outlets had an excess of phos-
phorus during this period, it would indicate that the amount
of phosphorus in the system is great enough: to provide more
phosphorus than the algae in the lakes can utilize. Such a
situation existed in Lakes Waubesa and Kegonsa during the
early 1940's.? While Lake Kegonsa supported about 135 metric
tons (dry weight) of algae, its outlet had concentrations of
more than 0.4 mg soluble PO,-P/1.

The main source of nitrogen and phosphorus to these lakes

was the secondary effluent of the Madison Sewage Treatment
Plant, which entered the Yahara River just above Lake Waubesa.
In 1958, however, the effluent from the treatment plant was
diverted around Lakes Waubesa and Kegonsa and piped to the

68


O Membrane Filtered Samples
A Autoclaved Samples

0.10

Soluble PO,-P (mg P/1)
oO
°
uw

0 : 0.05 0.10
Available P (mg P/1)

Fig. 5. Relationship between Available Phosphorus
(10-Day Growth Test with Selenastrum) and Soluble P0O,-P
in Madison, Wis. Lake Waters Collected 1/4/73

20


SECTION VII
COMPARATIVE CHEMICAL AND BIOASSAY ANALYSES

OF THE NUTRIENTS IN THE MADISON AREA LAKES
Introduction

The Madison area lakes, Lakes Mendota, Monona, Wingra,
Waubesa, and Kegonsa, have had a well documented history of
being troubled by algal and weed problems during the warm
seasons when the lakes also have the most appeal as recre-
ational sites for the general public. The displeasure of
the public with the conditions of the lakes has resulted in
a series of projects for the reversal of the eutrophication
of the lakes. These have included the diversion of treated
sewages from Lake Monona and the diversion of metropolitan
Madison's treated sewage from Lakes Waubesa and Kegonsa.
Thus, there has been a gradual reduction of man's influence
on these lakes. At the present time, there is a unique set
of circumstances taking place which allow us to evaluate the
effect of certain of man's influences on lakes with past
problem-clouded histories.

The Madison area lakes have a drainage area of 500 to 550
square miles of fertile farm lands, wooded slopes, and urban
areas. The various sewage diversion projects evaluated in
earlier reports! have lessened the influence of man to the
extent that the sewage of a population of more than 200,000
people in the watershed of Lakes Mendota, Monona, and Wingra
has been diverted around all of the area lakes. The lakes
continue to have both algal and weed problems, so further
diversion projects have been initiated by local populations.
We now have an opportunity to study and record the effect of
these new projects so that the end result will be the rela-
tive effect of 500 to 550 square miles of fertile farm land
drainage versus the sewages of various local populations, some
areas being sewered and some areas using septic tank disposal.
This unique situation will take place in a series of steps
that will allow for the collection of present-condition data
before each change takes place.

86


SECTION I

SUMMARY AND CONCLUSIONS
APPLICATION OF ALGAL ASSAYS TO INLAND LAKE RENEWAL PROJECTS

Chemical analyses of algae and water - Total P and Fe analyses
have been selected and evaluated.

Evaluation of algal biomass measurements - Data are presented
comparing the utilization of the different methods and for
converting the more economical techniques to dry weights.

Analyses of nutrients by algal growth - In excess of 300 lake
water samples have been tested with the AAP. In addition,
comparisons are given of results obtained with Selenastrum,
Microcystis, Anabaena, and the duckweed, Lemna minor,

Analyses of available phosphorus by one-day sorption tests
with algae - Eight different algae have been successfully
used to demonstrate the economy of this technique. This test
uses analyses of the P absorbed by algae rather than waiting
for a week or more for the algae to grow. The availability
of the soluble POs-P of lake waters is contrasted to the
unavailability of some total P compounds. In addition, the
rates of P sorption by different algae have been studied.

Nutritional status of in situ algae - The value of these
tests was demonstrated when the soluble POs-P of lake waters
was found to be hardly detectable in mid-summer, but analyses
of in situ algae indicated that Lakes Monona and Waubesa had
significantly higher levels of P available to the algae than
Lakes Mendota and Wingra. The algae demonstrate changes in
the content of P in lakes that do not appear as surplus P

in the water. Similar results have led to laboratory studies
of the availability of the P of various lake muds.

RATES OF PHOSPHORUS UTILIZATION BY ALGAE AND AQUATIC WEEDS

Comparative rates of P sorption by different types of aquatic
plants have been determined by three methods in order to
understand how the plants may compete for limited P sources.

1


Badfish Creek, which enters the Yahara River system below

the City of Stoughton. It is, therefore, possible to test
for differences in the available phosphorus concentrations
below each lake and the City of Stoughton, evaluate the
relative contribution of these sources, and determine whether
the effluent from the Madison Sewage Treatment Plant can have
any influence on the phosphorus economy (phosphorus available
versus phosphorus needed for algal growth) of the Yahara
River system. In turn, the potential effect of the available
phosphorus contributed to the Rock River by the Yahara River
can be evaluated. (A general diagram of the Madison lakes,
Yahara River-Rock River system is presented in Figure 13.)

Materials and Methods

In order to determine whether the ortho or the total phos-
phorus of the Yahara and Rock Rivers is available for the
test algae, and whether the system contains surplus available
phosphorus upstream from the sewage effluents of the sur-
rounding cities, the present study has utilized two types

of algal bioassays and chemical analyses: short-* and
long-term® bioassays to measure the available phosphorus

in water samples, in situ bioassays to determine limiting
or surplus phosphorus conditions,” and chemical analyses

of soluble ortho (stannous chloride method)’ and total
phosphorus.® The short-term bioassay requires incubation
for 1 day, in samples to be tested, of small quantitites of
P-limited Cladophora sp or Rhizoclontum sp collected from
Madison area lakes. The amount of extractable PO,~P from
algae after incubation in known concentrations of PO,~P is
compared with the amount obtained from algae after incuba~
tion in the different water samples, and is used to calcu-
late the concentrations of available phosphorus in the water
samples. The results of a typical test in which Cladophora
sp from the 3-meter depth of Lake Mendota was incubated in
different concentrations of ortho PO,-P in Gorham's medium’
are presented in Table 23 as the concentrations of extractable
(one hour, boiling water bath) PO,-P from the algae.

Similar direct relationships between concentrations of
available phosphorus and the concentrations of phosphorus

in algae can be obtained with total phosphorus analyses

of the algae. Since such tests merely measure the portion

69


data indicate that the two bioassays for available phos-
phorus agree very well with each other and with the analyses
for soluble POy-P. The total phosphorus analyses of these
samples were not sufficiently different from the soluble
PO,-P analyses to contrast with the bioassays.

In addition to water from the different river stations
sampled for bioassays and chemical analyses, collections of
tn situ algae were made from the Yahara River below Lake
Waubesa and Lake Kegonsa., These algae were extracted for
POy-P to determine if they had limiting or surplus phos-
phorus at the time of sampling. Cladophora sp collected
below Lake Waubesa had 0.12 mg extractable PO,-P/100 mg
algae, while the water sample contained 0.05 mg POy-P/1.
Sptrogyra sp from below Lake Kegonsa had 0.49 mg PO,-P/100 mg,
and the water sample contained 0.1 mg PO,-P/l. In contrast,
Rhizoclontum sp collected the same day from the 3-meter depth
of Lake Mendota had 0.075 mg extractable PO,-P/100 mg algae,
correlating with less than 0.02 mg PO,-P/l in the surface
waters of that lake.

The data from bioassays for in situ extractable PO,-P, growth
experiments, and chemical analyses indicate that the Yahara
River below Lake Kegonsa contained available P in excess of
that required by the lake algae during the late summer of
1971. Since there was considerably more P leaving Lake
Kegonsa than Lake Waubesa, it appears that Lake Kegonsa and
its drainage area increase the phosphorus content of the
Yahara River to such an extent that phosphorus is present
in the lake in excess of that which can be utilized by the
dense growths of algae or aquatic weeds present, and this
situation has not changed since 1943 despite the diversion
around Lakes Waubesa and Kegonsa of the secondary effluent
of the Madison Sewage Treatment Plant in 1958.

Comparison of the phosphorus contents of the outlets
of the Madison lakes -

Inasmuch as the tests in the Yahara River had indicated that
surplus phosphorus (0.12 mg PO,-P/1) was leaving Lake Kegonsa
during a period of the year when it would be expected that
algae would be reducing the phosphorus content of lake waters
to minimal concentrations, a comparison was made by bioassay

75


10.

ll.

12.

13.

14,

LIST OF TABLES

Comparisons of Total Phosphorus Analyses of Algae
Comparisons of Iron Analyses

Statistical Evaluation of the Results of Three
Technicians Making Biomass Measurements of Selenas-~
trum by Different Methods

Conversion of Biomass Measurements of Selenastrum
Grown in Gorham's Medium to Dry Weights

Comparisons of the Extractable PO4-P from Fresh
versus Dried Algae

Sorption of Phosphorus by P-Limited Cladophora sp
(Lake Wingra) from Lake Waters versus Creek Waters

Rates of Phosphorus Sorption by Aquatic Plants by
Analysis of the Medium versus Time

Comparative Rates of Phosphorus Sorption by
Cladophora sp and Gloeotrichia sp from Lake
Mendota Water

Effect of Competition by Gloeotrichia sp on the
Sorption of Phosphorus by Cladophora sp

The Effect of Lake Wingra Plankton on the Sorption
of Phosphorus by Cladophora sp

Release of Phosphorus from Disturbed in situ
Lake Muds

Sorption of Phosphorus by Aerobic Lake Muds
Comparisons of the Total Phosphorus Content of
Muddy Waters with the Extractable PO,-P of in situ
Algae

The Availability of Phosphorus from Dredged Muds
for Selenastrum capricornutum (AAP)

vii

Page

10

11

22

29

40

42

43

44

49

50

51

52


less soluble P0,-P than the now relatively fertile water in
the Yahara River. The in situ algae also indicated that
there was little change in the amount of P available at the
lagoon outlet, whereas the P content of the algae in the
Yahara River had increased at least fourfold (data of
11/8/72). Thus, during the summer the lagoon systems can

be looked on as being a minor source of available P to the
Yahara River, but not to Lake Monona; it had a minor diluting
effect during high nutrient periods after fall overturn.

Concern for the potential fertilization of the Yahara River,
and eventually Lake Monona, by the water of the Johnson
Street storm sewer caused a number of analyses to be made of
the sewer as a source of available P. This sewer receives
cooling water discharges from industries and has a rela-
tively high flow of warm water the year round. As has been
mentioned, during August and early September, the water
leaving Lake Mendota was nearly devoid of soluble PO,-P and
the algae at the outlet were P-limited. Tests between Lakes
Mendota and Monona indicated that there was soluble P0O,-P
entering the river from the sewer (as high as 0.1 mg P/1)
and the concentration of soluble P0,-P below the sewer was
slightly elevated (0.07 mg P/1). The algae collected in
this part of the river were Cladophora sp which were so
heavily coated with epiphytic diatoms that they appeared
brown to black. When the algae were separated it was found
that the untreated mixture had 0.067% extracted P; the
cleaned Cladophora had 0.075% P; and the diatoms had 0.025% P.
Thus, although different, all the algae were P-limited. To
test if the algae were alive and capable of absorbing P, if
it had been available to them, 20 mg samples were incubated
20 hours in 400 ml samples of Lake Mendota water (0.01 mg
PO0,-P/1) and Yahara River water from 200 meters below the
storm sewer (0.066 mg POy-P/1), and samples of each source
to which 0.04 mg P0,-P had been added. The extractable P0,-P
of the Cladophora plus epiphytes after such incubations was
0.078% P for either water sample despite the difference in
chemical analyses. However, samples containing the added
PO,-P had extractable PO,-P levels of 0.09 to 0.10% P,
indicating that, although these algae could not utilize some
of the P present below the storm sewer, they were capable of
absorbing PO,-P added to the samples. Further sorption tests
using Lake Mendota and Lake Wingra Cladophora sp and growth
tests of autoclaved samples using Selenastrum were carried
out to determine if the soluble PO,y-P of the Johnson Street
storm sewer was available P. Tests (9/14/72) with Lake
Wingra Cladophora indicated the extractable PO,-P of the

103


or river and extracted for PO,-P to determine if they had
limiting or surplus phosphorus at the time of sampling. It
has been shown that algae that are phosphorus-limited will
contain low concentrations of extractable PO,-P (less than
0.08 mg P/100 mg algae)*® or total phosphorus.'® Therefore,
these relatively simple analyses can be used to compare the
levels of phosphorus available to existing algal populations
in different environments or after different environmental
changes.

Results

Sources of phosphorus in the Yahara River -

Surveys’ of the phosphorus content of the Yahara River have
been carried out by comparing the results of bioassays for
available phosphorus (using both the one-day sorption-
extraction test and the long-term growth test) with chemical
analyses of soluble P0O,-P and total phosphorus.

Water samples were collected at two different times in
September of 1971 from the Yahara River below Lake Waubesa,
Lake Kegonsa, and the City of Stoughton and from the Badfish
Creek below the City of Oregon (containing combined effluents
from the sewage treatment plants of Madison and Oregon).
Rhizoclonium sp from the 3-meter depth of Lake Mendota was
used in a 22-hour sorption-extraction bioassay for available
phosphorus, and Selenastrum ecapricornutum (AAP) was used in

a long-term (9-day) growth test. The extractable PO,-P

from samples of Rhizoclontum (16 mg samples/500 ml) incu—
bated over night in controls with known concentrations of
phosphorus were used to calculate the concentrations of avail-
able phosphorus in the test waters. The results of these
calculations are presented in the first column of Table 25.

The amount of growth supported by the phosphorus of the three
Yahara River samples was also tested by adding Selenastrum

at a final concentration of 1,000 cells/ml to control cultures
of AAP medium containing different levels of PO,-P and to the
river waters. Since preliminary tests had indicated that the

73


surface runoffs to these two lakes during August (4 days had
rains in excess of 1 inch) did not cause the great increase
in the P of Lake Kegonsa. Therefore, the high levels of P
in Lake Kegonsa most probably are due to the septic tank
drainage from the summer population around this lake as com-
pared to the effects of the summer population around Lake
Waubesa which was in the process of being sewered. If there
is a spectacular decrease in the soluble P0,-P and P content
of the in sttu algae in Lake Kegonsa when the proposed sewers
replace the use of septic tanks around this lake, the com-
parative effect of surface runoffs from very fertile farm
lands, which will continue, and the septic tank drainage can
be conclusively demonstrated.

Lake Kegonsa demonstrates a tremendous capacity to assimilate
the soluble P added from Lake Waubesa and its smaller tribu-
taries, Halverson and Door Creeks, during the winter so that
relatively little soluble P0,-P leaves Lake Kegonsa, and the
algae at the outlet have comparatively low P values. This
situation also took place during the winter of 1943-44 when
Lakes Waubesa and Kegonsa were receiving the effluent of the
Madison Sewage Treatment Plant.° At that time, despite the
fact that the water entering Lake Kegonsa had 0.3 to 0.5 mg
PO,-P/1l (December, 1943 through April, 1944), there was

28% less P leaving the lake as either soluble P0,-P or total P.
In contrast, during the recent summer, when algae and aquatic
plants are considered to be very effective in removing
nutrients from lake waters, the soluble P0O.,-P and the P content
of the algae of this lake increased. During the summers of
1943 and 1944, the outlet of Lake Kegonsa contained as much
soluble PO,-P and total P as the inlet. Therefore, the sources
of P to Lake Kegonsa during the summer must be tremendously
more important than winter sources. The fact that the low-
lying cottages surrounding Lake Kegonsa are used mainly only
during the summer, as compared to the more permanent homes

on relatively higher grounds, tends to indicate that the
cottages on lower grounds may be the source of the excess P

to this lake during the summer. If this is the case, which
will be tested when the cottages are sewered, then the effects
of this source of P, which appear to not continue past November,
and the sewering of this population should have a measurable
effect on the lake during the first year.

Sources of Phosphorus _to the Madison Area Lakes

The value of bioassays for available P as compared to chemical
analyses for soluble PO,-P or total P is clearly demonstrated

99


the major sources of phosphorus in order to distinguish
between those that can be economically reduced from the
sources that are unmanageable. Only with such information
can a realistic evaluation of a situation be made before
costly. phosphorus removal programs are undertaken.

References

1.

Nutrients and Eutrophication: The Limiting Nutrient
Controversy. Symposium, American Society of Limnology
and Oceanography, Hickory Corners, Michigan, 1971.

W. K. Kellogg Biological Station, Michigan State Univ.

Massey, A., and J. Robinson. A Review of the Factors
Limiting the Growth of Nuisance Algae. Water and
Sewage Works 118:352-355, 1971.

Sawyer, C. N., J. B. Lackey, and A. T. Lenz. Investi-
gation of the Odor Nuisance Occurring in the Madison
Lakes. Madison, Wis. Governor's Committee Report.
1944. 137 p.

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

U.S. Environmental Protection Agency. Algal Assay
Procedure: Bottle Test. Corvallis, Oregon. Environ-
mental Protection Agency. 1971.

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

American Public Health Association. Standard Methods
for Water and Wastewater. 12th edition. New York.
Amer. Public Health Assoc. 1965.

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

Hughes, E. P., P. R. Gorham, and A. Zehnder. Toxicity

of a Unialgal Culture of Microcystis aeruginosa.
Can. J. Microbiol. 4:225. 1958.

84


eLt
Biologically Available Phosphorus (mg/L)

10

001

© Epilimnion

A Hypolimnion

Fig.

90 120 150 iho 210 2do 270

Time (Days)
63. Biologically Available Phosphorus - Larson Lake, 1973

3b0

330

360


Comparison of phosphorus determinations -

An analysis was conducted to compare the concentrations of
biologically available phosphorus as determined by the growth
bioassay, to soluble orthophosphate as determined by the
stannous chloride technique (Standard Methods). The chemical
analyses were conducted after lake samples were autoclaved.

A total of 149 paired determinations were made, and samples
from each of the lakes studied were included. The results
are shown in Figure 64.

Linear regression analysis indicated that the line of best
fit for this data had a slope of 0.98 and an intercept of .01,
indicating very nearly a one-to-one mean relationship between
the two techniques. However, as shown by the plot, there was
considerable variation in the data and the correlation coeffi-
cient was only 0.71. Thus, only about 50% of the variability
in the biologically available phosphorus can be attributed

to the orthophosphate concentration. The fact that the data
cluster about a line with a slope very near 1.0 lends further
Support to the use of orthophosphate determinations as a
measure of that portion of the total phosphorus pool which is
available for algal growth.

References

1. Peterson, J. 0., J. P. Wall, T. L. Wirth, and S. M. Born.
Eutrophication Contro: Nutrient Inactivation by Chemical
Precipitation at Horseshoe Lake, Wisconsin. Wisconsin
Department of Natural Resources. Madison, Wis. Technical
Bulletin No. 62. 1973. 20 p.

2. Algal Assay Procedure: Bottle Test. U.S. Government

Printing Office. Washington, D.C. 1972--795-146/1
Region No. 10. August 1971. 82). .

175


up by the wind storm and the Spirogyra had been covered by
the dredged muds, these in sttu algae had not been able to
absorb significant amounts of P from the different environ-
ments.

Laboratory bioassays with lake muds -

Since the ultimate test of the availability of a source of. P
is whether or not a plant can use it for growth, the avail-.-
ability of the P of the muds from the dredging operation was
tested using the growth of the green alga, Selenastrum capri-
cornutum (AAP). The alga was inoculated (50,000 cells/ml)
into Gorham's (=P) medium to which were added different levels
of PO,-P or dredged mud from Murphy's Creek. The growth of
Selenastrum after 12 days incubation in aerated cultures is
summarized as absorbance measurements in Table 14.

Table 14. THE AVAILABILITY OF PHOSPHORUS FROM DREDGED MUDS
FOR SELENASTRUM CAPRICORNUTUM (AAP)
50,000 cells/ml Gorham's (-P) medium 12 days incubation

Phosphorus P-concentration Growth attained
source (mg P/1) (Absorbance, 1 cm, 750 mi)
none 0.0 : 0.015
PO,-P 0.025 0.030
PO,-P 0.05 0.045
PO,-P 0.075 0.070
PO,y-P 0.10 0.090
mud 0.08 0.005
mud 0.20 0.005
mud 0.50 0.005
mud + POy-P 0.20 + 0.20 0.090

It is evident that the growth of this alga was dependent upon
the PO,-P added to the culture medium, twice as much Selen-_
astrum being produced with 0.02 mg PO4-P/1 as in P-free medium.

52


with analyses during a dredging operation. The phosphorus
status of in sttu algae during the operation and after lake
storms has been correlated to laboratory studies of the
availability of the P of lake muds. Proof of use of a
nutrient is best provided by growth responses of the test
alga. Such tests require relatively long incubations.
Shorter tests have been developed that rely on the use

of enzymatic responses or changes in the chemical composi-
tion of algae. All three types of responses have been used
in evaluating lake or river muds as sources of P for algae.
In addition, a discussion is presented of the advantage
rooted aquatic weeds will have over algae in eutrophic
aquatic environments which have been stripped of their
soluble P.

Results and Discussion

Release of phosphorus from lake muds -

It has been pointed out that in situ lake muds are anaerobic
and contain PO,-P in the interstitial waters." A series of
experiments were carried out following the release of P0O,-P
from lake muds disturbed as a simulation of the feeding of
fish or of wave action. A stainless steel colum, 30 cm in
diameter and 75 cm long, was embedded vertically in a lake
bottom such that the top extended over the water surface.
The mud at the bottom of the column was mixed by hand to a
depth of 10 cm. The soluble PO,-P in the water column was
measured before and after mixing to follow the release of P.
Tests were carried out in three lakes: Lake Wingra, 45 cm
depth, grey sandy silt; Monona Bay (Lake Monona), 50 cm depth,
grey sandy silt; University Bay (Lake Mendota), 45 cm depth,
black muck. The results of soluble P0,-P analyses are sum-
marized in Table 11 as the averages of duplicates.

It is evident that soluble PO,-P was released to the over-
lying waters from all three lake muds, the amounts of P
released ranging from 0.007 mg P/1 in Lake Wingra to about
0.04 mg P/l in Lake Mendota. However, in all cases the sol-
uble PO.-P contents of the enclosed water decreased with time
after initially high levels. When the muds were remixed after

48


OT

Table 3. STATISTICAL EVALUATION OF THE RESULTS
OF THREE TECHNICIANS MAKING BIOMASS MEASUREMENTS
OF SELENASTRUM BY DIFFERENT METHODS

Technician 3 | Technician 2 ;Technician 1

Dry weight Fluorometr Ceil count Absorbance 7
Centri- 1s 1% Haem Coulter®
fuged Gooch dil filt x10’/cec x10°/cc 750mu_ _ 6 00mu
413.3 66.0 45000 23000 3.43 2.83 0.21 0.24
410.0 0 46000 23000 3.50 2.90 0.221 0.24
413.3 133.3 47000 23000 3.32 2.92 0,21 0.24
420.0 100.0 45000 24000 3.18 3.10 0.21 0.24
416.7 133.3 48000 23000 3.69 3.23 0.21 0.24
413.3 0 48000 23000 3.11 3.37 0.21 0.24
Mean 414,4 72.1 46000 23000 3.37 3.06
St dev 3.45 61.2 1378 408 0.21 0.21
420.0 566.7 67000 12000 3.83 3.45 0.20 0.24
426.7 666.7 65000 18000 3.97 3.54 0.20 0.24
433.3 666.7 68000 22000 3.41 3.32 0.20 0.24
420.0 666.7 55000 21000 3.31 3.42 0.20 0.24
446.7 666.7 59000 14000 3.52 3.27 0.20 0.24
436.7 633.3 54000 22000 3.47 3.37 0.20 0.24
Mean 430.6 644.5 61000 18000 3.58 3.40
St dev 10.4 40.4 6150 4309 0.257 0.096
416.7 533.0 58000 26000 3.55 3.59 0.22 0.25
420.0 200.0 60000 24000 3.49 3.51 0.22 0.25
433.3 333.3 59000 25000 3.49 3.39 0.22 0.25
420.0 466.7 60000 26000 3.71 3.76 0.22 0.25
406.7 366.7 60000 25000 3.62 3.73 0.22 0.25
350.0 500.0 60000 25000 3.48 3.39 0.22 0.25
Mean 407.7 400.0 60000 25000 3.56 3.56
St dev 29.6 124.7 836.7 752.8 0.092 0.161
¥ 417.6 287.2 56000 22000 3.50 3.34 0.21 0.24
St dev & 11.77 372.2 8386 3606 0.116 0.255 0.01 0.005

@ Uncalibrated Coulter Counter Model A
Different sample used only for absorbance (105 mg/1)


Soluble PO,-P (mg P/1)

0.4

0.3

0.2

0.1

A Lake Mendota Cladophora sp
(0.18 mg PO«-P/100 mg Algae)

O Lake Wingra Cladophora sp
(0.029 mg PO,-P/100 mg Algae)

iT 1 j l
1 2 3 4

Incubation Time (Hours)

Fig. 10. Rates of Phosphorus Sorption by Cladophora:

P-Limited versus Surplus P
170 mg Algae/2,000 ml Lake Mendota Water

39


Thus, it would appear that when equal quantities of Cladophora
and Gloeotrichia are mixed they merely share any available P.

In other experiments, the effect of different amounts of
Gloeotrichia on the sorption of P by Cladophora was measured.
A summary is presented in Table 9 as the average amounts of
extractable PO,-P of duplicate 5 mg samples of Cladophora sp
incubated alone or with 9, 18, or 36 mg of Gloeotrichia sp
in Lake Mendota waters for 24 hours.

Table 9, EFFECT OF COMPETITION BY GLOFOTRICHIA SP
ON THE SORPTION OF PHOSPHORUS BY CLADOPHORA SP
10 mg Cladophora/1,000 ml Lake Mendota water
24 hours incubation

Extractable PO,-P of Cladophora sp
(mg PO,-P/100 mg algae)

Quantity of

Gloeotrichia Lake water Lake water
(mg/1) Lake water + 0.04 m P/l + 0.06 mg P/1L
none 0.07 0.20 -

9 0.06 0.19 0.21
18 0.06 0.13 0.17
36 0.06 0.14 0.19

It is apparent that the presence of the Gloeotrichia did not
affect the extractable P0,-P of the Cladophora in unsupple-
mented Lake Mendota water. When 0.04 mg PO,-P/1 was added
to the lake water, the higher quantities of Gloeotrichia
prevented the Cladophora from sorbing as much P as when in-
cubated alone. However, with an increase to 0.06 mg P/1l, the
Cladophora was able to sorb nearly maximal amounts of P even
in the presence of more than four times as much Gloeotrichia.

Further tests of the competition of algae for P were carried
out with the plankton and Cladophora sp from Lake Wingra.
There is usually a heavy bloom of plankton in Lake Wingra,

and it usually consists of a large number of different species
characterized by their small colonial forms. In a typical
test, the effect of live and killed plankton (17 mg/l) on the

43


the measurable growth of the algae will respond in a straight
line relationship. Cell counts with an electronic particle
counter or chlorophyll a measurements with a fluorometer are
relatively sensitive in comparison to cell counts with a
haemocytometer slide and microscope, absorbance, or dry
weight measurements. Examples of typical data are presented
in Table 24 as the growth of Selenastrum eapricornutum (AAP)
in different concentrations of ortho PO,-P in AAP medium® as
measured by the in vivo fluorescence of chlorophyll a after
5 days and cell counts after 12 days. Either method gives

Table 24. THE GROWTH OF SELENASTRUM CAPRICORNUTUM (AAP)
IN DIFFERENT CONCENTRATIONS OF PHOSPHORUS IN AAP MEDIUM

Average growth attained

Phosphorus After 5 days After 12 days
concentration in vivo chlorophyll a cell counts_
(mg P/1) (fluorescence units) (cells/ml x 107?)
none 9 7
0.025 223 180
0.05 630 360
0.075 880 480
0.10 1,260 600

a direct relationship between growth attained and the concen-
tration of phosphorus in the culture medium. The results of
the two types of bioassays when compared to chemical analyses
will indicate whether the ortho or total phosphorus of the
sample water is available for the algae tested. The phos-
phorus nutritional status of in situ algae can be determined
by sample analyses similar to those used in the short-term
bioassay. Collections of algae are taken from the test lake

72


vet
Biologically Available Phosphorus (mg/L)

10 E-——

al

O Epilimnion

4 Hypolimnion

—
‘9
] ~
01 ee \ R / ow Q
\ \ \
i \ !
y | / \ /
/
\ 1 \ / \
\ ! / \
\ / 4 \
.
+001 T T T T oH A T T T T cam
Jan, 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 30. Biologically Available Phosphorus - Snake Lake, North Basin, 1972


Let

10 e—
l_ O Epilimnion
A Hypolimnion

8
2 1.0 bk—
so)
3 =
g
°
Ej =
a,
on
°
s
oy
o

elf
a
CI L.
a =
S
x —
cal
Cal
ea
©
a
& .ol b—
a
°
a =

+001 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 23. Biologically Available Phosphorus - Horseshoe Lake, 1971


LAKE RESTORATION EFFECTS AS MEASURED BY THE
ALGAL ASSAY PROCEDURE: BOTTLE TEST

Bioassay data, including measurements of algal production,
identification of limiting nutrients and the determination
of biologically available nutrient concentrations, were
obtained for six study lakes over a period of two years.
This work was conducted as one element of a more compre-
hensive data collection program to evaluate lake renewal
techniques, particularly aluminum precipitation and hypo-
limnetic aeration. All assays were conducted on autoclaved
samples using the Algal Assay Procedure: Bottle Test.

Algal production in samples from Snake Lake and Horseshoe
Lake showed a significant reduction following treatment of
the lakes with aluminum. This reduction was most pronounced
in hypolimetic samples. This supports similar findings,
based on chemical analyses. Bioassays also showed a
distinct shift from nitrogen-limitation to phosphorus-
limitation in these lakes.

Two additional lakes, Long and Pickerel, were also treated
with aluminum. Bioassay data showed very little change as
a result of treatment. Algal production was relatively low
in all samples both before and after treatment, and biomass
was virtually always limited by phosphorus throughout the
study period.

Mirror and Larson Lakes were subjected to an aeration

schedule which included periods of total mixing and hypo-
limnetic aeration. Assay results for Mirror Lake showed

an increase in algal production in samples from the epilimnion
and a decrease in production in hypolimetic samples. A shift
from nitrogen-limitation to phosphorus-limitation was noted

in samples from the hypolimnion. Larson Lake showed a slight
increase in algal production in both surface and bottom waters
following aeration. A shift in nutrient limitation may have
occurred but the effect was less pronounced than that noted

in Mirror Lake.


6eT
Biologically Available Phosphorus (mg/L)

10

rT

=
°

TT

.
Rr

o
RP

001

O Epilimnion

A Hypolimnion

Jan.

30

T T T T v T T T
60 90 120 150 180 210 240 270

Time (Days)
Fig. 25, Biologically Available Phosphorus, Horseshoe Lake, 1973

T
300

mi
330

360


SECTION IV

THE RELEASE, SORPTION, AND AVAILABILITY

TO ALGAE OF PHOSPHORUS FROM LAKE MUDS
Introduction

The availability to algae of the P in lake muds has been ques-
tioned because we recently found phosphorus-limited algae
growing in intimate contact with lake muds containing phos-
phorus. It also has been shown by Swingle et al that the
muds of eutrophic fish ponds do not release enough avail-
able P to support the desired level of phytoplankton, but
that fertilization with only P would maintain the produc-
tivity of phytoplankton and harvestable fish crops. Our
studies with the duckweed, Lemna minor, have also indicated
that this rooted aquatic weed could not obtain available N
or P from aerobic lake muds.” In contrast to these results
with attached algae, phytoplankton, and duckweed, Martin

et al* have shown that the aquatic weed, Najas sp, obtained
the inorganic nutrients required for growth from lake muds
through a relatively weak root system. Our duckweed studies
have also indicated that ifthe roots of this plant could
penetrate mud layers that have become anaerobic, the plant
could obtain P for growth from the mud. It appears, there-
fore, that one must be careful in discussing the availability
of nutrients from lake muds because, while algae and aquatic
weeds appear to be alike in not being able to compete with
the forces holding P to aerobic lake muds, aquatic weeds
which can send roots into layers of anaerobic lake muds
which contain PO,-P in the interstitial waters can utilize
source of P that are not normally available to algae.

Layers of lake muds that are anaerobic contain PO,-P*

which should be available for the growth of algae. There-
fore, it was of interest to study under what conditions
available P was released from muds to surface waters. Both
tin sttu and laboratory studies were made of the release of
PO,-P from disturbed lake muds. These results were compared

47


10.

il.

12.

LIST OF FIGURES

Rate of Growth of Selenastrum in NAAM Medium

Growth of Selenastrum in Different Concentra-
tions of NO3-N in NAAM Medium

Growth of Selenastrum in Different Concentra-
tions of PO,-P in NAAM Medium and Lake Wingra
Water

Growth of Selenastrum in Different Concentra-
tions of Iron

Relationship Between Available Phosphorus (10-
Day Growth Test with Selenastrum) and Soluble
PO,-P in Madison, Wis. Lake Waters Collected
1/4/73

Comparison of the Soluble P0,-P of Lake Kegonsa
Waters and the Extractable P0O,-P of in situ
Algae at the Outlet

Concentration of Extracted PO,-P from Lake
Wingra Cladophora sp after One~Day Incubation
with PO,-P Added to Lake Wingra Water

Relationship Between Available Phosphorus (24-
Hour Sorption Tests) and Soluble P0O,-P in
Madison, Wisconsin Lake Waters

Sorption of PO,-P by Cladophora sp and Gloeo-
trichia sp in Different Volumes and Different
Incubation Times

Rates of Phosphorus Sorption by Cladophora:
P-Limited versus Surplus P

Total Phosphorus Content of Cladophora sp
Incubated with Different Phosphorus Levels

The Availability of Lake Mud Phosphorus for the
Growth of Duckweed (Lemna minor)

iv

16

17

20

24

27

28

31

39

55

67


Table 41. NUTRIENT LIMITATION IN SAMPLES
FROM LAKES SUBJECTED TO HYPOLIMNETIC AERATION

Number of samples
which responded
to_additions of:

Total
Lake Year Location N Pp N&P samples
Mirror 1971 epi 1 1 3 5
hypo 1 1 2 5
1972 epi 6 3 4 14
hypo 10 2 - 14
1973 epi - 4 4 8
hypo 1 2 5 8
Larson 1972 epi - 2 9 il
hypo 6 - 5 11
1973 epi - 10 11 22
hypo 2 7 12 21

166


SST

10e—

OEpilimnion
a Auypolimnion
2 1.0¢—
a =
ca]
ff =
Q
a
[7 —
nD
Q
=
ey
cae
qd
Gi
a
a
s
2.
tal
et
a
Li)
3)
a)
Oo .01
a
°
al
)
001 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 51. Biologically Available Phosphorus, Mayflower Lake, 1973


Table 21. AVAILABILITY OF THE PHOSPHORUS OF AEROBIC
LAKE MUD FOR THE GROWTH OF DUCKWEED (LEMNA MINOR)
2 fronds/50 ml Gorham's medium 3 weeks incubation

Average number

Source Concentration of fronds from
of phosphorus (mg P/1) triplicate cultures
PO,-P 0.0
PO,-P 0.025
PO,-P 0.05 . 7
PO,-P 0.10 19
PO,y-P 0.25 39
PO.,-P 0.50 45
PO,-P 1.25 75
Lake Wingra mud 0.15 5
Lake Wingra mud 0.75
Lake Wingra mud 3.0
Lake Wingra mud 7.5
hake Pangea mud 0.75 + 0.50 | 24

growth as 0.1 mg PO,-P/l. When PO,-P was added to lake muds
containing 0.75 mg P/l, there was a significant growth of the
duckweed, but not as much as occurred when 0.5 mg PO,-P/l were
tested in the absence of the mud. In order to demonstrate
that insolubility is not the factor involved in this unavail-
ability of the P of aerobic lake muds, a shark tooth, which
had been washed in warm running water for 45 days, was added
to a P-free culture, which after 14 days of incubation had
produced only 3 fronds. After an additional 12-day incuba-
tion there were 22 fronds in this culture. Similar tests for
the availability to duckweed of the N or Fe of aerobic lake
muds indicated that no available N could be detected in as
much as 5 ml of thick mud in a 50 ml culture (Table 22), but
aerobic muds were a significant source of Fe for duckweed.
The results of these tests were similar to results obtained
with a variety of algal species.}

64


two nutrients, such as N and Fe at the NAAM level, equals
or exceeds the growths attained in the control tests with
the highest level of P in NAAM medium, dilutions of the lake
water must be tested to obtain the concentration of avail-
able P. Lake waters frequently exceed the 0.1 mg P/1
concentration usually used in control tests. If algae do
not grow in lake waters to which are added N, P, and Fe

{or all the NAAM nutrients if other nutrients are suspected
to be limiting growth), it must be assumed that some toxic
factor is present and special tests will be required to
determine the toxic factor.

The relationship between the soluble P0,-P and available P
found with 10 Madison area lake water samples (autoclaved
or membrane filtered), which had been fertilized with N and
Fe at the NAAM levels in order to make the P of the lake
water the only limiting nutrient for the Selenastrum used,
is illustrated in Figure 5. Growth was measured after

10 days or more, and results with control cultures contain-
ing different levels of PO,~P were used to calculate the
available P of the lake water samples, It is evident that
for either the autoclaved or membrane-filtered lake waters
about 90% of the soluble P0O,-P was available P.

The value of using the growth of algae as a measure of the
fertility of water samples is that it demonstrates the level
of biologically available nutrients rather than mere total
analysis. The responses to spikes of nutrients can be used
to predict changes in the eutrophic level of the water with
changes in nutrient levels. The main disadvantage is the
length of time required for the algae to grow-~one week or
more,

NUTRITIONAL STATUS OF IN SITU ALGAE

The nutritional status of algae which have grown in a par-
ticular environment can be used to evaluate whether that
environment supplies limiting or surplus nutrients for the
growth of algae. In contrast to algal growth tests which
measure the biologically available concentration of a par-
ticular nutrient in a water sample and require a week or
more incubation period, relatively short-term tests can be
carried out by measuring changes in certain enzymatic
activities or chemical fractions of algae which have been
shown to reflect the status of the supply of certain nutri-
ents to the algae at the time sampled.

19


Table 18. THE EFFECT OF CONTACT TIME WITH AMMONIUM AND
NITRATE NITROGEN ON THE GROWTH OF DUCKWEED (ZEMNA MINOR)
Gorham's. (-N) medium

2 fronds/25 ml

Average number of fronds/culture
after 4 weeks

Contact time

(hours) -N 5 mg NHs-N/1_ 5 mg NO3-N/1
i 5 7 6
2 6 7 7
4 7 7 7
6 8 9 8
28 8 12 11
48 6 14 12

Table 19, THE EFFECT OF CONTACT TIME WITH DIFFERENT SOURCES
OF PHOSPHORUS ON THE GROWTH OF DUCKWEED (LEMNA MINOR)

2 fronds/25 ml

Gorham's (-P) medium

Average number of fronds/culture
after 4 weeks

Contact time 2.5 mg P/l
(hours) -P ortho pyro tripoly
1/2 4 5 4 4
1 3 4 4 3
2 4 5 4 4
4 3 8 6 4
6 4 10 9 6
8 3 12 8 6
24 3 14 26 12
28 4 28 14 17
48 4 17 16 17
52 5 27 27 22

62


8eT
Algal Production (mg/L Dry Wt.)

100

ce) Epilimnion

4 uypolimnion

T T
60 90

Fig. 34,

T T T T T T
120 150 180 210 240 270
Time (Days)

Algal Production - Snake Lake, South Basin, 1973

T
300

330

360


Algal Total P (mg P/100 mg Algae)

0 \ N i! ! 4
0 0.02 0.04 0.06 0.08 0.10 0.12

Phosphorus Added to Lake Mendota Water (mg P/1)

Fig. 11. Total Phosphorus Content of Cladophora sp
Incubated with Different Phosphorus Levels

24 Hours Incubation

55


The crude conversion table presented in Table 4 can be used
to convert one type of biomass measurement to another. From
the data in Table 3, it can be seen that these measurements
should all be thought of as having an accuracy of only 10%,
and one should not place much reliance on less difference in
experimental results.

A few precautions should be pointed out when using any of these
biomass measurements. It is obvious that, if the size or shape
of cells changes during different growth stages or nutrient
limitations, measurements of cell counts will not accurately
reflect changes in dry weight. It is known that cells of
Selenastrum, Chlorella, and Microcystis decrease in relative
size as cultures reach old or senescent stages. Therefore, the
conversion of cell counts to dry weights must be viewed as a
crude convenience. Similarly, the concentration of chloro-
phyll a in algae cultures will increase with increased dry
weight, but nutrient limitations and high light intensities
will cause the relative amount of chlorophyll a to decrease.

Thus, the rates of growth of algal cultures during the early
stages of growth can be readily followed and compared by
chlorophyll a measurements using fluorometry, but measurements
of final yields in waters containing different nutrient levels
may involve relatively large errors, Finally, increases in
absorbance or dry weight with increased age or nutrient supply
of algal cultures must be viewed with caution if the texture
of the cultures changes (clumping of cells) or inorganic pre-
cipitates form. Thus, there are problems involved with any
measurement of algal biomass and the conversion from one to
another, so it is always important to understand the normal
variability to be expected with these measurements and to apply
this information in evaluating the significance of results of
bioassays.

ANALYSIS OF NUTRIENT CONTENT OF WATER SAMPLES BY GROWTH OF ALGAE

The value of measuring the growth of algae in water samples is
that differentiation can be made between total nutrient contents
of water samples as obtained by chemical analyses and the nutri-
ents that are available to support the growth of algae under
controlled conditions. The concentration of any required nutrient
can be determined 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 have been presented as the "Algal Assay
Procedure" (AAP) by the Environmental Protection Agency.”

12


62

Table 6. SORPTION OF PHOSPHORUS BY P-LIMITED CLADOPHORA SP (LAKE WINGRA)
FROM LAKE WATERS VERSUS CREEK WATERS

19 hours incubation 15 mg algae/400 ml continuous shaking

Extracted : % of

Soluble PO,y-P Calculated Soluble P
. Added P PO,4-P (mg P/ available P as

Water Sample Source (mg _ P/1) (mg P/1) 100 mg algae) (mg P/1) available P
Lake Wingra 0.0 0.07 0.033 - -
" " 0.05 0.05 0.062 - -
" " 0.10 0.10 0.10 - -
" " 0.20 0.20 0.17 - -
Lake Waubesa 0.0 0.01 0.047 0.020 100
" " 0.10 0.11 0.11 0.11 100
Lake Kegonsa 0.0 0.074 0.093 _ 0.085 100
" " 0.10 0.17 0.17 0.20 100
Door Creek (at MN) 0.0 0.086 0.059 0.040 46
" " " " 0.10 0.19 0.096 0.090 47
Door Creek (at 12-18) 0.0 0.12 0.062 0.045 38
" " " " 0.10 0.22 0.11 0.11 50

'No soluble PO,-P detectable (less than 0.02 mg P/1).


6¢T
Algal Production (mg/L Dry Wt.)

100

O Epilimnion

a Hypolimnion

201 T Oo T T T T T v U q

Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)

Fig. 45. Algal Production - Pickerel Lake, 1972


Table 11. RELEASE OF PHOSPHORUS
FROM DISTURBED IN SITU LAKE MUDS
Muds stirred to 10 cm depth in 35 liter colum.

Soluble PO,-P in lake waters (mg P/1)

Time Lake Wingra Monona Bay University Bay
Pre-mix 0.011 0.009 0.025
0 - Time 0.018 0.042 0.063
5 Minutes 0.018 0.016 0.049
10 Minutes 0.014 0.012 0.037
15 Minutes 0.012 0.012 0.043
18 Minutes 0.020 0.021 0.053

15. minutes, there was another peak in PO,-P from the anaerobic
interstitial waters and the sorption of the P by the muds
under the aerobic conditions in the overlying waters.

Laboratory studies of this initial release of P were carried
out by carefully filling a 300 ml jar with mud, sealing the
jar, and later mixing with 3,000 ml of lake water from the
sampling sites. It was found that muds from Monona Bay re-
leased about 0.04 mg P/l within 5 minutes and muds from
University Bay released about 0.05 mg P/1l. The sorption of
PO,-P by these muds was followed by aerating the 3,000 ml
mud-water mixtures to establish aerobic conditions, adding
0.6 mg PO,-P, and following the concentration of PO,-P
remaining in solution with time, The results with muds from
Monona Bay and University Bay are summarized in Table 12 as
the percentage of PO.-P remaining in solution at different
times.

These data indicate that these muds which released PO.-P when
under anaerobic conditions were able to sorb added P when
tested under aerobic conditions. It is therefore assumed
that the decrease in soluble POs-P of in situ mixtures of
muds and aerobic waters was due to this ability of muds to
sorb P when changed from anaerobic to aerobic conditions.
Therefore, in natural situations, P could be released to the
overlying waters by disturbing anaerobic mud layers, but

aquatic plants would have to compete with aerobic muds for
the P. :

49.


(collected 11/20/72) to which were added the NAAM nutrients
except POw-P, The results of tests with different concen-
trations of PO4y-P added to these media are presented in
Figure 3 as the average growth attained (dry weight) after
7 days of incubation.

It can be seen that the growth of Selenastrum in either media
was directly related to the PO,-P concentration at least up
to 0.075 mg P/l. Therefore, the response of algae to limit-
ing concentrations of P will be similar whether the medium
is a natural water or synthetic culture medium, and standard
response curves generated in culture media can be used to
predict the available P concentration in media of unknown
composition as long as all other nutrients are in sufficient
supply.

The growth attained by Selenastrum in different concentrations
of iron was measured using either NAAM or, Gorham's medium
containing 1 mg EDTA/1 and adding iron as Fe-EDTA. The re-
sponse to growth-limiting concentrations of iron was similar
in either medium, but growth reached higher levels in
Gorham's medium than in NAAM when excess iron was present.
The relative response to different concentrations of iron was
the same whether measured by either fluorometry or absorbance
after 7 or 25 days incubation. The results of a typical
experiment in Gorham's medium are presented in Figure 4 as
the dry weights attained after 25 days incubation.

It is evident that the growth of Selenastrum is directly
related to the available iron content of the medium up to
concentrations near 0.02 mg Fe/l. These results have been
confirmed in other media and with Chlorella, Microcystis,
and Anabaena.

The application of algal growth tests to lake waters will
provide a measure of the potential nutrient status of the
lake water sample, will identify and measure algal growth-
limiting nutrients, and will record the biological response
to changes in concentrations of algal nutrients, In order
to provide such information, the growth of a test alga in
different concentrations of N, P, and Fe in control medium
is compared with growth attained in a lake water sample with
the following additions:

15


9€T
Algal Production: (mg/L Dry Wt.)

100

B
°

w
°

my

01

C O°
N
a O Epilimnion
= A Hypolimnion
T T | T T T T T T T T T
Jan. 30 60 90 120 150 180- 210 240 270 300 330 360

Fig. 32. Algal

Time (Days)
Production - Snake Lake, South Basin, 1971


growth can be eliminated in most surveys for sources of avail-
able phosphorus in natural waters. However, since the growth
of algae on the phosphorus in water samples is the ultimate
proof of the availability of phosphorus, the longer growth
tests must still be used occasionally to check on the results
obtained with the short-term bioassays or chemical analyses
of soluble ortho PO,4-P,

The tests on the Yahara River have shown that the water leaving
the lakes in the City of Madison, Lakes Mendota, Monona, and
Wingra, is relatively free of available phosphorus during the
late summer period when the algae and aquatic weeds would be
expected to have used what phosphorus was available. There-
fore, despite the large urban influence on Lakes Mendota,
Monona and Wingra, the phosphorus supplied to these lakes
apparently is held within these lakes and does not appear as
surplus phosphorus in the lake outlets during the peak periods
of aquatic plant production. In contrast, Lake Waubesa and
especially Lake Kegonsa have such large sources of phosphorus
that measurable quantities escape from the lakes as surplus
phosphorus in the lake outlets. Therefore, the fact is that
the Yahara River contains surplus phosphorus before the in-
fluence of the sewage effluents of the Cities of Stoughton,
Oregon, and Madison can become apparent. Thus, there are
significant sources of available phosphorus in Lakes Waubesa
and Kegonsa other than the Yahara River. In order to reduce
the available phosphorus concentration in the Yahara River
it would be as important to control these sources of avail-
able phosphorus as the phosphorus from the sewage effluents
of the Cities of Stoughton, Oregon, and Madison.

Speculations on the sources of phosphorus to Lakes Waubesa

and Kegonsa frequently mention the fact that for many years
these lakes were fertilized by the sewage effluent from the
City of Madison. A portion of this phosphorus was deposited

in the bottom of the lakes and could be a potential source

of phosphorus to the lake waters even after the sewage effluent
was diverted around these lakes in 1959. It would be expected
that the effect of phosphorus from this source would be most
pronounced in Lake Waubesa because this lake received the
sewage effluent directly from the treatment plant. However,
the fact that more phosphorus is found in the outlet of

Kegonsa than of Lake Waubesa indicates that the phosphorus
deposited in the lake muds during the years of heavy fertili-
zation by the Madison sewage effluent must not be as im-
portant a source of phosphorus as others that continue to add
phosphorus at the present time. Swingle et al!! have presented
good evidence that phosphorus deposited in muds of shallow

82


vot
Biologically Available Phosphorus (mg/L)

10¢-—

an
°

B

°
ra

OEpilimnion

Anypolimnion

001 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 57. Biologically Available Phosphorus - Mirror Lake, 1973


SECTION Iil
RATES OF PHOSPHORUS UTILIZATION

BY ALGAE AND AQUATIC WEEDS

Introduction

The need for information as to the relative rates of phos-
phorus utilization by algae and aquatic weeds became apparent
when it was found that certain algae become P-limited at
times during the summer.}/?/3 It was important to know how
algae and aquatic weeds would compete for P when ecosystems
are managed to limit the quantities of biologically avail-
able P. Since it has been shown that once algae or aquatic
weeds have absorbed P, it becomes unavailable to other plants
until the original plant is dead (live algae and aquatic
weeds do not excrete significant quantities of nutrients),
the rate at which different species of plants sorb P from
solution is as important as the relative amounts of P avail-
able to algae and aquatic weeds.' Therefore, the rate of
growth of different species of plants is not as important

as their rate of P sorption if the quantity of available P

is going to be used to control the biological productivity
of aquatic ecosystems.

The amount of growth attained by a plant with P absorbed

from a culture medium is the ultimate answer to the quantity
of available P in a culture medium. In order to utilize the
relatively rapid sorption process in conjunction with the

long incubation required for growth, algae or aquatic weeds
can be exposed to sources of P and then transferred to P-free
medium for growth. This process is also advantageous when
comparing the availability of different forms of a nutrient,
some of which may degrade to other forms during long incu-
bations. Results of this type of test have been presented

in studies with the green alga, Selenastrum capricornutum
(PAAP),! and the duckweed, Lemna minor,” in which it was

shown that Selenastrum could sorb maximal quantities of ortho-
and pyrophosphate in less than 2 hours whereas 4 to 6 hours
were required by duckweed to sorb significant quantities of

P from these two sources. Although the results of these tests
are based on the growth attained by the organisms, which is

36


higher than that calculated from fluorometric measurements
of chlorophyll a (0.018 mg P/l). The latter value is more
in line with the relatively low concentration of soluble
PO,-P found by chemical analysis (Table 26). In general,
the concentrations of available phosphorus correlate well
with the concentrations of soluble PO,-P of these samples.
Since these samples contain little phosphorus that is not
soluble PO,-P, no further correlations were made.

A comparison of the extactable PO,y-P and total phosphorus
of algae collected from the outlets of the different lakes
has also been made as a measure of the phosphorus available
to in situ algae. Cladophora sp were collected from ail
environments, and Spirogyra sp from two areas. The results
of extractions of PO,-P or of the total phosphorus of the
in sttu algae are presented in Table 27.

Table 27. PHOSPHORUS CONTENT OF IW SITU ALGAE
COLLECTED FROM THE OUTLETS OF THE MADISON LAKES

Averages of 2 to 6 samples (mg P/100 mg algae)

Extractable PO ~P Total P
Source Cladophora sp Spirogyra sp Cladophora sp
L. Mendota 0.065 - 0.16
L. Monona * 0.066 - 0.16
L. Wingra 0.038 - -
L. Waubesa 0.084 0.14 0.21
L. Kegonsa 0.14 0.40 0.28

The data on the amounts of extractable P0O,-P of the Cladophora
from the lake outlets indicate that algae from Lake Kegonsa
had relatively high values compared to the algae from the
other lakes. The extractable PO,-P of the Spirogyra samples
from the outlet of Lake Kegonsa also indicated that algae
from this environment had considerably more phosphorus avail-
able to them than to the Spirogyra from the outlet of Lake
Waubesa. Analyses of the total phosphorus of the Cladophora
indicate that algae from Lakes Mendota and Monona had similar
phosphorus contents, but Cladophora from Lakes Waubesa and

77


89T
Algal Production (mg/L Dry Wt.)

100

10

1.0

-O1

O spilimnion

A Hypolimnion N

90

Fig. 59.

To T T T T
120 150 180 210 240
Time (Days)

Algal Production - Larson Lake, 1972

300


The fact that the Anabaena responded to increased levels of
PO,-P by increased ethylene production indicates that it was
originally P-limited and was able to utilize the added PO,-P,
twice as much activity being measured in the presence of
0.005 mg P/l as in P-free medium. The Anabaena was not able
to utilize the 0.14 mg P/l in the form of mud under these
conditions, but because the P0,-P added to the mud was
utilized, the mud could not contain a toxic factor. Other
tests carried out in Murphy's Creek water to which P0O,-P or
lake muds were added also indicated that P-limited Anabaena
could not utilize lake mud P.

The bioassays carried out thus far have been with aerobic
suspensions of lake muds which contained no measurable
soluble POy-P. In order to determine if there was available
P in muddy waters which contained some soluble P0O,-P as well
as a greater amount of total P, tests were carried out with

a river water and the sorption of P by P-limited Cladophora.
The chemical composition of Cladophora after exposure to
different levels of P can be used as an indication that the
algae were able to sorb P under the conditions of the test.
Previous studies have indicated that the extractable P0O,-P
and extractable total P of Cladophora could be used to measure
the available P in culture media. In the present test,
Cladophora sp from Lake Wingra was incubated in 500 ml of
Lake Mendota water which was supplemented with different levels
of P0,-P. After incubation overnight, the algae were divided
into duplicate samples and the extractable PO,-P or total P
of the algae measured. The results are summarized in Table 16
as the average P concentrations of the algae.

The concentration of extractable P0O,-P or of the total P of
the samples of Cladophora sp from two locations were measured
after incubation in P0,-P supplemented Lake Mendota water and
20 or 40% dilutions of muddy Crawfish River water in Lake
Mendota water. The samples of known concentrations of PO,-P
in Lake Mendota water were to be control cultures which would
serve to determine the concentrations of available P in the
dilutions of Crawfish River water. In one series, two 5 mg
samples of a bright green Cladophora collected from a shaded
area of Murphy's Creek were incubated in 1,000 ml of supple-
mented Lake Mendota water. In the other series, two 10 mg
samples of pale yellow Cladophora from near the surface of
Lake Wingra were incubated in 500 ml of supplemented Lake
Mendota water. The average total P concentrations of dupli-
cate samples of the algae after 24 hours incubation are sum-
marized in Figure ll.

54


Kegonsa had increased phosphorus contents, the algal phos-
phorus concentration found at the outlet of Lake Kegonsa
being nearly double that at Lakes Mendota and Monona.

It is evident that, in the late summer, the phosphorus
concentration and eutrophication level of water leaving the
three lakes surrounded by the City of Madison is considerably
less than that of Lake Kegonsa, with Lake Waubesa having an
intermediate level.

The phosphorus content of the Rock and Yahara Rivers -

There has been some conjecture as to the effect of the phos-
phorus from the Madison Sewage Treatment Plant on the phos-
phorus economy of the Rock River. In order to establish an
effect, it would be necessary to know if any available phos-
phorus was present in the Rock River above the junction with
the Yahara River which brought the Madison Sewage Treatment
Plant effluent from the Badfish Creek. Samples for analyses
were taken at two stations on the Rock River above the junc-
tion with the Yahara, two stations on the Yahara River after
it received the waters of the Badfish Creek, and one station
on the Rock River below the junction with the Yahara River.
Two 24-hour bioassays were carried out using Cladophora sp
from Lake Mendota to sorb the available phosphorus from.the
test waters. One test measured the phosphorus sorbed by the
extraction of PO,-P and the other test measured the total
phosphorus of algae after incubation. In addition, growth
tests with Selenastrum were carried out. Thus, the results
of 3 types of bioassays could be correlated with chemical
analyses for soluble P0,-P and total phosphorus of the test
waters. The results of these tests are summarized in

Table 28.

The analyses for soluble P0,-P of the samples used in the
growth experiment are reported separately since there was a
general increase after the samples were processed for this
test. The growth test samples were autoclaved, gassed with
COz2 to dissolve any precipitate formed, aerated to remove
excess CO2 and raise the pH back to normal, and then filtered
(Whatman #2) to remove any precipitates that would interfere
with the electronic particle counting instrument used in
measuring the growth of the Selenastrum,

The analyses of samples of Rock River water upstream from the
junction with the Yahara River show that this river contains

78


Determination of dry weight -

Fluorescent values of Selenastrum cultures grown in NAAM
medium containing varying concentrations of phosphorus were
obtained for day 7 of incubation and related to the absor-
bance values at day 14 of incubation. The absorbance values
were then converted to ug/l dry weight, resulting in a linear
relationship between day-7 fluorescence and final dry weight.
The average day-7 fluorescent relationship to day-14 dry
weight is given by

X= 8.41 F
where X = dry weight at day 14 in mg/1
F = fluorescent units at day 7

Determination of biologically available N and P -

Fluorometric readings of the reference cultures were plotted
against the known concentrations of nutrient (N or P) in each
spike. By comparing the readings of the N+Fe- or P+Fe-spiked
lake samples to these curves, the concentration of either
nutrient which is available for algal growth was determined.

Presentation of data -

The results of bioassays for each lake wre plotted as a time
sequence to show: 1) the amount of algal biomass produced
in the samples of autoclaved lake water and the nutrient
which limited biomass production, and 2) the concentration

of biologically available phosphorus in the samples as deter-
mined by the growth assays.

Nutrient limitation was determined by amending the samples
with phosphorus and nitrogen, singly and in combination.

If a nutrient addition resulted in a significant increase

in biomass as compared to the unamended samples, then that
nutrient was identified as limiting. Limiting nutrients are
denoted on the plots by the symbols P, N, or PN adjacent to
the data points. The symbol PN is used to denote the condi-
tion in which the addition of neither nitrogen nor phosphorus

113


4.7 x 105m’. The maximum depth is 17 m and length of shore-
line is 1.8 km. The bottom sediments are primarily muck and
marl. Aquatic plant growth is lush around the shoreline.
For an undetermined number of years the lake received ef-
fluents from a nearby dairy and cheese processing plant
stimulating nuisance algal blooms in the mid-1960's. Horse-
shoe Lake was treated with alum in May 1970.

Long Lake - Langlade County ~ Long Lake is a slightly colored
lake of medium fertility. It has a surface area of 27.5 ha
and a maximum depth of 7.3 m. The lake is narrow with a
sinuous shoreline configuration. This shape, along with the
protection afforded by surrounding tall pines, encourages the
stagnation of surface waters during the summer and may con-
tribute to the occasional nuisance algal blooms that have
been reported. Long Lake was treated with alum in May 1972.

Pickerel Lake - Portage County - Pickerel Lake is a small,
clear-water lake of medium to high fertility. It has a
surface area of 21 ha and a maximum depth of 4.6 m. The
shoreline is fringed with rooted aquatics and algal produc-
tion is high during the summer season. The lake has been
subject to frequent winterkill during recent years. The
source of nutrient input is unknown at this time as there is
but one development on the lake, a church camp which does not
undergo heavy use. The lake was treated with liquid alum

on April 17, 1973.

Mayflower Lake - Marathon County (not treated) - (40 ha, 8m
maximum depth) Samples have been collected for bioassays as
part of a program to establish baseline data for this lake.
The lake has had problems with rooted aquatic vegetation and
algae, and future alum treatment is planned.

Lab studies on_ lake water samples treated with alum -

An experiment was done to determine whether 1) aluminum treat-
ment of lake-water samples in the laboratory would decrease
the amount of phosphorus available for, assimilation by AAP
organisms, and 2) introducing an amount of phosphorus (as PO,)
equivalent to that which was removed would stimulate growth
at original levels. Samples from Snake Lake (Vilas-Oneida
Counties) and Mirror Lake (Waupaca County) were used, repre-
senting soft and hard water lakes respectively.

116


As an example of the results one would get when the extract-
able P0,-P content of algae was compared to the total P of
algae, tests were carried out with P-limited Cladophora sp
from the surface of Lake Mendota (7/24/72) and P-surplus
Sptrogyra sp from University Bay Creek (7/24/72), a eutrophic
tributary of Lake Mendota. The extractable P0,-P of the
Cladophora was 0.071 mg P/100 mg algae (%P) and the total P
was 0.16%. The extractable PO,y-P of the Spirogyra was

0.42 mg P/100 mg algae and the total P of these algae with
surplus P was 0.51%. Thus, either type of analysis could be
used to describe the P-nutritional status of in situ algae.

As an example of how one can correlate data on the extract-
able POs.-P of algae with other environmental parameters, the
results of tests at the outlet of Lake Kegonsa are presented
in Figure 6. The concentrations of soluble PO,-P in the
water can be compared with the amounts of extractable P0,-P
from in situ algae (algae collected at the lake outlet) during
April through September, 1972, The species of algae tested
were Sptrogyra sp through mid-May, Ulothrix sp until early
June, and Cladophora sp through the rest of the summer and
fall.

It is evident that the extractable PO,-P from algae sampled
at the lake outlet reflected changes in the soluble P0O,-P

of the outlet waters, Peaks in either measurement occurred
in early May: and late summer, whereas relatively low values
occurred in mid-May and mid-June. Thus, it can be seen how
changes in the extractable P0,-P of in situ algae can be used
to evaluate changes in available P in a lake environment.

A note of caution: logical responses to changes in environ-
mental conditions can be followed using attached algae (such
as Sptrogyra, Ulothriz, Oedogonium, and Cladophora), but
planktonic algae do not always reflect environmental changes.
Under certain conditions the nitrogen-fixing blue-green algae,
Anabaena, Gloeotrichta, and Aphanizomenon, will be found to
be P-limited when the surface waters of lakes are relatively
devoid of P0,-P, but we have never found planktonic non-
nitrogen-fixing blue-green algae, such as Microcystis or
Coelosphaerium, to be P-limited under these conditions. The
latter algae have been found to be N-limited at certain times,
however. Also, one can never be sure whether planktonic
algae collected at the surface had not just recently come to
the surface and may reflect the nutritional state of sub-
surface algae.

23


8eT

100 -—
= O Epilimnion
A Hypolimnion
~ 10Rb—
2 =
i; =
A
a —
4
N\
ey
=
€1.0-—
§ —
Bal —
es)
3)
8 =
s
°
i. =
a
a
©
D
et
< abe
+02 T T T —T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Time (Days)
Fig. 44. Algal Production - Pickerel Lake, 1971


TLT
Biologically Available Phosphorus (mg/L)

10

01

001

Z O Epilimnion
= A Hypolimnion
fav
T T 3 T T T T T T T T T
Jan. 30 60 0 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 62. Biologically Available Phosphorus - Larson Lake, 1972


No. Page
28. The Phosphorus of the Rock and Yahara Rivers:

Bioassays versus Chemical Analyses 79
29. The Phosphorus of the Rock River and Some Tribu-

taries: Bioassays versus Chemical Analyses 80
30. The Algal Growth Attained, as Fluorometry Units,

in Autoclaved Samples of Madison Area Lake Waters 89
31. Comparative Analyses of the Yahara River and Tenney

Park Lagoon at Different Times 102
32. Comparison of Chemical Analyses of Phosphorus and

Available Phosphorus in Lake Waters and Creeks 105
33. Algal Bioassays Conducted 108
34. Lake Treatment with Aluminum 115
35. Phosphorus Analyses on Snake Lake Waters in mg/l 117
36. Growth Response of AAP Organisms to Nutrient

Removal and Spikes 118
37. Algal Response and Phosphorus Content of

Aluminum (III)-Treated Mirror Lake Waters 119
38. Average Algal Biomass Produced in Samples from

Lakes Treated with Aluminum 121
39. Nutrient Limitation in Samples from Lakes Treated

with Aluminum 122-123
40. Average Algal Biomass Produced in Samples from

Lakes Subjected to Hypolimnetic Aeration 158
41. Nutrient Limitation in Samples from Lakes Subjected

to Hypolimnetic Aeration 166
42. Miscellaneous Bioassay Results 173

43. Bioassay Results for Samples from Inlets to
Horseshoe Lake 174

ix


TET
Algal Production (mg/L Dry Wt.)

100

10

L Oepilimnion
- Anypolimnion
\
F A oe
Z \ , \ 0
o-, _-d 7
NP o . NP P
=
T T T ro T T T T T v T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 27, Algal Production - Snake Lake, North Basin, 1972


6eT
Biologically Available Phosphorus (mg/L)

10

-O1

001

© Epilimnion

A Hypolimnion

Biologically

T T
180 210
Time (Days)

Available Phosphorus - Snake Lake, South Basin, 1971


SvT
Biologically Available Phosphorus (mg/L)

10 E—

O Epilimnion

4 Hypolimnion

al b—
=
+01, °
+001 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Time (Days)
Fig. 41. Biologically Available Phosphorus - Long Lake, 1971


replicates of several different algae could be tested simul-
taneously under similar environmental conditions.

The amount of P sorbed by algae was measured by placing dup-
licate 5 mg samples of algae in 500 or 1,000 ml of water
containing different levels of P (0.02 to 0.1 mg P/1), in-
cubating the cultures overnight, and then analyzing the
algae for sorbed P and dry weight. The sorbed P was either
analyzed as the extractable P0,-P or as the total P of the
algae. In either case, results with control cultures contain-
ing no P were used in comparison with known concentrations
of P. The ease with which filamentous algae, such as Cla-
dophora, can be separated from planktonic species by the

use of a coarse screen makes it easy to compare the P sorbed
by different types of algae when in mixed cultures.

The analysis of extractable P0,-P of algae has been described
by Fitzgerald and Nelson.® The total P analysis procedure
was that of Gales et al.*® In well equipped laboratories
either method is readily carried out with about the same
expenditure of time, but the analysis of extractable PO,-P
requires less equipment and technical effort. When using
duplicate 5 mg samples of algae per 500 or 1,000 m1, either
type of analysis for sorbed P could detect differences caused
by 0.02 mg P/l in the culture medium after 18-24 hours incu-
bation. The volumes used in comparative tests are not im-
portant in rate studies since the effects of 0.01 or 0.02 mg
PO,-P added to 10 mg of algae can be detected whether the
algae are incubated in 100 or 1,000 mg. In addition, sorp-
tion tests with Lake Wingra Cladophora sp indicate that the
same rates of sorption take place in 400 ft candles of light
as in the dark.

Results and Discussion

Over 50 tests have been carried out measuring the rates of P
sorption by algae or aquatic weeds based on the loss of
soluble POy-P from the media. In a typical experiment,
Cladophora sp collected from Lake Mendota and found to con-
tain adequate P (0.18 mg extractable PO.-P/100 mg algae) were
compared with P-limited Cladophora sp (0.029 mg PO,-P/100 mg)
from Lake Wingra. Approximately 170 mg of either algae were
placed in 2,000 ml of Lake Mendota water which was supple-
mented with 0.4 mg PO,-P/l. Analyses of the PO,-P of the lake
water over a 4~hour period are summarized in Figure 10.

38


No.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

The Availability of PO,-P and Dredged Mud Phos-
phorus to Phosphorus-Limited Anabaena flox aquae
(Ind 1444) as Measured by the Reduction of
Acetylene to Ethylene

Changes in Composition of Lake Wingra Cladophora
sp after Incubation with Different Concentrations
of Phosphorus

The Effect of the Volume of Culture Medium on the
Number of Duckweed (Lemna minor) fronds produced
with Different Concentrations of Nitrogen and
Phosphorus

The Effect of Contact Time with Ammonium and
Nitrate Nitrogen on the Growth of Duckweed (Lemna
minor)

The Effect of Contact Time with Different Sources
of Phosphorus on the Growth of Duckweed (Lemna
minor)

Effect of Contact Time with Different Sources of
Iron on the Growth of Duckweed (Lemna minor)

Availability of the Phosphorus of Aerobic Lake
Mud for the Growth of Duckweed (Lemna mtnor)

Availability of the Nitrogen of Aerobic Lake Mud
for the Growth of Duckweed (Lemna minor)

The Extractable P0,-P of Cladophora sp Incubated
One Day in Different Concentrations of Phosphorus

The Growth of Selenaetrum capricornutum (AAP) in
Different Concentrations of Phosphorus in AAP
Medium

Comparisons of Phosphorus Analyses of Yahara River
and Badfish Creek: Bioassays versus Chemical
Analyses

Comparison of the Phosphorus Contents of the
Outlets of Madison Area Lakes: Bioassay versus
Chemical

Phosphorus Content of in situ Algae Collected from
the Outlets of the Madison Lakes

viii

53

56

61

62

62
63
64
65

71

72

74

76

77


Since growth tests require a relatively large amount. of
laboratory space for the number of flasks and long incu-
bations involved, it is frequently necessary to preserve
water samples for more appropriate times of analyses. In
situ algae may grow in some samples and compete with the

test algae of growth tests or cause confusing results, such
as when nitrogen-fixing algae might grow in tests of the
available N content of water samples, Therefore, it is con-
venient. to remove most organisms by membrane filtration |
(0.45 wu) or kill all organisms by autoclaving. Both of these
preservation techniques will affect the nutrient content of
the water samples. Membrane filtration removes particles
that are insoluble at the time of filtration. Since it has
been shown?’!°"1!that algae can utilize some forms of P, N,
and Fe that are relatively insoluble (shark teeth, hair,

and pyrite crystals), filtration of water samples from four
lakes in the Madison, Wisconsin area (January, 1973), which
were run after membrane filtration of the samples, indicated
all four of the waters were deficient in Fe (stimulation of
growth when Fe-EDTA was added), whereas there was no response
to added Fe when autoclaved samples were tested. It is known
that autoclaving makes available the P, N, and Fe of in sttu
algae, but because the available P of autoclaved and filtered
samples of the above-mentioned waters was nearly identical,
the availability of Fe in the autoclaved samples was not
thought to be due to the killing of the very sparse in situ
algae. Thus, these water samples must have originally con-
tained adequate available Fe which was removed by filtration.

There are several effects that autoclaving can have on the
nutrient status of water samples. The method used to pre-
serve water samples by autoclaving has been to autoclave the
samples, cool, gas with CO2 for 1/4 to 1/2 hour to resolu-
bilize some of the precipitated materials, and aerate for

8 to 12 hours to remove excess C02. As has been mentioned,
available P, N, and Fe can be released from in situ algae

in autoclaved samples... During July, 1972, 24 lake water
samples were tested for soluble P0O,-P, total P, and avail-
able P (sorption tests with Cladophora). Only two of the.
24 raw or filtered (Whatman #2) samples had more than

0.02 mg P/l as soluble PO,-P or available P. Six samples
that were autoclaved all had significantly higher soluble
POy-P than the raw samples, and in all 6 autoclaved samples
the available P was 2 to 4 times higher than the concentra-
tion of soluble P0,-P. Thus, both soluble PO,-P and other
forms of available P were released from the in situ algae by
autoclaving. The availability of certain forms of P other

32


In order to determine the nutrient content of the water samples,
the response of the test alga to known levels of the nutrient
in question must be measured. Therefore, the following set of
reference spikes was included with each test:

1 medium (-) P*

2" (-) P + 0.025 mg/l P
3 " (-) P + 0.05 mg/l P
4 " (-) P + 0.075 mg/l P
5 " (-) P_+ 0.186 mg/l P
6 medium (-) ND

7 " (-) N+ 0.25 mg/1 N
8 " (-) N+ 1.0 mg/l N
9 " (-) N+ 4,2 mg/l N

asynthetic nutrient medium made without phosphorus.
Synthetic nutrient medium made without nitrogen.

The phosphorus spike of all treatments was.added as K2HPO,;
the nitrogen spike as NaNO3; and the iron as FeCl; +

Na2EDTA - 2H20.

Because of spatial limitations, it was necessary to use sample

volumes of 20 cc in 50 cc Erlenmeyer flasks, resulting in
surface to volume ratios within the range specified.

Incubation -

Each experimental setup was incubated at 24°C, with illumina-—
tion of 400 ft C. Flasks were swirled daily.

Harvesting techniques -

Although many of the samples assayed were relatively fertile,
the concentrations of cells produced were normally insufficient
to permit direct gravimetric determination of algal biomass.
Therefore, biomass was determined indirectly by measuring
fluorescence of in vivo chlorophyll a. ‘Two types of fluorome-
ters were used, a Turner (III) and an Amnico unit.

Tests conducted during the initial stages of the project
established that maximum fluorescent readings were obtained
from cultures which had been incubated for 5-9 days and that
readings often decreased significantly after day 9. As a
result, a standard incubation period of 7 days was selected,
and all cultures were harvested at that age.

112


weights could be filtered. This is a typical result with
certain algal cultures, but the technique is usually very
effective with planktonic algae collected from the field.

The fluorometry measurements indicate that for relatively
dense algal cultures it is best to dilute the sample rather
than use a 1% neutral density filter. This is due to the
self-shading of dense cultures.

Manual cell counts using haemocytometer slides are the most
laborious of all these meaSurements. However, the presence
of contaminating algae or debris in the samples can be de-
tected and evaluated by this method. Also, it can be used
with samples containing large amounts of debris that might
interfere with some of the other techniques. The use of
electronic particle counters makes cell counting less of a
chore, but is not applicable to samples with large amounts
of debris. The use of an uncalibrated electronic particle
counter is justified if one develops conversion factors to
convert readings to actual cell counts or dry weights.

The measurement of absorbance (optical density) of algal cul-
tures is one of the easier techniques to be used. It is
preferable to use a wave length of light (750 mu) which does
not rely on chlorophyll absorption, since cultures which are
deficient in some essential nutrients lose their chlorophyll
with increased incubation and, like fluorometry, give low
readings for actual final yield measurements. This method
should not be selected because of its apparent precision.
Absorbance measurements are easy but the apparent precision
of the measurements is due to a lack of differentiation be-
tween similar cultures. It is a relatively crude measurement,

Table 4. CONVERSION OF BIOMASS MEASUREMENTS
OF SELENASTRUM GROWN IN GORHAM'S MEDIUM TO DRY WEIGHTS

A concentration of Selenastrum of 100 mg/l (dry weight)
is equivalent to:
Absorbance (1 cm cells)

750 mu - 0.20
600 mu - 0.23

Fluorometry
Turner - 1,300,000 units
Aminco - 3,200 units

Cell counts 8,400,000 cells/ml

11


SECTION If

CHEMICAL AND BIOASSAY METHODS FOR NUTRIENT ANALYSES
CHEMICAL ANALYSES OF WATER SAMPLES

Soluble PO.-P was most frequently analyzed by the stannous
chloride method? using al cm cell. Analyses of 0.02 mg P/1
or more were considered to be significant. Occasional
analyses with the ascorbic acid method! using 10 cm cells

in Beckman DU supported the values obtained with the stannous
chloride method and extended the lower limits to at least
0.002 mg P/l.

Total P analyses were by the H2S0,-persulfate-autoclave
method.! The lower limit for these analyses was considered
to be 0.02 mg P/1l.

TOTAL PHOSPHORUS CONTENT OF ALGAE

Comparisons have been made of two methods of analysis for
the total P of three different types of algae containing dif-
ferent amounts of P.

H2S0,-HNO; digestion - Dried and weighed samples of algae
were digested with 1 ml concentrated H,SO, and 5 ml concen-
trated HNO; until clear, neutralized, and analyzed.

H280,-persulfate digestion - Dried algae plus 1 ml 10N H2S0,4,
0.4 g potassium persulfate, and 30 ml H20 were autoclaved for
30 minutes, neutralized, and analyzed. The results of the
analyses are presented in Table 1.

It is evident from the results presented in Table 1 that
either digestion method can be used for analyses of the
total P of algae even when there is nearly a tenfold varia-
tion in the total P content of the algae.


No.

13.

14.

15.

16.

17.

18,

19,

20-22.
23-25.

26-28,

29-31.

32-34.

35-37.

38-40.

General Diagram of the Madison Lakes, Yahara
River-Rock River System

Relationship Between Soluble PO.y-P of Lake
Waters and Available P - Growth Tests

Comparison of the Soluble PO.u-P of Lake Mendota
Waters and the Extractable POy-P of in situ
Algae at the Outlet

Comparison of the Soluble PO,-P of Lake Monona
Waters and the Extractable PO,-P of in situ
Algae at the Outlet

Comparison of the Soluble P0,-P of Lake Waubesa
Waters and the Extractable P0O,-P of in situ
Algae at the Outlet

Comparison of the Soluble P0,-P of Lake Kegonsa
Waters and the Extractable PO,~P of in situ
Algae at the Outlet :

Comparison of the Soluble PO,-P of Madison Area
Lakes, Fall and Winter, 1972-73

Algal Production - Horseshoe Lake, 1971-73

Biologically Available Phosphorus -
Horseshoe Lake, 1971-73

Algal Production - Snake Lake, North Basin,
1971-73

Biologically Available Phosphorus - Snake Lake,
North Basin, 1971-73

Algal Production - Snake Lake, South Basin,
1971-73

Biologically Available Phosphorus - Snake Lake,
South Basin, 1971-73

Algal Production - Long Lake, 1971-73

Page

70

91

92

93

94

95

96
124-126

127-129

130-132

133-135

136-138

139-141
142-144


yet
Algal Production (mg/L Dry Wt.)

loo}.
Le O Epilimnion
4A Hypolimnion
10e-—
1.0
lke
01F T T T T T T T T 1 T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 20. Algal Production - Horseshoe Lake, 1971


Table 31. COMPARATIVE ANALYSES OF THE YAHARA RIVER
AND TENNEY PARK LAGOON AT DIFFERENT TIMES

In situ
algal P
Soluble Available P (extractable
Date and PO,-P (sorption) PO,-P)
sample area (mg P/1) (mg P/1) (% P)
8/4/72
Mendota outlet 0.02 0.02 0.06
Lagoon inlet 0.01 0.02 0.07
Above Lagoon outlet 0.02 - 0.07
Lagoon outlet 0.04 0.04 0.20
Below Lagoon outlet, 50 M 0.02 - 0.10
Below Lagoon outlet, 300 M 0,02 0.02 0.06
8/21/72
Mendota outlet 0.018 - 0.05
Lagoon outlet 0.044 - 0.11
9/14/72
Mendota outlet 0.016 - -
Lagoon inlet 0.018 - 0.072
Lagoon outlet 0.030 - 0.10
Below Lagoon outlet, 300 M 0.022 - 0.072
11/8/72
Mendota outlet 0.12 0.14 * 0.30
Lagoon inlet 0.12 0.12 0.26
Above Lagoon outlet _ 0.12 0.14 -
Lagoon outlet 0.06 0.04 0.17
Below Lagoon outlet, 300 M 0.12 0.08 0.39

102


Table 17. THE EFFECT OF THE VOLUME OF CULTURE MEDIUM
ON THE NUMBER OF DUCKWEED (LEMNA MINOR) FRONDS PRODUCED
WITH DIFFERENT CONCENTRATIONS OF NITROGEN AND PHOSPHORUS

Supplemented Gorham's medium 2 fronds/culture

Average number fronds/culture
after 3 weeks

Culture medium 25 mi 50 ml 100 mi

Gorham's (-N) 11 11 10
Gorham's (-N)

+ 0.25 mg NO3-N/1 12 19 22
Gorham's (=N)

+ 0.5 mg NO3-N/1 13 23 36
Gorham's (-N)

+ 1.0 mg NOs-N/1 23 40 58
Gorham's (-N)

+ 2.0 mg NOs-N/1 300 51 88
Gorham's (-P) 4 3 4
Gorham's (-P) 5 5 5

+ 0.025 mg POy-P/1
Gorham's (-P) 5 6 9

+ 0.05 mg PO,-P/1
Gorham's (-P)

+ 0.10 mg PO,-P/1 5 17 28
Gorham's (-P) 22 39 63

+ 0.20 mg PO,-P/1

the essential nutrient were counted after 3 or 4 weeks of
incubation. The results of typical experiments are presented
as the average numbers of fronds produced after exposures to
different sources of N, P, and Fe, in Tables 18, 19, and 20,
respectively.

The data comparing NH,-N and NO3-N under these conditions
indicate that either source of N was available and required
between 6 and 28 hours for maximal sorption to take place.
When the different sources of P were compared, ortho- and
pyro-P appeared to be sorbed slightly faster than tripoly-P,

61


Growth (Dry Weight, mg/1)

62.5. 4

50.0 --

37.5
Onaam
25.0 [-
A Lake
12.5 -
0 i mn i I
0 0.05 0.10 0.15 0.20

PO,-P Concentration (mg P/1)

Fig. 3. Growth of Selenastrum in Different Concentrations
of POw-P in NAAM Medium and Lake Wingra Water
plus NAAM(-P) Nutrients
7 Days Incubation Mean and 95% Confidence Limits

16


Set
Biologically Available Phosphorus (mg/L)

-001

10

Be
°
|

w

°
rary

OEpilimnion

Aunypolimnion

Jan.

30

Fig. 31.

T T T T T T T T T

60 90 120 150 180 210 240 270 300
Time (Days)

Biologically Available Phosphorus - Snake Lake, North Basin, 1973

330

360


Sorption and growth - Plants are exposed to sources of P

for relatively short periods, washed, and subcultured in
P-free medium for 1-4 weeks. This technique has been used
with Selenastrum and the duckweed, Lemna minor. Results
indicate that maximal amounts of P are sorbed by Selenastrum
in less than 2 hours, whereas duckweed requires 4 hours.
Either plant can utilize pyrophosphate as rapidly as ortho-
phosphate, but tripolyphosphate requires slightly longer
exposures.

Analysis of medium versus time - Approximately 100 mg of
algae are added to media or lake water containing 0.4 mg
P/l. Analyses of the supernatant for remaining P are taken
for 1 to 6 hours. Results indicate that P-limited
Cladophora sorb POy-P at a rate of 0.05 mg/100 mg algae/
hour, whereas Cladophora with adequate P sorb P at a rate
of 0.02 mg/100 mg algae/hour. The rates for Rhizoclonium
and Gloeotrtchia were 0.07 and 0.05 mg/100 mg algae/hour,
respectively. In contrast, the rates for duckweed (Lemna
minor) and tips of Myrtophyllum sp were 0.003 and 0.008 mg/
100 mg/hour, respectively.

Increase in extractable PO,-P or total P - Duplicate 5 mg
samples of algae are added to 1,000 ml media with different
levels of P. After 24 hours incubation the extractable P0,-P
or total P of the washed algae is measured. The minimum
detectable available P concentration is about 0.02 mg P/1l.
These techniques can readily be used with mixtures of algae
in the same medium. Results when Cladophora and Gloeotrichia
are mixed indicate the algae compete for limited quantities
of P, but do not exclude each other.

THE RELEASE, SORPTION, AND AVAILABILITY TO ALGAE
OF PHOSPHORUS FROM LAKE MUDS

Experiments carried out in situ and in the laboratory indi-
cate that naturally layered lake muds in three lakes release
soluble PO.-P when disturbed. However, when these muds are
suspended under the aerobic conditions of the overlying
waters, they sorb the phosphorus (P) released within about

20 minutes. The significance of these data is that P released
from disturbed muds will be competitively sorbed by both muds
and plants.

It was shown that the supernatant waters of a dredging -opera-
tion contained 0.13 mg soluble PO,-P/1. However, the in situ
suspended fine grey sand-to-silt sorbed 50% of the soluble
PO,-P per day.


cet
Biologically Available Phosphorus (mg/L)

10oF-
a O Epilimnion
Zz 4 Hypolimnion
1.0 —
4a O~
~
oa ~~
ee eed o~ ~~o
=
.01
L.
+001 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 29. Biologically Available Phosphorus - Snake Lake, North Basin, 1971


Soluble Ortho-Phosphate (mg/1)

1.0-—

a °
of ° °
= ° 000 0 0
°
fe) ° °
o
°
o0.1-— oo ° °
r 0 6° °
a ° 8 tome) (0)
0°
(oho) 6 ° °
8 8 °
= ° 00 9 O%® 00 0 °
° o°f a ° °
°° 8 ee
L- ao ° aad
° °
@ ° °
-01-— 000 © °
- ° ° °
oot tl tuil | iil | iii]
.001 ol 0.1 1.0

Estimated Biologically Available Phosphorus

Fig. 64.

Comparison of Phosphorus Determinations

176 #US. GOVERNMENT PRINTING OFFICE:1974 S46-316/258 1-5


Phosphorus Nutrition of Algae

Several methods are available to differentiate between algae
which have surplus P and those that are P-limited. Algae

and aquatic weeds containing adequate or surplus P will
release more than 0.08 mg PO,-P/100 mg (dry weight) of plant
material when extracted in e boiling water bath for 1 hour. ?
Also, the total P content of plant tissues can be used as a
relative measure of the availability of environmental sources
of P to the aquatic plants.*75 The results of these tests can
be confirmed by measuring the alkaline phosphatase activity
of the algae or aquatic weeds. It has been found that plants
that are P-limited will have as much as 25 times more alkaline
phosphatase activity as plants grown with surplus P.

The selection of the method to be used to evaluate the P
nutritional status of algae will depend upon the situation.
The amount of PO,~P extracted from algae using a boiling
water extraction requires the least amount of equipment.
However, the ease with which one can carry out total P
analyses using the H2SO,-persulfate-autoclave method allows
one to readily carry out a large number of analyses by either
method. Both methods require a measurement of the dry weight
of the algae used in the tests. One usually dries the algae
after the extraction procedure, but algae can be dried and
then weighed and extracted at a convenient time, much like
the procedure used in total P analyses. Comparisons of the
results of fresh extracts versus extracts of dried algae

have been made when algae contained surplus P and when algae

were P-limited. The results of representative tests are sum-
marized in Table 5,

The results of these comparisons show that the calculated
concentration of extractable PO,-P from fresh algae is highér
than when dried algae are extracted. This is due to the loss
of soluble compounds from the fresh algae during the extrac-
tion procedure and a resulting lower dry weight measurement.
However, the interpretation of the results, whether the algae
had surplus or limiting supplies of available P, would only
be confused when algae are near the border line between the
two conditions. Either sequence of the extraction procedure
could be used as long as it is applied systematically to all
samples in the series of tests to be compared.

21


8ST

Table 40.

AVERAGE ALGAL BIOMASS PRODUCED IN SAMPLES

FROM LAKES SUBJECTED TO HYPOLIMNETIC AERATION*

_Epilimnion Hypolimnion
Dec- Mar- June- Sept- Dec- Mar- June- Sept-
Lake Year Feb May Aug Nov Feb May Aug Nov
1971 1.6 23.
Mirrorb 1972 5.1 3.0 0.15 5.4 307. 138. 127. 47.
1973 9.1 5.5 4.5 3.1 8.8 16.
c 1971
Larson 1972 2.6 0.69 1.1 2.0 1.5 0.76 9.0 7.2
1973 3.6 1.5 1.4 15. 4.1 13.

a.

All values given in mg/l dry weight.

Daeration schedule - 8/10/72-9/8/72, hypo only; 9/19/72-11/21/72, total lake;
1/30/73-3/8/73, hypo only; 3/30/73-4/22/73, total lake;
6/21/73-8/28/73, hypo only.

Aeration schedule - 2/2/73-3/16/73, hypo only; 4/25/73-9/20/73, hypo only.


nutrients indicate there were differences in the growth of
algae in water from Lakes Mendota and Monona when samples
were collected during high flows versus low flows. Much
higher growths were achieved in the samples from the high-
flow period. This is a logical response since growth in
these lake waters was more limited by N or Fe, and surface
runoffs apparently supply these nutrients during the winter
in much the same manner as in the summer. The fact that
there was little difference in the growth of the algae be-
tween high- and low-flow samples after they had been spiked
with a combination of N and Fe indicates that surface run-
offs at this time of year do not add significant quantities
of P, which is the limiting factor when N and Fe are both
added. There was little response to added N and Fe in the
Lakes Wingra and Kegonsa samples because during the winter
these lakes have very low levels of P. This is an interesting
condition in the case of Lake Kegonsa because the flow from
Lake Waubesa contains relatively high amounts of available P
and additional P.comes from the smaller inlets to this lake,
Halverson and Door Creeks.

The relationship between the soluble P0O,-P and available P
of these lake waters calculated from the data of samples
spiked with a combination of N and Fe for both membrane-
filtered and autoclaved samples is summarized in Figure 14.
It is evident that in these lake waters, P analyzed as
soluble PO,-P is available to all three algae.

The data presented from growth tests of algae in lake waters
have demonstrated: how the selected algae can be used to
compare the fertility of different lakes; how the lakes will
respond to additions of N, P, and Fe; the effect of high-
versus low-flow periods during the winter on the available
amounts of N and Fe in some of the lakes which are limited
by the N and Fe levels; and the lack of effect of surface
runoffs during winter in lakes which are limited by P.

The Soluble PO.-P of Lake Waters
and P Content of In Sttu Algae

The results of analyses of the soluble P0,-P of the lake water
and the extractable PO,-P of the in situ algae are presented
in Figures 15-18. Rainfall data are also presented in Figure 1
Fall and winter (1972-1973) analyses of soluble PO,-P are pre-
sented in Figure 19. The early in sttu algae analyzed were
Ulothriz sp and Spirogyra sp with Cladophora sp appearing in
late May and continuing through September.

90


cET
Algal Production (mg/L Dry Wt.)

100b—

O Epilimnion

4 Hypolimnion

01
Jan.

30

60 90

Fig. 28.

T T T T T T
120 150 180 210 240 270
Time (Days)

Algal Production - Snake Lake, North Basin, 1973

T
300

330

T
360


lakes is not an available source of fertilizer. Fish ponds
that had been heavily fertilized for 15 years with N-P-K
fertilizers to maintain productivity of crops of fish were
tested to determine which fertilizers could be regenerated
from the very fertile bottom muds. It was found that no
significant decrease in production of fish resulted from
omitting both nitrogen and potassium from the fertilizer
mixture during a four-year experimental period. Omission of
phosphorus, however, caused a decrease in production. Thus,
except for phosphorus, adequate nutrients were available
from these fertile pond bottoms. Therefore, the important
sources of phosphorus that are supplying surplus quantities
of phosphorus to Lakes Waubesa and Kegonsa are probably not
related to past fertilization of these lakes but are the
result of present sources of phosphorus. The effect of inter-
ceptor sewers (under construction) partially around Lake
Waubesa may indicate the contribution of phosphorus from the
many homes along the lakeshore. Further study of the Lake
Kegonsa area may indicate the relative importance of develop-
ing a sewer system versus directing around the lake a creek
that is known to carry a relatively high load of nutrients,
as proposed in the 1944 study of Sawyer, Lackey and Lenz.?

The Rock River and two of its tributaries, the Crawfish and
Bark Rivers, contain available phosphorus above Lake Koshkonong
(more than 0.2 mg P/1l). In addition, the Rock River contains
more than 0.1 mg available P/1 below the lake and above its
junction with the Yahara River, which indicates that the Rock
River already contains phosphorus in excess of that which
could be used by the algae and aquatic weeds of the river or
Lake Koshkonong, even before the introduction of the effluents
from Stoughton, Oregon, and Madison. Therefore, although the
Yahara River adds available phosphorus to the Rock River, the
significance of this phosphorus is questionable since the
river already contains more phosphorus than can be used by

the plants present during this time of the year (September,
1971).

The data from bioassays and chemical analyses presented indi-
cate how nutrient surveys can be used to determine whether
lakes contain available phosphorus in excess of that which
could be used by the algae or aquatic weeds present. Ifa
lake is shown not to have surplus available phosphorus during
the summer period, as in the case of the lakes in the City
of Madison, Lakes Mendota, Monona, and Wingra, it would be
expected that further reductions in the sources of phosphorus
to these waters might reduce the biomass of algae present.
When a lake contains surplus phosphorus for the amount of
algae and aquatic weeds present, it is important to determine

83


Samples of in sttu Cladophora sp from 2 lakes, collected
after wind storms had stirred up considerable mud, were
found to be P-~limited even though the surrounding waters
contained as much as 0.8 mg total P/l. It was also shown
that Spirogyra sp that had grown through layers of dredged
mud containing 0.09% P were P-limited.

Laboratory tests of the availability of the P of lake muds
under aerobic conditions indicate that Selenastrum capri-
cornutum (PAAP) cannot grow on such sources of P, and the
N2-fixing capacity of P-limited Anabaena flos aquae (Ind
1444) is not benefited by the P from lake muds. Overnight
incubation of P-limited Cladophora sp in Lake Mendota water
supplemented with both soluble PO4-P and total P from muddy
Crawfish River water indicated, by increases in the total P
content of the algae, that it is only able to use the soluble
PO.-P of the Crawfish River water.

Other studies with the duckweed, Lemna minor, have also
shown that this aquatic weed cannot utilize the P of aerobic
lake muds but can absorb P from lake muds when their roots
are able to penetrate presumably anaerobic areas. There-
fore, it would appear that the growth of rooted aquatic
weeds would dominate a eutrophic aquatic environment from
which the soluble POs-P was removed.

DUCKWEED (LEMNA MINOR) FOR NUTRITIONAL BIOASSAYS

Duckweed (Lemna minor), a rooted aquatic plant which- can be
readily cultured under different environmental conditions,
has been used in nutritional bioassays. Short-term exposures
for sorption in conjunction with 3- to 4-week incubations for
growth have been used to show how labile compounds can be
tested as available sources of essential nutrients. Tests
indicated that ortho- and pyrophosphate could be sorbed in

4 hours while 6 hours were required to sorb tripolyphosphate.
Tests under similar conditions indicated that between 6 and
28 hours were required for duckweed to sorb maximal amounts
of either NH4y-N or NO3-N. Tests with Fe as FeCl; alone or

in the presence of citric acid or EDTA indicated that only
with EDTA present was the Fe available. Significant sorption
of Fe took place with 8-hour exposure when EDTA was present.

Growth experiments lasting 3 weeks in which the availability
of the N, P, or Fe of aerobic lake muds was tésted indicated
that duckweed could not obtain either available N or P under
these conditions, but could utilize Fe from the muds.


Table 30. THE ALGAL GROWTH ATTAINED, AS FLUOROMETRY UNITS,
IN AUTOCLAVED SAMPLES OF MADISON AREA LAKE WATERS
Averages of triplicate cultures

No spikes NAAM level of N and Fe added
High flows Low flows High flows Low flows
1/18/73 1/30/73 2/12/73 2/22/73 1/18/73 1/30/73 2/12/73 2/22/73

Selenastrum (1,000 cells/ml)

Mendota 1.5 1.6 0.1 0.2 7.8 8.7 4.1 4.7
Monona 0.8 0.9 0.4 0.3 5.4 5.5 3.8 5.4
Wingra 0.04 0.02 0.02 0.05 0.06 0.03 0.09 0.05
Waubesa 1.1 1.2 1.3 1.2 3.0 4.0 3.6 3.5
Kegonsa 0.7 0.4 0.3 0.9 0.8 0.6 1.3 1.3
& Mteroeystis (50,000 cells/ml)
Mendota 0.05 0.03 0.02 0.1L 1.6 1.8 0.7 0.7
Monona 0.03 0.03 0.03 0.06 0.9 1.0 0.7 1.0
Wingra 0.04 . 0.1 0.03 0.08 0.05 0.2 0.09 0.2
Waubesa 0.04 0.04 0.04 0.03 0.5 0.6 0.5 0.7
Kegonsa 0.04 0.05 0.05 0.1 0.1 0.4 0.2 0.4
Anabaena (50,000 cells/ml)
Mendota 0.7 1.0 0.03 0.6 1.9 1.5 0.08 1.1
Monona 0.7 0.4 0.07 0.5 1.3 1.5 0.06 1.0
Wingra 0.06 0.04 0.04 0.2 0.1 0.08 0.04 0.2
Waubesa 0.5 0.2 0.06 0.5 0.8 0.6 0.3 0.9
Kegonsa O.1 0.1 0.04 0.3 0.4 0.3 0.09 0.3



BK
.
o

RAIN

WATER - Soluble POy-P (mg P/1) (Inches)
Oo
°
a
T

eh d cdl seal

°
.
°
00
T

ALGAE - Extractable P
(mg PO.-P/100 mg Algae)

0.05 1 1 rt 1 L
Apr May June July Aug Sept

Fig. 17. Comparison of the Soluble PO,-P of Lake Waubesa
Waters and the Extractable P0O,-P
of in situ Algae at the Outlet

94


maintained in triple-strength, autoclaved nutrient medium.
The age of the culture used for inoculation purposes ranged
from 7 to 21 days.

Inoculum preparation - The inoculum was prepared by centri-
fuging a portion of a 7- to 21l-day-old culture of Selenastrum.
eapricornutum, discarding the supernatant, and resuspending
the cells in a solution of NaHCO; (45 mg/l in glass-distilled
water) to give a final concentration of 1000 cells/ml when
added to the sample flasks.

Synthetic medium -

The composition of the synthetic nutrient medium is as follows:

Macronutrients

The following salts (biological or reagent grade)
in milligrams per liter of glass-distilled water:

Concentration Concentration

Compound (mg/1) Element (mg/1)
NaNO; 25.5 N 4.2
K2HPO, 1.0 P 0.186
MgCl2 5.7 Mg 2.9
MgSO, * 7H20 14.7 s 1.9
CaClz * 2H20 4.4 c 2.1
NaHCO 3 15.0 Ca 1.2

Na 11.0

K 0.469

Micronutrients

The following salts (biological or reagent grade)
in micrograms per liter of glass-distilled water:

Concentration Concentration
Compound (ug/L) Element (ug/L)
H3BO3 185. B 32.460
MnCl2 264. Mn 115.3
anCl2 32.7 Zn 15.6
CoCl2 0.780 Co 0.354
CuCl2 0.009 cu 0.004
Na2MoO, * 2H20 7.260 Mo 2.87
FeCl; 96.0 Fe 33.0
Naz2EDTA + 2H20 300.0

110


of the plant remains in an aerobic environment, such as occurs
in normal growth in a lake. Consequently, an experiment was
carried out comparing the availability of the P of lake muds

to duckweeds floating so far above the muds that they could
only absorb P which passed through an aerobic column of medium
with duckweeds whose roots could penetrate the mud and pre-
sumably extend into any anaerobic environment developed in the
mud, Two fronds were placed in 20 cm culture tubes containing
Gorham's (-P) medium. To four tubes containing 25 ml of Lake
Wingra mud (2.0 mg total P/tube), 30 ml of Gorham's (-P) medium
were added so the duckweed floated 8 cm above the surface of
the mud. The second set of mud tubes contained only 5 ml of
Gorham's (-P) medium so the duckweed floated only about 1 cm
from the mud surface. The results of the experiment as the
average number of fronds per test condition after 6 weeks
incubation are graphically summarized (next section, Figure 12).

Little growth (5 fronds) of the duckweed took place in the
P-free Gorham's medium, but 27 fronds developed in the presence
of 0.005 mg PO,-P. It is evident that no available P was
released to the overlying medium in the case where the mud
containing 2 mg of total P was overlain by 30 ml of Gorham's
(-P) medium since the duckweed in these tubes had no more
growth than those with only Gorham's (-P) medium, There was
considerable growth (27 fronds) of the duckweed whose roots
penetrated the mud layers which were presumed to have developed
an anaerobic environment during the long incubation period

(6 weeks). Observations of these cultures for an additional

8 weeks also indicated that even with additional incubation

the duckweed in cultures in which the roots could not touch

the mud layers did not grow any farther.

These tests thus indicate how tests can be carried out to
contrast the availability of P in lake muds to algae or float-
ing aquatic weeds versus that to aquatic weeds whose roots can
penetrate to anaerobic layers of mud. As one would expect
from chemical studies of the release of PO,-P to the overlying
aerobic waters of lakes, there is available P in lake muds
that are under anaerobic conditions. One should be aware,
however, of the fact that PO,-P released from anaerobic lake
muds into an aerobic environment can be competitively sorbed
by muds under the aerobic environment. ®

57


Table 20. EFFECT OF CONTACT TIME WITH DIFFERENT SOURCES
OF IRON ON THE GROWTH OF DUCKWEED (LEMNA MINOR)
2 fronds/25 ml Gorham's (-N, -EDTA) medium

FeCl; 1 ng/l

Contact time 0.025 mg 0.100 mg citric acid 1 mg/l EDTA
(hours) -Fe Fe/l Fe/l 0.025 mg Fe/l 0.025 mg Fe/l

1 3 4 5 5 6

4 5 4 4 4 6

8 4 4 4 6 10

24 4 6 6 4 10

28 5 6 6 3 16

48 6 4 4 7 14

51 4 4 4 5 14

68 6 7 5 5 16

but duckweed could utilize tripoly- as well as ortho- or
pyro-P when enough exposure time was allowed. The tests with
different sources of Fe indicated that duckweed could only
utilize the Fe in the presence of EDTA, with maximal growth
occurring after about 1 day exposure. These results illus-
trate that short-term exposure periods for the comparative
sorption of essential nutrients can be used in conjunction
with the relatively long incubation time required for maximal
growth.

When an essential nutrient is to be tested and there is no
problem with potential conversion of the nutrient to another
form, simple growth bioassays can be carried out in suitable
control cultures and with the source of nutrient. The results
of a typical experiment testing the availability of the P of
lake muds under aerobic conditions are presented in Table 21
as the average number of fronds developed per triplicate
culture. In this type of test, 2 fronds were added to 50 ml
of supplemented Gorham's medium which contained either lake
mud or different concentrations of P. The depth of the cul-
tures under these conditions was such that the roots of the
duckweed were touching the lake mud on the bottom of the
flasks.

It is evident that under the conditions of this test, a sig-
nificant increase in frond numbers occurred with a concentra-
tion of 0.1 mg PO,-P/1 in the control cultures, but that as
much as 7.5 mg P/l in the form of mud did not support as much

63


ANALYSES OF THE TOTAL IRON CONTENT OF SAMPLES

Comparisons have been made of results using two digestion pro-
cedures and a direct method for analyzing the iron content of
samples. The iron analysis procedure was to: add 1 ml of
"PerroZine" iron reagent (Hach Chemical Co. - Note: Caution
should be used with this commercial product because it has only
a limited shelf-life.) to a 25 ml sample, heat in a boiling
water bath, and measure the purple color produced. Iron in the
range of 0.1 to 1.6 mg/l could readily be analyzed using 1 cm
tubes with this colormetric analysis. The results of the com-
parison of H2S0,-HNO3; or H2SO,-persulfate digested samples
with direct analyses of an iron ore and an alga are presented
in Table 2.

It is evident that these samples did not require a previous
digestion step before analyses using this reagent. The rela-
tively large standard deviation, 6, using the suspension of the
iron ore, taconite, is believed to be mainly due to sampling
errors due to difficulty in keeping the different-sized par-
ticles evenly suspended. Similar tests with Microcystis,
Cladophora, Pithophora, and soil extracts also showed that
direct analyses of samples were adequate for iron determinations,

ALGAL BIOMASS MEASUREMENTS

The applicability of the different methods of measuring the bio-
mass of algal cultures depends upon the concentration of algae.
Relatively dilute cultures can be readily measured using cell
counts (haemocytometer or electronic particle counter) or the
fluorescence of chlorophyll @. More concentrated cultures can
be measured using absorbance or dry weight measurements. Ease
of use makes fluorometry and absorbance the usual measurements
of choice. As a demonstration cf the precision within each of
these measurements and the factors to be used in converting each
to dry weights, a series of replicate (6) tests were carried out
using each of the biomass measurements with Selenastrum grown

in Gorham's medium. The results of these tests are presented

in Table 3 as the raw data, means, standard deviations, and 95%
confidence limits.

The techniques used for dry weights, centrifuging a known volume
and weighing the pellet in tared pans or filtering a known
volume through a tared gooch crucible (glass filter pad), are
both normally effective measurements. However, the Selenastrum
culture (Gorham's medium) used for these tests was particularly
slimy and caused the gooch filters to plug before reliable

8


eSTt
Biologically Available Phosphorus (mg/L)

10—-—

O01

001

O Epilimnion

4 Hypolimnion

Jan.

30

Fig.

48.

T T T T T T T
90 120 150 180 210 240 270
Time (Days)

Biologically Available Phosphorus - Pickerel Lake, 1972


Soluble PO.-P (mg P/1)

0.16

°
ad
N

°
°o
co

0.04

A Selenastrum (Membrane Filtered Samples)

A Selenastrum (Autoclaved Samples)

O Anabaena (Autoclaved Samples)

e@ Microcystis (Autoclaved Samples)

A A
A
A
03°e A
A
L ea of «
A
8°
A
r Ne fo)
e A
wee a
Ae
OA
1 1 1 1 l
0 0.04 0.08 0.12 0.16 0.20

Available P (mg P/1)
Fig. 14. Relationship Between Soluble PO,-P

of Lake Waters and Available P - Growth Tests
January and February, 1973

91


Three species of algae have been selected for use in growth
tests. They are the green alga, Selenastrum capricornutum
(AAP), and the blue-green algae, Microcystis aeruginosa (AAP)
and Anabaena flos aquae (AAP). All three grow readily in the
algal assay medium (NAAM). In order to evaluate the nutrient
content of water samples, it is necessary to measure the re-
sponse of the test alga to different levels of the nutrients

in question, Thus, standard curves of the response of the
alga to a range of concentrations of a nutrient in NAAM medium
are compared to the growth of the alga in the test water.

Since the growth of the alga in the test water may be limited
by some other nutrients than the one to be measured, the test
waters are usually spiked with certain nutrients when measuring
the available content of a particular nutrient. In the Midwest,
spikes of N, P and Fe have been found to give the most sig-
nificant results, and combinations of these nutrients are used
in measuring nutrient contents of test waters.

In a typical experiment using Selenastrum the final yield of
this alga would be measured in NAAM medium (to demonstrate

that the inoculum used was viable) and in different levels of
N, P, and Fe in NAAM medium, The various media would be placed
in Erlenmeyer flasks, inoculated so that each culture would
have 1,000 cells of Selenastrum per ml, and incubated under

400 ft C of light at 24°C until maximum growth occurred.

The rate of growth of Selenastrum in NAAM medium is presented
in Figure 1 as the fluorescence attained after different times
of incubation. These data indicate that a logarithmic rate of
growth occurred between days 1 to 3 or until the growth reached
about 1.5 mg/l (150,000 celis/ml); linear growth occurred
between days 3 to 7; and, finally, maximal yield (37 mg/1)
after. 9 days. A general rule would be that, if the culture was
visibly green in color, it no longer was in the log-phase of
growth, The maximum specific growth rate of Selenastrum in
NAAM was 2.6/day, and the standard deviation of the data at
day 4 (2.54 fluorescent units) was 0.41 (7.6 + 1.2 mg/l).

The relationship between the growth of Selenastrum and the
concentration of NO3-N in NAAM medium is presented in Figure 2
as the dry weight attained after 7 days in cultures containing
different concentrations of NO3-N. It can be seen that in NAAM
there is a direct relationship between the growth attained and
the concentration of NO3-N at least up to 2 mg N/l. In more
concentrated media, such as Gorham's medium, this direct rela-
tionship continues to as high as 20 mg N/1.

As a demonstration of the growth attained by Selenastrum versus

the concentration of PO,-P in culture media, comparative tests
were carried out in NAAM and in autoclaved Lake Wingra water

13


to or greater than that of field collections of Cladophora sp,
measurements were made of the amount of P that each of these
algae could sorb when limited or surplus quantities of PO,4-P
were added to mixtures of the two algae. In the first test
approximately 12 mg samples of algae alone or together were
placed in 1,000 ml of Lake Mendota water containing either no
measurable P, or with the addition of 0.04, 0.08, or 0.12 mg
PO,-P. The Cladophora and Gloeotrichia were recovered from
the culture media by using a large-mesh screen for the Cla-
dophora and a fine plankton net for the Gloeotrichia. The
extractable POy-P of the algae after 20 hours incubation
alone or together in the different media was measured and is
summarized in Table 8.

Table 8, COMPARATIVE RATES OF PHOSPHORUS SORPTION
BY CLADOPHORA SP AND GLOEFOTRICHIA SP FROM LAKE MENDOTA WATER
12 mg algae/1,000 ml Lake Mendota water

Extractable PO,-P
(mg P/100 mg algae)
after 20 hours incubation

Lake water
Lake +0.04 mg +0.08 mg +0.12 mg

Algae tested water PO4-P/1 PO,-P/1 PO,y-P/1L
Cladophora-alone 0.08 0.19 - -
Gloeotrichia-alone 0.40 0.55 - -
Cladophora in mixture 0.09 0.13 0.18 0.18
Gloeotrichia in mixture 0.40 0.43 0.66 0.66

It can be seen that the Cladophora used were relatively P-
limited (low extractable P0,-P values after incubation in
unsupplemented lake water) when compared to the Gloeotrichia.
When tested alone in the presence of 0.04 mg POy-P either alga
was able to sorb P such that their extractable PO.,-P increased
considerably. When the algae were incubated together with
0.04 mg PO«-P/1 neither alga prevented the sorption of P by
the other alga, and the result was that both had only limited
increases in their extractable POs-P. In the presence of

0.08 mg PO.-P/1 both algae were able to sorb what appeared

to be maximal quantities of P since no further increases in
extractable PO,-P occurred in the presence of 0.12 mg POy~P/1.

42


The nutrient control projects in the Madison area are:

1) diversion of the sewage effluents of the Villages of
Waunakee and DeForest (accounting for 10% of the inorganic
nitrogen and 36% of the phosphorus entering Lake Mendota)
from the Lake Mendota watershed to a metropolitan sewer
system by-passing the Madison area lakes (1971); 2) collec-
tion of sewages from cottages and homes (presently using
septic tanks) surrounding Lake Waubesa and disposal to the
by-pass sewer system: construction started in 1972 and will
be conducted in two stages, each affecting different popu-
lations; and 3) collection, treatment, and by-pass disposal
of sewages of the cottages and homes (septic tank disposal)
of the Lake Kegonsa area: a sanitary district has been
created, but construction has not been started.

Studies have been made by the principal investigator and
others associated with the Water Chemistry Program of the
University of Wisconsin, using chemical and bioassay tech-
niques, to follow the changes in the nitrogen and phosphorus
nutritional status of algae in the surface waters of Lakes
Mendota and Monona throughout the algal-growing seasons of
1968 to the present. These data can be used to compare
with results in 1973 and later, to determine if any changes
can be correlated with the diversion around the Madison lakes
of the sewage effluents of the Villages of Waunakee and
DeForest in contrast to the continuing effects on this deep,
stratified lake caused by drainage from 250 square miles of
fertile farm area.

Data collected in September, 1971" indicated that, before

the sewers were built around Lake Waubesa, the concentration ~
of soluble PO,-P and available P was 0.05-0.07 mg P/l at the
outlet of Lake Waubesa. However, after the sewer construction
of 1972, the levels of soluble PO,-P and available P in Lake
Waubesa did not exceed 0.03 mg P/l throughout August and
September of 1972. Thus, there is an indication that the con-
version of septic tanks to sewerage and diversion had a sig-
nificant effect on the level of P available in Lake Waubesa
despite normal runoffs from the fertile farm lands of the area.
Further data from this area as the sewerage program continues
should substantiate the results obtained in 1972.

The results of analyses of Lake Kegonsa and its algae provide
spectacular data, since it is the only lake of the five-lake
chain which had increasing levels of soluble PO,-P and avail-
able P during July, August, and September (to more than

0.1 mg P/l) when the other lakes were practically devoid of
soluble PO.-P. Thus, urban and farm land surface runoffs

87


Nitrogen Nutrition of Algae

Changes in the environmental supply of N to in sttu algae
can be followed by measuring the ammonia absorption rate

of the algae in the dark. Algae that are limited by the
supply of available N are able to absorb NH,-N in the dark
four to five times more rapidly than plants with adequate
N.” .fhus, one can evaluate the fertilizing effect of rains
on N-limited algae. Cladophora samples collected from the
surface and 0.7-meter depth of Lake Mendota (June 19, 1972)
had an average NH,-N absorption rate of 22 ug N/10 mg algae
(dry weight)/hour and had a yellow-green color. After a
rain during the night of June 19, the Cladophora had an
average absorption rate of 6 ug N/10 mg/hour and were bright
green. Similarly, a series of rains during mid-July, 1972,
caused: the NH,-N absorption rate of the Cladophora of
Lake Mendota to decrease from 25 to less than 5 yg N/10 mg/
hour, the algae to turn from yellow to green, and, with con-
tinued growth in the presence of excess N, the algae became
coated with epiphytic diatoms and blue-green algae.®

ANALYSIS OF AVAILABLE P BY SORPTION TESTS WITH ALGAE

As an alternative to waiting for algae to grow using ab-
sorbed P, one can measure the P absorbed. This can be done
by incubating algae in the water sample to be tested and
measuring the increase in the P of the algae by either ex-
traction or total P analyses. The amount of P in algae
after incubation in known concentrations of POu-P is compared
with the amount obtained from algae after incubation in the
different water samples and is used to calculate the concen-
trations of available P in the water samples. The algae must
be alive because the absorption process is physiological
rather than mere adsorption: a mixture of P-limited diatoms
from Lake Wingra (12/26/72) was found to readily absorb
PO,-P; the extractable PO.-P after overnight incubation in-
creased from 0.04% P in control (-P) cultures to 0.30% P

in cultures containing 0.1 mg P/1l, whereas diatoms that had
been killed by two cycles of freezing-thawing had only ex-
tractable PO.-P levels of 0.013 and 0.014% P after overnight
incubation in control and 0.1 mg P/l cultures, Field algae
to be used do not have to be freshly collected in order to be
used to absorb available P from test solutions, Algae §eta-
dophora sp or mixed diatoms) stored in refrigerators (4 °C)
for as.long as 6 months were still capable of absorbing

25


09T
Algal Production (mg/L Dry Wt.)

100

10

-O1

7 /
y \
| g@-----0 \ bx
= \ I
\ |
\ |
\ |
_E_— \
r \ py o---3;
| \ vo PN
aa a
= \ / \ a O Epilimnion
Y \ ea A
\ An 2 Hypolimnion
PN Yoo
= N N
L N
[— a
T T T > T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360

Time (Days)
Fig. 53. Algal Production - Mirror Lake, 1972


Sel
Algal Production (mg/L Dry Wt.)

/
100—
Zi O Epilimnion
—_ A Hypolimnion
/
[— /
/ P
ep. / /
1oL— a /
F 3 /
P P /
1.0 7
- nO
— PN >~o"
P
abe
-
01 T T T T T T T T T T T T
Jan. 30 60 90 120 150 180 210 240 270 300 330 360
Time (Days)
Fig. 21. Algal Production - Horseshoe Lake, 1972


Table 2. COMPARISONS OF IRON ANALYSES
Iron content
Digested _ samples
No. of H2SO,4-HNO3 H2S0,-persul fate Direct analysis
Iron source samples Ave 62 Ave 62 Ave 62
Taconite
suspension 8 0.368 0.057 0.456 0.152 0.406 0.175
(mg Fe/100 ml)
Chlorella
pyrenotdosa n
(Wis 2005) 8 0.070 0.008 0.072 0.005 0.067 0.008
(% Fe)

a
Standard deviation


The P in the form of dredged mud was not available for the
growth of Selenastrum-under these aerobic conditions, a con-
centration of 0.5 mg lake mud P/1l giving less growth than
0.025 mg POy-P/1l. When PO.-P (0.2 mg P/1) was added to a
culture with 0.2 mg mud P/1, the Selenastrum was able to grow
on the added PO.,-P, showing that the lack of growth with
other mud samples was not due to the mud being toxic to the
alga.

The availability of the P of dredged lake muds was also tested
with P-limited N2-fixing blue-green algae. It has been shown
that the rate of fixation of N2 by certain blue-green algae
is dependent upon whether the algae have adequate P. The
exposure of P-limited algae to available PO,-P for as little
as 15 minutes will allow the algae to absorb enough P so that
increased capability of the algae to fix Nz can be measured
by the acetylene-reduction method. In a typical test, 12 mg
samples of Anabaena flos aquae (Ind 1444), which had been
cultured under limiting P conditions, were added to 225 ml of
Gorham's (-P) medium to which was added PO,-P or mud from

the Murphy's Creek dredging operation. After 2 hours incu-
bation in the light, the algae were centrifuged and the
ethylene produced by triplicate 4 mg samples of the Anabaena
in 1 ml volumes was measured after 30 minutes exposure to
acetylene in the light. The results as the average n moles

of the ethylene produced/mg algae/30 minutes are summarized
in Table 15.

Table 15, THE AVAILABILITY OF PO,-P AND DREDGED MUD PHOSPHORUS
TO PHOSPHORUS-LIMITED ANABAENA FLOS AQUAE (IND 1444)
AS MEASURED BY THE REDUCTION OF ACETYLENE TO ETHYLENE

2 hours incubation Gorham's (-P) medium
Concentration Ethylene produced

Phosphorus source (mg P/1) (n M/mg algae/30 min)
none i 0.0 1.6
PO,4-P 0.005 3.3
PO,-P 0.01 5.4
PO4-P 0.02 10.8
dredged mud 0.14 1.6
dredged mud + PO,-P 0.14 + 0.02 4.2

53


Table 39. NUTRIENT LIMITATION IN SAMPLES
FROM LAKES TREATED WITH ALUMINUM

Number of samples
which responded
to additions of:

Total
Lake Year Location N P Né&P_ samples

Horseshoe 1971 epi - 1 1 3

(treated 5/70) hypo - 2 - 3

1972 epi - 3 1 6

hypo - 3 - 5

1973 epi 1 4 1 7

hypo 2 3 2 7

Snake, 1971 epi 2 - 1 3

North Basin hypo 1 - - 1
(treated 5/72)

1972 epi 2 5 6 14

hypo 2 9 2 13

1973 epi - 5 5 10

hypo - 8 2 10

‘Snake, 1971 epi 1 ~ - 1

South Basin hypo - - - 0
(treated 5/72)

1972 epi 1 7 - 9

hypo 1 5 2 9

1973 epi - 6 3 10

hypo - 10 - 11

122


and ground waters. During periods of high flow, after rains
or snow melts when the creeks contained surface runoffs, the
soluble PO,y-P was generally available: in 30 water tests,
26 had available P.

During low flow periods when creek flow was mainly ground
water, the available P of 30 water tests was below 30% of
the soluble PO,-P and the majority of the tests had no
available P. Detailed tests have not determined whether
the unavailability of the ground water P was due to inter-
ference in PO,-P analyses by arsenic, but occasional in-
creases in availability after periods of room temperature
storage or autoclaving have been noted, so at this time
final conclusions can not be made.

References

1. Lawton, G. W. Limitation of Nutrients as a Step in
Ecological Control. In: Algae and Metropolitan Wastes.
Cincinnati, Taft Sanitary Engineering Center, Technical:
Report W61-3, U.S. Public Health Service, 1961. p. 108-
117.

2. Fitzgerald, G. P., and G. F. Lee. Use of Tests for
Limiting or Surplus Nutrients to Evaluate Sources of
Nitrogen and Phosphorus for Algae and Aquatic Weeds.
Madison, Water Chemistry Program Report, University
of Wisconsin-Madison, 1970. 31 p.

3. Fitzgerald, G. P. Bioassay Analysis of Nutrient Avail-
ability. In: Nutrients in Natural Waters, Allen, H. E.,
and J. R. Kramer (ed.). New York, John Wiley and Sons,
1972. p. 147-170.

4. Fitzgerald, G. P., S. L. Faust, and C. R. Nadler.
Correlations to Evaluate Effects of Wastewater Phos-
phorus on Receiving Waters. Water and Sewage Works.
120(1): 48-55. 1973.

5. Sawyer, C. N., J. B. Lackey, and A. T. Lenz. Investi-
gation of the Odor Nuisance Occurring in the Madison
Lakes. Madison, Wis., Governor's Committee Report.
1944, 137 p.

6. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Burris.
Acetylene Reduction Assay for Determination of Phosphorus
Availability in Wisconsin Lakes. Proc. National Acad.
Sci. 66:1104-1111. 1970.

106


Table 33. ALGAL BIOASSAYS CONDUCTED

a
No. assays

No. assays®

prior to. after

Lake name - Treatment and date treatment treatment
nutrient inactivation

Horseshoe with aluminum (5/70) 16
nutrient inactivation

Snake with aluminum (5/72) 10 19
nutrient inactivation

Long with aluminum (5/72) 5 16

. nutrient inactivation
Pickerel with aluminum (4/73) 25 6
Mayflower not treated 5 0
. hypolimmetic aeration

Mirror (1/73) 21 7
hypolimnetic aeration

Larson (2/73) 16 18

Horseheaa drawdown/winter aeration 5 0

(?)

Windfall aquatic plant mgmt 5

Shadow NA 6

White winter aeration 1

?Bach assay consists of 2 samples, one from
the epilimnion and one from the hypolimnion.

108


No.

41-43.

44-46.
47-49.

50.
51.

52-54,
55-57.

58-60.
61~63.

64.

Biologically Available Phosphorus - Long Lake,
1971-73

Algal Production - Pickerel Lake, 1971-73

Biologically Available Phosphorus -
Pickerel Lake, 1971-73

Algal Production ~- Mayflower Lake, 1973

Biologically Available Phosphorus - Mayflower
Lake, 1973

Algal Production - Mirror Lake, 1971-73

Biologically Available Phosphorus - Mirror
Lake, 1971-73

Algal Production - Larson Lake, 1971-73

Biologically Available Phosphorus - Larson
Lake, 1971-73

Comparison of Phosphorus Determinations

vi

Page

145-147

148-150

151-153
154

155
159-161

162-164
167-169

170-172
176


10.

ll.

Gerloff, G. C. Evaluating Nutrient Suppleis for the
Growth of Aquatic Plants in Natural Waters. In:
Eutrophication: Causes, Consequences, Correctives.
Washington, D.C., Nat. Acad. Sci., 1969. p. 537-555.

Swingle, H. S., B. C. Gooch, and H. R. Rabanal.
Phosphate Fertilization of Ponds. In: Proc. 7th
Annual Conference, S.E. Assoc. Game and Fish Comnm.,
Hot Springs, Oct. 1963. p. 213-218.

85


the P measured as soluble P0,-P in the creek samples and the
spikes of P0,-P added were only about 50% available. In
general, we have found that only 20 to 75% of the soluble
PO,-P of river or creek waters which have not been modified
by being below lakes or lake-like widespreads was available P
whether measured in short-term sorption tests or growth tests
using Selenastrum.

The volumes and incubation times used in sorption tests do
not appear to be important factors as long as control cultures
with different concentrations of POy-P are incubated under
the same conditions as samples to be tested. As examples,

the extractable PO,-P from samples of algae incubated in
volumes from 100 to 1,000 ml containing different amounts of
added PO,-P for 3 to 20 hours are presented in Figure 9. The
data show that whether Lake Wingra Cladophora is incubated

in 100 ml of Gorham's-P medium for 20 hours, or in 1,000 ml

of Lake Mendota water (7/14/72) for 4 hours, or whether Lake
Mendota Gloeotrichia sp (7/31/72) is incubated in 200 ml
Gorham's-P medium for 30 hours, there is a direct relationship
between the extractable PO,-P of the algae and the concentra-
tion of PO,-P in the medium tested.

WATER SAMPLE PRESERVATION

The treatment to be given water samples before evaluating
their nutrient content will depend upon the test to be used.
Usually sorption tests require so little time and space to
carry out that tests can readily be carried out on samples

as fast as they are received or even in the process of bring-
ing them to the laboratory, if necessary. Since the usual
handling procedures in harvesting sorption tests consists

of removing the test algae with a forceps or coarse plankton
net funnel, most planktonic in situ algae in the water sample
would not be harvested and analyzed. However, their presence
in the sample during the sorption incubation may have an
effect on the results because of competition with the test
algae. In a previous report (1972) the effects of various
planktonic algae (Gloeotrichia and mixed plankton) on the

P sorbed by Cladophora was reported: the extracted P0,-P

of the Cladophora incubated alone going from 0.024% P in

Lake Wingra water to 0.075% P in lake water plus 0.04 mg
PO,-P/1, whereas in the presence of 17 mg/l of mixed plankton
from Lake Wingra the extracted P0,-P of the Cladophora went
to only 0.033% P in lake water plus 0.04 mg PO,-P/l. The
majority of in sttu algae can readily be removed from water
samples by plankton nets or centrifugation, and samples can
be stored for short periods in the dark or under refrigeration.

30


OST

Algal Production

(mg/L Dry Wt.)

100

H
°

01

O Epilimnion

A Hypolimnion

Jan.

30

Tv T

T T T T
90 120 150 180 210 240
Time (Days)

Fig. 46. Algal Production - Pickerel Lake, 1973

T
270

T
300

T
330

360


Snake Lake - Several liters of autoclaved lake water (February,
1972) were treated with 10 mg Al/l using commercial liquid
alum, and the pH was raised back to 7.5 by adding NaOH.

The flocculant precipitate was settled for several days before
the supernatant liquid was decanted for analysis. Reactive-
phosphorus was determined spectrophotometrically (molybdate-
stannous chloride) on raw, autoclaved, supernatant, and
supernatant plus added phos,hate waters. Biologically avail-
able phosphorus was determined by triplicate AAP analysis on
the autoclaved, treated, treated-reconstituted, and standard
media waters. In conjunction with the AAP analyses, NO3, POy,
and Fett+ spikes were added to determine whether algal growth
would be stimulated by these nutrients. A stimulatory
response to a phosphate spike was interpreted as phosphorus
limitation in the algal culture. Algal growth was measured
fluorometrically, and was related to dry weight of organisms.

Table 35 shows that about 83% of the reactive phosphorus was
removed by A1(III) treatment and subsequent settling of the
floc. Biologically available P appeared to be reduced by an
even greater amount. While severe toxicity was not a problem
(moderate growth in supernate + phosphate); there was a re-
duction in the growth rate. The Mirror Lake data show this

Table 35. PHOSPHORUS ANALYSES
ON SNAKE LAKE WATERS IN mg/l

Reactive P
(spectro- Biologically
photometric) Available P (AAP)

Raw water 0.094 -
Autoclaved water 0.118 0.12
Supernate of treated water 0.020 0.002
Supernate + phosphate 0.110 0.058

effect also. Table 36 shows the fluorometric response of
cultures. The data show a substantial growth potential for
the autoclaved lake water (89.6 mg dry weight of organism
per liter) and algal growth was stimulated by nitrogen addi-
tions. Aluminum treatment resulted in a large decrease in

117


0.12

1 1

o ol wv
o oO

° °

1
N
i=)

°
°

0.06 F

o co o

(t/a SW) d-"Od eTaNTOS - YALYM

-_—s
_—%
“>
fa %
°°

1
i=}
ial
i=}

25

o

(eebtw Su 0oT/d-"0d bur)
d@ eTqeqoea3xg - gvDIV

0.20 F

i
wn
a

.

o

0.10 fF

Aug Sept

July
Comparison of the Soluble PO,4~P of Lake Mendota

May June

Apr

15.

Fig.

~-P

Waters and the Extractable PO,

of in situ Algae at the Outlet

92


than soluble POx-P is well documented in this laboratory.
One-day sorption tests with P-limited Cladophora indicated
that, when tested at 0.10 and 0.15 mg P/1, all of the P of
pyrophosphate (2.4% soluble ortho PO,-P) and tripolyphosphate
(1.6% soluble ortho P0,-P) was available, whereas only 0.5%
of a concentration of 1.0 mg P/1l of phosphate (0.4% soluble
ortho PO,-P) was available. Also, Cladophora was able to
obtain available P equivalent to at least 0.07 mg P/l from

a shark tooth in 1 liter of Lake Mendota water in a 2-day
sorption test.

In contrast to the increase of soluble and available P caused
by, autoclaving lake water samples containing in sttu algae,
autoclaving muddy river water samples caused a decrease of
soluble PO,-P in 11 of 12 samples. This loss in soluble PO,-P
in muddy samples is believed to be due to sorption of P by

the mud,?

The general conclusions that can be drawn about water sample
treatments are: :

Raw samples - The nutrient content of untreated stored samples
may change because of the growth of algae. Also, in situ
algae can compete with test organisms in either sorption or
growth tests. For sorption tests the effects of in sttu

algae can be removed by reducing their numbers to below com-
petitive. status by crude filtration through plankton nets

or coarse filter papers. Minimal changes appear to take

place in samples stored refrigerated in the dark.

Preserved samples - Membrane filtration will remove competi-
tive organisms to nutrition tests as well as insoluble nutri-
ents which might or might not be available nutrients. Such
treatments allow the measurement of nutrients soluble at the
time of treatment, that is, what is left in water samples
after algae and bacteria are removed. The filtration of
samples of more than a few hundred mi, however, requires
very special equipment not normal to routine laboratories.
Autoclaving causes a release of nutrients from in situ algae
and an increase in the sorption of nutrients by muds. How-
ever, the total available nutrient content of water samples
(the availability of nutrients in the water and contributed
by the death of in situ algae) can be measured after auto-
claving. Samples to be stored for long periods could be
crudely filtered to remove the majority of in situ algae or
muds and then autoclaved as a compromise of the effort re-
quired and the integrity of results.

33