EPA-660/3-75-034
JUNE 1975
Ecological Research Series
Proceedings: Biostimulation/and/Nutrient
Assessment Workshop
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Jgemj
Corvallis, Orana 97330
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RESEARCH REPORTING SERIES
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U.S. Environmental Protection Agency, have been grouped into
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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 STUDIES
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 formation, 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
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EPA-660/3-75-034
JUNE 1975
PROCEEDINGS: BIOSTIMULATION - NUTRIENT
ASSESSMENT WORKSHOP
October 16-17, 1973
Corvallis, Oregon
Sponsored by
Eutrophication and Lake Restoration Branch
Pacific Northwest Environmental Research Laboratory
National Environmental Research Center
Corvallis, Oregon
Program Element 1BA031
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For Sale by th^ National TechnTcaF Information Service,
U.S. Department of Commerce, Springfield, VA 22151
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ABSTRACT
The workshop was held to bring together those investigators in
the Environmental Protection Agency who are actively engaged in re-
search relating to biostimulation and nutrient assessment to present
the results of their studies. The papers presented were concerned
with the results of algal assays conducted on various waters and
wastes to determine their biostimulatory effects as well as the re-
sults of other research relating to the assessment of nutrients and
their effects on the aquatic ecosystem.
This report was submitted by the Pacific Northwest Environmental
Research Laboratory of the Environmental Protection Agency. The
conference was held October 16-17, 1973.
11
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CONTENTS
Page
RESEARCH PERTAINING TO DETERMINATION OF ATP IN SOILS AND SUBSURFACE 1
FORMATIONS
William R. Duffer, William J. Dunlap, and James McNabb
THE EFFECT OF MASS TRANSPORT ON BIOSTIMULATION OF ALGAL GROWTH 4
James W. Falco, Pat C. Kerr, Michael B. Barren, and
Donald L. Brockway
TOXICITY OF ZINC TO THE GREEN ALGA SELENASTRUM CAPRICORNUTUM 28
AS A FUNCTION OF PHOSPHORUS OR IONIC STRENGTH
Joseph C. Greene, William E. Miller, Tamotsu Shiroyama, and
Ellen Merwin
ALGAL ASSAYS FOR THE NATIONAL EUTROPHICATION SURVEY 44
Albert Katko
FREQUENCY ANALYSIS OF CYCLIC PHENOMENA IN FLOWING STREAMS 53
Pat C. Kerr, James W. Falco, R. Marie Stead, and
Donald Brockway
THE EFFECT OF HIGHER TROPHIC LEVEL COMPONENTS IN AN AQUATIC 87
ECOSYSTEM MODEL
Ray R. Lassiter
THE USE OF ALGAL ASSAYS TO DETERMINE EFFECTS OF WASTE DISCHARGES 113
IN THE SPOKANE RIVER SYSTEM
William E. Miller, Joseph C. Greene, Tamotsu Shiroyama, and
Ellen Merwin
EFFECT OF NITROGEN AND PHOSPHORUS ON THE GROWTH OF 132
SELENASTRUM CAPRICORNUTUM
Tamotsu Shiroyama, William E. Miller and Joseph C. Greene
THE USE OF IN SITU ALGAL ASSAYS TO EVALUATE THE EFFECTS OF 143
SEWAGE EFFLUENTS ON THE PRODUCTION OF SHAGAWA LAKE
PHYTOPLANKTON
Paul D. Smith
THE DEVELOPMENT OF A STANDARDIZED MARINE ALGAL ASSAY PROCEDURE 174
FOR NUTRIENT ASSESSMENT
David T. Specht and William E. Miller
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CONTENTS (CONTINUED)
Page
GROWTH REQUIREMENTS OF ENTEROMORPHA COMPRESSA AND 213
CQDIUM FRAGILE
Richard L. Steele
GREAT LAKES NUTRIENT ASSESSMENT 226
Nelson A. Thomas, Katherine Hartwell, and William E. Miller
ASSESSING TREATMENT PROCESS EFFICIENCY WITH THE ALGAL 244
ASSAY TEST
Richard E. Thomas and Robert L. Smith
UTILIZATION OF ENERGY BY PRIMARY PRODUCERS IN FOUR PONDS 249
IN NORTHWESTERN FLORIDA
Gerald E. Walsh
HETEROINHIBITION AS A FACTOR IN ANABAENA FLOS AQUAE BLOOM 275
PRODUCTION
Llewellyn R. Williams
CONTRIBUTORS 318
IV
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RESEARCH PERTAINING TO DETERMINATION OF ATP
IN SOILS AND SUBSURFACE FORMATIONS
William R. Duffer, William J. Dunlap
and James McNabb
The National Water Quality Control Research Program and the
National Ground Water Research Program at the Robert S. Kerr
Environmental Research Laboratory are concerned with the determi-
nation of ATP as a measure of bioactivity in soils and subsurface
formations. Such measurements are needed in investigations
pertaining to land application of waste waters and degradation of
ground water.
Lee e_t_ aj_ have developed a method for ATP determination that
is moderately successful for sediments and surface soils.'' ' This
method involved acid extraction of soil or sediment samples, treat-
ment of the extract with cation exchange resin, and determination
of ATP in the extracts by the 1 uciferin-1 uciferase bioluminescence
assay. The effect of substances in the extract which interfere
with the ATP-luciferin-1 uciferase reaction is reduced or eliminated
by dilution. However, our preliminary investigations indicate that,
because of the presence of high levels of interfering substances
in extracts of many terrestrial materials, the Lee method lacks the
sensitivity required for meaningful analysis of low activity
samples, such as those originating in subsurface regions or surface
regions receiving noxious wastes.
-------
Present research efforts have been directed toward improvement
of the sensitivity of the luciferin-luciferase bioluminescence assay
for ATP in terrestrial materials. We have attempted to develop
relatively simple and rapid column chromatographic procedures for
purification of ATP in acid extracts of soil materials. Acid
solutions of ATP uniformly labeled with 14c were chromatographed on
columns containing various chromatographic stationary phases. ATP
in eluate fractions was detected and quantitated by liquid scintil-
lation spectrometry. Cation exchange, molecular exclusion chroma-
tography, and ion retardation were employed in efforts to retain
interfering substances on the column while rapidly eluting ATP.
These efforts were unsuccessful because of failure to achieve
satisfactory separation of ATP from impurities on reasonably sized
columns or because of unacceptable loss of ATP due to sorption on
the columns. Chromatographic procedures employing strongly and
weakly basic anion exchange resins, neutral polymeric adsorbents,
and ion retardation resin were utilized in efforts to rapidly elute
interfering substances while retaining ATP on the columns, with
subsequent elution of purified ATP. Procedures employing anion
exchange resins and neutral polymeric adsorbents were generally
unsuccessful due to very strong sorption of ATP on the columns.
Such sorption resulted in the need for unacceptably lengthy or
drastic procedures for ATP elution. ATP was found to be effectively
retained on Bio Rad AG 11 A8 ion retardation resin when applied to
-------
the column in 0.6 N ^$04 solution and during subsequent water
elution, and appears to be relatively easily eluted from such
columns by 0.2 M to 1 M NH4C1 and Tris Hydrochloride* Experiments
in our laboratories indicate that NH4C1 and Tris HC1 in concentra-
tions required for ATP elution from the ion retardation resin
interfere to some extent with the luciferin-luciferase reaction.
Nevertheless, this procedure appears to offer some promise for
improving the sensitivity of ATP analysis in terrestrial material,
and is being further investigated.
Doxtader has developed a butanol extraction method for ATP
in soils.'2' This may give a cleaner extract which may be more
easily purified for greater sensitivity. We are awaiting details
of this method and will examine it experimentally if our present
avenue of approach is blocked or if it appears more likely to
provide the simplicity and sensitivity desired.
REFERENCES
1. Lee, C. C., R. F. Harris, J. D. H. Williams, D. E. Armstrong, and
J. K. Syers. Adenosine Triphosphate in Lake Sediments: I.
Determination. Soil Sci. Soc. Amer. Proc. 35:82-86. 1971.
2 Ausmus, B. S. The Use of the ATP Assay in Terrestrial Decomposition
Studies. Bull. Ecol. Res. Comm. (Stockholm) 17:223-234. 1973.
*Tris (hydroxymethyl) aminomethane hydrochloride
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THE EFFECT OF MASS TRANSPORT ON BIOSTIMULATION OF ALGAL GROWTH
James W. Falco, Pat C. Kerr, Michael B. Barron
and Donald L. Brockway
INTRODUCTION
The role of mass transport in regulating nutrient availability
and the concomitant effect on biological activity has received little
emphasis in ecological studies even though this process could be
the over-all rate limiting step in many natural ecosystems. Data
indicating the importance of mass transport dates to work reported
by James1 in 1928. The growth rate of aquatic plants was stimulated
by the increased availability of carbon resulting from either an
increased flow rate of the medium or an increased rate of molecular
diffusion. Myers'2 data indicate the presence of resistance to
diffusion of C02 between the atmosphere and the cell wall of the
green alga, Chlorella pyrenoidosa. Similar results were reported
o
by Kerr et a!0 for the blue-green alga, Anacystis nidulans. This
diffusion resistance in turn regulated the growth of both algae.
Diffusion resistance to oxygen and/or organic substrates has
been reported for yeast4 and bacterial films5>6'7'8'9. Jernelov and
Lann10 reported that shaking increased the rate of biological
methylation of mercury in both eutrophic and oligotrophic lake
sediments. Thus diffusion resistance and turbulence are important
in regulating the transport of any material to or from the cell.
Biological uptake is generally mediated, or catalyzed, by surface
enzymes, which have different kinetics for the uptake of different
materials. Organisms vary widely in size and shape and thus in
surface to volume ratio. It should be possible to apply to such
biological systems certain of the quantitative theories governing
the effectiveness of catalyst mass. These mathematical expressions
may then be incorporated into ecosystem (or organism) models.
In this paper, a model of the uptake of major nutrients limited
by a diffusional resistance around an algal cell is proposed. A
series of experiments to demonstrate these limitations is discussed.
4
-------
Finally, a model for internal resistance that may limit uptake by
colonial algae (and aggregated algal cells) is also developed.
Also treated is the case of a single limiting nutrient diffusing
independently of other material gradients with a non-linear reaction
uptake step at the cell wall boundary.
The mathematical formulations used are analogous to those used
by Toor11 and Hudson12 in solving transient concentration profiles
caused by step changes in boundary conditions of coupled reacting
and diffusing materials. The formulations differ in geometry from
those used by Toor and Hudson.
The formulations used in the aggregate model to describe diffu-
1 3
sion into a mass of cells are similar to those developed by Thiele
to describe the diffusion into and reaction occurring inside catalyst
grains.
The solutions developed are not restricted by the requirement of
commutativity of diffusivity and rate coefficient matrices as are
previous solutions by Wei14, but can be applied to any set of coupled
constituents.
EXPERIMENTAL
To demonstrate the effect of mass transfer limitations on
uptake of major nutrients and growth rate of algae, a series of
comparative experiments was undertaken.
Chlorella pyrenoidosa was cultured in pH 5.5 Benson-Fuller
nutrient solution containing one mt/l of Hunter's trace element
solution. The cultures were grown at 22°C under 2000 fc white light
(a cool white fluorescent plus incandescent source) in a water-cooled
reciprocating shaker. Cultures were gassed with 1, 3, or 57= C02
in air (Linde research grade gases) and were shaken at 0, 17, or
33 rpm with an amplitude of 9.4 cm. Capillary tubing or 27-gauge
hypodermic needles, or both, were used to meter and equalize the
o-as flow to cultures.
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Aliquots were removed aseptically from each culture at zero
time and at 24-hour intervals thereafter. The algae were enumerated
by direct microscopic counts at 1500 X using a Petroff-Hauser
Bacteria Counter. The cultures were checked for bacterial contamina-
tion daily by direct microscopic examination. Any contaminated culture
was discarded.
MODEL FOR EXTERNAL MASS-TRANSFER RESISTANCE TO NUTRIENT UPTAKE
In modeling the uptake of nutrients by an algal cell, we
separated the cell/nutrient solution system into three zones.
First, throughout the bulk of the liquid surrounding the cell,
material is turbulently mixed; consequently the nutrient concentra-
tions are uniform in this first zone. Secondly, in a narrow zone
adjacent to the cell wall, there is no convective mixing and thus
transport of materials is by diffusion only (Figure 1). This region
BOUNDARY LAYER/BULK LIQUID
INTERFACE
CONCENTRATION AT
CELL WALL (C0)
BOUNDARY
LAYER
BULK LIQUID (C,)
Fig. 1. Model of nutrient diffusion into an algal cell.
-------
is called the boundary layer. Lastly; at the liquid/cell-wall
interface, removal of nutrients from the boundary layer proceeds
by a kinetic uptake step.
Nutrient transport within the three zones may be described by
two boundary conditions and one ordinary differential equation:
.Jf° - \~/ I _ (-1 p
^ dr f dr J ~ U' R°
(C) = (d), r = R! (2)
A [D] ^&- = V(R), r = R0 (3)
where A = surface area of the cell (pm2)
(C) = column matrix of nutrient concentrations (g-atom/pirf)
(d) = column matrix of nutrient concentrations in the
bulk liquid (g-atom/pm )
[oj = matrix of diffusion coefficients (p,m /sec)
r = distance from the center of an algal cell (pm)
R! = distance from the center of the cell to the boundary
layer-bulk liquid interface (pm)
R0 = radius of an algal cell (pm)
(R) = column matrix of specific uptake rates (g-atom/pm sec)
V = volume of an algal cell (pm0)
Steady-state uptake during a cell cycle has been assumed in order
to obtain equation (1), which is a steady-state mass balance. All
three equations have been written in matrix form to include the
possibility of nutrient uptake coupling between various nutrients
either in the diffusion term or in the kinetic uptake term in
equation (3).
The only unknown in the three equations is the nutrient uptake
matrix in equation (3). If one assumes pseudo-first order uptake
during a cell cycle, the rate of uptake would be proportional to
the concentration present at the cell wall. The justifications for
-------
making such an assumption are (1) the actual non-linear kinetic
terms can be linearized to give an apparent first-order rate, and
(2) an analytical solution that permits clear discrimination of
coupling effects can be obtained. If the uptake rate is assumed
to be
(R) = [K] (c) (4)
where [K] = matrix of rate coefficients (sec"1), the solution to
equations (1), (2), and (3) is
where [ij = identity matrix.
As a test case, the boundary layer concentration profiles for
Cyclotella nana, a diatom for which published data on phosphorus
uptake rates are available15, were calculated. Assuming that the
effective radius of an algal cell is 2.65 [m and the boundary layer
thickness is approximately 2.65 pm, various profiles result depending
on Y, the ratio of D to K, as shown in Figure 2. The scalar form of
equation (5) has been assumed, and thus coefficients are the scalar
diffusion coefficients of phosphorus in water and the scalar specific
uptake rate of phosphorus by C_. nana. This would occur if phosphorus
uptake is limiting and completely uncoupled from the rates of trans-
port and uptake of other materials.
If we assume that phosphorus concentration is below the half
saturation constant, t^, the rate coefficient, K, can be estimated
as
V
i
(6)
K = 2.
m
where V = maximum uptake rate
K = half-saturation constant
-------
3.174 3.705 4.232
FIGURE 2
BOUNDARY LAYER CONCENTRATION PROFILES AS A FUNCTION
OF RADIUS AND RELATIVE UPTAKE RATE (D .
-------
For phosphorus uptake by £. nana K is reported by Fuhs15 to be
53.1 sec"1 where uptake rate has been converted to g-atoms per
unit volume of cell per sec. If the diffusion coefficient is
assumed to be 100 Mm2 /sec (a reasonable estimate), Y is 1.88 pin3.
Referring to Figure 3, this corresponds to an effective reduction in
phosphorus uptake of 4070 as compared to the case of no diffusional
resistance. This is a minimum figure for reduction in uptake
rate since it has been assumed that the entire surface area is avail-
able for nutrient uptake. In fact, only a portion of the surface
occupied by enzyme sites is actually involved. More serious limita-
tions on nutrient transport would therefore exist than is indicated
by the above calculations.
If uptake rates at higher concentrations are considered, non-
linear Michaelis-Menten kinetics must be used, i.e.,
m
In this case the form of the solution to equations (1), (2),
and (3) is the same as in the linear case but the solution is more
complex. Equation (5) becomes
1 1 ' B + d (8)
where
B =
m ( ° 1\ / m x\
3D
v RI ) v
0 RO - RI
r, 3W
KO RI
Ro3 '
r Vm /R°'Ri\ /Km + Cl\-1
OT-> \TJ / \ D ^ / J
L JL) Kj^ KQ -1
0 Ro - RI
R 3R
K0 K]_
VO TJ "D
U . x-K-O -^-1 \
m 1 ( }
""" " 3n VR 2R /
JU KO KI
(9)
10
-------
100
o
os w
w o
O H
H (/)
IH
Q tfl
W W
W
H fa
(X, H
7.0
FIGURE 3
REDUCTION IN UPTAKE AS A FUNCTION OF DIFFUSIONAL RESISTANCE
-------
Figure 4 shows a graphical means of estimating B as well as an
illustration of nutrient uptake as function of external concentration.
The slanted lines represent the relationship between the nutrient
concentration in the bulk liquid, nutrient concentration at the cell
surface, and the resulting rate of diffusion. To find the cell surface
concentration for a given bulk concentration and diffusion rate, find
the slanted line whose X intercept corresponds to the bulk concentration.
Then follow the slanted line upward to the point at which the Y
coordinate equals the diffusion rate. A perpendicular dropped from
that point to the X axis indicates the cell surface concentration.
The curved line represents the rate of uptake by the cell as
a function of concentration at the cell wall. As stated by equation
(3), at steady state the rate of uptake must equal the rate of
diffusion or transport to the cell wall. The superimposed graphs of
diffusion and uptake therefore represent a graphical solution to
the equation. If, for example, the bulk phosphorus concentration is
2.3 X 10~21 g-atom/iW3 , the point at which the rate of diffusion
equals the rate of uptake may be found by following the appropriate
slanted line to its intersection with the curved uptake line. The X
coordinate of the point is the resulting cell wall concentration (C0)
(2.02 X 10~21) and the Y coordinate is the resulting rate of diffusion
and uptake (0.78 X 10~14 g-atom/hr cell), which is equal to B/R03.
Since the rate of uptake approaches a limiting value (V )
as cell wall concentration increases and the diffusion rate is
linearly proportional to the concentration difference (C^-Co),
diffusion limitations become less important as nutrient con-
centration increases. Figure 5, showing the ratio of cell wall
concentrations (C0) to bulk concentrations (Cx) as a function
of bulk concentration, illustrates this point.
12
-------
1.2
1.0
w
-------
1.0
0.8
0.6
o
0.4
0.2
I
J
3.0
0 1.0 2.0
Cx [('g atom/pm3) X 10"21]
FIGURE 5
RELATIVE CONCENTRATION OF PHOSPHORUS AT THE CELL WALL COMPARED TO BULK CONCENTRATION
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A MODEL FOR INTERNAL MASS TRANSFER RESISTANCE
TO NUTRIENT UPTAKE IN CELL AGGREGATES
In natural ecosystems, algal cells often aggregate into colonies.
In such aggregates, nutrients must diffuse past the outer layer of
cells before being taken up by the innermost cells. In modeling
nutrient uptake in a colonial system, it is assumed that the aggre-
gate acts as a homogeneous sphere that absorbs nutrients as they
diffuse to the center of an effective sphere. It is further assumed
that external resistance to movement of material is small compared
to internal diffusional resistance. Thirdly, it is assumed that the
packing of spherical algae cells within an aggregate is optimal;
and thus for a large number of cells within an aggregate, the volume
fraction of cells is 0.707.
Including the above assumptions, the following equations des-
cribe steady-state uptake of nutrients over a single cell cycle
[K] (C) = 0 , r SR0 (10)
(C) = (C0), r = Ro (11)
r-0
where Vf = volume fraction of algae.
We further assumed that linear kinetics describe the uptake of
nutrients, and full coupling of constituents is permitted.
The solution to this set of equations is
(C) = ° Sinh {Vf
sinh {V; [D]-1 [K]} RO (C0) (13)
Figure 6 shows this solution for the scalar form of equation (13),
which would be applicable to the case of a single limiting nutrient
with no diffusional coupling.
15
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cr>
1.0
0.8 -
0.6
olu
0.4 -
0.2 -
0.0
Y - 0
2.0 2.4
Figure 6
CONCENTRATION PROFILE WITHIN AN ALGAE CELL AS A FUNCTION
OF RADIUS AND RELATIVE UPTAKE RATES (D KV
-------
Now, the rate of nutrient uptake at steady-state must be equal
to the rate of diffusion of nutrients into the aggregate
coth {{V [D]-1!*])* R0} - 3 [°][BK]| (Co) (14)
"- i > v _f KQ j
The rate of uptake that would occur if there were no diffusional
limitations is simply
R = | TT Ro3 V/ [Kj (C0) (15)
Thus the relative rates of uptake can be related to the maximum
uptake rate by a matrix of efficiency coefficients,
PI] -
coth {{[D?1 Vf [K]}% R0} - [I]} (16)
f
which is valid for any matrices [Dj and [Kj . Figure 7 shows the
relationship between efficiency and y* the ratio of D to K, for
the scalar case of a single limiting nutrient and a single algal cell
of 5.29 |_un diameter. If we assume an uptake coefficient, K, for
phosphorus of 53.1 sec"1 and a diffusion coefficient, D, of 1QO |im2 /
sec, y i-s equal to 1.88 and the relative uptake rate is approximately
807o of the uptake rate with no diffusion resistance. Table 1 shows
the effect of increasing the number of algae cells in a cluster assuming
the same properties of the algae aggregate as for the single cell.
17
-------
1.0
w
0.4 -
w 0.6 -
g
tn
tu
M 0.2
0.0
1.0
2.0
3.0
4.0
5.0
Figure 7
INTERNAL EFFICIENCY AS A FUNCTION OF
RELATIVE UPTAKE RATE (D
TABLE I
REDUCTION IN UPTAKE EFFICIENCY
WITH INCREASING SIZE OF AGGREGATE
Number of Cells
Across the Diameter
of a Cluster
T] (efficiency)
1
3
5
10
.562
.411
.174
18
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RESULTS AND CONCLUSIONS
Figures 8, 9, 10, and 11 show the growth of C_. pyrenoidosa
at three concentrations of C02 and three shake rates. Higher
growth rates are attained at the high shake rates in every case.
Table 2 lists the ranges of variances in each experiment. No
overlap in variance exists for any of the compared data points,
indicating significant statistical difference in any pair of curves.
Table 3 lists the number of doublings at each shake rate divided
by the number of doublings obtained at the same COg concentration
but at zero shake rate at the end of a 72-hour growth period. A
three to four-fold stimulation of biological activity is indicated
for cultures grown at a shake rate of 17 rpm versus cultures grown
in still vessels. No further stimulation occurred when the shake
rates were increased to 33 rpm. Apparently the major boundary layer
resistances are eliminated at the lower shake rate.
In interpreting the results of these experiments it is necessary
to develop some criterion for quantitating the level of turbulence
in the culture. Such a measure is a Reynolds number, which is
defined as
N.
average velocity of shaking X a characteristic
dimension x density of fluid
Re viscosity of fluid
If an effective dimension of 2.5 cm (the depth of the culture)
is used, the Reynolds numbers corresponding to the two shake rates
in the experiments are 1930 and 3746 respectively. It would be
difficult to extrapolate these data to comparable Reynolds numbers
in the field since turbulence is induced by oscillating motion
at extremely low Reynolds numbers and since boundary layer thickness
is probably more dependent on absolute velocity rather than Reynolds
number. However, Fuhs15 has reported significant differences between
phosphorus uptake in shaken laboratory cultures and uptake in lakes
at comparable concentration. This observation coupled with our
19
-------
X
CO
f-l
T-l
-------
4J
o
II
X
CO
i-<
iI
-------
X
00
V
CJ
§
u
§
0
24 48
TIME (HRS)
FIGURE 10
CONCENTRATION OF ALGAE AS A FUNCTION OF TIME
(C02 CONG = 3%)
22
-------
X
co
S
H
I
U
O
1
0
24 48
TIME (MRS)
72
FIGURE 11
CONCENTRATION OF ALGAE AS A FUNCTION OF TIME
(C02 CONG = 5%)
23
-------
TABLE 2
RANGES OF COEFFICIENTS OF VARIATION OF AVERAGED
DATA POINTS FOR COMPARATIVE EXPERIMENTS
Carbon Dioxide Concentration
Shake Rate (RPM)
0
17
17,
4-19%
9-31%
37o
2-327,
20-617,
57,
5-347o
22-627,
TABLE 3
RELATIVE INCREASE IN ALGAL GROWTH RATE
COMPARED TO ZERO SHAKE RATE DURING A 72-HOUR PERIOD
Carbon Dioxide Concentration
Shake Rate (RPM)
17
33
17o
4.2
4.0
37o
2.8
5%
3.2
24
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results indicates that large diffusional resistances can limit uptake
of nutrients in natural aquatic ecosystems.
The data collected thus far are insufficient to determine
boundary layer thickness and overall mass transfer resistance shown
in the mathematical development of the external diffusion resistance
model. The limited data indicate that, as predicted by the model,
the effect of turbulence on increasing cell growth is less at higher
C02 concentrations. As more data are obtained at intermediate
shake rates and at lower nutrient concentrations, accurate esti-
mates of boundary layer thickness (Rx R0) and mass transfer
resistance D/K will be obtained.
SUMMARY
A model is proposed to account for mass transport limitation
to the uptake of major nutrients. A thin film diffusion resistance
layer is hypothesized. In an example using Cyclotella nana (a
diatom), diffusion resistance was calculated to result in a maximum
decrease of 407= in phosphorus uptake rate. Transport limitation
was demonstrated experimentally. The growth of Chlorella pyrenoidosa
(a green alga) was found to be stimulated by shaking the cultures.
Using similar mathematical techniques, the internal resistance
to the uptake of nutrients in algal cell aggregates was also modeled.
25
-------
REFERENCES
1. James, W. 0. The Effect of Variations of Carbon Dioxide Supply
upon the Rate of Assimilation of Submerged Water Plants. XIX.
In: Experimental Researches on Vegetable Assimilation and
Respiration. Proc. Roy. Soc. CIII-B;l-42, 1928.
2. Myers, J. The Growth of Chlorella pyrenoidosa under Various
Culture Conditions. Plant Physiol. _l£:576-589, 1944.
3. Kerr, Pat C., Doris F. Paris, and D. L. Brockway. The Inter-
relation of Carbon and Phosphorus in Regulating Heterotrophic
and Autotrophic Populations in Aquatic Ecosystems. U. S.
Environmental Protection Agency, Washington, D. C. 16050 FGS.
July 1971. 53p.
4. Borkowski, J. D., and M. J. Johnson. Experimental Evaluation
of Liquid Film Resistance in Oxygen Transport to Microbial
Cells. Applied Microbiol. 15:1483-1488, 1967.
5. Kalinske, A. A. Effect of Dissolved Oxygen and Substrate
Concentration on the Uptake Rate of Microbial Suspensions.
Journal WPCF. 43_: 73-80, 1971.
6. Gulevich, W., C. E. Renn, and J. C. Liebman. Role of Diffusion
in Biological Waste Treatment. Env. Sci. Tech. 2^:113-119, 1968.
7. Mueller, J._A., W. C. Boyle, and E. N. Lightfoot. Oxygen
Diffusion through Zoogloeal Floes. Biotech, and Bioeng. X:
331-358, 1968.
8. Hartmann, L. Influence of Turbulence on the Activity of
Bacterial Slimes. Journal WPCF. 3£:958-964, 1967.
9. Whalen, W. J., H. R. Bungay III, and W. M. Sanders III.
Microelectrode Determination of Oxygen Profiles in Microbial
Slime Systems. Environ. Sci. Technol. .3:1297-1298, December
1969.
26
-------
10. Jernelov, A., and H. Lann. Studies in Sweden on Feasibility
of Some Methods for Restoration of Mercury-Contaminated
Bodies of Water. Env. Sci. Tech. _7: 712-718, 1973.
11. Toor, H. L. Dual Diffusion-Reaction Coupling in First
Order Multicomponent Systems. Chem. Engrg. Sci. 20:941,
1965.
12. Hudson, J. L. Transient Multicomponent Diffusion with
Heterogeneous Reactions. Amer. Inst. Chem. Engng. J.
13.: 961, 1967.
13. Thiele, E. W= Relation between Catalytic Activity and
Size of Particle. Ind. Eng. Chem. 3_1:916, 1939.
14. Wei, J. Intraparticle Diffusion Effects in Complex Systems
of First Order Reactions. I. The Effect in Single Particles.
Journal of Catalysis. 1_:526, 1962.
15. Fuhs, G. W. , Susanne D. Demmerle, E. Canelli, and M. Chen.
Characterization of Phosphorus-Limited Plankton Algae.
In: Nutrients and Eutrophication: The Limiting-Nutrient
Controversy, Likens, G. E. (ed.). Lawrence, Allen Press,
Inc., 1972. p. 113-133.
27
-------
TOXICITY OF ZINC TO THE GREEN ALGA
SELENASTRUM CAPRICORNUTUM AS A FUNCTION OF
PHOSPHORUS OR IONIC STRENGTH
Joseph C. Greene, William E. Miller,
Tamotsu Shiroyama, and Ellen Merwin
INTRODUCTION
Direct evaluation of waste toxicity through biological assay, involving
the use of living organisms as water quality indicators, often is
necessary in addition to any chemical examination. Kahn (1964) advocated
the use of algae as water quality indicators and suggested the further
development of the algal bioassay. In February 1969, a group consisting
of government, university and industrial laboratories began research to
develop a reliable and reproducible algal assay. As a result of this
work, the Algal Assay Procedure Bottle Test (US EPA, 1971) was developed
and has been refined sufficiently to be offered now for wider use in
connection with eutrophication and other related algal production problems.
The test has been used to determine the effects of secondary and
tertiary wastewater effluents in lakes and river systems (Miller,
et. al., 1971) as well as to determine growth limiting nutrients in
natural waters (Maloney, et. al., 1972). In addition, it has been
used to evaluate the effect of various waste treatment processes on
the amount of algal growth in treated effluent and to determine
growth limiting nutrients in each type of effluent.
The study described herein is concerned with evaluating the ability of
the test to identify the potential toxicity of heavy metals in a
defined inorganic medium.
METHODS
The Algal Assay Procedure Bottle Test (US EPA, 1971), with modifications,
was used in the study. The test alga was Selenastrum capricornutum Printz.
28
-------
Modifications included the following: (1) In addition to the zinc
nutrient requirement of the test medium (Table I), zinc spikes were
added (as ZnCl2) in increasing 10 ug/1 increments. Three replicate
flasks containing algal assay mediurn without zinc additions served as
controls and one flask was prepared for each elevated zinc concentration;
C2) each flask was inoculated to give a final concentration of 10,000
cells/ml from a seven-day-old culture of S_. capricornutum; (3) assays
were carried out in 500 ml polycarbonate Erlenmeyer flasks containing
100 ml of total sample; and (4) all polycarbonate Erlenmeyer flasks
and laboratory glassware used for test preparation and algal culture
were leached of heavy metal contaminants with a 10 percent solution of
nitric acid.
EXPERIMENTAL DESIGN
Zinc-Phosphorus Interactions
The following tests were designed to determine if a relationship existed
between zinc and phosphorus concentrations, in a defined inorganic
medium, that would affect the 14-day maximum growth yield of S_.
capricornutum. One-fourth strength algal assay medium containing 0.047
mg P/l was prepared and dispensed into seven one-liter graduated
cylinders. Phosphorus (as K^ H PO,) was added to each of the seven
cylinders to give final concentrations of 0.047, 0.093, 0.139, 0.186,
0.279, 0.372 and 0.465 mg P/l. To another set of eight cylinders,
one-half strength medium, also containing 0.047 mg P/l, phosphorus was
added to give final concentrations of 0.047, 0.093, 0.139, 0.186, 0.372,
0.558, 0.774 and 0.930 mg P/l. This resulted in 15 distinct media--
phosphorus test substrates into which selected concentrations of zinc
were added.
29
-------
CO
o
Element
(yg/i)
N
P
Mg
S
C
Ca
Na
K
B
Mn
Zn
Co
Cu
Mo
Fe
Cl
EOT A*
100%
4200.000
186.000
2904.000
1911.000
2143.000
1202,000
11001.000
469.000
32.460
115.374
15.691
0.354
0.004
2.878
33.051
6587.000
300.000
AAM
75%
3150.000
139.000
2178.000
1433.000
1607.000
901.000
8250.000
351.000
24.345
86.530
11.768
0.265
0.003
2.158
24.788
4941.000
225.000
Concentration
50%
2100.000
93.000
1452.000
955.000
1071.000
601.000
5500.000
234.000
16.230
57.687
7.845
0.177
0.002
1.439
16.525
3294.000
150.000
25%
1050.000
46.000
726.000
477.000
535.000
300.000
2750.000
117.000
8.115
28.843
3.922
0.088
0.001
0.719
8.262
1647.000
75.000
12.5%
525.000
23.000
363.000
238.000
267.000
150.000
1375.000
58.000
4.057
14.421
1.961
0.044
0.000
0.359
4.131
824.000
37.000
*Sodium Salt
Table I. Chemical composition of the algal assay culture media.
-------
Zinc-Ionic Strength Interactions
In order to determine what relationship existed between zinc and ionic
strength and their combined effect on the 14-day maximum yield of SL
capricornutum, the following tests were designed. Algal assay medium
(100 percent) and four dilutions, resulting in ionic strength of 12.5,
25, 50, and 75 percent, were prepared (Table I). In these media,
phosphorus, the primary limiting nutrient, increased proportionately
with the increase in ionic strength. Four additional nutrient media
were prepared which ranged in ionic strength from 25 to 100 percent of
the normal algal assay medium. Each of these media contained 0.047
mg P/l.
DISCUSSION AND RESULTS
Heavy metals are normally found in the environment and some perform
critical biological functions. As by-products of various industrial
processes, however, these substances are likely to become associated
with biological materials in toxic concentrations. The state of
chemical combination of a pollutant determines its chemical behavior
in the aquatic environment. Ionic species, such as heavy metals, tend
to be either held in solution or establish solubility or ion exchange
equilibria with suspended materials. The introduction of heavy metals
into receiving waters and their effects on the biota depend upon a
variety of complex responses governed by several basic factors: (1)
nature of the metal; (2) heavy metal concentration; (3) environmental
characteristics of the receiving system; (4) presence of other
toxicants; and (5) exposure time.
Little is known about the role of zinc in phytoplankton production.
Only scant information is available concerning those concentrations
which most efficiently stimulate growth and those which are most toxic.
31
-------
Zinc is an essential micro inorganic element required in trace amounts
for algal growth (Wiessner, 1962; Walker, 1954). Under conditions of
elevated concentration, however, the normal enzyme-zinc mechanisms may
be significantly altered, giving rise to toxicity. Antagonistic or
synergistic interactions may also alter zinc toxicity. Ernst (1968)
reported a negative correlation in Thlaspi alpestre spp calaminare
between intercellular zinc accumulation and the phosphate concentration
in the medium. Furthermore, Whitton's (1970) studies on the effects
of zinc, copper and lead to Chlorophyta from receiving waters cultured
in artifical nutrient medium, suggested that a medium, rich in nutrients
such as phosphorus, may lower the toxic effects of heavy metals.
In fourteen bioassays, conducted in 25 percent AAM containing ortho-
phosphorus concentrations ranging from 0.047 to 0.465 mg/1, 40.4 ± 9.9
yg Zn/1 were required to produce ^95 percent inhibition of the 14-day
maximum yield (>_ 95 percent I,,) of S_. capricornutum (Figure I). These
data suggest that phosphorus did not significantly alter the level of
zinc needed to produce >_ 95 percent I,, of the test al-ga. Furthermore,
a twenty-fold increase in orthophosphorus concentration in 50 percent
AAM also failed to alter the level of zinc necessary to produce > 95
percent 1^ of the test alga (Figure II). The results of these tests
indicate that a statistically significant effect of phosphorus upon
zinc's toxicity to S_. capricornutum does not exist. However, the
average concentration of zinc producing _> 95 percent I,, in 25 and 50
percent AAM was shifted from 40.4 to 68.0 yg/1. respectively. This
decrease in zinc toxicity, observed during the study of zinc-phosphorus
interactions, led us to consider ionic strength as a major factor
responsible for the toxicity of zinc upon the growth of S_. capricornutum.
An important part of the chemical environment of a bioassay procedure
is the relative concentration of related ions (i.e., Na, K, Mg, and Ca)
32
-------
10
10
xlO
o»
2
I
10
10
-i
IOT
i I I I I I I I I I I I
I I I I I I I I I
25% ALGAL ASSAY MEDIUM
CONTAINING-
.047 mg P/L
.093
.186
.465
III:
o
- o:
o
o
i i i r i
i
i
10 20 30 40 50 60 70 80 90 100 110
jug ZINC / L
FIGURE I. Graph showing the effect of zinc on the
14-day maximum yield of S_. capricornutum
cultured in 25% algal assay medium.
-------
10
10
10
I
h-
10
10
-I
: I I I I I II I I I
I I I I I I I I I I I I I
50% ALGAL ASSAY MEDIUM
CONTAINING-
.047 mgP/L
.093
.186 "
.558
.930
20 30
40 5O 60
,pg ZINC/L
70 80 90 100 110
FIGURE II.
Graph showing the effect of zinc on the
14-day maximum yield of S_. capri cornutum
cultured in 50% algal assay medium.
-------
present in the medium. This may be in part due to the effect of ionic
strength upon the efficiency of solute uptake and perhaps water loss
(i.e., osmo-regulation) within the algal cells (Cairns, et al., 1972).
In addition to the direct osmotic effects on S_. capricornutum, total
salts play an important role in influencing the distribution of the
chemical species present. Any chemical reactions which are dependent
on mass law equilibria will be dependent on the total ionic strength
of the solution. Changing the total salt concentration will change
the ionic strength which, in turn, decreases the effective concentration
of the zinc present in solution. Because heavy metals can be bound to
varying degrees by ligands and particles, absolute concentration of a
metal does not necessarily mean that amount which has an effect on the
test alga. In Figure III the zinc concentration required to achieve
similar inhibitory growth effects (>_ 95 percent I,-) increased with
the increase of specific conductance (ionic content) of the test
substrates. Figure IV is a three-dimensional response surface which
illustrates the increased requirement for zinc (ug/1) in medium of
increasingly greater specific conductance (ionic strength). Normal-
ization of the data shown in Figures III and IV, obtained by dividing
the average concentration of zinc required to produce ^> 95 percent I,.
by the micrograms per liter of total ions in the medium (Figure V),
showed that a constant percentage (0.0056) of zinc relative to total
ion content was responsible for >^ 95 percent I,. in test substrates
ranging between 7.9 to 61.0 umhos/cm conductivity.
The effect of increasing specific conductance can also be seen in
Figure VI. However, the maximum algal growth potential in these media
was limited by purposely adding only 0.047 mg orthophosphorus/1. This
was done to determine if biogenic sites were acting simultaneously with
increased specific conductance to decrease the effective concentration
of zinc required for >_ 95 percent I,, as shown in Figure III. An
35
-------
10
10
10
_ \
- N
- \
10
10
-I
I i i in i i ii i
ALGAL ASSAY MEDIUM
umhos/cm
61.0
45.8 _.---
3O.5
15.3
7.9 _.._.._..- 12.5
% AAM
. IOO.O
_ 75.0
. 50.O
- 25.0
1 \
j
_
1 I
i
I i I I I I I I I I I I I 1 J I I I I
C 2O 4O 60 8O IOO I2O I4O I6O I8O 200
jjg ZINC/L
FIGURE III. Graph showing the effects of zinc on the
14-day maximum yield of S_. capricornutum
cultured in media of different total salt
concentrations (ionic strength).
-------
ZINC-ALGAL ASSAY MEDIUM INTERACTIONS
(EXPERIMENTAL)
CO
~ N
Z
X or
<
>- z
< UJ
o
200
100 % Algal Assay Medium
166 pg Zn/l Causing »95% Inhibition
5 Number of Replicate Tests
. Represents the theoretical level of zinc (>jg/l) that will cause
produced by the control cultures.
% inhibition of a 14 day maximum yield of S. coprlcornutum when compared to the maximum yield
Concentration of zinc (>jg/l) contained In the algal assay medium.
Represents ^95% inhibition of the 14 day maximum yield of S. caprlcornutum.
FIGURE IV. Effects of ionic strength and zinc interactions and
their combined effect on the maximum yield of S_. capricornutum.
-------
ZINC-ALGAL ASSAY MEDIUM INTERACTIONS
CO
03
% MEDIUM 12.5
UPS/LI TOTAL IONS 3581
( >l/ohmfl«} CONDUCTANCE 79
IMMl 99 PERCENT REDUCTION OF MAXIMUM YIELD (ALGISTASIS).
ALL CULTURES WERE INOCULATED TO GIVE A FINAL CONCENTRATION OF 10,000 ctill/ml.
FIGURE V. Effects of ionic strength and zinc and their
combined effect on the 14-day maximum yield
of S. capricornutum.
-------
I03 r
bl I I I I I I I I I I I I I I I I I
10
_ \
I
h-
10"
10"
.047 mg PHOSPHORUS / I in
.Mmhos/cm %AAM
61.0
IOO
45.8 _._._.-. 75
30.5 50
15.3 25
I I I I I I I I I I I I I S I i i i i I I I
20 40 60
8O 100 120 140 160 180 200
jjg ZINC/ I
FIGURE VI. Effects of zinc on the 14-day maximum yield of
S. capricornutum cultured in phosphorus limited
T.047 mg/1) algal assay media.
-------
important mechanism of toxic action is thought to be the poisoning of
enzymes. Under conditions of elevated concentration the normal enzyme-
zinc mechanisms may be significantly altered. In the presence of
greater numbers of algal cells (biogenic sites) more enzyme-active sites
should be available to tie-up zinc ions. A comparison of Figures III
and VI indicate that, within the limits of these tests, there was no
significant effect on zinc toxicity caused by the differences in cell
numbers.
From the data presented in this paper the ratio of ionic strength as
lamhos/cm to the concentration of zinc required to obtain >_ 95 percent
algistasis was determined. The ratio (2.72 ± 15 percent) between
ionic strength and _> 95 percent algistatic zinc concentration is
significant at the 95 percent confidence level. However, we accept
data within ± 20 percent to be statistically valid. The factor of
2.72 ± 20 percent multiplied by the ionic strength (ymhos/cm) of a
test substrate should indicate the level of zinc in yg/1 that would
inhibit >_ 95 percent growth of the test alga provided other antagonistic
or synergistic constituents are absent. These data implicate ionic
strength as the dominant factor regulating zinc toxicity upon the
growth of S_. capricornutum. Sensitivity of the test alga to zinc is
shown to be inversely proportional to the increase in ionic strength
of the test substrate. Ion pair formation may be a significant process
by which the availability of zinc ion in waters of higher ionic strength
is altered. This may be due to ion pair formation with some of the
more common cations present in a receiving water such as calcium,
magnesium and sodium. Research is underway to determine if one of
these more common cations can be identified as the major constituent
causing the decrease of the effective zinc concentration in medium of
higher ionic strength.
40
-------
We recognize that the inability to generalize results from
specific observations is a major stumbling block in the use of
laboratory developed ecological information for environmental
management. We are also aware of the risk of extrapolating from
one organism to another and from one geographical locality to
another. Hutchinson and Stokes (1973) demonstrated that four
species of Chlorophyte alga isolated from metal-contaminated water
are tolerant to the heavy metals found in those waters. However,
these heavy metals are highly toxic to laboratory strains of the
same or closely related species. The greatest scientific benefit
will result if intensive research is focused on relatively few
kinds of organisms. A large body of information on the physical
and chemical requirements of S. capricornutum has been produced.
Also, Selenastrum is easy to culture in artificial medium or natural
water and, consistently reliable and reproducible growth responses
have been obtained with it. For these reasons, we believe S. capri-
cornutum is an excellent test species for the determination of
pollutant toxicities.
SUMMARY AND CONCLUSIONS
The Algal Assay Procedure Bottle Test, with S_. capricornutum as the
algal test species, can be used to identify potential toxicity of heavy
metal concentrations in natural waters containing sufficient quantities
of nutrients essential for algal growth. Utilization of the AAP Bottle
Test indicates that S_. capricornutum is highly sensitive to elevated
zinc concentrations. The results of the research discussed in this
paper indicate:
1. Phosphorus (0.047 to 0.930 mg/1) does not significantly
affect the toxicity of zinc to S_. capricornutum.
2. Algal cell numbers (biogenic surface area) did not
significantly affect the concentration of zinc required
to produce >_ 95 percent inhibition of the 14-day maximum
yield.
41
-------
3. Ionic strength is the dominant factor regulating zinc
toxicity apon the growth of S_. capricornutum. Sensitivity
of the test alga to zinc is inversely proportional to the
increase in ionic strength (specific conductance) of the
test substrates.
4. The factor of 2.72 ± 20 percent multiplied by the ionic
strength (ymhos/cm) of a test substrate will indicate the level
of zinc in yg/1 that would inhibit _> 95 percent growth of the
test alga provided other antagonistic or synergistic constituents
are absent.
REFERENCES
Cairns, J., G. R. Lanza and B. C. Parker. 1972. Pollution Related
Structural and Functional Changes in Aquatic Communities with
Emphasis on Freshwater Algae and Protozoa. Proc. Acad. Nat.
Sci. Philadelphia 124(5):79-127.
Ernst, W. 1968. Die Einfluss der Phosphatversorgung sowie die
Wirkung von ionogenem und Chelatisiertem Zink auf die Zink - und
Phosphatoufnahme einiger Schwermelallpflanzen. Physio!. Plant.
21:323-333.
Hutchison, T. C. and P. M. Stokes. 1973. Metal Toxicity and Algal
Bioassays. Symposium on Water Quality Parameters, Canada
Centre for Inland Waters, Burlington, Canada.
Kahn, K. R. 1964. Potentiality of Algae in the Bioassay of Microchemical
Pollutants in Water Systems. Environ. Health 6:274-277.
Maloney, T. E., W. E. Miller and T. Shiroyama. 1972. Algal Responses
to Nutrient Addition in Natural Waters. I. Laboratory Assays.
Special Symposia, Volume I, Nutrients and Eutrophication. Amer.
Soc. Limnol. and Oceanog.
Miller, W. E. and T. E. Maloney. 1971. Effects of Secondary and
Tertiary Wastewater on Algal Growth in a Lake-River System.
Jour. Water Poll. Cont. Fed. 33(12).
United States Environmental Protection Agency. 1971. Algal Assay
Procedure Bottle Test. National Eutrophication Research Program,
Corvallis, Oregon. 82 p.
42
-------
Walker, J. B. 1954. Inorganic Micronutrient Requirements of
Chlorella II. Quantitative Requirements for Iron, Manganese, and
Zinc. Arch. Biochem. Biophys. 53:108.
Whitton, B. A. 1970. Toxicity of Zinc, Copper and Lead to Chlorophyta
from Flowing Waters. Arch. Mikrobiol. 72:353-360.
Wiessner, W. 1962. Inorganic Micronutrients. In: Physiology and
Biochemistry of Algae (R. A. Lewin, ed.). Academic Press,
New York. pp. 267-279.
43
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ALGAL ASSAYS FOR THE NATIONAL EUTROPHICATION SURVEY
Albert Katko
INTRODUCTION
The National Eutrophicatlon Survey (NES) was Initiated by the U.S.
Environmental Protection Agency in 1972 with the primary mission of
identifying and investigating those lakes and reservoirs in the con-
tiguous United States which were in danger of eutrophicatlon caused
by phosphorus inputs from municipal sewage treatment plant discharges.
In its entirety, the NES effort included sampling of each lake
included in the Survey, the tributaries and outlets of each lake, and
municipal waste treatment facilities which impacted each lake either
by direct discharge to the water body or to a tributary of the water
body.
The specific objective, with regard to the approximately 800 lakes
eventually to be included in the Survey, is to determine whether nutrient
reduction, particularly phosphorus, would significantly improve water
quality. To accomplish this the following information must be developed
for each lake or reservoir:
1. The present trophic status of the lake and its water quality
characteristics.
2. The sources and controllability of nutrients with particular
emphasis on nutrients originating from municipal waste treat-
ment facilities.
3. Identification of the nutrient (phosphorus, nitrogen, other)
which limits primary productivity.
44
-------
Upon meeting the objectives and making an evaluation of each lake
on a case-by-case basis, joint EPA - State recommendations will be
developed for corrective or preventive action.
As of January 1, 1974, sampling programs will be in progress or have
been completed in all 27 states east of the Mississippi River involving
some 485 lakes and 2,500 stream sampling sites.
To determine the trophic condition of the lakes, the Survey has two
helicopters equipped for sampling, each with a team directed by a limnolo-
gist. The helicopter teams measure and record the depth, temperature, pH,
dissolved oxygen, turbidity, and conductivity with submersible probes. In
addition, depth-integrated water samples are taken for chlorophyll and phyto-
plankton analysis. Three complete circuits of sampling sites are made on
each lake during the sampling year.
National Guardsmen sample the significant tributaries to each lake once
a month for a year, and sewage treatment plant (STP) effluent samples are
collected at the same interval and over the same period by the plant opera-
tors.
Algal assays are being conducted to aid in determining the relative
trophic status of the study lakes, and to determine the growth limiting
nutrient, nitrogen or phosphorus, at the time of sampling.
This report briefly summarizes the procedures used in the Survey algal
assays and preliminary findings based on analysis of the first round of data,
45
-------
METHODS
One set of samples for algal assay is collected by the helicopter
team from a lake at a time during the year when the lake is unstratified.
Although each lake is sampled three times during the year for other
parameters, time and space limitations prohibit performing more than one
assay for most lakes. The method for collecting the assay samples is
based on the number of stations sampled at each lake. Each assay sample
consists of a five gallon composite of one to four depth-integrated samples
from the various stations. On large lakes with many stations, one assay
sample is composited for each set of up to four stations which are grouped
on the basis of the judgment of the field limnologist.
After collection, the lake samples are air-mailed to the laboratory
in Corvallis and stored at -23.3° C until the assays can be performed.
The samples are prepared as outlined in Figure 1 and according to the
procedures described in the Algal Assay Procedure Bottle Test (EPA 1971).
The various nutrient additions and the chemical analysis performed on
each sample are given in Table 1.
All treatments plus controls are set up in triplicate for each lake
water. The test organism is a green alga, Selenastrum capricornutum. The
cultures are incubated for 14 days at 24 ±2° C under continuous lighting
of 400 ft-C on gyratory shakers and growth responses are monitored by
periodic cell counts made with an electronic particle counter equipped with
a mean cell volume computer. Mean cell volume determinations are converted
to equivalent dry-weight values.
46
-------
Plater
Growth & Biomass
Monitoring
.1
I
Autoclave
'ChemX 5,
AnalJ
Fil
^ Pi ^p^n^fi ^
Spike
Innoculate
:er
^ /Chen.
Unal.
Data Reduction & Analysis
Figure 1. Assay flow chart
Table 1
CHEMICAL PARAMETERS & NUTRIENT SPIKES USED IK ASSAY
Chemical Analysis
Conductivity
pH
Inorganic Carbon
Alkalinity
Kjeldahl - N
NH3 - N
N02 & N03 - N
Total P
Ortho P
Eulfate
Total Hardness
Calcium
Mapnesium
Sodium
Potassium
Chloride
Silicon
Iron
Manganese
Spike Cones,
Control
1.0 N
0.-01 P
0.02 P
0.05 P
0.025 P & 0.5 N
0.05 P & 1.0 N
47
-------
RESULTS
During the 1972 sampling year, lakes in 10 northeast and northcentral
states were sampled, and algal assays were completed on samples from 206
lakes in that area. The productivity range in the control samples (ambient
nutrients) from these lakes was very great with yields ranging from less
than 0.1 mg/1 dry weight to 248 mg/1. Generally speaking, those water bodies
which were most enriched were nitrogen-limited while those which had the
best water quality were phosphorus limited, although there were exceptions.
Occasionally, nitrogen and phosphorus together were required to substan-
tially stimulate a growth response. In a few other cases, algal growth
was either limited by the lack of an unidentified constituent or by the
presence of toxicant and there was no response to additions of nitrogen,
phosphorus, or combinations of the two.
Nutrient limitation for the 206 lakes assayed is summarized in Table
2 by state. As determined by the assay, approximately 57% of all the
lakes were P limited, 37% were N limited, 1% were limited by some other
element or toxicant, and the limiting nutrient could not be conclusively
identified by the remaining 5%. However, the sampling was biased in that
the majority of the surveyed lakes received municipal waste effluents.
DISCUSSION
The algal assay procedure is a useful tool in evaluating the trophic
condition and the limiting nutrient in a body of water: however, other
48
-------
TABLE 2
ALGAL ASSAY SUMMARY FOR 1972 NESP LAKES AND RESERVOIRS
State
Connecticut
Maine
Massachusetts
Michi gan
Minnesota
New Hampshire
New York
Rhode Island
Vermont
Wisconsi n
Totals
Number of P
Limited Lakes
3
7
1
26
23
4
19
1
7
26
117
Number of N
Limited Lakes
3
2
7
31
0
6
1
0
18
76
Number Limited by
Another Element
0
0
0
0
2
0
0
0
0
0
2
Number for Which
Data not Conclusive
2
0
0
1
8
0
0
0
0
0
11
Total
8
9
8
35
64
4
25
2
7
44
206
% of all
56.9
36.9
1 .0
5.2
100.0
-------
biological and chemical parameters should also be examined when the
assay results are evaluated for a particular lake.
One of the difficulties, which has not been resolved completely,
is how the assay results can be related to actual field conditions
and to lake problems or to lake classification. The control yields
(representing ambient nutrient levels) should be related to the trophic
condition of the lakes, and, in general, they are. A lake which produces
a control yield of 1 mg/1 or less is generally in acceptable to very
good condition, while higher yields indicate conditions ranging from
slightly eutrophic to very eutrophic. The highest yield observed in the
Survey to data was 248 mg/1, and it is not difficult to interpret such
extreme algal assay results; however, the subtle differences are more
difficult to interpret. It would be desirable to be able to pinpoint
differences in that narrow yield range between oligotrophic and eutrophy
which represents but a small segment of the assay yield scale, and also
to be able to predict what benefit to a lake, if any, could be expected
from nutrient reductions which would result in a change from 20 mg/1
yield to 10 mg/1 yield. Hopefully, as more assay and field data are
assembled by the National Eutrophication Survey and National Eutrophi-
cation Research Program, some of these questions will be answered.
Some problems have been encountered in our program during sample
transport and treatment which should be noted. Sometimes, significant
50
-------
decreases in conductivity, alkalinity, phosphorus, and nitrogen were
observed. The massive logistics of the sampling program make it impos-
sible or economically unfeasible to freeze or ice a large number of
samples for shipment, and samples often are in transit from three days
to eight days. The preparation procedure, particularly the autoclaved-
filtered treatment, occasionally has produced anomalies in the water
chemistry. Losses of nitrogen and phosphorus or dissolved minerals have
occurred.
Since significant changes in water chemistry can occur during sample
transport and processing, it is particularly important to have additional
knowledge of the chemistry of each water body at the time of sampling.
When these changes and anomalies were observed, little reliance was placed
on the assay results.
SUMMARY
The algal assay bottle test has been a useful tool in aiding the
evaluation of the trophic state of surface waters and in determining the
limiting nutrient for lakes included in the National Eutrophication Survey
Control yields, in general, have shown a relationship to the trophic
state of the lakes in the Survey but must be carefully evaluated in terms
of the other parameters and information available.
Thus far the assays have indicated that the majority of the lakes
surveyed were phosphorus limited. As more assay data and corresponding
51
-------
field data are collected, and we are better able to relate the meaning
of the assay results to field situations, the test will be even more
valuable to the Survey.
52
-------
FREQUENCY ANALYSIS OF CYCLIC PHENOMENA IN FLOWING STREAMS
Pat C. Kerr, James W. Falco, R. Marie Stead,
and Donald Brockway
INTRODUCTION
In an effort to determine those conditions that have the
greatest effect on biological activity in a flowing stream, field
studies > ' have been conducted to monitor concentrations of
nutrients, including various species of carbon, nitrogen, and
phosphorus. Interpretations of temporal and spatial variations
in these data, together with supporting laboratory results and
theoretical hypotheses, have been used as the basis for both
qualitative and mathematical models of flowing aquatic ecosystems.
Most attention to date has been directed toward long-term
effects in flowing streams; variables have been averaged over
relatively long reaches of streams and over long intervals of time.
In contrast, we investigated the cyclic non-random variations in
major nutrient concentrations and bacterial populations in oligo-
trophic first-order mountain streams. Cyclic phenomena in these
fast moving streams have three important implications. First, if
valid long-term models are to be developed based on field data,
sampling periods must be planned so that averaged data reflect the
true long-term behavior of the ecosystem rather than short-term
fluctuations. Secondly, since water quality and biological activity
in these streams are closely coupled to dynamic conditions in the
surrounding watershed4, the parameters that are coupled to watershed
conditions and can thus be manipulated by altering land management
policy must be determined. Lastly since the long-term fate of major
nutrients is directly affected by short-term cycles, the natural
frequencies of these variations must be known to adequately describe
long-term behavior.
53
-------
DESCRIPTION OF FIELD SITES
The data investigated in this study were taken during a field
study conducted jointly by the U. S. Environmental Protection Agency
and the International Biological Program at the U. S. Forest Service
Coweeta, North Carolina Hydrologic Station. Since sampling site and
procedures have been discussed in a previous report5, only a brief
discussion of the two data sets analyzed will be presented.
Sampling station 6-S is located on a primary stream directly
upstream of a weir pond. The watershed surrounding the stream has
been perturbed by clear-cutting that was done six years prior to
the beginning of the study. Sampling station 18-S is also located
on a primary stream directly upstream of a weir pond. The watershed
surrounding this stream is a climax eastern deciduous forest.
Sampling for parameters shown in Table 1 was done at least once a
week, and usually twice a week. The data used in this study was
accumulated over 64 weeks of sampling.
Table 1. INDEX OF MONITORED PARAMETERS'
PH
Total Organic Carbon (TOC)
Total Inorganic Carbon (TIC)
Carbon Dioxide (C02)
Bicarbonate (HC03~)
Total Phosphorus (TP)
Ortho-Phosphorus (PQi=)
Nitrate Nitrogen (N03~)
Dissolved Oxygen (DO)
Sediment Bacteria (Sed. Bact.)
Suspended Bacteria (H20 Bact.)
54
-------
FREQUENCY ANALYSIS
The method used to determine major frequencies of cyclic varia-
tions in nutrient concentrations was Fourier transformation of the
two data sets collected over a 64-week interval starting with the
first week of the study. When samples were taken on different days
of two consecutive weeks, values were interpolated to obtain esti-
mates of concentrations at even weekly intervals. Since Fourier
transforms have not been widely applied to environmental studies,
a brief geometric analogy demonstrating the application of Fourier
analysis will be given. Most of the material describing Fourier
transform has been abstracted from tests by Morse and Feshback
and Kreyszig7.
Consider a plane, i.e., a two dimensional vector space, con-
taining two vectors A and B, which are 90 apart (Figure 1). Since
these vectors are perpendicular, they are linearly independent.
This means that, if the length of vector B is changed, the component
of the vector _A + B in the direction of vector^A remains unchanged.
This statement is not true if _A is not perpendicular to _B, as
illustrated in Figure 2. Two vectors are perpendicular (orthogonal),
if their inner product, _A _B, defined by equation (1), is zero.
A jJ = |A| IB) aw (A,B) (1)
Mathematical functions can be treated as vectors in an n-
dimensional function space. Addition of vectors in this case is
defined simply as arithmetic addition of functions instead of vector
addition (as illustrated in Figure 2). An inner product is defined
as follows.
If the functions _/! (x) , _/2(x)< /n(x) ^ space F, then
T
fi ' _/j = ^ w(x) /t(x) /-(x) dx (2)
o
55
-------
en
CP)
Figure 1
ILLUSTRATION OF TWO PERPENDICULAR VECTORS
AND VECTOR ADDITIONS
-------
A+B|
A+B
Figure 2a
ILLUSTRATION OF INDEPENDANCE
OF PERPENDICULAR VECTORS
Figure 2b
ILLUSTRATION OF DEPENDANCE OF VECTORS
WHICH ARE NOT PERPENDICULAR
-------
where w(x) = a weight function
T = constant interval of integration
Two functions are orthogonal if their inner product is zero.
Now, just as any vector in a two dimensional vector space can
be written as the vector addition of two perpendicular vectors that
span the space, any function in an n-dimensional function space can
be written as the sum of n orthogonal functions that span the function
space. More precisely, if /(x) is in a function space spanned by
/i (x) , /s (>0 j ° /nOO ) then
fOO = i|1 Ai/i(x) (4)
where all A. are constants.
In treating non-cyclic phenomena, collected data are usually
fit to an n " order polynomial
f(x) = A + Bx + Cx2 4- ... + Dxn (5)
that is presumed to describe the process. In terms of the function
space concept, data are fit to a function in some n-dimensional
polynomial space spanned by a set of n orthogonal polynomials.
Often /(x) is then broken up, i.e., decomposed into its orthogonal
components.
For cyclic phenomena, the data are fit into a Fourier series of
sine and cosine functions
n n
/(x) = S A, cay (k TT x) + E B, sin (k rr x) (6)
k=0 K k=l R
In this case /(x) is a function in a function space spanned by 1,
ca? (TTX) , cos (2rrx) , ... aw (nrrx) , sin (rrx) , ... sin (nrrx). It is easy
to define an inner product for which these functions are orthogonal.
If, in equation (2),
w(x) = 1 (7)
T = 1
The above trignometric functions have the following properties
58
-------
\ COS (krrx) sin (krrx) dx = 0
(8)
\ cos (knx) sin (mrrx) dx = 0 (9)
<_>
o
1
cos (krrx) cos (mrrx) dx = 0 (10)
1
r
\ sin (knx) sin (irtnx) dx = 0 (11)
o
k 7^ m
Thus sin (knx) and cos (krrx) , for k = 1, 2, . . . n, are a set of
orthogonal functions that span a 2n dimensional function space.
Equations (8) through (11) are valid only when k and m are integers.
Finally
1 1
^ sin2 krrx dx = V COS2 knx dx = -y- (12)
o o
A Fourier series (equation 6) may be fit to a data set using
2S f 01
grations:
values for A, and B, , estimated by performing the following inte-
dx = _/(x) _/ (13)
k
B = 2 /(X) sin (knx) dx = _/(x) _/ (14)
o
which follows from equation (6) and equations (8) through (12) .
59
-------
Fourier transforms are extensions of Fourier series used when
large numbers of frequencies are involved. In this case the summa-
tion in equation (6) becomes an integral
/(x) = A + V [A(krr)
° TT J
d(kTT)
and equations (13) and (14) become
A(krr) = \ /(x) mr(krrx) dx
+ B(kn) sin (krrn)]
(15)
(16)
B(krr)
= \ /(x) sin (krrx) dx
(17)
A in equation (15) is interpreted as the mean value of the data
set. Substituting
1 , ix -ix)
sin x = -JT (e - e '
1 ix -ix)
COS x = -y- (e - e '
,10.
(18)
,1nx
(19)
where
i = /"-I . (20)
into equation (15), one obtains another form of the Fourier transform:
/(x) =
iWX
dx
(21)
where w = 2rrk.
Both forms of the Fourier transform appear frequently in the
literature and should be recognized as equivalent. Since in this
preliminary analysis only the magnitude of variation in the parameters
was required, absolute values of A(w) and B(w) were added to give
the total amplitude of variation at a frequency disregarding phase
60
-------
angle. Throughout the remainder of the paper, these values are
referred to as Fourier coefficients.
RESULTS
A computer program that performs fast Fourier transforms,
developed by Cooley and Tukey8, was used to analyze data taken
at Coweeta, stations 6-S and 18-S. Since data were taken at
weekly intervals, the highest frequency that could be detected was
26 cycles per year. The total analysis time period of 64 weeks
was chosen to insure the detection of any annual cycles.
Figures 3-13 show variations in Fourier coefficients as func-
tions of frequency for various parameters at station 6-S. A large
annual cycle is evident in each case, with the notable exceptions
of total organic carbon and suspended bacteria. These annual cycles
are the dominant frequency for dissolved oxygen, nitrate-nitrogen,
total phosphorus, and ortho-phosphate. The annual frequency is
the only significant nitrate-nitrogen frequency.
Figures 14-23 show variations in Fourier coefficients as
functions of frequency for various parameters at station 18-S.
Again in all cases except suspended bacteria, a large annual cycle
is present. At station 18-S, carbon dioxide, bicarbonate, total
phosphorus, and ortho-phosphate exhibit dominant annual cycles.
DISCUSSION AND CONCLUSIONS
Tables 2 and 3 show a summary of major frequencies in parameters
measured at stations 6-S and 18-S respectively. Significant non-
random fluctuations occur in major nutrients and bacterial concen-
trations in both streams. Both tables also indicate that
total inorganic carbon, total organic carbon, total
phosphorus, suspended bacteria and sediment bacteria
exhibit cyclic variations at high frequencies, and
nitrate-nitrogen exhibited significant cyclic varia-
tions only at low frequencies.
61
-------
12
H
§
fe
10
STATION 6-S
I .] 1 i
I
8 10
12
14 16 18 20 22 24 26 28
FREQUENCY (CYC/YR)
Figure 3
FOURIER TRANSFORM FOR pH
62
-------
Ol
CO
25.0
20.0
S 15.0
8
CJ
w
10.0
5.0
STATION 6-S
I
I
I
I
I
I
I
10 12 14 16 18 20
FREQUENCY (CYC/YR)
Figure 4
FOURIER TRANSFORM FOR TOTAL ORGANIC CARBON
I
22 24 26 28
-------
01
50
H 40
H
H
R 30
w
H
8
20
10
I I
I I
I I I I
I I
STATION 6-S
I
I
8 10 12 14 16 18
FREQUENCY (CYC/YR)
20
22 24 26
28
Figure 5
FOURIER TRANSFORM FOR TOTAL INORGANIC CARBON
-------
40
oi
H
Z
w
fa
fa
W
O
w
O
fa
30
20
10
STATION 6-S
10 12 14 16 18
FREQUENCY (CYC/YR)
Figure 6
I
20 22 24 26 28
FOURIER TRANSFORM FOR CARBON DIOXIDE
-------
60*
cr>
o-i
40|
w
H
O
fa
§
w
20l
STATION 6-S
8 10 12 14 16 18
FREQUENCY (CYC/YR)
20
22 24 26 28
Figure 7
FOURIER TRANSFORM FOR BICARBONATE
-------
160
T I
140
120
glOO
w
80
Pi
w
8
60
40
20
1 - 1
T 1 1 T
STATION 6-S
I
_l
I
10 12 14 16 18 20
FREQUENCY (CYC/YR)
Figure 8
22 24 26 28
FOURIER TRANSFORM FOR TOTAL PHOSPHORUS
67
-------
80
(Tl
cx>
60
H
II
U
M
o 40
OS
o
PL4
20
STATION 6-S
10 12
14
FREQUENCY (CYC/YR)
Figure 9
I
16 18 20 22 24 26 28
FOURIER TRANSFORM FOR ORTHO-PHOSPHATE
-------
12.0
I I I I I I I I T
I I r
CT>
10.0
X
H
IS
w
M
§
u
w
M
eei
t>
O
8.0
6.0
4.0
2.0
STATION 6-S
68 10 12 14 16 18 20
FREQUENCY (CYC/YR)
Figure 10
22 24 26 28
FOURIER TRANSFORM FOR NITRATE-NITROGEN
-------
40
i r
STATION 6-S
30
W
S 20
B
10
10 12 14 16 18
FREQUENCY (CYC/YR)
20
22 24 26
28
Figure 11
FOURIER TRANSFORM FOR DISSOLVED OXYGEN
70
-------
10.0
8.0
n
o
x
H
6.0
w
o
^ u 4.0
w
2.0
1 i
2 4
I I
1 | T
T T
STATION 6-S
i j
i i i i i i
8 10 12 14 16 18
FREQUENCY (CYC/YR)
Figure 12
FOURIER TRANSFORM FOR SEDIMENT BACTERIA
20 22 24 26 28
-------
rxj
6.0
5.0
X
H
8
o
(d
4.0
3.0
2'°
1.0-
i i r
i i i r
STATION 6-S
I
8 10 12 14 16 18
FREQUENCY (CYC/YR)
20
22 24 26
28
Figure 13
FOURIER TRANSFORM FOR WATER BACTERIA
-------
10
Co
M 6
o
o
te.
STATION 18-S
J_
I
10 12 14 16 18 20
FREQUENCY (CYC/YR)
Figure 14
FOURIER TRANSFORM FOR pH
22 24 26 28
-------
25,
W
O
o
20
H
n 15
10
I I I
J L
J I L.
* ' '
10 12 14 16 18
FREQUENCY (CYC/YR)
T5_
20 22 24 26 28
FOURIER TRANSFORM FOR TOTAL ORGANIC CARBON
-------
100
80
w 60
M
O
1-1
fe
O
o
M 40
I
20
STATION 18-S
I
_L
8 10 12 14 16
FREQUENCY (CYC/YR)
18
20 22 24 26 2
Figure 16
FOURIER TRANSFORM FOR TOTAL INORGANIC CARBON
-------
40
30
H
Z
W
M
0
§ 20
u
10
STATION 18-S
10 12 14 16 18
FREQUENCY (CYC/YR)
Figure 17
FOURIER TRANSFORM FOR CARBON DIOXIDE
20 22 24 26 28
-------
50
E-i
Z
w
M
O
Fn
w
O
Pi
40
30
20
10
STATION 18-S
I
I
I
10 12 14 16
FREQUENCY (CYC/YR)
18 20 22 24 26 28
Figure 18
FOURIER TRANSFORM FOR BICARBONATE
77
-------
200
STATION 18-S
oo
W
H
H
Cd
o
H
g
O
150
100
50
10 12 14 16 18 20
FREQUENCY (CYC/YR)
Figure 19
22
24 26 28
FOURIER TRANSFORM FOR TOTAL PHOSPHORUS
-------
100
STATION 18-S
80
60
w
o
o
40
20
10 12
14
16 18 20
22
24 26 28
FREQUENCY (CYC/YR)
Figure 20
FOURIER TRANSFORM FOR ORTHO-PHOSPHATE
79
-------
10.0
03
o
CD
o
X
H
U
M
Fu
W
O
8.0
6.0
4.0
2.0
STATION 18-S
10 12 14 16 18
FREQUENCY (CYC/YR)
20 22 24 26
28
Figure 21
FOURIER TRANSFORM FOR NITRATE-NITROGEN
-------
25
STATION 18-S
20
H
2
W
ft.
ft.
W
O
U
15
10
I
I
I
I
I
I
_L
6 8 10 12 14 16 18 20
FREQUENCY (CYC/YR)
Figure 22
FOURIER TRANSFORM FOR DISSOLVED OXYGEN
22
24 26 28
81
-------
CD
r\>
no
X
H
2
w
M
O
H
fa
fa
W
O
u
o
fa
2.0k
STATION 18-S
1.5
l.Of-
0.5
I
J
I
I
I
I
I
I
10 12 14 16 18
FREQUENCY (CYC/YR)
Figure 23
20
22 24 26 28
FOURIER TRANSFORM FOR SEDIMENT BACTERIA
-------
Table 2. MAJOR OSCILLATORY FREQUENCIES OF MONITORED CONSTITUENTS AT STATION 6-S
Frequency (cyc/yr)
Constituent
pH
TOG
TIC
C02
HC03~
TP
P04 =
N03~
DO
Sed.
Bact .
H20
Bact.
1
X
X
X
X
X
X
X
X
X
2-3
X
X
X
X
X
X
X
X
X
X
5-6
X
X
X
X
X
X
X
X
8-10
X
X
X
X
X
X
X
11-13
X
X
X
X
X
X
X
14-17
X
X
X
1
X
X
X
19-22
X
X
?
X
X
X
24-25
X
X
?
X
X
X
26
X
oo
CO
-------
Table 3. MAJOR OSCILLATORY FREQUENCIES OF MONITORED CONSTITUENTS AT STATION 18-S
Frequency (cyc/yr)
Constituent
PH
TOG
TIC
C02
HC03~
TP
P0i =
N03~
DO
Sed.
Bact.
H20
Bact.
1
X
X
X
X
X
X
X
X
X
X
2-4
X
X
X
X
X
X
X
X
X
5-7
X
X
X
X
X
X
X
8-10
X
X
X
X
X
X
X
11-13
X
X
X
X
X
X
14-17
X
X
X
X
X
X
18-20
X
X
X
X
21-22
X
X
X
X
X
24-25
X
X
X
X
X
CO
-pi
-------
Water bacteria, sediment bacteria, and all forms of inorganic
carbon appear to cycle more rapidly in the perturbed stream (6-S)
than in the stream surrounded by the climax forest (18-S). The
inverse is true for nitrate-nitrogen, dissolved oxygen, and total
organic carbon. These differences are very pronounced for nitrate-
nitrogen and suspended and sediment bacteria. Based on preliminary
data, total inorganic carbon, total organic carbon, total phosphorus,
and bacterial concentrations are interacting parameters.
This preliminary study demonstrates the cyclic phenomena
occurring in streams and illustrates the application of Fourier
transforms in the analysis of such cyclic phenomena. Future work
should involve the determination of phase angles of oscillation of
major frequencies of nutrient cycles and the quantitative investi-
gation of couplings between nutrient and biological cycles implied
in this preliminary study.
SUMMARY
Data sets containing measurements of the concentrations of
major nutrients and bacteria in two primary oligotrophic streams
over a 64-week period were Fourier transformed. The transform
coefficients as function of frequency indicate that non-random
variations occurred in all measured parameters. Changes in nutrient
and bacterial cycling patterns caused by clear-cutting of a water-
shed are illustrated by the differences between patterns in two
streams, one in a climax forest and one in a clear-cut watershed.
ACKNOWLEDGMENTS
Data used in this paper was collected as a joint effort of the
Southeast Environmental Research Laboratory, U. S. Environmental
Protection Agency, and the International Biological Program, Coweeta
site.
85
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REFERENCES
1. MacCrimmon, H. R. and J. R. M. Kelso. 1970. Seasonal Variation in
Selected Nutrients of a River System. Fisheries Research Board
of Canada 27:837-846.
2. Kelso, J. R. M. and H. R. MacCrimmon. 1969. Diel and Seasonal Variations
in Physiochemical Limnology, Speed River, Ontario. Water Resources
Research 5_: 1388-139 4.
3o Wright, J. C. and I. K. Mills. 1967- Productivity Studies on the Madison
River, Yellowstone National Park. Limnol. Oceanogr. 12:568-577.
4o Todd, R. L., Pat C. Kerr, W. A. Swank, D. L. Brockway, J. Douglas,
and C. D. Monk. 1973. Chemoautotrophic Nitrification and Nitrate
Losses in an Eastern Deciduous Forest Floor Ecosystem. Presented
at the Annual Meeting of the American Society of Limnology and
Oceanography. Salt Lake City, Utah, June 10-14.
5. Kerr, Pat C., D. L. Brockway, Doris F. Paris, J. T. Barnett, Jr.,
and Marilyn F. Cox. 1974. The Fate of Carbon, Nitrogen, and
Phosphorus in Inland Surface Waters. U.S. Environmental
Protection Agency. (In press)
6. Morse, P. M. and H. Feshback. 1953. Methods of Theoretical Physics,
Vol. I. McGraw-Hill Publishing Co., New York. 19 p.
7. Kreyszig, E. 1965. Advanced Engineering Mathematics. John Wiley
and Sons, Inc., New York. 48 p.
8. Cooley, J. W. and J. W. Tukey. 1967. An Algorithm for the Machine
Calculation of Complex Fourier Series. Math. Comp. 19(90):
297-301.
86
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THE EFFECT OF HIGHER TROPHIC LEVEL COMPONENTS
IN AN AQUATIC ECOSYSTEM MODEL
Ray R. Lassiter
INTRODUCTION
Within-season patterns of phytoplankton blooms, grazer and
predator population peaks, fluctuations in decomposer activity,
and variations in material cycling are prominent features of
aquatic ecosystem dynamics. The processes of translocation and
transformation of materials are expected to occur differently
in systems that differ in patterns of these blooms and peaks.
To provide a framework for studying material transformations
and translocations in varying aquatic ecosystems a mathematical
model was constructed and used in simulations. Simulations
were run representing situations with and without consumers.
After observations have been made of the fluctuation patterns of
relatively well-understood materials, i.e., major nutrients, the
model will be used to assess the probable behavior of various
pollutants in systems with different magnitudes of fluctuations
in the component populations.
This paper describes the rationale basic to such a model
and compares results from versions of the model with and without
higher trophic levels. It also compares the present model with
a precursory model, in this context, the present model is referred
to as the "enhanced model."
THE MULTIPLE SPECIES MODEL CONCEPT AND A PRECURSORY MODEL
Many mathematical models lump all organisms of a single general
type, in the researcher's view, into one compartment, e.g., phyto-
plankton. The multiple species model, in contrast, represents
87
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individual types of organisms as individual compartments. Eggers
and Male2 stated that models with species differentiation are
necessary for questions concerning competition, predation, selection,
and adaptation to be considered.
A model was developed1 in which six algal "species" were rep-
resented. Included also was a simple, dynamic, inorganic chemical
equilibrium system plus a set of organic chemicals not directly
involved in the equilibria among the inorganic chemicals. The
specific growth rates and gaseous exchange rates used in the model
were temperature dependent. The algae were dependent upon light,
carbon, nitrogen, and phosphorus for growth. Because of differences
in temperature optima and differences in uptake efficiency and growth
rates, the individual species competed with varying degrees of success
as the season progressed. The result (Figure 1) was a seasonal
pattern of "blooms" reminiscent of a growing season observable in
natural waters (Figure 2). Note an early spring peak of growth
when most nutrients were present in inorganic form, and a later
season competition for cycling nutrients as the nutrient levels and
the temperature changed.
To find the effect of physiological type upon the pattern of
phytoplankton production, species 1 was removed from the simulation.
The results are shown in Figure 3, A and B. Figure 3B shows the
simulated phytoplankton standing crop through time with species 1
absent, and Figure 3A is the corresponding portion of Figure 1
redrawn to the same scale as Figure 3B. Note the same general
pattern of total phytoplankton biomass. Removing one species did
not greatly alter the total phytoplanktonic biomass production early
in the season even when the species removed accounted for nearly
the entire bloom when included in the simulation. The property
of the model giving rise to this behavior cannot exist outside
a multiple species model. This is a necessary property of models
to be used to investigate situations in which species respond
differently to various pollutants.
88
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X
oo
oo
O
oo
<
O
0.25
0.20 -B
0.15-
Total
Species 1
Species 2
Species 3
Species 4
Species 5
Species 6
Figure 1
1000 2000 3000 4000 5000
TIME (hours)
Simulated phytoplankton biomass through a growing season.
150
100-
Q_
O
01
O
50-
0'
50 100 150 200 250 300 350 400
TIME (days)
Figure 2. Pattern of chlorophyll during 1969 on a Potomac River sampling
station. Data approximated from Figure 39 of O'Connor et al.
89
-------
0.25
0.20 H
0.15 -
0.05 -
0.00
_ Total
-- Species 1
- Species 2
- Species 3
Species 4
-- Species 6
500 1000 1500
TIME (hours)
2000
CD
ii
X
oo
00
-------
THE ENHANCED MODEL
Many aspects of the enhanced model are identical to those of
the precursory model. However, several differences and additions
require some elaboration. These aspects include algal, bacterial,
and zooplankton growth, a coupling structure comprised of the food
web and other transfers and transformations, and the stoichiometric
relations for the various transformations of materials. The structure
is shown schematically in Figure 4. The main concepts are discussed
below. The detailed system equations will be found in Appendix 1
and Appendix 2.
MODEL COUPLING STRUCTURE
Respiration
Respiration
Uptake
Inorganic chemicals
Predaceous
zooplankton
(1 species)
Grazing
zooplankton
(2 species)
Phytoplankton
(6 species)
Unassimilated
Bacteria
(1 species)
Death
Excretion
Death
Excretion
Secretion
Organic chemicals
I
Figure 4. Schematic representation of the enhanced model,
91
-------
Algal Growth
As in the precursory model, algal growth was made a function of
light, temperature, and the concentration of CO^, ammonia nitrogen,
and mono- and dihydrogen phosphate ions in the water. The Michaelis-
Menten model was used as the basis for the growth rate terms used
in both cases. However, in the precursory model, growth rate was
made a function of a product of three terms, each of the form
C/CK + C), in which C is the concentration of a major nutrient, and
K is the concentration of the same nutrient that results in half
maximal growth. Although frequently used, this form has several
disadvantages1. In the enhanced model, growth rate is a function of
the minimum of the set of terms {^/(Ki+Cx), CW(K2+C2), C3/(K3+C3)}
where subscripts refer to specific nutrients (see Appendix 1). Using
this construction, the growth rate constant as found by experimentation
appears to be more nearly applicable to the multiple factor situation.
Bacterial Growth
Bacteria were not explicitly present in the precursory model.
Instead, organic compounds were assumed to decay at rates proportional
to their concentrations. In the enhanced model bacteria were included
in order to transform organic carbon, nitrogen, and phosphorus into
their respective inorganic forms. In general, bacteria utilize inorganic
ions in addition to organic compounds; but for this model bacterial
growth rate was not made an explicit function of inorganic ions. Any
inorganic ions assimilated were assumed to be used immediately after
they were released from the organic form. The growth rate of bacteria
was made a function of the minimum of the set of terms {Cg/(K5+C5),
C6/(K6+C6), (^/(Kv-K^)}, i-e" a function of the concentrations of
organic compounds only (for subscripts, see Appendix 1).
Zooplankton Growth
Zooplankton were not represented in the precursory model.
Consequently no effect of grazing or other effect of higher trophic
levels could be studied. In the enhanced model, a Michaelis-Menten
92
-------
expression is used to describe satiation, i.e., the reduced grazing
rate per unit prey with increasing prey density. The specific growth
rate (|i) term for predator i is
m
= a J-i PJJBJ
1 1 K± + .f-^.B.
A
in which \i is maximal growth rate,
p is a weighting constant defining the predation
rate by the i predator on the j prey type
relative to the rate on any other prey type,
B is biomass of prey,
K is the concentration of prey as modified by
the weight, p, that results in half maximal
predator growth, and
m is the number of prey types.
If the predator feeds by random encounter of the prey individuals,
regardless of species, the p.. are all equal.
The Coupling Structure of the Model
The dynamic part of the model is a set of highly coupled
differential equations, which in turn are coupled to a set of
algebraic equations for chemical equilibrium. The equations are
coupled in that terms for disappearance of a material (living or non-
living) in one equation have corresponding terms for appearance
in one or more other equations. For example, the disappearance of
dissolved inorganic material may be accompanied by an appearance of
algae, and the disappearance of algae may be accompanied by appear-
ance of zooplankton and organic material. Disappearances are negative
terms and appearances positive terms in the differential equations.
All couplings are shown in the appendices. Many of the terms
involved in the couplings contain stoichiometric coefficients,
n.., in which i indicates a source (reactant) and j indicates a
93
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product. These will be discussed in the following section. Examples
of the couplings are discussed below.
If Ga is the vector of growth rates of algae, all yields are
100% efficient (Y = 1), and n is the vector of stoichiometric
2 a
coefficients relating moles of C02 to moles to algae , then the loss
rate of C02 due to algal growth is -n aGa. If the vector of respira-
tion rates of heterotrophic organisms is -Rz, then the corresponding
rate of change for dissolved C03 is n, RZ and for dissolved 02 is
-n, v, R,. If the vector of excretion loss rates of organic carbon
I }.L _ ^4
from zooplankton is Ez, then rate of increase of organic carbon is
n ^ Ez. (For further definition of the symbols, see Appendix 1.)
The food web relationships are represented by growth rates,
predation rates that sustain those growth rates, and other trans-
formations occurring as a result of predation. Of the total growth
rate of species i, (p^, Equation 1), the fraction F^-, given by
P B
F = lk k /ox
ki SB
is attributable to predation upon species k.
Accounting for the yield (Y-^), the biomass of predators able
to take prey (B-j^), and the stoichiometric coefficient relating a
mole of prey to a mole of predator (n/.), the predation rate on
an individual of species k is given by
m
pik Bk -
P = £ n' B
k 1=1 ki i Y / m / - m
Yi ,S,p.. B/ -K. +.Z, p. . B.
]=l^ij J i j=l ij j
P !
94
-------
Alternatively, since the growth rate of the i predator is
m
.En p. . B.
J=l 13 J
K. + .£.. p. . B.
=
substitution into equation 3a results in
n P G
= \ 7
"
or using Equation (2)
Pk
The transfer of unassimilated algae to the organic pool and
the stoichiometry of the transformation are discussed in the following
section along with other aspects of stoichiometry.
When the system is completely coupled, and when no net losses
or gains to the system are included, there should be conservation of
mass. To check on proper system coupling and stoichiometry in
computer models, one might leave off net system gains and losses
until conservation of mass is achieved, then add export and import
rates to simulate open system dynamics.
Stoichiometric Computations
One aspect of ecosystem modeling that is often neglected is the
Stoichiometric relationships that must be maintained in transforma-
tion of elements from one form to another. Cordeiro et al.4 and
Verhoff and Smith have simulated systems in which complete cycles
were modeled and stoichiometry was considered. This aspect of
system modeling is especially important when material cycling is
studied. Thus the following method of computing the Stoichiometric
coefficients was developed for the enhanced model.
95
-------
Because a unit of consumed material may differ in atomic ratios
of the principal elements from a unit of consumer protoplasm,
assimilation of the consumed material is incomplete. That is, some
of the consumed material must necessarily be released by the con-
sumer in order to maintain its atomic ratios. This situation also
sets the minimum rate of consumption per unit increase in consumer.
The actual rate is increased by a factor for inefficiency of
assimilation. This factor may equal one as is assumed in this
model for assimilation by algae, but in general it will be greater
than one.
To illustrate the computation of a stoichiometric coefficient,
let algal composition be C100 N15 P^ and zooplankton composition be
Ceo N16 P-L . The ratios of numbers of atoms in a mole of zooplankton
to those in a mole of algae form the set:
f 80 16 1 \
Uoo~ ' "is" ' ~TT
The largest member of this set is the stoichiometric coefficient
representing the minimum number of moles of algae required to supply
a one-mole increase of zooplankton:
f 80 16 11 16
n = max J L =
n _
' i r ~
az UOO ' 15 ' 1 \ 15
or stated in another way, the reciprocal of the largest member (15/16)
is the number of moles theoretically available for consumer biomass
production per mole phytoplankton consumed. The quantity actually
required is greater than the minimum calculated above because of
assimilative inefficiencies. If the assimilative efficiency is
y (moles consumer biomass produced per mole theoretically
Z3.
capable of being produced), the stoichiometric coefficient for
this transfer is
n* =
az y
J za
and the overall coefficient
n = n' n*
az az az
96
-------
The rate of consumption of an individual of phytoplankton
species k, P , may now be written more compactly. Defining the
rC
terms F, . G. from Equation (3c) as /L . , in which i = 1, 2, ...,
and r is the number of predators, the equation becomes
in which TL. is the vector of stoichiometric coefficients for
transfer of the k1-'1 prey to the it'1 predator, and
A, . is the vector of weighted prey biomass values, as
weighted by the vulnerability of the ktn prey to
the i*-*1 predator.
These computations relate moles of algae to moles of zooplank-
ton. However, since the elemental composition of algae is assumed
to be different from that of zooplankton, some of the material must be
transformed to substances other than zooplankton. For example,
carbon, at 100 atoms per mole of phytoplankton and 80 atoms per
mole of zooplankton, must be liberated by zooplankton at the rate
of 20 atoms per mole of algae eaten simply to maintain the integrity
of the zooplankton protoplasmic composition. To illustrate, assume
that the liberated carbon becomes organic carbon. A stoichiometric
coefficient is needed for this transfer. The stoichiometric co-
efficient may be expressed as
_ moles of organic carbon produced ,-.
a^s moles of predator formed
z
in which n p is the stoichiometric coefficient relating the
a£s
appearance of organic carbon to the formation of
zooplankton biomass from the assimilation of
phytoplankton .
This coefficient may be used with the growth rate of the predator
to compute the rate of production of organic carbon. Other co-
efficients may be computed to use with other variables, each
coefficient being the partial derivative of organic carbon with
97
-------
respect to the variable of interest. A coefficient with the units
specified above may be obtained as follows:
x n -x
i az 2 /7\
V^ = x
az5 X3
in which xx is the number of carbon atoms per mole of phytoplankton,
n is the number of moles of phytoplankton taken per mole
az
of predator produced, the stoichiometric coefficient
as defined in Equation (4),
x2 is the number of carbon atoms per mole of predator,
and
x3 is the number of carbon atoms per mole of organic
carbon.
The numerator, x n -x , represents the number of carbon atoms pro-
' i az 3 ^
duced as organic carbon per mole of predator formed. Referring to
the definitions of x1 , x2, and Xg , the units for the stoichiometric
coefficient may be determined by
a , , m, , , a
1--J 1 - (--1
\ mn/ \m0/ \ mP/
U(n
Hlg
in which U(n p ) is read "the units of the term (n p ),"
a is the number of carbon atoms,
m-|_ is moles of phytoplankton,
m2 is moles of zooplankton,
trig is moles of organic carbon.
When simplified the units nig/m2 match those specified in Equation (6),
Equation 7 may be applied as well to any by-product material
as to organic carbon. If used as a general formula,
xx is number of atoms of any element per mole of some
consumed material,
naz is number of moles of the material that are consumed
per mole of consumer produced,
98
-------
x2 is number of atoms of the element per mole of consumer,
x3 is number of atoms of the element per mole of the by-
product,
and for generality -
n x replaces n p , the subscript a^s signifying that the
*23 az5
transfer occurs because organism 2 eats organism (or
material) 1 with material 3 appearing as a byproduct.
Where two or more by-products (e.g., both organic carbon and C02 )
result, this formulation becomes more involved but no different
in principle.
The rate of appearance of organic carbon is
E = n D G
aP5 z
and, in general, for the rate of appearance of material x from
predator 2 feeding on prey 1
E = n , Go
x a^x ^
This equation states that for each mole of prey eaten or predator
formed, n ^ moles of by-product are produced.
The computation of these coefficients remains applicable for
at least two additional model refinements. First, in the enhanced
model each species in a given trophic level is assumed to be of the
same elemental composition and no organism is assumed to feed on
more than one trophic level. Therefore any consumer eats food all
of the same elemental composition. This is clearly a gross over-
simplification. The method of computing the stoichiometric coeffici-
ents, n. ., will remain applicable for a model not restricted in this
manner. Second, in this and most models, the atomic ratios of the
principal elements in the organisms are assumed to remain constant
over time. However, the compositions of single-celled organisms
are known to be, to some degree, a function of the concentration
of nutrients in the external medium. Metazoans change body com-
position with age and with varying accumulation of carbohydrates
99
-------
and fats as energy storage. Future models might include such
factors, and the above method of computing stoichiometric coeffici-
ents will apply to these model refinements as well.
The Effect of Higher Trophic Levels
The role of consumers has often been overlooked or minimized
in aquatic ecosystem models. To investigate this aspect of system
functioning, trial computer runs were made with and without con-
sumers included in the model and with all other factors the same.
Figure 5A shows the phytoplankton biomass from a run without con-
sumers. The enhanced model without the consumers is similar to
the precursory model, yet the results are very different (compare
Figure 1 with Figure 5A). These differences arise partly from
differences in the growth models, as previously described, but
mostly from the relatively low natural death rates for phyto-
plankton used in the enhanced model. The low phytoplankton death
rates were used to allow the effects of death rates from zoo-
plankton grazing to show up more distinctly.
The results obtained using the model with consumers are
strikingly different from those obtained without consumers.
Figure 5B shows the phytoplankton biomass from a run with con-
sumers. The two periods of rapid reductions in biomass
resulted from zooplankton grazing. Figure 6 shows total phyto-
plankton biomass and biomass of the two grazer populations as
simulated by the model. The two periods of decline in phyto-
plankton correspond to the periods of maximum rate of increase
of the zooplankton. Zooplankton declined because they reduced
their food supply and because temperature became unfavorable.
The initially obvious differences in standing crop of algae
are not the only differences observable when zooplankton are added
to the model. Differences in nutrient cycling can be noted for any
nutrient. For example Figure 7 A shows the concentration of total
inorganic carbon. The dips in the curve correspond to periods of
rapid phytoplankton growth (hence inorganic carbon uptake). The
100
-------
X
00
00
O
QQ
O
-------
0.4
0.3-
CD
ii
X
co
oo 0.2 H
O
DO
0.1 -
0.0
Phytoplankton
-- Consumer 1
- Consumer 2
I
500
T
1000 1500 2000
TIME (hours)
2500
3000
Figure 6. Total phytoplankton biomass and the biomass of each of two
species of primary consumer. Note the correspondence of
maximum rate of increase of consumer corresponding to
maximum rate of decrease of phytoplankton.
general curved appearance in the graph reflects the varying solu-
bility of COs as temperature varies. A comparison with Figure 7B
shows the greater irregularity associated with the situation in
which zooplankton participate in nutrient cycling.
Another manifestation of the role of consumers is the bacterial
dynamics. Figures 8, A and B, show the bacterial standing crop
without and with consumers, respectively. Without consumers an
initial peak was followed by stabilization at a relatively low
level. The reason for this stabilization was limitation of
bacteria by nitrogen. When consumers were added, nutrients were
made available at higher rates and increased amounts as evidenced
by several growth periods and a higher maximum standing crop.
However, there was greater variation in the production of organic
102
-------
X
o
CD
Sc
-------
0.8
CD
p i
X
0.6-
0.2-
o
1.0
O
CQ
X 0.8 H
CO
CO
0.6-
0.4-
0.2-
0.0
o
-------
material throughout the growing season, as evidenced by fluctua-
tions in bacterial standing crop. Also the bacterial population
declined near the end of the season because the consumers virtu-
ally eliminated the phytoplankton populations, thus reducing
the rate of organic material production. The reduction in bacterial
population further resulted in a low rate of transformation of
aquatic materials to inorganic nutrients for phytoplankton growth.
The specific growth patterns predicted by experimental computer
simulations cannot be expected to correspond directly to any
natural situation. However, the changes in growth patterns brought
about by including consumers in the simulation illustrate the
importance of higher trophic levels in ecosystem modeling. In
addition to their direct effect on prey populations, consumers
play a significant role in nutrient cycling and therefore in
setting the patterns and levels of abundance of all types of aquatic
organisms.
Models such as the one described are useful in studying the
effects of various nutrient regimes on biomass production and
growth rate limitation. Biomass limitation occurred in all simula-
tions. The only limit to biomass production was nutrient avail-
ability; therefore a maximum biomass was reached when utilization
reduced nutrients to concentrations at which population loss
rates equalled or exceeded uptake rates. Zooplankton consumers,
by recycling nutrients, may slightly increase the upper limit for
phytoplankton (Figure 5, A and B) and bacteria (Figure 8, A and B)
but on the other hand they can ultimately reduce the standing
crop (Figure 6).
CONCLUSIONS
Dynamic models considering many factors, e.g., a food web,
stoichiometric relations, and conservation of mass, can be built
and used effectively to investigate aquatic ecosystem functioning.
This model indicates that consumers are important in the
dynamics of every system component examined.
105
-------
Such models have potential in studying many phenomena asso-
ciated with aquatic ecosystems and the effects of pollution in
these systems. The effect of removing species or otherwise altering
such systems may be considered. Such models may be used to plan
large scale experiments or studies with ecosystems. They can aid
in determining the types and quantities of measurements needed
and can provide a framework for data analysis.
REFERENCES
10 Lassiter, R. R. and D. K. Kearns. Phytoplankton Population Changes
and Nutrient Fluctuations in a Simple Aquatic Ecosystem Model.
In: Modeling the Eutrophication Process, Proceedings of a
Workshop Held at Utah State University, Logan, Utah, September
5-7, 1973 (E. J. Middlebrooks, Donna H. Falkenborg, and T. E.
Maloney, eds.). Utah State University and U. S. Environmental
Protection Agency. Logan, Utah. PRWG136-1. Utah Water
Research Laboratory, College of Engineering, Utah State
University. November 1973. p. 131-138.
2. Eggers, D. M. and L. M. Male. The Modeling Process Relating to
Questions about Coniferous Lake Ecosystems. In: Proceedings -
Research on Coniferous Forest Ecosystems - A Symposium (J. F.
Franklin, L. J. Dempster, and R. H. Waring, eds.). Washington,
U. S. Government Printing Office, 1972. #0101-0233. p. 33-36.
3. O'Connor, D. J., R. V. Thomann, and D. M. Di Toro. Dynamic Water
Quality Forecasting and Management. U. S. Environmental
Protection Agency, Washington, D. C. EPA-660/3-73-009.
170 p.
4. Cordeiro, C. F., W. F. Echelberger, and F. H. Verhoff. Rates of
Carbon, Oxygen, Nitrogen, and Phosphorus Cycling Through
Microbial Populations in Stratified Lakes. In: Modeling
the Eutrophication Process, Proceedings of a Workshop Held at
Utah State University, Logan, Utah, September 5-7, 1973
(E. J. Middlebrooks, Donna H. Falkenborg, and T. E. Maloney,
eds.). Utah State University and U. S. Environmental
Protection Agency. Logan, Utah. PRWG136-1. Utah Water
Research Laboratory, College of Engineering, Utah State
University. November 1973. p. 111-120.
5. Verhoff, F. H. and F. E. Smith. Theoretical Analysis of a
Conserved Nutrient Ecosystem. J. Theoret. Biol. 33:131-147,
1971.
106
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APPENDIX 1
THE SYSTEM OF DYNAMIC EQUATIONS
The equations of the model are summarized below to indicate
the couplings. For explanation of the terms refer to Appendix 2,
Dissolved Oxygen (C^)
dCn
dt -J.V~.LB ~ ci) + nia (Ga + s) " nih(Rh + E7s)
Dissolved C02 (C2)
dC2 _ ,. fr
- C2) - n2a (Ga + S) + n2h (Rh + E75)
Dissolved Ammonia (C3)
dt *
Dissolved Phosphate (C4)
Total Organic Carbon (C5)
i. = n (D +S)+n,D,+nr)E -n , G,
dt sa^ a ' 5h h aPE z eb b
Total Organic Nitrogen (Cs)
§. = n D + n , D, + n n E - n , G,
dt ea a eh n aV& z eb b
Total Organic Phosphorus (C7)
dC7
- - = n D + n D, + n 0 E - n G
dt va a ?h h aF7 z 7b b
2
Phytoplankton (B^, i=l, 2, ..., 6)
dB.
^ = G. - D. - P.B.
dt i ! ! !
107
-------
Bacteria (B7)
dB7
67 - D7 - R7
at
Herbivorous Zooplankton (B8 ,B9)
dB,
I = G. - D. - P.B. - R.
dt J ] J J J
Carnivorous Zooplankton (B10)
dB,
dt
G10 ~ D10 - R10
APPENDIX 2
EXPLANATIONS OF TERMS USED IN APPENDIX 1
The terms comprising the equations summarized in Appendix 1
are presented below in a somewhat expanded form. Some deviation
from the notation employed in the text will be noted. For example
where the text uses Ga as a vector of growth rates, G is used
below as a sum of growth rates. The whole terms nxaGa (text)
and nxaGa (Appendix 1) are the same where nxa is a constant vector,
the case for the actual equations used and presented in Appendix 1.
C is the molar concentration of dissolved oxygen in water when
the water is saturated with oxygen.
C2s is the molar concentration of dissolved C02 in water when the
water is saturated with C02.
Da is the sum of non-predatory death rates of all phytoplankton
species. It is assumed to be proportional to the biomass of
each species:
b
D = Z k. ,B.
a i=l id i
in which k is the death rate of the ith phytoplankton species,
108
-------
D. is the non-predatory death rate of the i1- population (see Da)
Di = kid V i = l> 2> " 10-
D7 is the non-predatory death rate for bacteria, assumed to be
proportional to the bacterial biomass:
D7 = k7d B7
in which kj is the death rate constant.
E is the rate of production of organic carbon (see Equation 7) .
E7 is the rate of decomposition of carbonaceous material. It is
the rate of removal of carbonaceous material minus the rate
of assimilation of carbonaceous material into bacterial
biomass. The term for organic material x is
E = n ,G, - G,
?x xb b b
in which n , is the stoichiometric coefficient relating moles
of organic material x removed per mole of bacteria
produced, and
G, is the growth rate of bacteria (see text, BACTERIAL
GROWTH).
The particular coefficients are
n * for organic carbon,
nsb for organic nitrogen, and
n?b for organic phosphorus
corresponding to decomposition rates E7B, E7S, and
E77 .
Ga is the growth rate of six species of phytoplankton:
6.4
G = E M,. TT
109
-------
G- is the growth rate of the ith species of phytoplankton:
4
G. = M-.
> x J-2
...
For both G and G.
3. 1
ia, is the maximal specific growth rate for the
ith phytoplankton species,
C- is the water concentration of the ^ nutrient,
and
K-J is the concentration of the )th nutrient for which
half maximal growth rate is attained by the ith
species.
G. (j = 8, 9, or 10) are zooplankton growth rates (see Equation
3a ff).
GV is bacterial growth rate:
* 7
7 ^ 1=5
in which ^ is the maximal specific growth rate for bacteria,
and
K. is the concentration of nutrient i for which
half maximal growth rate is attained.
k]_ is reaeration rate for dissolved oxygen.
k2 is reaeration rate for dissolved C02.
n is the number of moles of 02 produced per mole of phytoplankton
produced or per mole of organic carbon secreted by phyto-
plankton.
n is the number of moles of C02 used per mole of phytoplankton
produced or per mole of organic carbon secreted by phytoplank-
ton.
n3a is the number °f moles of ammonia used per mole of phytoplank-
ton produced.
110
-------
n4a is the number of moles of phosphate used per mole of phyto-
plankton produced.
n a is number of moles or organic carbon produced per mole of algae
dying from non-predatory causes or per mole of organic carbon
secreted.
n a is number of moles of organic nitrogen produced per mole of
algae dying from non-predatory causes.
n is the number of moles of organic phosphorus produced per
mole of algae dying from non-predatory causes.
n is number of moles of 02 used per mole of heterotrophic organism
catabolized in respiration or per mole or organic carbon decom-
posed.
n , is number of moles of C02 produced per mole of heterotrophic
3 n
organism catabolized in respiration or per mole of organic
carbon decomposed.
n , is number of moles of ammonia produced per mole of heterotrophic
organism catabolized in respiration or per mole of organic
nitrogen decomposed.
n , is number of moles of phosphate produced per mole of hetero-
trophic organism catabolized in respiration or per mole of
organic phosphorus decomposed.
n , is number of moles of organic carbon produced per mole of
5 n
zooplankton dying from non-predatory causes.
n is number of moles of organic nitrogen produced per mole of
s in
zooplankton dying from non-predatory causes.
n , is number of moles of organic phosphorus produced per mole
of zooplankton dying from non-predatory causes.
(See also the explanation for E7) .
p. is the predation rate on an individual of species i (see
Equations 3 and 5).
Ill
-------
R, is the sum of the respiration rates for heterotrophs. It is
h
assumed to be proportional to temperature per unit biomass
over the temperature range used. The term is
10
R, = E k. TB.
h i=y 1R i
in which k is the respiration rate concentration (time"
X T"1), and
T is temperature, °C.
R. is the respiration of the jth zooplankton species (j = 8, 9, or 10),
the jth term in the sum of terms defining R .
R7 is the respiration rate for bacteria, the term in the sum of
terms for R. for which i = 7.
h
S is assumed to be a constant rate per unit biomass. The term
is
6
S = S k. B.
i=l 1S i
in which S is secretion rate for all phytoplankton combined,
k. is the secretion rate constant for the i^h
is
phytoplankton species, and
j_t
B. is the biomass of the i n phytoplankton species.
112
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THE USE OF ALGAL ASSAYS TO DETERMINE EFFECTS OF WASTE
DISCHARGES IN THE SPOKANE RIVER SYSTEM
William E. Miller, Joseph C. Greene, Tamotsu Shiroyama
and Ellen Merwin
INTRODUCTION
The Washington State Department of Ecology (Cunningham and Pine,
1969) and the U.S. Environmental Protection Agency, Region X (Schmidt
and Kreizenbeck, 1973) defined three major water pollution problems
within the Spokane-Coeur d'Alene River drainage basin. They were (1)
nutrient over-enrichment, (2) toxicity (heavy metals), and (3) low
dissolved oxygen.
Corrective water quality management practices have been initiated
to maintain and/or obtain class A water quality standards throughout the
Spokane River system, below Post Falls, Idaho, for both dissolved oxygen
(8.0 mg/1) and total coliform content (medium of 240--20% not to exceed
1,000). This goal is to be met by upgrading the City of Spokane's
sewage treatment plant (STP) from primary to secondary treatment. While
secondary treatment would enable compliance with the class A dissolved
oxygen and total coliform bacteria criteria, it would not solve the
nutrient enrichment pollution problem which exists downstream from the
STP.
Assessment of the nutrient enrichment problem in the Spokane River
Basin is complicated by the occurrence of heavy metals (predominantly
zinc) in the upper reaches of the Coeur d'Alene Lake drainage basin.
Mine tailings and metallic sulfide minerals have entered the Coeur
d'Alene Lake basin for over 80 years (Rabe, Wissmar, and Minter, 1973).
The greatest development in mining has taken place along the south fork
of the Coeur d'Alene River between Kellogg and Mullan, Idaho. In this
area are located the principal mineral deposits of the Spokane Basin,
113
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including the largest silver mine and the first and third largest lead
producing mines in the United States. Ores from the mines are reduced
in several mills, usually located near the larger producing mines. A
large lead smelter located at Kellogg, Idaho, and an electrolytic zinc
plant and cadmium refinery reduces the ore and concentrates to refined
metals ready for market (U.S. Department of the Army, Corps of Engineers,
1952). Eighty-three percent of the zinc entering the Spokane drainage
basin is either from mining and smeltering wastewater effluents or is
leached from mine tailings (Schmidt, et. al., 1973). Proposed and
existing smelter wastewater treatment measures are currently being
upgraded and implemented by the Bunker Hill Mining Company at Smelterville,
Idaho, in an effort to solve the existing heavy metals pollution problem.
The impact of the proposed municipal and industrial wastewater
treatment measures upon the growth of algae in the Spokane river system
has been under considerable debate. Waste effluents are usually compli-
cated mixtures of organic and inorganic substances. The interaction of
these effluents on the growth of planktonic algae in a multiple use
river system are not well defined. The effects of phosphorus, ionic
strength and zinc interaction upon the growth of Selenastrum capricornutum
Printz in defined inorganic culture media have been reported (Greene,
Miller, and Shiroyama, 1974). Those results indicated that ionic
strength, expressed as ymhos/cm can be multiplied by a factor of 2.72 to
obtain the concentration of zinc (yg/1) that will inhibit growth 95% or
more of the test alga; the sensitivity of S. capricornutum to zinc is
inversely proportional to the ionic strength of the test substrate; and
a twenty-fold increase in phosphorus content of the test water did not
affect zinc toxicity.
Phosphorus and nitrogen yield relationships for S. capricornutum
grown in defined inorganic culture media have been determined (Shiroyama,
Miller and Greene, 1974). Factors for converting the orthophosphorus
and total soluble inorganic nitrogen (TSIN = NCL + NO., + NHj content of
114
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a test substrate into maximum yield of the test alga have been determined.
Each yg/1 of orthophosphorus in waters containing > 10 yg P/l, when
other constituents are not growth limiting, will support 0.43 mg dry
wt/1 of $_._ capricornutum. Waters containing less than 10 yg phosphorus/1
generally support less than 0.10 mg dry wt per microgram of orthophos-
phorus. One microgram/1 of TSIN in waters which are not growth limited
by constituents other than inorganic nitrogen will support 0.038 mg dry
wt/1 of the test alga. These factors can be used to identify potential
eutrophication problem sites. However, it should be recognized that
constituents other than nitrogen or phosphorus may be growth limiting.
The calculated theoretical productivity potential must be verified by
actual algal assay analysis to determine (1) the presence of other
growth limiting nutrients, (2) the presence of toxicants such as heavy
metals and (3) if the chemical analyses for nitrogen and phosphorus
within the test substrates are realistic.
This paper is concerned with the results of algal ass-ays performed
on water collected at seven Spokane River and tributary sites (Fig. 1)
to evaluate the impact of industrial (smelter and mining) and domestic
waste effluents on the growth of planktonic algae. The goals of the
assay were to determine (1) the nutritionalnitrogen and phosphorus--
status of the Spokane River system, (2) the critical nutrient responsible
for the support of algal growth within the Spokane River system and (3)
the algistatic concentration of zinc that would prevent the growth of
planktonic algae.
METHODS
The Algal Assay Procedure, (AAP) Bottle Test, August 1971, using
Selenastrum capricornutum Printz as the test alga, was used to assess
the algal growth response of the Spokane River samples. Prior to assay-
ing, the samples were autoclaved to solubilize the nutrients in the
indigenous biomass, carbonated with a mixture of one percent carbon
dioxide in air until the original pH was obtained, and filtered through
115
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cr>
BOWLe
PITCHER1
PARK
MULLAN
Figure 1. Spokane River Basin sampling sites
-------
a 0.45 micron porosity membrane filter to remove participate material
which would interfere with using an electronic particle counter for
measurement of algal biomass in the inoculated test samples. Results of
the algal assay are reported as the maximum yield of the test alga (mg
dry wt/1) obtained during a 14-day incubation period-
Seven sampling sites (Fig. 1) were chosen in the Spokane River
Basin which represented varying types of water quality with respect to
both nutrients and the presence of toxic materials. The water sample
collected at the seven mile station in the Spokane River was divided
into two aliquots; control and control passed through Dowex Al exchange
resin (sodium form) to remove selected divalent and polyvalent heavy
metal cations. After passage through the ion exchange resin, calcium
and magnesium, equivalent to that in the original water sample, were
added back to the water. A mixture of trace metals (AAP Bottle Test,
1971) was also added back to the water. Pretreatment with 1.0 mg/1 ethylene
diamine tetra acetic acid (EDTA) sodium salt was made prior to the assay of
the water sample collected at Riverside State Park.
DISCUSSION AND RESULTS
Nutrients enter the Spokane River basin from several major sources
including domestic, industrial, agricultural and groundwater intrusion.
Population increases and lack of adequate wastewater treatment facilities
have been identified as the major causes of increasing amounts of nitrogen
and phosphorus entering the Coeur d'Alene and St. Joe River systems
(Funk.et.al., 1973).
Nitrogen trends in the Spokane River system indicate increasing
nitrate concentrations due to groundwater accretions. The industries,
the Spokane sewage treatment plant, and tributaries confluent with the
Spokane River were relatively insignificant direct sources of nitrate
nitrogen compared to the groundwater (Schmidt and Kriezenbeck, 1973).
117
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Total soluble inorganic nitrogen levels in the natural waters of
the Spokane and Coeur d'Alene drainage systems range from a low of 0.001
mg/1 at Post Falls, Idaho, to a high of 0.918 mg/1 at Smelterville,
Idaho. A conversion factor of 0.038 multiplied by the micrograms total
soluble inorganic nitrogen per liter in the test water, (Shiroyama,
Miller and Greene, 1974) can be used to predict the 14-day maximum yield
of S. capricornutum if nitrogen is the primary limiting nutrient and no
toxic consituents are present. Theoretically, therefore, enough nitrogen
is present in these waters to support 0.04 to 34.88 milligrams dry
weight of the test alga during the 14-day culture period.
Miller, Maloney, and Greene, (1973) established productivity sub-
groups for 14-day cultures of S. capricornutum as low productivity
(0.00-0.10 milligrams dry weight per liter), moderate productivity
(0.11-0.80 milligrams dry weight per liter), moderately high productivity
(0.81-6.00) milligrams dry weight per liter), and high productivity
(greater than 6.00 milligrams dry weight per liter).
Nitrogen productivity potential within the Spokane drainage basin
ranges from low to high with the greatest potential in the reach below
Riverside State Park to Long Lake Dam. Control of nitrate nitrogen
within the lower Spokane River basin is complicated by groundwater
inflow containing high concentrations of nitrate nitrogen. The control
of nitrogen productivity is also limited by economics and the presence
of blue-green nuisance algae and other microorganisms capable of fixing
atmospheric nitrogen. These factors led us to concentrate on phosphorus
as the controlling constituent to regulate p.lanktonic algal productivity
within the Spokane River system.
118
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The south fork of the Coeur d'Alene River at Mullan, Idaho, is a
low nutrient high quality water, uncontaminated by mine waste discharge.
An autoclaved and filtered sample of the south fork at Mullan, collected
in November 1972, had a conductivity of 49 ymhos/cm and contained 7 yg
orthophosphorus/1, 3 yg TSIN/1 and 18 ug Zn/1. The orthophosphorus and
inorganic nitrogen content indicated that, theoretically, it would
support algal growth in amounts not greater than 0.11 mg dry wt (0.038
times 3 yg TSIN/1). The actual algal yield in the sample was 0.03 mg
dry wt/1 (Figure 2). The addition of 20 yg P/l to the sample produced
0.31 mg dry wt/1 of S. capricornutum. The predicted algal yield for
this phosphorus concentration (27 yg P/l) in the presence of all other
essential nutrients is 11.6 mg (27 X 0.43) dry wt/1 of the test alga.
The failure of the test water to support this amount of algal growth
indicated that the south fork of the Coeur d'Alene River may become
limited by other essential nutrients when sufficient phosphorus is added
to the test water- Tne addition of 1000 yg N/l and 20 yg P/l increased
algal growth to 8.2 mg dry wt/1 or 71 percent of the expected yield of
11.6 mg dry wt/1. This indicated that when the nitrogen and phosphorus
requirements for algal growth were satisfied, either other essential
nutrients or the presence of toxic materials limited maximum algal
growfcl) in the test water. The ionic strength (49 ymhos/cm) of this
water multiplied by 2.72 indicated 133 yg Zn/1 would inhibit algal
growth greater than or equal to 95 percent. The 18 yg Zn/1 contained in
the test water could limit algal growth by 13 percent (Table 1) when
sufficient nutrients are introduced. Thus, 84 percent (71 plus 13) of
the algal yield obtained with the addition of 1000 yg N and 20 yg P can
be accounted for. Interaction of constituents or the absence of other
essential nutrients may be responsible for the other 15 percent of
anticipated yield.
119
-------
10
o»
2
1
10
10"
"2
| i |
1 ' 1
1000 jjg N/L
20 jug P/ L
N plus P
1
1
0
Figure 2.
2
8
10 12 14 16
DAYS
Effect of various nutrient additions to South Fork Coeur d'Alene
River water, Mullan, Idaho on the growth of S. capricornutum
120
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Table 1. CALCULATED PERCENT ZINC
TOXICITY
Sampling Site
So. Fork Coeur d'Alene at Mullan
So. Fork Coeur d'Alene at Smelterville
Coeur d'Alene at Lane
Coeur d'Alene Lake (driftwood pt. )
Spokane River at Post Falls
Spokane River at Seven Mile Bridge
Spokane River at Long Lake Dam
yg/i
Zn
18
7500
2470
118
107
75
18
Specific
Conductivity
ymhos/cm
49
382
152
54
49
163
169
% calculated*
Zn. toxicity
13
>100
>100
80
80
17
4
*yg/l Zn -e- (2.72 x ymhos/cm x .95) This calculation is valid for zinc
only and does not take into account interaction with other heavy metals.
The Coeur d'Alene Lake watershed is the main contributor of zinc to
the Spokane River; contributing about 83 percent of the total monthly
load during September 1972 low flow conditions (Schmidt and Kreizenbeck,
1973).
Samples collected at the south fork of the Coeur d'Alene River at
Smelterville, the Coeur d'Alene River at Lane, and Coeur d'Alene Lake
(off Driftwood Point) in November 1972 contained concentrations of
dissolved zinc ranging from 7500 yg/1 at Smelterville to 118 yg/1 off
Driftwood Point. The algal assay growth responses of these waters
reflects the calculated toxicity of zinc on the growth of S. capricornutum
(Table 1). Each of these autoclaved and filtered test waters supported
less than 0.10 mg dry wt/1 of the test alga. This growth response was
within the anticipated yield based on the orthophosphorus content in the
samples collected at the Coeur d'Alene River at Lane and at Coeur d'Alene
Lake (off Driftwood Point). The sample collected at Smelterville con-
tained 96 yg P/l. This concentration of phosphorus, in the presence of
all other essential nutrients and the absence of toxic materials, should
121
-------
have supported 41.3 mg dry wt/1 of S. capricornutum. This theoretical
(96 times 0.43) 41.3 mg dry wt/1 maximum yield (calculated for the south
fork of the Coeur d'Alene River at Smelterville) indicates that this
water could have a high phytoplankton growth productivity potential if
its zinc content (7500 yg Zn/1) is reduced below the algistatic level.
An abundance of periphytic algal growth was observed in the River during
the November 1972 sampling. This suggests to the authors that these
periphyton may have a greater tolerance to high concentrations of zinc
than phytoplankton.
The autoclaved and filtered (November 1972) water sample collected
from the Spokane River at Post Falls contained 8 yg P/l, 1 yg TSIN/1 and
107 yg Zn/1. The nitrogen and phosphorus nutrient content of this water
is essentially the same as that found in the autoclaved and filtered
Coeur d'Alene Lake (off Driftwood Point) water sample. Water collected
at both sampling sites exhibited similar algal growth response to the
addition of nitrogen and phosphorus (Figure 3). The average dissolved
zinc (112 yg/1) in these water samples could override the effect of
additional phosphorus loading, up to 20 times (160 yg P/l) greater than
that presently found, upon the growth of planktonic algae (Greene ejt aj_,
1974). This suggests that zinc toxicity may be the predominant growth
limiting factor for planktonic algae in the Spokane River between Post
Falls and Nine Mile Bridge.
The Spokane River samples collected at Riverside State Park and
Seven Mile Road bridge reflect the nutrient loading of the City of
Spokane's sewage treatment plant (STP). The Spokane STP is the major
contributor of phosphorus to the Spokane River. Cunnningham and Pine
(1969) cited an increase from 54 kilograms orthophosphorus per day (120
pounds per day) in the upstream water to 1418 kilograms per day (3100
pounds per day) immediately downstream from the STP. This is approx-
imately a 26-fold increase of orthophosphorus in the receiving water.
122
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10
10
en
2
I
I-'
£10°
10
10
-2
CONTROL
1000 jug N/L
20 jug P/ L
N plus P
6
8
SO 12 14 16
Figure 3.
DAYS
Effect of various nutrient additions to Spokane River water
Post Falls, Idaho on the growth of S. capricornutum
123
-------
In September 1972 the Spokane Sewage Treatment Plant contributed 57
percent of the total monthly phosphorus loading to the Spokane River
(Schmidt, et aj_, 1973).
The maximum 14-day algal yield obtained in the November 1972 (auto-
el aved and filtered) Spokane River water sample collected at Seven Mile
Road bridge was 0.11 mg dry wt/1 of S. capricornutum (Figure 4). This
was 0.3 percent of the expected yield of 36.6 mg dry wt/1 that could be
supported by 85 yg P/l. The predicted yield for this phosphorus level
is valid when either no toxicant is present in the water or when other
nutrients are not growth limiting. The November 1972, Seven Mile Road
bridge sample contained 75 yg/1 of dissolved zinc. This level of zinc
could limit algal growth 17 percent providing interaction with other
heavy metals does not contribute to the overall toxicity.
Figure 5 shows the effect of removing the 75 yg Zn/1 from the Seven
Mile Road bridge sample (by cation exchange) upon the growth of the test
alga. Algal growth yield in the treated sample was increased to 15.4 mg
dry wt/1. This represents 80 percent of the theoretical yield of 19.3
mg dry wt/1 calculated for the 510 yg/1 TSIN content of the test water.
Zinc removal from the test water increased productivity approximately
160 fold. The addition of 40 and 100 yg Zn/1 back to the metal stripped
sample again inhibited algal growth.
The effect upon zinc toxicity of the addition of 1.0 mg EDTA/1 to
an autoclaved and filtered Riverside State Park water sample was also
evaluated. This water sample contained 115 yg P/l, 509 yg TSIN/1 and
125 yg Zn/1. The yield in the untreated sample was 0.12 mg dry wt/1 of
S. capricornutum. This represents less than 1 percent of the expected
yield of 19.4 mg dry wt/1 based on the growth limiting TSIN content of
the test water (Figure 6). After chelation with 1.0 mg EDTA/1 this test
water supported 21.7 mg dry wt/1 of the test alga. These data indicate
that zinc and other heavy metals are primarily limiting planktonic algal
growth within the Spokane River system. This also reinforces our hypo-
thesis that zinc interaction downstream from the Spokane sewage treat-
124
-------
10
10'
10
I
H*
10
10
-2
CONTROL
1000 jug N/ L
20 jug P/ L
N plus P
, L
8
10 12 14 16
DAYS
Figure 4. Effect of various nutrient additions to Spokane River water,
Seven^le Road Bridge, Washington on the growth of S. capricornutum
125
-------
O»
2
1
10
I0
10
10
"2
CONTROL
METALS REMOVED
__ METALS REMOVED + IOO>igZn/L
METALS REMOVED + 40-ugZn/L
16
Figure 5.
DAYS
Effect of zinc removal from feven Mile Road Bridge water on the
growth of S. capricornutum
126
-------
10
10
CONTROL
1.0 Mg/L EDTA
10
o»
2
I
£10°
o
10'
f- /
-2
10
Figure 6.
1
1
I
I
> 2 4 6 8 10 12 14 16
DAYS
Effect of EDTA addition to Spokane River water, Riverside
State Park, Washington on the growth of S. capricornutum
127
-------
ment plant prevents massive algal growth relative to the nutrient
loading from the Spokane sewage treatment plant.
Natural decomposition, and/or complexing of zinc by organic compounds
or some other Zn removal mechanism, downstream from the Spokane sewage
treatment plant reduced the concentration of zinc from an average of 112 yg/1
immediately downstream from the treatment plant to less than 20 yg Zn/1
in the (November 1972) Long Lake Dam water sample. The growth response
of S. capricornutum, reflecting the reduced zinc level within the Long
Lake Dam water sample, is shown in Figure 7. The maximum yield obtained
in this sample was 14.9 mg dry wt/1. The theoretical yield, based on
the orthophosphorus content of 46 yg/1, is 19.8 mg dry wt/1. The lower
than anticipated growth response to the addition of nitrogen and phosphorus
indicates that either some essential nutrient other than phosphorus may
be limiting maximum algal growth or that a toxic substance, other than
zinc, may be present in this water. However, a growth response of 14.9
mg dry wt/1 of S. capricornutum in the algal assay indicates that Long
Lake is a highly productive body of water.
SUMMARY AND CONCLUSIONS
Algal assays were used to define the effects of heavy metal and
domestic wastewater effluents upon the potential growth of planktonic
algae within the Spokane River Basin. Results of these assays led to
the following conclusions:
(1) Nitrogen and phosphorus additions stimulated algal growth in
waters collected in the upper reaches of the south fork of the
Coeur d'Alene River at Mullan, Idaho. Stimulatory effects of
these nutrients decreased proportional to the zinc concen-
trations in waters collected downstream from Smelterville,
Idaho.
128
-------
10
10
I
£10°
10
10
-2
CONTROL
1000 jug N/L
20 jug P/ L
- ^^^ - r \j jji'
N plus P
8
10 12 14 16
DAYS
Figure 7. Effect of variousn utrient additions to Long Lake Dam water
on the growth of S. capricornutum
129
-------
(2) Algal growth potential in the Spokane River from Post Falls,
Idaho, to Riverside State Park, Washington, is regulated by
the average dissolved zinc content of 112 yg/1.
(3) A 20-fold increase in orthophosphorus loading to the Spokane
River system upstream from Riverside State Park would have
little effect upon the growth of planktonic algae unless the
zinc content of these waters is reduced.
(4) A natural reduction of zinc from 112 yg/1 at the Spokane STP
to 20 yg/1 at Long Lake Dam, 23 kilometers downstream from the
treatment plant, enabled algal growth to increase proportionally
to the orthophosphorus content of the water.
(5) Assessment of nutrient enrichment problems, complicated by the
occurrence of heavy metals (predominantly zinc), can be accom-
plished through use of algal assays.
(6) The addition of chelators (EDTA) and the use of cation exchange
resins to remove heavy metal toxicity from test waters prior
to algal assay holds considerable promise. These two pretreat-
ment methods gave similar assay results on waters collected
within the Spokane River system.
130
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REFERENCES
Cunningham, Richard K. and Roland E. Pine. 1969. A Preliminary
Investigation of the Low Dissolved Oxygen Concentrations That
Exist in Long Lake Located Near Spokane, Washington. Washington
State Water Pollution Control Commission. Technical Report
No. 69-1.
Environmental Protection Agency. 1971. Algal Assay Procedure Bottle
Test. National Eutrophication Research Program, National
Environmental Research Center, Corvallis, Oregon. 82 p.
Funk, William H., Fred W. Rabe, Royston Filby, Jon I. Parker,
James E. Winner, Larry Bartlett, Nancy L. Savage, Paul F. X.
Dunigan, Jr., Neil Thompson, Richard Condit, Paul J. Bennett,
and Kishor Shah. 1973. Biological Impact of Combined Metallic
and Organic Pollution in the Coeur d'Alene-Spokane River Drainage
System. Washington State University and University of Idaho,
Joint Report (B-044 Wash and B-015 IDA).
Greene, Joseph C., William E. Miller, Tamotsu Shiroyama, and Ellen
Merwin. 1974. Variation of Zinc Toxicity to the Green Alga
Selenastrum capricornutum Printz As a Function of Phosphorus
or Ionic Strength. (In preparation)
41V~l-Ill_a I -/ / O
Algal Assays.
or Ionic Strength. (In preparation)
Miller, William E., Thomas E. Maloney and Joseph C. Greene.
Algal Productivity in 49 Lakes as Determined by Algal
Water Research 8:667-679.
Rabe, Fred W., R. C. Wissmar, and R. F. Minter. 1973. Plankton Popula-
tions and Some Effects of Mine Drainage on Primary Productivity
of the Coeur d'Alene River, Delta, and Lake. Water Resources
Research Institute University of Idaho, Technical Report A-030-IDA.
Schmidt, Bill and Ron Kreizenbeck. 1973. September 1972 Spokane
River Basin Survey Report. Presented at Spokane Feb. 20, 1973.
Shiroyama, Tamotsu, William E. Miller, and Joseph C. Greene. 1974.
A Method for Predicting the Maximum Yield of Selenastrum
capricornutum Printz in Phosphorus and Nitrogen Limited Waters.
(In preparation)
U. S. Department of the Army, Corps of Engineers. 1952. Columbia
River Basin Study.
131
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EFFECT OF NITROGEN AND PHOSPHORUS ON THE
GROWTH OF SELENASTRUM CAPRICORNUTUM
Tamotsu Shiroyama, William E. Miller
and Joseph C. Greene
INTRODUCTION
Phosphorus and nitrogen have been established as the two most
critical nutrients related to algal productivity and eutrophication. In
1919, E. Naumann indicated that phosphorus and nitrogen play a major
role in the eutrophication process. More recently, Vollenweider (1968)
reiterated Naumann's viewpoint. Sawyer (1968), Weiss (1969), Miller et.
al. (1971, 1973), and others have also shown that phosphorus and nitrogen
are important in algal productivity. This paper discusses the growth
stimulating and/or limiting effects of phosphorus and nitrogen upon
Selenastrum capricornutum Printz. cultured in a defined medium.
METHOD
The Algal Assay Procedure Bottle Test (EPA, 1971) was used exclu-
sively throughout this investigation. The assays were carried out in
500-ml Erlenmeyer flasks containing 100 ml of the algal assay medium
(AAM). Dibasic potassium phosphate (KpHPO*) and sodium nitrate (NaNO^)
salts were used as sources of phosphorus and nitrogen respectively. All
assays were conducted in triplicate and each flask was inoculated from a
seven-day-old culture of S. capricornutum grown in AAM to give a final
concentration of 1000 cells/ml. Inoculated flasks, without the added
phosphorus or nitrogen, served as controls.
132
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The cultures were incubated at 24+2°C under continuous "cool-white"
fluorescent lighting of 400 ft-c (4304 lux) and continuously shaken at
110 oscillations per minute for a maximum of 14 days. Counts were made
with an electronic particle counter equipped with a mean cell volume
computer. Algal cell were counted on days 3, 5, 7, 10, and 14.
RESULTS AND DISCUSSION
Phosphorus,
By maintaining the nitrogen concentration at 4.200 mg/1 (nitrogen
concentration provided in MM), the response of S. capricornutum showed
that, when nitrogen was not growth-limiting, the maximum yield increased
proportionally with each phosphorus addition. This relationship between
the biomass (mg/1 dry wt.) produced with each concentration of phosphorus
as a function of time (days) is shown in Figure 1. A linear relationship
exists between maximum yield (mg/1) and phosphorus concentration (Figure
2) up to a phosphorus concentration of approximately 0.300 mg/1. Above
this concentration, a nutrient other than phosphorus becomes growth-
limiting. These results show that AAM, having a phosphorus concentration
of 0.186 mg/1, is definitely phosphorus limiting for the growth of S_^
capricornutum. Also, an increase or decrease in the concentration of
phosphorus concomitantly affects the maximum growth rate. For example,
by the fifth day at which the growth rate was at its maximum, the phos-
phorus increase from 0.006 to 0.012, to 0.023, to 0.046, to 0.092, to
0.186 mg/1 resulted in a maximum growth rate increase from 0.871 to
1.07, 1.19 to 1.31, and to 1.42 day , respectively.
A linear regression analysis of phosphorus versus algal biomass
(mg/1 dry wt.) showed that on days 3, 5, 7, 10, and 14, the correlation
between the two variables did not become significant until day 5 (Figure
3). This same statistical analysis also demonstrated that the growth
133
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80 -
NITROGEN = 4.2 MG/L
£40-
.012 P
.006 P
8 10 12 14 16 18 20 22
Figure 1. Effect of phosphorus additions in AAM on the growth
of S. capricornutum
134
-------
NITROGEN = 4.2 MG/L
(MAX. YIELD)
co
en
130
110
\
90
^70
CC
Q
50
30
10
.024
o
I.I.I. I, I I , I 1 I I I , I , I , I , I , 1 , I I
.096
.168
.240 .312
P-MG/L
.384 456
Figure 2. Maximum yield of S. capricornutum i.n MM with
phosphorus additions
-------
130
110
PHOSPHORUS CURVE
(NITROGEN = 4.2MG/L)
CO
(D
90
£70
o:
Q
50
30
10
.012
rDAY 14
--DAY 10
L DAY 07
- DAY 05
.048
.084
.120 .156
P-MG/L
.192
Figure 3. The relationship of biomass and phosphorus addition
in AAM of S. capricornutum
-------
responses for days 7, 10, and 14 were nearly identical and the correlation
coefficients between phosphorus and biomass for the five different days
were 0.622, 0.995, 0.999, 0.998, and 0.999, respectively.
The linearity of the phosphorus curve (Figure 2) makes it possible
to utilize the slope in predicting the maximum yield of the alga. In
the presence of other essential nutrients and in the absence of any
toxicants, .001 mg/1 of orthophosphorus in water containing > .010 mg P/l
will yield 0.43 mg/1 dry weight of the alga.
Nitrogen
The effect of nitrogen upon the growth of S. capricornutum was also
evaluated. The phosphorus concentration was maintained at the level
used in MM (0.186 mg/1) and the nitrogen concentrations were varied
from 0.016 to 4.200 mg/1 (Figure 4). A comparison of the growth rates
found during the phosphorus additions (Figure 1) and by the nitrogen
additions (Figure 4) indicate that these two nutrients do produce similar
results. For example, the maximum growth rate for nitrogen was achieved
by the seventh day, but during phosphorus addition this rate was achieved
by the fifth day. When the nitrogen was increased from 0.016 to 0.064
to 0.132 to 0.524 to 2.10 mg/1, the growth rate increased correspondingly
from 0.496 to 0.613 to 0.778 to 0.977 and to 1.034 day'1, respectively.
The t-test distribution analysis on the maximum algal yield showed
that with lower concentrations of nitrogen the yield did not become
significant until nitrogen levels of between 0.032 and 0.064 mg/1 were
reached (Figure 5). At concentrations of nitrogen greater than 2.10
mg/1, with phosphorus being the limiting growth factor (0.186 mg/1), the
maximum yield became less significant.
The linear regression analysis showed, as it did with phosphorus, a
definite linear relationship between maximum yield and nitrogen concen-
137
-------
PHOSPHORUS = O.I86 MG/L
80
70
60
50
40
30
20
10
00
. 4.2 N
* 2.IN
.05 N
.524N
.262N
.I32N
.064N
.0-3 2-N-
i i i
1 . 1
1 I 1
2 4 6 8 10 12 14 16 18 20 22
DAYS
Figure 4. Effect of nitrogen additions in AAM on the growth of
$ capricornutum
138
-------
GJ
UD
80
60
40
20
7
6
5
4
3
2
I
PHOSPHORUS = 0.186 MG/L
(MAX. YIELD)
. i . I . i . ,,i I .
.l.l.l.i.l.i.i
.03 .09
.15 .21
.27 .60
N-MG/L
.8 3.0
4.2
5.4
Figure 5. Maximum yield of S. capricornutum in AAM-with nitrogen
additions.
-------
tration (Figure 5). This relationship was linear at nitrogen concentra-
tions of 0.032 to 2.10 mg/1, above which, the phosphorus concentration.
became growth-limiting thereby preventing further utilization of nitrogen
by the algal cell. Figure 6 shows the linear relationship for days 5,
7, 10, and 14 where the correlation coefficients between nitrogen and
biomass (mg/1) were 0.949, 0.998, 0.999, and 0.999, respectively.
Figure 5 was used for ascertaining the predictive yield factor for
nitrogen. Assuming the essential nutrients and toxic conditions stated
earlier for orthophosphorus, .001 mg/1 of total soluble inorganic nitrogen
(N02+N03+NH3) will produce a maximum yield of .038 mg/1 dry weight of
the alga.
CONCLUSION
Growth rates of SL capricornutum, grown in Algal Assay Medium,
revealed that the major uptake of phosphorus and nitrogen occurred in
the first five days of growth.
The higher growth rate observed with phosphorus, as compared to
nitrogen indicates that the test alga assimilates phosphorus more rapidly
than nitrogen.
There is a definite linear relationship between biomass produced
and the amount of phosphorus and nitrogen present.
The determination of phosphorus and nitrogen requirements for the
alga, indicate that when all other essential nutrients are present and
in the absence of any toxicants, the maximum growth responses of the
alga can be predicted. Waters containing > .010 mg/1 orthophosphorus
will yield 0.43 milligrams dry weight of the alga per .001 mg P/l.
Similarily for each .001 mg/1 total soluble inorganic nitrogen will
yield .038 milligrams dry weight of the alga. Actual yield is considered
statistically significant within +20 percent of the predicted yield.
140
-------
NITROGEN CURVE
(PHOSPHORUS = 0.186 MG/L)
- DAY 14
* - DAY 10
- DAY 07
- DAY 05
0.3 0.6 0.9 1.2
N-MG/L
1.5
1.8
2.1
4.2
Figure 6. The relationship of biomass and nitrogen additions
in AAM of S. capricornutum
141
-------
REFERENCES
Environmental Protection Agency. 1971. Algal Assay Procedure:
Bottle Test. National Eutrophication Research Program, National
Environmental Research Center, Corvallis, Oregon.
Miller, W. E., T. E. Maloney, and J. C. Greene. 1973. Algal
Productivity in 49 Lake Waters as Determined by Algal Assays.
(In Press)
Miller, W. E. and T. E. Maloney. 1971. Effects of Secondary and
Tertiary Sewage Effluents on Algal Growth in Waters of a Lake-
River System. J. Water Poll. Con. Fed. 43(12):2361-2365.
Naumann, E. 1919. Nagna Synpunkter Agaenda Limnoplanktons Okologie.
Svensk. Bot. Tidsker. 13:129-163.
Sawyer, C. N. 1966. Basic Concepts of Eutrophication. J. Water
Poll. Con. Fed. 38(5):737-744.
Shapiro, J. and R. Ribeiro. 1965. Algal Growth and Sewage Effluent
in the Potomac Estuary. J. Water Poll. Con. Fed. 37(7):1034.
Vollenweider, R. A. 1968. Scientific Fundamentals of the Eutrophica-
tion of Lakes and Flowing Waters, with Particular Reference to
Nitrogen and Phosphorus as Factors in Eut>rophication. Technical
Report to the Organization for Economic Cooperation and Develop-
ment, Committee for Research Cooperation. 159 p.
Weiss, C. M. 1969. Relation of Phosphates to Eutrophication. J.
Amer. Water Works Assoc. 61(8):387-391.
142
-------
THE USE OF IN. SITU ALGAL ASSAYS TO EVALUATE THE EFFECTS OF SEWAGE
EFFLUENTS ON THE PRODUCTION OF SHAGAWA LAKE PHYTOPLANKTON
Paul D. Smith
INTRODUCTION
Shagawa Lake is a eutrophic lake in northeastern Minnesota near
2
the Canadian Border. It has an area of about 9.2 km , a mean depth
of 5.7 m and a maximum depth of 13.7 m. About 65% of the surface inflow
to the lake is from the Burntside River which drains oligotrophic
Burntside Lake; the remainder comes from several minor streams (Malueg,
et al. 1973). Because the region is primitive geologically and its
lakes are predominantly oligotrophic, Shagawa Lake contrasts sharply
by being one of the more productive water bodies in Minnesota (Megard
1970). Paleolimnetic studies (Bradbury and Waddington, 1973) indicate
the lake is a victim of cultural eutrophication as a result of past
logging and mining on its shores and by virtue of its function over the
past 70 years as a receptacle for the domestic wastes of the city of
Ely (pop. 5,000).
Shagawa Lake was chosen by the Federal Water Pollution Control
Administration as a suitable candidate for a lake restoration demonstration
project. It was desired to demonstrate the restoration of a eutrophic
lake which owed its problems to the input of domestic secondary sewage.
The method to be tested was advanced wastewater treatment for phosphorus
removal as a means to drastically reduce the phosphorus loading of the
lake and concomitantly its productivity.
Prior to the decision in 1971 to build the tertiary sewage treatment
facility at Ely, several years of data were collected to characterize
the limnology of Shagawa Lake. Also, a pilot tertiary treatment
143
-------
plant was built in 1967 as a part of a special series of experiments
designed to determine if this approach would work. Included in that
preliminary work was a number of in situ algal assays. Three of these
assays included the measurement of algal photosynthesis and this
paper will discuss the results of those experiments as they pertain
to the use of in situ algal assays.
METHODS
The studies were conducted in basins floating in Shagawa Lake. Three
large basins holding 150,000 gallons each were constructed using
neoprene impregnated nylon. They measured 12.2m x 12.2m at the surface,
1.5m x 1.5m at the bottom and were 6.1m deep. The bottoms of the
basins were sealed to prevent any exchange activity while at the
surface the basins were entirely open to the atmosphere. Buoyant
support was provided by a tubular aluminum float system. In addition,
five small basins capable of holding 80 gallons each were constructed
using 100-gallon capacity plastic bags supported by styrofoam collars.
Various mixtures of Shagawa Lake water, Burntside River water and
treated sewage effluent were placed in the basins along with an
inoculum of the current Shagawa Lake plankton. The three large basins
were filled simultaneously with lake water drawn from a point source
to ensure all basins received an identical inoculum of plankton;
additives were metered-in mechanically throughout the filling process.
The small basins were filled manually. Two levels of tertiary treatment
were tested: one designed specifically to remove phosphorus (99+%)
by chemical coagulation (alum) and filtration (i.e., the CF-system), and
a more elaborate scheme which incorporated a column of activated
charcoal, and cation and anion exchangers (i.e., the AE-system, see
Figure 1) capable of removing over 90% of all the nutrients in Ely's
secondary wastewater (Brice and Powers, 1969). The small basins were
sampled using a long plastic tube to remove a vertically integrated
144
-------
Primary
Settling Tank
High Rate
Trickling Filter
Activated
Charcoal
(CF)
Coagulation
Settling
Filtration
01
i
0
(CO
Cation
Exchange
(CE)
Anion
Exchange
(AE)|
L Tertiary System
Secondary
Settling Tank
(SE)
Chlorination
Discharge
FIGURE 1.
Sewage treatment facility block diagram
-------
sample. The large basins were sampled at four depths in the center and
a composite prepared for all analyses.
Chlorophyll a_was determined in duplicate by the method of UNESCO
(1966) correcting for phaeopigments (Lorenzen, 1967). Primary productivity
was measured in duplicate (small basins) or triplicate (large basins)
using the light- and dark-bottle oxygen method (Strickland, 1960)
with a six hour in sjitu incubation period.
Each day that productivity was measured in the basins the rates of
gross photosynthesis observed were compared statistically. The day's
data were arranged using a one-way classification scheme (according to
sewage type), an analysis of variance was performed and linear comparisons
made between selected pairs of treatment means using the method of
Scheffe1 (Snedecor and Cochran, 1967; Guenther, 1964). Rate differences
were declared significant at the 0.05 probability level (i.e. where a = 0.05),
The ratio of photosynthesis to respiration (P/R) was used to quantify
the relationship between autotrophy and heterotrophy in the basin
plankton and was calculated as follows:
3
daily gross photosynthesis (mq C/m /day)
- ^
24-hour community respiration (mg C/m /day)
RESULTS
The Large Basin Experiments
The objectives of these experiments were to evaluate the effectiveness
of the pilot tertiary plant in reducing the capacity of Ely's secondary
sewage effluent to stimulate phytoplankton primary production and to
observe the effect of different effluent mixtures on the specific
photosynthesis rates of Shagawa Lake algae.
146
-------
Experiment 1
In 1969 the filling of the basins began June 18 and sampling was initiated
June 20 when the basins were full. Planktonic photosynthesis was
measured every other day until the experiment was terminated July 3.
Lake water was mixed with treated sewage effluent in the ratio of
95 parts Shagawa Lake water to 5 parts effluent. Table 1 shows the
composition of the mixtures in the large basins.
Table 1. Composition of the large basins in Experiment 1
Basin
Effluent
SE CF
AE
%Shagawa Lake Mater
Filtered Unfiltered
Secondary 5 - -
CF-tertiary 5 -
Control - 5
95
95
95
A 5% enrichment was first tested because preliminary experiments
(Megard, 1969) had indicated this low a concentration could cause a
measurable response and, indeed, it was sufficient to cause a
significant increase in phytoplankton primary production.
Figure 2A shows that chlorophyll a^was most abundant in the basins
o
which received sewage additions. A maximum concentration of 46 mg/m
occurred after 9 days in the basin that contained 5% secondary effluent.
A peak of 28 mg/m was reached after 7 days in the basin that contained
CF-tertiary effluent. In contrast, chlorophyll ^concentrations in the
control basin remained fairly stable in the range of 13-20 mg/m3.
147
-------
CO
50j-
40
to
10
1.2
1.0
-g 0.8
ro
a-
3 *
EXPERIMENT I
D 5% Secondary Effluent
A 5% Tertiary Effluent (CF)
o 5% Filtered Shagawa Lake Water
* = low incident light
FIGURE 2. A. Chlorophyll a_ concentrations,
B. Gross Photosynthesis,
0.6
0.4
0.2
01
o
rr
0
II
13 *
n
u
1
C.
D.
I 3 5 7 9 II 13*
Day
Specific gross photosynthesis,
Ratio of daily gross photosynthesis to 24-hour
community respiration (P/R)
-------
Rates of gross photosynthesis (Figure 2B) were significantly greater
in the basins containing sewage effluents and rates in the basin with
secondary effluent were consistently higher than those in the basin
with CF-tertiary effluent.
Figure 2C shows that specific gross photosynthesis (mg C/mg chl a/
day) was significantly higher in the basin containing secondary effluent
during the first 5 days of experiment 1 while values in the tertiary and
control basins were quite similar. After day 5 there was no apparent
treatment effect on this parameter.
The P/R ratios plotted in Figure 2D indicate a higher degree of autotrophy
was present in the basins receiving sewage effluents. The mean ratio
in the secondary basin (1.9) was only slightly greater than in the
CF-tertiary basin (1.7). The mean ratio in the control basin was 1.3.
Experiment 2
The filling of the basins began July 15, 1969 and sampling was initiated
July 16. Planktonic photosynthesis was measured every other day until
the experiment was terminated July 29. Table 2 shows the composition
of the mixtures in the basins. Lake water was mixed with treated
sewage effluent in the ratio of 90 parts lake water to 10 parts
effluent.
149
-------
Table 2. Composition of the large basins in Experiment 2
Basin °,
SE
Secondary 10
CF-tertiary
Control
I Effluent
CF AE
-
10
-
% Shagawa
Filtered
-
-
10
Lake Water
Unfiltered
90
90
90
This 10% mixture was used to further test the effectiveness of the
tertiary treatment system to reduce the capacity of secondary effluent
to stimulate algal growth.
As in experiment 1 there was a treatment effect readily apparent in
the photosynthesis data (Figure 3A) while the cholorphyll a^ data
(Figure 3B) showed a lag in response.
The CF-tertiary treatment of the secondary effluent severely altered
the capacity of the secondary sewage to stimulate planktonic primary
productivity, and the mean rate of daily gross photosynthesis in the
CF-tertiary basin was 85% less than the rate in the secondary basin,
and 59% less than the mean rate in the control basin. There is reasonable
doubt, which will be discussed later, that this was accomplished solely
by the removal of certain nutrients from the secondary sewage.
Chlorophyll a_ and gross photosynthesis all declined in a similar manner
in both the control basin and CF-tertiary basin, but the numerical
values for these parameters were significantly lower in the CF-tertiary
basin in which there was also an unusually low balance between autotrophy
and heterotrophy as indicated by the P/R ratios in Figure 3C.
150
-------
2.or
o
o
to
£ 1.0
\
u
0>
0
XA-^
I I I I
2 4 6 8 10 12* 14
Day
C
3
2
o
o
o:
l
-
D\
^n
\
n
°^n°
- V
A K
\ 1 1
/n
nx
/
/
/
^oo
A A
1 1
\-«
\
\
\
v
\ / A
\/
V
i i
0 2 4 6 8 10 12* 14
Day
EXPERIMENT 2
a 10% Secondary Effluent
A 10% Tertiary Effluent (CF)
0 10% Filtered Shagawa Lake Water
* = low incident light
E
\
o>
50
40
30
20
10
0
120
100
80
B
D D
AAA-
I I I
1
2 4 6 8 10 12 14
Day
o|
O
o»
£
40
20
0
D
11
1 1
24 6 8 10 12* 14
Day
FIGURE 3.
A. Gross photosynthesis, C. Ratio of daily gross photosynthesis to 24-hour
R community respiration (P/R),
D- Chloropyll a_ concentrations,
D. Specific gross photosynthesis
151
-------
Specific rates of gross photosynthesis in the secondary basin were
in general less than those in the control basin except for day 2
(Figure 3D). This was probably related to a density factor to be
discussed later. While the mean specific rate of gross photosynthesis
in the CF-tertiary basin (43 mg C/mg chl a/day) was 20% lower than
the mean rates in the secondary and control basins (54 and 55 mg
C/mg chl a/ day, respectively), it was still indicative of the presence
of phytoplankton in reasonably good physiological condition, judging
from in situ photosynthesis experiments with lake phytoplankton
(Megard, 1969 and Megard and Smith, 1973). Thus it would appear that
there was a factor in the CF-tertiary effluent that proved detrimental
only to a discrete portion of the phytoplankton early in the experiment
(i.e. prior to the first sampling). It may have been a normal
constituent of the effluent that became disruptive at the 10% level
of enrichment, or a transient material which entered the tertiary
treatment system. It remains unidentified.
The Small Basin Experi ment
The purpose of this experiment was to assess the impact that different
tertiary effluents might have on the future productivity of Shagawa
Lake. To achieve this some alterations were necessary in the experimental
design. The most significant change was the use of Burntside River water
as a base medium. Because of the unfeasibility of transporting the
volume of Burntside River water necessary for filling the 150,000-gallon
basins, the experiment was conducted using 80-gallon basins. Table
3 defines the composition of the basins.
152
-------
Table 3. Composition of the small basins
Basin
Effluent
SE
CF
AE
% Shagawa Lake Water Burntside
Filtered Unfiltered River
Water (1%)
Secondary 5 - - - 1
CF-tertiary 5 - 1
AE-tertiary - - 5 - 1
Burntside R. - - 1
Shagawa Lake - 99 1
94
94
94
99
-
All basins received 1 part Shagawa Lake water to provide a plankton
inoculum; three basins received 94 parts Burntside River water plus 5
parts of a sewage effluent (SE, CF or AE); a fourth basin received 99
parts Burntside River water to serve as a control and the fifth basin
received 99 parts of filtered Shagawa Lake water. The latter basin
was included to test the assumption of the experimental design that the
basin containing Burntside River water plus secondary sewage was, in
fact, a model of Shagawa Lake. If true, similar responses would be
anticipated in these two basins.
The basins were filled on August 5, 1969, and sampled immediately.
Planktonic photosynthesis was measured every other day until the
experiment was terminated on August 13.
In general the biological activity in the five 80-gallon basins followed
two distinct patterns, one common to the Shagawa Lake basin and its
experimental model, the secondary basin, and the other common to the
153
-------
AE-tertiary, CF-tertiary and control basins. In the former group
the parameters were indicative of a rapidly growing algal population;
that is, the rates of chlorophyll a_ synthesis (Figure 4A), gross
photosynthesis (Figure 4B), and their P/R ratios (Figure 4C) were
all high relative to the other basins.
Regarding primary productivity there was no statistically significant
difference (p = 0.05) between the secondary basin and the Shagawa
Lake basin after day 3; mean daily gross photosynthesis rates in
the secondary (0.82 g C/m3/day) and Shagawa Lake (0.83 g C/m3/day)
basins were nearly identical and three times as great as the mean
rate in the control basin (0.26 g C/m /day). There was no apparent
stimulation of productivity in the CF- or AE-tertiary basins (Scheffe's
test, 5% level of significance). Phytoplankton productivity in the
CF-tertiary basin after day 3 was 65-74% less than in the secondary
basin while it was 75-82% less in the AE-tertiary basin.
Chlorophyll a^ data corroborated the productivity data for all basins.
It did appear from chlorophyll a_ and gross photosynthesis data (cf. Figures
4A and 4B) that a negative response was occurring in the AE-tertiary
basin after day 5, but this could not be confirmed statistically.
Specific gross photosynthesis rates (Figure 4D) in general increased
through day 7- The secondary basin did exhibit significantly higher
rates on two separate days which resulted in its mean rate (39 mg C/mg
chl/day) being 30% greater than the means of the other four basins
which were essentially identical (i.e. 30-32 mg C/mg chl/day, see
Table 6).
In general the P/R ratios followed patterns similar to those in the large
basins in that the ratios achieved their greatest values in the basins
154
-------
ro
e
\
CP
e
60
50
40
30
20
10
0
3579
Day
O
o
B
3* 5 7
Day
o
a:
3k
2k
o
-a
a
o
CP
E
60
50
40
30
20
10
0
FIGURE 4.
5
Day
7
A.
B.
C.
MEDIUM BASIN EXPERIMENT
D 5% Secondary Effluent
0 5% Tertiary Effluent (CF)
5% Tertiary Effluent (AE)
A 99% Burntside River Water D.
A 99% Filtered Shagawa Lake Water
* = low incident light
Chlorophyll a_ concentrations,
Gross photosynthesis,
Ratio of daily gross photosynthesis to 24-hour
community respiration (P/R),
Specific Gross photosynthesis
155
-------
showing the highest primary productivity. The mean P/R ratios in
the secondary and Shagawa Lake basins were, respectively, 43% and 29%
greater than in the control basin. The P/R ratios in the CF-tertiary
basin were very similar to those in the control, while the ratios in
the AE-tertiary basin began to lag after day 5 correlating with its
chlorophyll decline, indicative of a waning phytoplankton population.
DISCUSSION
The Large Basin Experiments
Daily Gross Photosynthesis
Secondary sewage effluent from the Ely municipal wastewater treatment
plant exhibited a definite capacity to stimulate phytoplankton primary
production. Two iji situ large-basin experiments were conducted and
in each phytoplankton productivity was statistically the greatest, by
a highly significant margin (1% level of significance), in the basin
receiving secondary sewage effluent. On the basis of mean gross
photosynthesis rates the stimulation of productivity in the basin
containing 10% secondary was 1.9 times that observed in the basin
with 5% secondary (Table 4).
Table 4. A comparison of mean gross photosynthesis rates in
experiments 1 and 2.
Experiment
1
2
°i
10
Ltt .
5
10
C
0.50
0.58
CF
g C/m /day
0.59
0.24
SE
0.94
1.57
Basin
CF SE
% Stimulation
18 88
171
156
-------
The effect of CF-tertiary treatment was to reduce the capacity of
secondary wastewater to stimulate phytoplankton production, but it also
appeared to introduce a factor deleterious to some algae where an
effluent concentration of 10 percent was used.
In experiment 1 (5% mixture) there was stimulation of productivity
in the CF basin but on the basis of mean gross photosynthesis rates it
was 70 percent less than in the secondary basin (Table 4). In experiment
2 (10% mixture) mean gross photosynthesis in the CF-basin was 59% less
than in the control basin. This reflected a substantial loss of
chlorophyll a_ in the CF-basin that occurred during the filling process
and was apparent in the first day's samples (Figure 3B). Since the
chlorophyll ^concentration in the composition water (i.e. Shagawa
Lake water) was monitored hourly it is known that the low chlorophyll
level in the CF-basin was due to a loss in that basin as opposed to gains
realized in the control and secondary basins. The concentrations of
phaeopigments in all three basins were nearly identical at the start
of the experiment suggesting the chlorophyll was lost through sedimentation
and not cellular lysis.
The idea of an inhibitory factor in the CF-effluent is important enough that
some reference should be made to four experiments not included in this
paper. In August, 1969, a 3% mixture of CF-tertiary effluent stimulated
phytoplankton productivity while mixtures containing 10% and 20%
CF-effluent exhibited the inhibitory effect described above (Smith, 1973).
In September, 1969, an additional experiment was conducted using a 10%
mixture of CF-effluent but in this case no deleterious effect was observed
(Brice, unpublished data). These results indicated the inhibitory factor
was either of a transitory nature, being absent by September, or it was
to some degree selective towards the kind of algae it acted upon.
Further investigation of the phenomenon was not pursued because of time
and manpower limitations and the fact that the concentration necessary to
bring about the effect was five-to-ten times greater than the lake
would experience under actual full-scale loading conditions. However, the
157
-------
results do emphasize that investigators must consider possible toxic factors
when engaging in experimental work with treated domestic wastewater
effluents, a fact that has been documented elsewhere as well (Middlebrooks
et a!., 1971; Dunstan and Menzel, 1971).
P/R Ratios---
The P/R ratio serves to quantify the relationship between production and
consumption in an ecosystem (Odum, 1961) and was used here to determine
the relative level of autotrophy (vs. heterotrophy) in each basin.
The general effect of sewage enrichment in the large basins was to
enhance autotrophy. The secondary basin consistently produced the
highest mean P/R ratios in each experiment (Table 5). Examining the data
it was apparent that where productivity was stimulated and the levels
of both photosynthesis and respiration (Smith, 1973) increased
along with biomass, the increase in photosynthetic activity was greater.
This agrees with the findings of Ryther (1954) who demonstrated that
the P/R ratios of Dunaliella euchlora cultures were a function of the
nutrient status of the algae with photosynthesis rates declining
more rapidly than respiration rates at the onset of nutrient deficiency.
Table 5. Photosynthesis/respiration (P/R) ratios observed in
two large basin experiments. Mean (range).
Experiment
1
2
%
Effluent
5
10
Control
1.3(1.0-1.8)
1.4(1.2-1.8)
Basin
Tertiary
1.7(1.2-3.1)
1.0(0.5-1.4)
Secondary
1.9(1.2-3.0)
2.2(1.6-2.9)
158
-------
The situation in the CF-tertiary basin was inconsistent. Photosynthesis/
respiration ratios were enhanced in experiment 1 (5% mixture) but they
were distinctly depressed in experiment 2 (10% mixture). Inasmuch
as 1) the P/R ratios in the secondary and CF-tertiary basins of
experiment 1 were very similar and-2) the P/R ratios in the control
basins of experiments 1 and 2 were basically identical, it is evident
that the community in the CF-basin in experiment 2 was in a stressed
situation relative to the CF-effluent.
Specific Gross Photosynthesis
While P/R ratios can be indicative of the health of an algal population,
they are at best conservative estimates where a natural population
is concerned due to the influence of heterotrophic respiration. A more
common reference is specific photosynthesis, here related to chlorophyll a_
(i.e. mg C fixed/mg chl a/day). While this parameter, too, is not
entirely definitive, it focuses on the photosynthetic system and has been
demonstrated repeatedly to be a good general index of the physiological
condition of natural algal populations and artificial cultures
(Hepher, 1962; Fogg, 1966; Glooschenko and Curl, 1971).
In the large basin experiments the effect of secondary effluent was to
cause an initial stimulation of specific photosynthesis which later
waned, while the effect of CF-tertiary treatment was to remove any capacity
to stimulate.
It is not certain what specific factors were responsible for the observed
declines in the secondary basins. There is evidence from both basin and
lake studies that suggests it was related in some manner to the density
of the algae. In experiments 1 and 2 the rapid decline in specific
photosynthesis in the secondary basins coincided with a moderate
increase in chlorophyll ^concentration. With respect to Shagawa
159
-------
Lake, the data in Figure 5 show that mean specific photosynthesis, which
had been increasing throughout the summer, exhibited a substantial decline
in mid-August when the density of the late summer blue-green algal boom
D 3
jumped from 28 mg/m of chlorophyll a^ to 67 mg/m at the onset of an
exponential growth phase.
Specific photosynthesis has been found to vary considerably in some
lakes (Tailing, 1957; Megard, 1970; and Elster, 1965) and very little
in others (Tailing, 1965). There are many factors which can influence
specific photosynthesis rates such as water temperature, the nutritional
status and photic history of the algae (Tailing, 1966). The importance
of plankton density has also been discussed in a number of papers. Some
have found it to be a significant factor (Wright, 1960; Elster, 1965)
while others have concluded it was not (Megard, 1970; Tailing, 1965). Thus
it is apparent that the manner in which the element of plankton concentration
functions must be dependent on other factors involved.
Generally speaking, the mean rates of specific photosynthesis observed
in each experiment (Table 6) did not differ largely from one another
(i.e. ± 7-22%).
Table 6. Mean rates of specific gross photosynthesis
(mg C/mg chl a/day)
Experi-
ment
1
2
*
%
Eff.
5
10
5
Month
June
July
August
SE
32
54
39
CF
30
43
30
Basin
AE C SL
29
55
30 30 32
Shagawa
Lake
in situ
-
-
24
Small Basin Experiment
160
-------
CTl
o
o
o
E
o
I*
100
i i
Photosynthesis
801 Chlorophyll a_
60-
40
20
0
/\
/ \
/ \
i \
\
IOO
80
60
40
20
10 20 30 10 20 30 10 20 30 10 20 30
Jun Jul Aug Sep
0
rO
ol
o
FIGURE 5. Maximum specific photosynthesis and chlorophyll a_
in Shagawa Lake, 1970 (Megard and Smith, 1973).
-------
However, over the entire 1969 experimental period, the mean rates
of the control basins showed a pattern of variation similar to that
observed in the lake during the summer of 1970 (Megard and Smith, 1973),
with elevated rates during July and lower rates in June and August
(cf. Table 6 and Figure 5). Where there was stimulation of specific
photosynthesis (i.e., in the secondary basins and Shagawa Lake) it
appeared to be dampened by a density-related factor. In terms of
3
chlorophyll a_, a concentration of about 27 mg/m appeared significant
because above this level algae in the secondary basin fixed carbon at
specific rates similar to those in the control basin.
The Small Basin Experiment
This experiment (5% mixture) is discussed separately from the large
basin experiments because it was conducted using a different format.
While four of the five basins used contained Burntside River water,
a fifth was filled with filtered Shagawa Lake water. The latter basin
was compared to the secondary basin to test the assumption that the
mixture containing secondary effluent and Burntside River water was,
in fact, an experimental model of Shagawa Lake. The AE- and CF-
tertiary basins were compared to the secondary and control basins to
assess the impact these tertiary effluents might have on the fertility
of Shagawa Lake.
Figure 6 shows the mean gross photosynthesis rates in each basin.
It is readily apparent that CF-tertiary treatment (i.e. phosphorus
removal) was sufficient to thoroughly destroy the capacity of secondary
effluent to enhance phytoplankton primary productivity. This is con-
sistent with the facts that 1) the Burntside River is essentially
devoid of soluble reactive phosphorus (SRP) throughout the year
162
-------
CTi
CO
0 O.I
g C/m3/day
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1
BR
;.57 AE1
CF
5% SE
:?99% SL V
FIGURE 6. Mean rates of gross photosynthesis in the small basin
experiment (BR = Burntside River, SL = Shagawa Lake,
AE, CF, SE = sewage types)
-------
(i.e. £0.001 mg/1) thus making orthophosphate a likely growth limiting
factor, 2) the concentration of SRP in the CF-tertiary effluent was only
0.008 mg/1 and hence contributed only 0.0004 mg/1 of SRP to the assay
system, an amount unlikely to cause a measurable stimulation of growth,
and 3) CF-tertiary effluent would not support the growth of Selenastrum
capricornutum without the addition of ortho-phosphate (Miller and Maloney,
1971). The results of this experiment were further supported by the
in situ algal assays of Powers et al. (1972) which demonstrated, using
phytoplankton indigenous to Shagawa Lake, that phosphorus and nitrogen
together were growth limiting in Burntside River water at the times
tested during the summers of 1969 and 1970.
The use of photosynthesis measurements did not offer an early insight
into the responses of the subject phytoplankton as was the case in the
large basin experiments. This was due in part to the sensitivity of the
oxygen method which requires at least 5 pg/1 of chlorophyll to
permit incubation times of 6 hours or less.
The specific gross photosynthesis rates observed were of interest. While
the Shagawa Lake and secondary basins supported high growth rates,
specific photosynthesis was similar in all basins except for a modest
stimulation in the secondary basin. This observation supports those of
Tailing (1966) and Megard (1970) that specific photosynthesis rates of
mixed natural algal populations that are actively growing are not
necessarily greater than those that are not. Thus specific photosynthesis
rates are not certain guides to the nutritional status of a mixed
phytoplankton population, because what is being measured is a mean weighted
according to the abundance and activity of several contributing populations,
The data collected served to validate the basic assumption that the
secondary basin was an experimental model of Shagawa Lake, for throughout
the experiment there was generally close agreement between the parameters
164
-------
measured in the secondary and Shagawa Lake basins. Thus, it may be
concluded that a full-scale CF-tertiary treatment system has the potential
to significantly reduce the trophic status of Shagawa Lake.
The Significance of Phaeopigments
Only recently have methods been developed to permit routine analyses
to be made for phaeopigments (i.e. phaeophytin and phaeophorbide) in water
samples being analyzed for chlorophyll a_ (Yentsch and Menzel, 1963;
Lorenzen, 1967).
Yentsch (1965J demonstrated the importance of correcting chlorophyll
a_ analyses for interference from phaeopigments, particularly with
deep water samples or samples near discontinuity layers. He showed that
failure to apply this correction can result in sizeable underestimates of
specific photosynthesis on the part of viable algae at those depths.
Phaeopigments were analyzed routinely in these experiments and it
was found that they could introduce considerable error into chlorophyll a_
determinations in batch-tests such as are described here. In experiment
2 on day 6 the Strickland and Parson trichromatic equation indicated the
chlorophyll ^concentration in the secondary basin to be 45 yg/1. In
fact, 44% of this value (i.e. 20 yg/1) were phaeopigments (Figure 7).
Obviously the failure to compensate for such an error would seriously
affect the accuracy of any calculated values of specific photosynthesis.
Table 7 gives a comparison of specific photosynthesis rates calculated
from corrected and uncorrected chlorophyll ^values in both the
165
-------
EXPERIMENT 2
Chlorophyll^, (uncorrected)
Chlorophyll o_ (corrected)
l\_/w
80
E 60
x
E* 40
20
0
rnueupiyiiiciiib
Chi. g_ (corr.) + Phaeopigments
^-"^,.\
/' ^*""". -v.>.
// .' .
I 1 1 1 1 1 1
2 4 6 8 10 12 14
Day
FIGURE 7. Chlorophyll a^ and phaeopigments in the secondary
basin (10% mixture)
166
-------
Table 7. A comparison of specific photosynthesis rates showing
the effect of correcting chlorophyll a^ analyses for phaeopigment
interference (experiment 2)
Day
2
4
6
8
10
12*
14
Control
Uncor-
rected
54.9
37.8
37.8
46.6
40.8
30.6
43.6
mg C
Basin
Cor-
rected
60.2
46.8
47.0
65.8
56.0
43.8
68.6
fixed/ing chlorophyll a/
%
Change
10.6
23.8
24.3
41.2
37.2
43.1
57.3
Uncor-
rected
101
39.6
33.7
34.8
31 .5
20.1
28.8
day
Secondary
Cor-
rected
116
44.0
46.7
50.5
43.4
29.2
48.1
Basin
°/
10
Change
14.9
11.1
38.6
45.1
37.8
45.3
67.0
*Low incident light
secondary and control basin of experiment 2. The concentrations of
phaeopigments in both basins were essentially the same on the initial
day of sampling (Figures 7 and 8) and were typical of levels
observed in Shagawa Lake during the summer growing season (Megard and
Smith, unpublished data).
Thus it behooves investigators to be cautious with regard to
chlorophyll a_ analyses. Lorenzen (1967) suggests, as a guide for
screening samples, that where the ratio
.665
-665
(before acidification)
(after acidification)
1.6
167
-------
EXPERIMENT 2
cr>
00
30
^ 20
e I0
^mui upny 1 1 u
. Chlorophyll CL
r naeopigmenis
Chi. a_ (corr.) +
MMMW
|MM^
-^"^.
-V.
^--
r I i i
\ UMUUF f CCICU /
(corrected)
Phaeopigments
- »^.
^.«- ... *
** «».
i i i
0
8 10 12
14
Day
FIGURE 8.
Chlorophyll a and phaeopigments in the control basin
(10% mixturej
-------
the presence of phaeopigments warrants quantitative consideration. The
familiar empirical equations for chlorophyll a_ were doubtlessly generated
using preparations essentially free from phaeopigments or other degradation
products and thus the presence of these entities would detract from the
accuracy of any chlorophyll a_ equation inasmuch as the degradation products
of the pigment do have significant extinction coefficients in the 663 - 665
nm range. As a result equations such as those of UNESCO (1966) or Strickland
and Parsons (1968) will over-estimate the presence of chlorophyll a_
and on the other hand they will underestimate the collective concentration
of chlorophyll a_ and degradation products due to the extinction coefficients
of the latter being smaller than those of the former in the 663 - 665 nm
range (Figures 7 and 8).
CONCLUSIONS
1. Ely's secondary sewage effluent possessed a definite capacity to
stimulate algal growth and specific photosynthesis; the use of CF- and
AE-tertiary treatment significantly reduced that capacity.
2. CF-tertiary effluent can be detrimental to some phytoplankton in
mixtures >10% wastewater.
3. Specific photosynthesis rates (i.e. assimilation ratios) are not
thoroughly reliable indicators of mixed-population activity. It is
recommended their use in assays as a response indicator be approached
3
cautiously where chlorophyll a_ concentrations exceed 27 mg/m .
4. There is good probability a full-scale CF-tertiary wastewater
treatment plant at Ely would result in a significant reduction in
the trophic status of Shagawa Lake.
169
-------
RECOMMENDATIONS: BATCH ASSAY TECHNIQUE
1. Large basins requiring many hours to fill should be sampled during
the filling process to define initial responses,
2. The length of batch assays should be at least 10 days to allow
the full growth potential of the test system to be realized.
3. Photosynthesis measurements can give a much earlier indication
of treatment effects than chlorophyll a_.
4. Chlorophyll a_ measurements should consider phaeopigment interference,
ADDENDUM
A full-scale tertiary wastewater treatment facility has been constructed
at Ely. It began operation January 1, 1973, and is principally a
phosphorus removal plant employing lime precipitation and not alum which
was used in the pilot plant studies. Algal assays by W. E. Miller and
coworkers, NERC, Corvallis, using 100% tertiary effluent from the full-
scale plant showed no signs of toxicity (personal communication).
ACKNOWLEDGMENTS
I wish to thank Dr. R. 0. Megard for his guidance in the field
work. R. M. Brice, Chief of the Shagawa Lake Project, together
with M. D. Schuldt and other staff members contributed valuable
advice and cooperation. Thanks, also, to D. W. Schults, D. P.
Larsen, K. W. Malueg and W. E. Miller for their critical reviews
of the manuscript.
170
-------
REFERENCES
Bradbury, J. P. and J. C. B. Waddington. 1973. The Impact of
European Settlement on Shagawa Lake, Northeastern Minnesota,
U.S.A. Limnological Research Center, University of Minnesota,
Minneapolis. Contribution No. 112. 32 p.
Brice, R. M. and C. F. Powers. 1969. The Shagawa Lake, Minnesota
Eutrophication Research Project. Pages 258-269 in Proceedings
of the Eutrophication-Biostimulation Assessment Workshop.
Pacific Northwest Water Laboratory, Corvallis, Oregon.
Dunstan, W. M. and D. W. Menzel. 1971. Continuous Cultures of
Natural Populations of Phytoplankton in Dilute, Treated
Sewage Effluent. Limnol. and Oceanogr. 16:623-632.
Elster, H. J. 1965. Absolute and Relative Assimilation Rates in
Relationship to Phytoplankton Populations. Mem. 1st. Ital.
Idrobiol. 18 Suppl:79-103.
Fogg, G. E. 1966. Algal Cultures and Phytoplankton Ecology.
University of Wisconsin Press, Madison. 126 p.
Glooschenko, W. A. and H. Curl. 1971. Influence of Nutrient
Enrichment on Photosynthesis and Assimilation Ratios in
Natural North Pacific Phytoplankton Communities. J. Fish.
Res. Bd. Canada. 28:790-793.
Guenther, W. C. 1964. Analysis of Variance. Prentice-Hall,
Englewood Cliffs, N. J. 199 p.
Hepher, B. 1962. Primary Production in Fishponds and its
Application to Fertilization Experiments. Limnol. and
Oceanogr. 7:131-136.
Lorenzen, C. J. 1967. Determination of Chlorophyll and
Phaeopigments: Spectrophotometric Equations. Limnol. and
Oceanogr. 12:343-346.
Malueg, K. W., R. M. Brice, D. W. Schults and D. P. Larsen. 1973.
The Shagawa Lake Project. U. S. Environmental Protection
Agency, National Environmental Research Center, Corvallis,
Oregon. EPA-R3-73-026.
Megard, R. 0. 1969. Algae and Photosynthesis in Shagawa Lake,
Minnesota. Limnological Research Center, University of
Minnesota, Minneapolis. Interim Report No. 5. 20 p.
171
-------
. 1970. Lake Minnetonka: Nutrients, Nutrient Abatement,
and Photosynthetic System of the Phytoplankton. Limnological
Research Center, University of Minnesota, Minneapolis.
Interim Report No. 7. 210 p.
Megard, R. 0. and P. D. Smith. 1973. Mechanisms that Regulate
Growth Rates of Phytoplankton in Shagawa Lake, Minnesota.
Limnol. and Oceanogr. 19:279-296.
Middlebrooks, E. J., D. B. Porcella, E. A. Pearson, P. H. McGauhey,
and G. A. Rohlich. 1971. Biostimulation and Algal Growth
Kinetics of Wastewater. J. Water Poll. Control Fed.
43:454-473.
Miller, W. E. and T. E. Maloney. 1971. Effects of Secondary
and Tertiary Wastewater Effluents on Algal Growth in a Lake-
River System. J. Water Poll. Control Fed. 43:2361-2365.
Odum, E. P. 1961. Factors Which Regulate Primary Productivity and
Heterotrophic Utilization in the Ecosystem. Pages 65-71 i_n_
Algae and Metropolitan Wastes. U. S. Dept. Health, Educ.,
Welfare, Public Health Service. Pub. No. SEC TR W61-3.
Powers, C. F., D. W. Schults, K. W. Malueg, R. M. Brice, and M. D.
Schuldt. 1972. Algal Responses to Nutrient Additions in
Natural Waters. II. Field Experiments, p. 141-156 TJT_ G. E.
Likens, ed. Nutrients and Eutrophication. Am. Soc. Limnol.
Oceanogr. Spec. Symp. 1.
Ryther, J. H. 1954. The Ratio of Photosynthesis to Respiration
in Marine Plankton Algae and its Effect on the Measurement of
Productivity. Deep Sea Res. 2:134-139.
Smith, P. D. 1973. Studies on the Influence of Sewage Effluents
on Phytoplankton Productivity in Experimental Ponds. M. S.
Thesis. University of Minnesota, Minneapolis. 124 p.
Snedecor, G. W. and W. G. Cochran. 1967. Statistical Methods.
6th ed. Iowa State University Press, Ames, Iowa. 593 p.
Strickland, J. D. H. 1960. Measuring the Production of Marine
Phytoplankton. Fish.Res. Bd. CaYiada. Bull. 122. 172 p.
Strickland, J. D. H. and T. R. Parsons. 1968. A Practical
Handbook of Sea Water Analysis. Fish. Res. Bd. Canada.
Bull. 167. 311 p.
172
-------
Tailing, J. F. 1957. Photosynthetic Characteristics of Some
Freshwater Plankton Diatoms in Relation to Underwater
Radiation. New Phytologist 56:29-50.
1965. The Photosynthetic Activity of Phytoplankton in
East African Lakes. Int. Rev. Ges. Hydrobiol. 50:1-32.
. 1966. Photosynthetic Behavior in Stratified and
Unstratified Lake Populations of a Planktonic Diatom. J.
Ecology 54:99-127.
UNESCO. 1966. Determination of Photosynthetic Pigments in
Seawater. Monogr. Oceanogr. Methodol. 1. New York, N. Y.
69 p.
Wright, J. C. 1960. The Limnology of Canyon Ferry Reservoir:
III. Some Observations on the Density Dependence of
Photosynthesis and Its Cause. Limnol. and Oceanogr.
5:356-361.
Yentsch, C. S. 1965. The Relationship Between Chlorophyll and
Photosynthetic Carbon Production with Reference to the
Measurement of Decomposition Products of Chloroplastic
Pigments. Mem. 1st. Ital. Idrobiol. 18 Suppl:323-346.
Yentsch, C. S. and D. W. Menzel. 1963. A Method for the
Determination of Phytoplankton Chlorophyll and Phaeophytin
by Fluorescence. Deep Sea Res. 10:221-231.
173
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THE DEVELOPMENT OF A STANDARDIZED MARINE
ALGAL ASSAY PROCEDURE FOR NUTRIENT ASSESSMENT
David T. Specht and William E. Miller
INTRODUCTION
The need for a means to assess the biological availability of
nutrients in estuarine and marine waters is no less urgent than for
freshwaters. Estuaries are particularly valuable and sensitive resources
(Ketchum, 1971, Inman and Brush, 1973) for which no standardized primary
producer bioassay for water quality exists. Algal bioassays have been
developed to suit specific purposes, but most are narrow in design and
require highly specialized personnel to operate and evaluate (Atkins,
1923; Smayda, 1970; Goldman, Tenore and Stanley, 1973; Skulberg, 1964;
Glass, 1973; Dierberg, 1972; McPhee, 1961; Tarzwell, 1971; Mitchell,
1973; Tallqvist, 1973; Fitzgerald, 1972).
Inasmuch as estuaries are the "nurseries" of virtually all commer-
cially important marine shellfish, fish, and their food organisms, it
becomes vitally important to be able to accurately assess their capabil-
ities for primary productivity and to determine and predict the impact
of the addition of various nutrients and wastewaters upon that produc-
tivity (Edmondson and Edmondson, 1947; Goldman, Tenore and Stanley,
1973; Thomas, 1971; Ryther and Dunstan, 1971; Mitchell, 1973; Tallqvist,
1973(b); O'Sullivan, 1971; Apollonia, 1973; Waite and Mitchell, 1972;
Preston and Wood, 1971; Ryther and Guillard, 1959; Ryther, 1954; Ketchum,
1971).
For reasons of simplicity and consistency, the protocol for the
Marine Algal Assay Procedure follows closely that of the widely accepted
Freshwater Algal Assay Procedure: Bottle Test (AAP) (National Eutrophi-
cation Research Program, 1971). This is particularly true of the physical
facilities required,incubation conditions and methodology.
174
-------
Establishment of Criteria for Bioassay Organism
Unlike freshwater systems, estuaries in particular pose the problem
of interpretation of the effect of varying salinities on the effect of a
given concentration of a nutrient on the biomass production of a given
alga. While no algal species was thought to be unaffected by varying
salinity, the most ideal candidate would tie one that responded in a
linear, predictable manner to such changes. A species was desired that
would meet this important criterion as well as be genetically stable,
reproduce primarily asexually, and not clump or tend to adhere to the
walls of the flasks.
Other major problem areas to be considered were the array of factors
that govern the consistency of growth response, such as light intensity,
temperature, influence of pH, relative size of inoculum, and selection
and modification of a standard stock medium in which intracellular
nutrient carryover in the inoculum would be minimal.
A significant consideration is the ease with which algal biomass
production could be measured. Consistent with the AAP, all results
would be converted to equivalent dry weight, defined by standard gravi-
metric methods (NERP, 1971).
Selection of Candidate Test Species
Considering the established criteria, we selected candidate algal
species on several bases, looking for three classes of algae. To demon-
strate biostimulation, we evaluated Dunaliella tertiolecta (DUN clone)
and Nannochloris atomus, two unicellular green algae. To look at toxicity
or inhibition possibilities, we are considering Isochrysis lutheri,
Cyclotella menenghiana, and Thalassiosira pseudonana, typical crustacean
or bivalve food organisms. We also hope to look at nitrogen fixing
175
-------
marine blue-green algae, such as Coccochlon's elabens or Tichodesirnium
sp. (Taylor, Lee and Bunt, 1973). These algae represent three taxonomic
classes: greens, diatoms, and blue-greens. Thus far, most of the work
has centered around the green flagellate, Dunaliella tertiolecta Butcher
(DUN clone), kindly provided by Dr. John Ryther, Woods Hole Oceano-
graphic Institution. This alga has shown the most linear response for
every parameter examined so far. A distinct advantage of Dunaliella is
that it requires no outside source of vitamins (Provasoli, 1963). Some
comparison test runs of field samples have been conducted with Thalassio-
sira pseudonana Hasle and Heindal (CN clone) provided by Dr. Paul Margraves
of the University of Rhode Island, but more background work is needed on
salinity-nutrient interaction. After the needed information on the
diatom is assembled, more intensive efforts will be made in search of a
suitable blue-green alga.
Development of Media Response Curves
The initial work with Dunaliella was performed using a modified
version of Burkholder's Artificial Sea Water (ASW) (Burkholder, 1963).
This is one of several potentially amenable media which will support
algal growth at relatively low nutrient levels (Kester, Duedall, Connors,
and Pytkowicz, 1967; Lyman and Fleming, 1940) (see Table 1).
Salinity, Tolerance and Salt Interaction
Although it was assumed that all algae would be affected to some
degree by the salinity of the media or samples, it was desirable to find
the alga with the most linear response and tolerance to manipulation of
salinity, primarily between 5 0/00 (ppt) and 35 °/00. In order to
establish linearity of response, levels of salinity chosen were 5, 8,
12, 16, 20, 24, 30, and 35 °/°o (Figure 1). The initial salinity tests
176
-------
TABLE 1. MARINE ALGAL ASSAY PROCEDURE SUMMARY
I. TEST ORGANISM: Dunaliella tertiolecta Butcher (DUN clone)
available from the Eutrophication and Lake Restoration Branch,
PNERL, EPA. 200 SW 35th St., Corvallis, OR 97330; or WHOI.
II. ENVIRONMENTAL CONDITIONS: In general, follow exactly the
ALGAL ASSAY PROCEDURE BOTTLE TEST (NERP, EPA, August, 1971)
with the following exceptions:
TEMPERATURE: 18 - 20°C
SAMPLE SIZE: 100 ml in a 500 ml Erlenmyer flask
INOCULUM: Inoculate with 1 ml washed (w/ 20 °/oo ASW
less N & P, by serial centrifugation) 5-8
day old culture at a concentration of 10,000
cells/ml (to give a final concentration in
the flask of 100 cells/ml, equivalent to
approximately 0.03 mg dry wt/Z-).
III. BASAL MEDIUM: Modified Burkholder's Artificial Seawater (ASW)
with NAAM levels of the following nutrients; N, P, Fe, and Na2EDTA
Use Analytical Reagent or Reagent Grade chemicals, and double
glass distilled water.
Compound
NaCl
Na2S04
NaHC03
KCL
KBr
H3B°3
MgCl2'6H20
SrCl2-6H20
CaCl2-2H20
H20 to
grams /I
23.48
3.92
0.19
0.66
0.096
0.026
10.61
0.04
1.469
1,000 ml
grams/4Z
93.92
15.68
0.76
2.64
0.384
0.104
42.44
0.16
5.876
4,000 ml
Filter the above through prewashed 0.45 urn membrane filter.
177
-------
FOR DILUTION TO VARIOUS SALINITIES: (4 liter batches)
Salinity. %
35
30
24
20
16
12
8
5
ASW stock, I
4.0
3.428
2.742
2.285
1.828
1.371
0.914
0.571
HgO, I (glass distilled)
0.0
0.571
1.257
1.714
2.171
2.628
3.085
3.428
For any given final salinity, mix well, adding the following
NAAM levels of nutrients:
NaN03 102 mg/4Z batch (4.2 mg N/Z)
K2HP04 4.176 mg/4Z batch (0.186 mg P/Z)
Na2EDTA 1200 yg/4Z batch (300 yg/Z)
*NAAM trace metal mix (minus Fed-)
filter through 0,45 ym membrane filter,
add AFTER filtration, sterilized FeCl3, 384 yg/4Z batch (33.05 yg Fe/Z).
Add the following: 0.0928 g H3B03; 0.208 g MnCl2'4H20; 0.016 g
ZnCl2; 0.714 mg CoCl2«6H20; 0.0107 mg CuCl2»2H20; 3.63 mg Na2MoO
2H20; make up to 500 ml, adding 1 ml of this concentrate to
each liter of media.
178
-------
o first run
A second run
slope 5.521
r=.942, 14° freedorr
12 16 20
SALINITY
24
30 35
°/
oo
Figure 1. Dunaliella tertiolecta Burkholder's ASW + NAAM nutrients
-------
were made with rather high levels of nutrients, but later nutrient
response trials showed essentially the same linearity at much lower
levels. The data thus produced provided the basis for the three-
dimensional plots such as Figures 2, 3, and 4, which portray response
"surfaces" according to salinity-nutrient interactions. These responses
appear to be predictable, within limits (Figures 2, 3, and 4).
Temperature and Light
Several trials were made at 24 +_ 2°C, the standard temperature used
in the freshwater Algal Assay Procedure. However, responses were both
irregular and too rapid for accurate assessment; and replication was
particularly poor. A suggested temperature of 18°C was found to yield
satisfactory results in these same terms (McLachlan, 1960).
Light levels of 400 ft-c + 10 percent (1300 yw/cm ; 4300 lux)a
proved satisfacotry for Dunaliella, and no reason was seen to change
this from the freshwater Algal Assay Procedure (NERP, 1971).
The work so far with Thalassiosira has been done at 20 + 2°C, with
o ~
no shaking, and 550 ft-c +_ 10 percent (1850 yw/cm ; 5920 lux), according
to data provided by Larsen (1973) in his work with this species. Good
replication has been obtained under these conditions.
The energy level output of a bank of six 48 inch "cool white" fluores-
cent lamps (GE 40 watt, @ 60 Hz) was approximately 1300 yw/cm (range,
380-760 nm) at a distance of 26 3/4 inches, as measured with a ISCO
model SRC spectroradiometer. Using the same measurement geometry, a
Weston Model 756 Illumination Meter read 400 footcandles. All reflecting
surfaces were matte white.
Therefore, utilizing a calibrated illumination meter with a footcandle
readout, one may, by adjusting the height of the lights, achieve a known
energy level output of 1300 yw/cm .
For further discussion of the problems of the differences in absorption
of light by photosynthesizing organisms and by man's eye and their
measurement, see Tyler (1973).
180
-------
100
r = .818'
10°fr
slope 1069.698
slope 352.816 r=.940
r = .950
/
/
\\J 1
slope
M
r
1
>x
X
i
1025.819
? r=.961
^^^ 16°fr
slope 1228.043
r= .986
16°fr
0.05
^/A 16
^/^ 20^
.010
._ ).005
^T3.0025
Figure 2. Growth of Puna lie 1la at various salinities and phosphorus
concentrations in ASW. Dry weight at day 10.
-------
CO
(V)
slope 8.479
r=.983
16°fr
slope 9.445
r=.978
16°fr slope 10.195
r=.981
J6°fr
slope 9.972
??r=.977
16°fr
1.0
Figure 3. Growth of Dunaliella at various salinities and nitrate
nitrogen concentrations in ASW. Dry weight at day 10.
-------
CO
CO
100,
slope 40.346 slope 67.208
= 993 r=.988
16°fr
slope 64.544
r=.996
sbpe 61.237
r=.989
16°fr
0.01
Figure 4. Growth of Puna liella at various salinities and ammonia nitrogen
concentrations in ASW. Dry weight at day 10.
-------
Biomass Determination
Several methods of growth measurement, indirect or direct, may be
employed. Direct cell counting, using a haemocytometer with a microscope,
is relatively accurate at high population levels, but very slow. Gravi-
metrically determined dry weights are likewise time consuming and may be
at variance with calculated dry weights if substantial bacterial growth
occurs in samples, or substantial amounts of ferric-magnesium hydroxide
forms in the sample. Also, accurate dry weights are difficult to obtain
at low algal biomass concentrations.
In the development of this test, electronic particle counters were
used to provide numerical cell counts and volumetric measurements (mean
cell volume) of the samples. Samples were diluted at a ratio of 1:10 or
1:100, depending on the cell population density, with filtered isotonic
saline (1% Nad) electrolyte. Periodic cross checks for accuracy were
made by haemocytometer counts and actual gravimetric dry weights. Since
Dunaliella is substantially larger than Selenastrum capricornutum (AAP
test species), aperture current and amplification settings used for
Dunaliella are generally one-half that used to count Selenastrum in the
freshwater assay procedure. Generally, the electronic particle counter
was found to be the most feasible method of dealing with large numbers
of samples, but may not be economically justified for casual or infrequent
assays.
Nutrient Response
The initial assumption was that estuarine waters are nitrogen
limited for algal growth (Ryther and Dunstan, 1971; Ryther and Guillard,
1959; Goldman, Tenore, and Stanley, 1973; Welch, 1968; Eppley, Carlucci,
Holm-Hansen, Kiefer, McCarthy, and Williams, 1971; Thomas, 1970). One
recent sampling survey of the Yaquina estuary showed that while water
184
-------
from the lower bay was predictably nitrogen limited for algal growth,
water from the upper bay and its tributaries was shown to be phosphorus
limited for algal growth. The Marine Algal Assay Procedure, using
Dunaliella. can serve to define this boundary, which can change not only
with the season, but with each tide, and fluctuations of tributary
inflow.
With respect to this situation, the response curves developed for
Dunaliella show that the alga will respond to concentrations at least as
low as 2.5 yg P/l, 10 yg ammonia N/l, and 50 yg nitrate N/l in defined
medium.
These curves were developed from additions (spikes) ranging from
2.5 to 50 yg/1 in five steps for phosphorus (Figure 5), 10 to 1,000 yg in
five steps for nitrate nitrogen (Figure 6) and ammonia nitrogen (Figures
7 and 8). All assays were conducted in triplicate along with controls.
Nitrilotriacetic acid (NTA) was examined as a possible nitrogen source
(Erickson, Maloney and Gentile, 1970; Rudd, Townsend, and Hamilton,
1973; Sturm and Payne, 1973; Mitchell, 1973), or for possible growth
depressing or enhancing effects by chelation in actual estuaries field
samples. There were no significant yield differences between control
and spikes ranging from 50 to 1,000 mg/1 NTA nitrogen (expressed as
nitrogen, 7.5% N by weight).
Although ammonia nitrogen was more stimulatory than nitrate
nitrogen in our ASW (Paasche, 1971), there was a growth rate difference,
but no yield difference, in spiked field samples (Figures- 15, 16, 18,
and 19).
In Burkholder's ASW 5 °/00 to 35 °/00 salinity, Puna1iell a produces
an average of 1.076 mg dry weight per yg of phosphorus, 0.0318 mg dry
weight per yg of nitrate nitrogen, and 0.0796 mg dry weight per yg of
ammonia nitrogen (Table 2).
185
-------
5%o ASW
-i-NAAM nutrients .
-phosphorus
O +0 mg P/L
-K0025mg P/L
+ .005 mg P/L -
+ .01 mg P/L f
.025mg P/L -
.05mg P/L -
10
I6%o ASW
+NAAM nutrients
- phosphorus
O +0 mg P/L
.0025 mg P/L
KOOSmg P/L?
.025mgP/L
4.05mg P/L
MA022373
0 4
DAYS
DAYS
IOJr
10"
12
DAYS
20%» ASW
+ NAAM nutrients
-phosphorus
O +0 mg P/L
O +.0025mg P/L
0 -H.005mgP/L:
A + .01 mg P/L
O +.025mg P/L -
V 4.05mg P/L -
MA 022373
. I i . . I . .
I03
10°
16
20
24
10
10"'
35%o ASW
+NAAM nutrients
- phosphorus
O +0 mg P/L
O 4.0025 mg P/L
Q t.005mg
A 4.01 mg P/L j
O -t-.025mg P/L
V 4.05mg P/L
MA 030973R
_L
12
DAYS
16
20 24
Figure 5 Growth response of Dunaliella tertiolecta
186
-------
5%» ASW H
+ NAAM nutrients^
- nitrogen
O + O mg N/L
O -KOImg N/L _
+.05mg N/L --
A+ .10 mg N/L -
O -r .50 mg N/L '
V + 1.0 mg N/L "
MA 030973
I , , , I , , .
I03rr
10'
£10°
o
10
10
I6%» ASW :
+ NAAM nutrients '
nitrogen
low N0| -N-
O + 0 mg N/L
O -KOIrng N/L -
D +.05mg N/L :
A + .10 mg N/L -
O + .50mg N/L ;
V + 1.0 mg N/L
MA 030973
DAYS
12
DAYS
16
20
24
I03F
I02
o>
X
IOC
10
20%0 ASW
+ NAAM nutrients
- nitrogen
low N0| -N-
O + 0 mg N/L
O + .0lmg N/L .
D +.05mg N/L
A+ .10 mg N/L
O -t- .50mg N/L
V + 1.0 mg N/L
MA 030973
_L
_L
I03rr
12
DAYS
16
2O
24
10'
£lO°
O
10"
10
35%oASW :
+ NAAM nutrients -
- nitrogen
low NOj -N-
O + 0 mg N/L
O -i-.OImg N/L
D + .05 mg N/L
A+ .10 mg N/L -
O -t- .50mg N/L
V + 1.0 mg N/L
MA 030973
i,!,,,!,,,
12
DAYS
16
20
24
Figure 6« Growth response of Dunaliella tertiolecta
187
-------
10 L I I I I I I I I I I I I I I I I |[l I
10'
£io°
o
10"
5%oASW
+ NAAM nutrients .
-nitrogen
lowNH*-N-
O + 0 mg N/L
O + .01 mg N/L
Q + .05mg N/L
A -t-. I mg N/L
O -r .5mg N/L
^7 + I.Omg N/L
MA 032373
i J ^ ^ 1 , ^
0 4
12
DAYS
16
20
24
20%oASW _:
+ NAAM nutrients -
-nitrogen
lowNH*-N-
O * Omg N/L
O + .01 mg N/L _
a 4 .05mg N/L r
A +. I mg N/L
.5mg N/L -
I.Omg N/L '
!6%oASW
-tNAAM nutrients
-nitrogen
lowNH*-N-
O + 0 mg N/L
O 4 .01 mg N/L
.05mgN/L
A + . | mg N/L
O -r.Smg N/L
V + I Omg N/L
MA 032373
I , , , I , , , I , , ,
0 4
I03
I02
10°
10'
20 24
DAYS
_L
35%oASW
+NAAM nutrients"
-nitrogen
lowNH^-N-
O + 0 mg N/L
O +.0lmg N/L .
D -K05mgN/L
A + . I mg N/L .
O4.5mg N/L
V + I.Omg N/L
MA 032373
I ' - I ' i
12
DAYS
16
20 24
Growth response of Punaliella tertiolecta
188
-------
00
X.
£
i
>
Q
10
O
O
1C?
Calculated Regression Line
Salinity Slope Cor.Coef (r) 16°Freedom
O
O
m
A
I6%o
20%o
35%o
66.7
78.1
77.0
73.2
.998
.995
.993
.985
MA 032373
d.5
NHj-N-,mg./L
7.5
Figure 80 Growth response of Dunaliella tertiolecta, day 14
-------
TABLE 2. BIOMASS PRODUCED PER UNIT OF NUTRIENT BY DUNALIELLA
AT DAY 14 IN DEFINED MEDIA
mg dry weight/yg of nutrient P:N ratios
Nutrient
Salinity
C ol
3 Ao
16%0
20%0
35%0
P
0.557
+0.158
0.930
+0.240
1.170
+0.164
1.129
+0.232
N03" - N
0.0096
+0.0014
0.0308
+0.0394
0.0331
+0.0379
0.0315
+0.0361
NH4+ - N
0.0747
+0.0075
0.0844
+0. 0058
0.0766
+0.005
0.0765
+0.0193
P:N03" - N
1:58
1:30.2
1:35.3
1:35.8
P:NH4+ - !\
1:7.5
1:11.0
1:15.3
1:14.8
Average* 1.076 0.0318 0.0796 1:33.8 1:13.5
*Average is calculated from the 16 %0, 20%0, and 35%0 data only.
Replication
One especially outstanding characteristic of the use of Dunaliella
was the consistency of data replication. An example of this is the
result of the investigation of NTA as a nitrogen source in natural water
samples. NTA appeared to have little, if any, effect on the growth of
Dunaliella, either stimulatory or inhibitory. In the experiment, the
control and four levels of NTA (0.05 to 1.0 mg/1 as N) with three repli-
cates each gave a total of fifteen replicates for each of six natural
waters (all Oregon coastal estuaries). In all cases, a t-test (13
190
-------
degrees of freedom) showed no significant difference between dry weight
from controls and samples spiked with varying levels of NTA. The normal-
ized standard deviation for the entire run of 90 flasks was less than +
15 percent.
Standard Inoculum Level
In order to approach natural phytoplankton population levels and
avoid problems of nutrient carry-over in a healthy inoculum, the size of
the inoculum was reduced from an initial 1,000 cells/ml (0.3 mg/1 dry
weight) to 100 cells/ml (0.03 mg/1 dry weight). Although there was a
detectable lag in growth of about two days, growth rate was unaffected
and the final yield was virtually identical at day 10 or 12 (Figure 9).
The inoculum was dispensed in a 1 ml volume.
Field Tests
The majority of field work has been done with water from the Yaquina
River, an estuary at Newport, Oregon (Figure 10). It is relatively near
and has been studied extensively by other EPA and Oregon State University
personnel. Some samples were also obtained from several sites in Puget
Sound and assayed in cooperation with the Washington State Department of
Ecology.
The ability of Puna!iell a to respond to low level nitrogen spikes
in a natural water sample is shown in Figure 11, which illustrates the
shift in potential nutrient limitation from nitrogen to phosphorus in a
sample from the Toledo station in the Yaquina estuary. As the sample
was presumed to be nitrogen limited, nitrate was added in incremental
steps of 0.02, 0.1, 0.2, 0.5, and 1.0 mg/1 as nitrogen to try and find
the point at which all the phosphorus would be exhausted, shifting the
sample to phosphorus limited growth (0.001 to 0.05 mg P/l and N + p
combinations were also added in overall examination of the sample). The
growth response at day 7 started to decline just below 0.5 mg/1 total
nitrogen, plateauing at about 23 mg dry wt/1 with the 1.0 mg N/l spike.
191
-------
10 L I I I I I I I I I ' I I I I I [ I I I I
10
0 4
5%oASW + Full NAAM nutrients _
inoculum strength cells/ml
o 100/ml
A 250/ml
Q 1000/ml
IA 030973
I . . . I , . , I i i i I , i
IO3 L | I I | ' I I | I I I I I I ' I ' ' ' I
12
DAYS
16 20 24
20%o ASW+ Full NAAM nutrients
Inoculum strength cells/ml
o 100/ml
250/m,l
0 1000/ml
20 24
16 %o ASWtFull NAAM nutrients
inoculum strength cells/ml
o 100/ml
A 250/ml
Q 1000/ml
IA 022373
, , , I , , . I i , i I i , ,
12 16
DAYS
20 24
35%o ASW+ Full NAAM nutrients
inoculum strength cells/ml
o 100/ml
& 250ml
a 1000/ml
Figure 9. Growth response of Dunaliella tertiolecta.
192
-------
Charlie's Dock
(16.0 miles)
O.S.U Small
Boat Dock
(1.5 miles)
Toledo Public
Boat Landing
( 7.0 miles)
Yaquino River
Bridge
(21.5 miles)
Elk City
Boot Dock
(19.5 mites)
Elk Creek
Bridge
(21.5 miles)
MILL CREEK
Nautical Mile
Figure 10. Marine algal assay field sampling sites, Yaquina Estuary,
Newport, Oregon.
-------
en
e
-------
The sample was at that point limited by phosphorus. To demonstrate this
point, we examine the biomass produced by the 0.05 mg P + 1.0 mg N/l
spike which yielded approximately 36 mg dry wt/1. If all points on the
plot not limited by phosphorus (excludes the 1.0 yg N/l spike) are
considered, a line of very good fit can be plotted (r = 0.986, t = 26.7,
19 degrees of freedom, significant at the 0.1% level) showing that about
p
97 percent (r ) of the change in dry weight can be attributed to a
change in the level of nitrogen. To show the advent of phosphorus
limitation, a curve can be fitted (by the least squares power curve
method) to the increasing N only spikes (r = 0.987), showing a plateau
of approximately 23 mg dry wt/1 defined by the 0.5 and 1.0 mg N/l spikes.
The multiple levels of spikes show that when nitrogen is limiting,
growth responses are linear with respect to the spike level until some
other nutrient or physical factor becomes limiting.
Samples from Puget Sound were collected and assayed by personnel
from the Washington State Department of Ecology and EPA Region X.
Figure 12 compares the relative growth of the different samples. Day 10
dry weights plotted against inorganic nitrogen accounted for 83 percent
of the variation in dry weight, indicating that nitrogen was probably
the limiting nutrient factor for algal growth.
In a set of Yaquina Bay samples taken on November 1, 1972, one day
after the onset of heavy winter rains, phosphorus was found to be the
limiting nutrient at the Burpee sampling site. Downstream stations
(Toledo and OSU Dock) appeared growth limited by nitrogen (Figures 13
and 14). The bioassay was repeated after a storage period of six weeks.
Nitrogen limitation in the Toledo station was confirmed and the
phosphorus limitation was reaffirmed, indicating that the apparent
phosphorus limitation was real and was not a condition related to
decreased salinity. It was instead probably caused by the increased
nitrogen content of the rainwater runoff.
195
-------
10
ID'2
A.-Mud Bay
B.-DabobBay
C.-Oyster Bay
D.-Oakland Bay
E.-S.Hood Canal
(POTLATCH)
Filtered-No Spikes
I I I
8
12
DAYS
16
20 24
Figure 12 Algal assay of Puget Sound sites, Spring 1973,
196
-------
10'
10'
1O
,0
o Control
o +.05 P
A+1.O N
0+Q5P+1QN-
significantly different from control
0
3
Ddays
1O
14
Yaquina Bay, OSU Dock, I I/I /72 33.8%o salinity
1Cr
10
10'
o
0
o Control
o +.05 P
A +1.0 N
0+.O5P+1ON
significantly different from control
3days
1O
14
Yaquina Bay, OSU Dock, II/I/72 33.8%0 salinity
(sample stored 6 weeks)
o Control
+.05 P
A +1.0 N
0+.O5P+1ON
* significantly different from control
10'
days
Yaquina Bay,Toledo, M/l/72 24%osalinity
A+1.0 N
0+.05P+1ON
significantly different from control
days
Yaquina Bay,Toledo, II/I/72 24%0salinity
(sample stored 6 weeks
Figure 13. Growth response of Dunaliella tertiolecta
197
-------
1O -
o Control
o +.05 P
A+1.0 N
GH-.05P+1.0N
significantly different from control
days
Yaquina Bay, Burpee, I I/I/72,!7%o salinity
10=
10'
10°
0
©Control
o +.05 P
A+1.0 N
GH-.05P+1QN.
+ significantly different from control
3
5 days
10
14
Yaquina Bay, Burpee, II/I/72 I7%osalinity
(sample stored 6 weeks)
10
15 20 25 30 35
Yaquina Bay,8 August I972
x IO"2mg./L total N
A
O
%o Salinity
Figure 14. Growth response of Puna lie 1la tertiolecta
198
-------
Figure 14 shows the plot of dry weights from controls of samples
taken at the same stations in August, 1972. The data are plotted on two
different axes, according to inorganic nitrogen content or according to
salinity. A linear regression of the nitrogen line yields a correlation
coefficient (r) of 0.965 (Student's t, 9.882, with 7 degrees of freedom,
significant at the 0.1 percent level), indicating that approximately 93
2
percent (r ) of the change in dry weight can be attributed to a change
in the amount of nitrogen.
A similar statistical assessment of the salinity plot yields a
correlation coefficient (r) of 0.708 (students t, 2.655, with 7 degrees
of freedom, significant at the 5 percent level) indicating that only 50
2
percent (r )
in salinity.
2
percent (r ) of the change in dry weight could be attributed to a change
Samples were taken on June 28, 1973, in Yaquina Bay (Figure 10)
from the OSU Dock station to the head of tidewater at Elk City, and
approximately 2 miles farther up each of the two main tributaries, Elk
River and Yaquina River. Salinities ranged from 30.8 °/00 (49,300 ymho
conductivity) to less than 2.8 °/00 (down to 65 umho) in the Yaquina
River sample (see Table 3 for chemical analyses of samples [Environmental
Protection Agency, 1971].
Given time to adjust physiologically. Puna!iel la grew in these
essentially freshwater situations and gave a statistically significant
indication of the growth limiting nutrient (Figures 15 and 16). In this
case, it is evident from the growth curves that nitrogen was the growth
limiting nutrient at the OSU Dock Station (Figure 15). The situation at
the Toledo station (14 °/00 salinity) is somewhat indistinct, but a t-
test shows that growth in the phosphorus spiked sample is significantly
higher (at the 5 percent level) than the control and those spiked with
nitrate or ammonia nitrogen. This indicates that phosphorus is the
199
-------
Table 3. CHEMICAL PARAMETERS OF YAQUINA ESTUARY
AND TRIBUTARY SURFACE GRAB SAMPLES, 28 June 1973,
AT OR NEAR HIGH WATER, ON INCOMING TIDE
Sampling Site
Parameter
g NH4+ -N- mg/1
0 N03" -N- mg/1
Tot. sol. P mg/1
Ortho-P mg/1
Sulfate, mg/1
Sol. Fe, yg/1
Sol. Mn, yg/1
Salinity, °/00
Conductivity, umho
Tot. Sol. C, mg/1
Sol . reactive Si , mg/1
OSU Small
Boat Dock
0.036
0.120
0.035
0.03
3100
240
20
30.76
49,200
24.5
.68
Toledo Public
Boat Landing
0.018
0.129
0.015
0.013
896
100
27
14.14
23,200
15.8
2.2
Burpee
0.076
0.416
0.015
0.011
256
100
34
2.882
5,800
9
4.6
Charlie's Dock
0.06
0.430
0.015
0.019
10
140
11
<2.84
208
5.4
4.8
Elk City
0.038
0.498
0.015
0.009
3
140
13
<2.84
69
4.7
5.25
Elk River,
Upstream
0.025
0.315
0.015
0.011
<2.5
160
10
<2.84
67
4.8
5.4
Yaquina River,
Upstream
<0.001
0.560
0.01
0.008
4
140
9
<2.84
65
4.5
5.5
-------
CONTROLS
O Yoquina.OSU Dock
O Toledo
B Burpee
A Charlie's Dock
O Elk City
V Elk River
> Yaquina River
MA 070373
I , , , I , , , l , , ,
10s
16
20
24
O significantly different from
control (O) and N spikes (m,A)
at day 8 and 10 at the 5 % level .
'Yaquino Bay,Toledo, I4.l%«
O Control
O +0.05 mg P/L
a +l.0mg NO^ -N-/L
A +1.0mg NHj -N-/L
O + 0.05mg P/L + I.Omg NOj -N-/L
V +0.05 mg P/L + I.Omg NH* -N-/L
*significantly different from control
MA 071373
12
DAYS
16
20 24
10s
I
I-:
*
10°
10
10"'
Yoquino Bay, OSU Dock 3Q8%o
O Control
0 +0.05 mg P/L
0 +l.0mg NO^ -N-/L
& -H.Omg NHJ -N-/L
O + 0.05 mg P/L -H.OmgNOj -N-/L
V +0.05mg P/L + I.OmgNH»-N-/L
*significantly different from control
MA07I373
Burpee, 2.9%= salinity
O Control
+O.05mg P/L
+I.Omg NO^ -N-/L
& + I.Omg NHJ -N-/L
O +0.05mg P/L + I.Omg NOj -N-/L
V +O.05mg P/L + I.Omg NH* -N-/L
significantly different from control
MA 070313
l/M 19
Figure 15 Growth response of Dunaliella tertiolecta.
201
-------
I03rr
'I
Charlie's Dock, <2.8%.>
O Control
0 +0.05 mg P/L
+ l.0mg NOj -N-/L
H.Omg NHJ -N-/L
+ 0.05mgP/L+lOmgMOj -M-/L
V +O.O5mg P/L + I.Omg NHt -N-/L
^significantly different from contro
MA 070373
, , I
IO3 L i i i i i i i I ' i i I i i ' I
!0Z
10"
ElkCity,<2.8%»
O Control
O +0.05 mg P/L
a +1.0 mg NOj -N-/L
A -H.Omg NHJ -N-/L
O +0.05mgP/L+IOmg NO j -N-/L
V +-O.05 mg P/L 4-1.0 mg NH* -N-/L
*significantly different from control
MA 070373
12
DAYS
16
20 24
I03F
Elk Ri»er,<23%»
O Control
O +0.05 mg P/L
a +l.0mg NOj -N-/L
^ +l.0mg NHJ -N-/L
O +0.05mgP/L+I.OmgNOj -N-/L -
V + 0.05 rng P/L+1.0 mg NH+-N-/L-
*significantly different from control
MA 070373
' ' I ' i ' 1 ' I i
10"
IOC
10
-|ii
12
DAYS
16
2O
24
Yaquina River,Upstreom,<2.8%o
O Control
O +0.05 mg P/L
n +l.0mg NOj -N-/L
* +l.0mg NHj -N-/L
O fO.OSmgP/L+IOmgNOj .. .
V +0.05mg P/L + 1.0 mg NH*-N-/
*significantly di?f arent from control
MA 071373
.. I ... 1 ... I
12
DAYS
16
20 24
Figure 16. Growth response of Dunaliella tertiolecta.
202
-------
growth limiting nutrient. At the Burpee station (Figure 15), phosphorus
was shown to be the growth limiting nutrient (2.9 °/00 salinity, 5800 ymho),
The remainder of the stations, all essentially freshwater (208-65 ymhos
conductivity), showed a marked lag in growth patterns. All samples
responded in a statistically significant manner to phosphorus spikes,
but not to nitrogen. Replication with Puna1iel la was not as good in
waters of very low salinities as in those of higher salinities, but more
experimentation should yield a workable test protocol to establish
acceptable replicability.
Five southern Oregon coastal estuaries (Figure 17) were sampled on
July 25, 1973, from Coos Bay on the south to Yaquina Bay on the north.
These estuaries, sampled on the flood tide at or near high water, were
nitrogen limited with the exception of the Umpqua River. This sample,
11.6 °/°° salinity, responded in a statistically significant manner to
phosphorus spikes, but not to nitrogen spikes (Figures 18 and 19; see
Table 4 for chemical analyses of samples [Environmental Protection
Agency, 1971].
A non-marine application of the Marine Algal Assay Procedure using
Dunaliella is exemplified by results obtained with samples of water from
Abert Lake, a highly alkaline (50,000 ymho conductivity) drainage sink
in southeastern Oregon. Attempts to assay this sample using Selenastrum
capricornutum Printz of the Freshwater Algal Assay Procedure failed when
the algal inoculum was killed by the high alkalinity. However, after an
initial adjustment time lag, Dunaliella showed nitrogen to be the growth
limiting nutrient (Figure 20).
SUMMARY
The effort to design and evaluate a marine version of the Algal
Assay Procedure: Bottle Test has shown the suitability of Dunaliella
tertiolecta Butcher (DUN clone) as a highly versatile and consistent
bioassay organism for nutrient assessment in marine, estuarine, and some
alkaline freshwater situations.
203
-------
Table 4. CHEMICAL PARAMETERS OF 6 OREGON
ESTUARY SAMPLES AFTER MEMBRANE FILTRATION
Surface Grab Samples, 25 July 1973,
on Incoming Tide at or near High Water
Sampling Site
Parameter
rxj NH4+ -N- mg/1
"^ NO," -N- mg/1
Total P mg/1
Ortho-P mg/1
Sulfate mg/1
Sol . Fe, pg/1
Sol. Mn, yg/1
Salinity, °/oo
Conductivity, ymho
Tot. Sol. C, mg/1
Sol . reactive Si , mg/1
Coos Bay at
North Bend
0.145
0.044
0.03
0.028
3200
240
40
31.18
47,500
25
0.4
Coos Bay at
Horsefall Rd. Bridge
0.118
0.019
0.035
0.028
3300
240
40
31.14
47,500
26
0.4
Umpqua River
at Reedsport
0.027
0.003
0.01
<0.001
1500
80
20
11.62
19,500
13
2.5
Siuslaw River
at Florence
0.059
0.009
0.01
0.01
2500
120
20
21.97
35,000
18
0.72
Alsea River
at Waldport
0.103
0.082
0.035
0.036
3400
240
30
33.09
50,400
25
0.84
Yaquina River
at Newport
0.100
0.11
0.035
0.03
3700
240
30
32.88
50,300
25
0.72
-------
Figure 17.
Marine algal assay field sampling sites
Oregon coastal estuaries (not to scale)!
205
-------
Coos Bay
at North Bend, Or.
Control
+OO5mg.P/L
tl.Omg. NO§ -N-/L
*1.Omg. NH^ -N-/L
+OO5mg.P/L*1.Omg. NOJ-N-/L
*O.05mg P/L+1.0mg.NH^-N-/L
, I , . . I , , , I , ,
Siuslaw River
at Florence, Or
O Control
O »005mg P/L
Q *1.0mg NOo -N-/L
A *10mg NhC -N-/L
O *005mg.P/Lt10mg. NOVN-/L
~ OOSmg P/Lt10mg. NH^-N-/L
l_i
8 12 16
DAYS
I
o
cr
Q
1O"
I'M
10 o
.,,3
10'
t
10
o:
Q
10
Coos Bay at
Horsetail Road Bridge,
O Control
O »QO5mg.P/L
O »1.Omg. ND§ -N-/L
A «1.Omg. NH; -N-/L -i
O tO.05mg.P/L.1.0mg.NO§-N-/L
-- 1
8 12 16
DAYS
20 24
Umpqua River
at Reedsportpr
Control
OOSmg.RL
»1.0mg NO| -N-/L
»1.0mg. NH^ -N-/L
*005mg.Rt tLOrng. NC«-N-/L
*0.05mgP/L»1.0mg.NH4-N-/L
M i , M , i , I . i i
0
S 12 16 20 24
DAYS
Figure 18. Growth response of DunalieUa tertiolecta.
206
-------
10"
10
Alsea River
at Waldport, Or.
O Control
O *QO5mg.P/L
13 -lOrng. NOj -N-/L
A .I.Omg. Nhtf -N-/L
O »OO5mg.P/t..1.Omg NC4-N-/L
V .O.OSmg.P/L.I.Omg NH^-N-/L
I . . . I , . , I , . . I , . ,
0
d :1 16
DAYS
Yaquina River
at Newport, Or
O Control
O *OO5mg.P/L
D «1.Omg. NO| -N-/L
A .rOmg NH^ -N-/L ]
O *O.O5mg.Rt*10mg.NO3-N-/L
^ +O.O5mg.P/L»1Dmg.NH^-N-/L 1
i i . I i , , I . , , I , , ,
IO
24
in0
IO
10
10"
O Coos Bay at North Bend
O Coos Bay at Horsefall Rd Br -
Q Umpqua River at Reedsport
A Siuslaw River at Florence
O Alsea Rwer at Waldport
^ Yaquina River at Newport
Sampled
I
I
71 25/73
, i I ,
I
12
ie
20
24
Figure 19.
DAYS
Growth response of Puna liella tertiolecta,
207
-------
1X3
o
CD
10"
I
I-'
10
10"
Abert Lake.Ore.
G +.05P + I.ON
A + I.Omg/L N
O + .05mg/L P
G Control
Filtered only
Conductivity ;50,OOOMmho _|
12
DAYS
16
20
24
xlO1
o>
S
I
10
10"
0 4
Abert Lake,Ore.
O-t-.05P4l.ON
±+ I.Omg/L N
O+ .05mg/L P
O Control
Autocloved and
filtered
Conductivity=50,000 Mmho
_L
I
I
12
DAYS
16
20
24
Figure 20. Growth response of Dana1leila tertiolecta
In alkaline fresh water
-------
REFERENCES
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180:491-493.
Atkins, W. R. G. 1923. The Phosphate Content of Fresh and Salt Waters
In Its Relationship to the Growth of Algal Plankton. J. Mar. Biol.
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Burkholder, P. 1963. Some Nutritional Relationships Among Microbes of
the Sea Sediments and Waters. In: Symposium on Marine Microbiology
(C. H. Oppenheimer, ed.). Thomas", Springfield, pp. 133-150.
Dierberg, Forrest E. 1972. Development of an Algal Assay for Estuarine
Water Quality. MS Thesis, University of North Carolina, Dept. of
Environmental Sciences and Engineering.
Environmental Protection Agency. 1971. Methods for Chemical Analysis
of Water and Wastes, 1971. Analytical Quality Control Laboratory,
Water Quality Office, Cincinnati, Ohio. Pub!. #1602007/71.
Edmondson, W. T. and Y. H. Edmondson. 1947. Measurements of Production
in Fertilized Salt Water. Jour. Mar. Res. 6:228-245.
Eppley, R. W., A. F. Carlucci, 0. Holm-Hansen, D. Kiefer, J. J. McCarthy,
and P. M. Williams. 1971. Evidence for Eutrophication in the Sea
Near Southern California Coastal Sewage Outfalls, July, 1970.
California Oceanic Fisheries Investigations Reports. UCSD 10P20-90.
Erickson, S. J., T. E. Maloney, and J. H. Gentile. 1970. Effect of
Nitrilotriacetic Acid on the Growth and Metabolism of Estuarine
Phytoplankton. J. Water Poll. Contr. Fed. 42(8) Pt. 2:R329-R335.
Fitzgerald, G. P. 1972. Bioassay Analysis of Nutrient Availability.
In: Nutrients in Natural Waters (Herbert E. Allen and J. R.
Kramer, eds.). Wiley Interscience, N.Y. pp. 147-170.
Glass, Gary E. 1973. Bioassay Techniques and Environmental Chemistry.
Ann Arbor Science Publ., Inc. Ann Arbor, Mich.
Goldman, Joel C., Kenneth R. Tenore, and Helen I. Stanley. 1973.
Inorganic Nitrogen Removal from Wastewater: Effect on Phytoplankton
Growth in Coastal Marine Waters. Science 180:955-956.
Inman, Douglas L., and Birchard M. Brush. 1973. The Coastal Challenge.
Science 181:20-31.
209
-------
Kester, Dana R., Tver W. Duedall, Donald N. Connors, and Ricardo M.
Pytkowicz. 1967. Preparation of Artificial Seawater. Limnol. and
Oceanogr. 12(1):176-179.
Ketchem, Bostwick H. 1971. Population, Natural Resources, and Biological
Effects of Pollution of Estuaries and Coastal Waters. IJK Man's
Impact on Terrestrial and Oceanic Ecosystems (William H. Mathews,
Frederick E. Smith, and Edward D. Goldberg, eds.). MIT Press,
Cambridge, Mass. pp. 59-70.
Larsen, D. P. 1973. Personal communication.
Lyman, J. and R. H. Fleming. 1940. Composition of Seawater. J. Mar.
Res. 3:134-146.
McLachland, J. 1960. The Culture of Dunaliella tertiolecta Butcher -
A Euryhaline Organism. Can. J. Microbiol. 6:367-379.
McPhee, Craig. 1961. Bioassay of Algal Production in Chemically Altered
Waters. Limnol. and Oceanogr. 6:416-422.
Mitchell, Dee. 1973. Algal Bioassays for Estimating the Effect of
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O'Sullivan, A. J. 1971. Ecological Effects of Sewage Discharge in the
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-------
Rudd, J. W. M., B. E. Townsend, and R. D. Hamilton. 1973. Discharge
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211
-------
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212
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GROWTH REQUIREMENTS OF ENTEROMORPHA COMPRESSA
AND CODIUM FRAGILE
Richard L. Steele
INTRODUCTION
Enteromorpha compressa, other species of Enteromorpha, and members
of the Ulvales are known to grow abundantly in areas where organic pollution is
rampant (Perkins and Abbot, 1972; Levring, et al., 1969) and where salinity
changes occur frequently. Research on E. compressa, a world-wide species,
was begun with the purpose of developing a rapidly growing alga that could
be used to detect effects of pollution and also become an easily manipulated
bioassay tool. Codium fragile, a recent invader to the East Coast, has
also become an obnoxious pest. First described from Long Island, New York,
in 1957, it has become one of the most common sea weeds that one encounters
along eastern shores from Maine to New Jersey. In Shinnecock Bay, Long
Island, standing crops of 11,000 grams/square meter have been reported
(Moeller, 1969). Aside from ecological imbalances encountered, the most
deleterious effects are on the shellfish industry. Scallops, mussels, and
oysters are no match for the buoyant plants that are swept by tides and
currents and are subsequently deposited on shore where they die (Ramus,
1971) . Control of both of these plants is dependent on knowledge of the
biology of the plant.
MATERIALS AND METHODS
For this study, clean, actively growing plants of Codium fragile
were obtained from either Newport, R.I., or Stonington Harbor in Connecti-
cut. Plants of Enteromorpha compressa were obtained at low tide from
213
-------
Charlestown Pond, Charlestown, R.I. Cultures of unialgal E_. compressa and
C. fragile were started by growing in unenriched sea water, with GeO
, T £
(6-10 mg/L) and 10 ml penicillin G per liter (100,000 units per ml), added
to retard growth of diatoms (Lewin, 1966), bluegreen algae and bacteria.
All cultures of both species were started with tips 1 cm long. Plants were
grown at a day length of 16 hr./8 hr. light/dark cycle at 18°C ± 2°C.
Illumination was 450-600 ft. C under cool white fluorescent lamps and the
culture medium was changed every 4 days.
Experimental media is a formulation, TM-6, devised at the National
Marine Water Quality Laboratory for trace metal studies and is of the
following formulation, Table 1. In all cases silicon was omitted from the
medium. For appropriate experiments, one or several of the ingredients
were omitted. In experiments dealing with trace metals, the medium was
further treated by passing through columns containing the sodium form of
carefully purified (Davey, et_ al_. , 1970) chelex-100 (Bio-Rad). Metals
were then added back in known concentrations for each experiment.
Other enrichments were tested by adding back to media deficient in
a particular ion. Phosphorus was removed from the artificial sea water
by treating with an excess of Fed (100 mg/L) and subsequently filtering
the media through .22 y Millipore filters.
Light, temperature, and salinity preference studies were performed
in sea water that had been passed through the chelex-100 column. Tempera-
ture studies were done over the range of 0° to 30°C. and light experiments
were done over the range of 50 ft. C to 1200 ft. C. For both species,
growth was measured as increase in length of the excised tips 1.0 cm
original length. All transfers were made with teflon coated forceps,
rinsed after each transfer with 18 mohm deionized water.
214
-------
Table 1. FORMULATION OF GROWTH MEDIUM TM-6
1.
2.
3.
*4.
5.
6.
*7.
8.
9.
Compound
Na Cl
K Cl
Mg S0k-7ti 0
Ca (as Cl)
Na N03
K2HP04
Na2SiO'3'9H20
Fe (as Cl)
Vitamins
Thiamine HC1
Biotin each ml
B12
Stock Solution
21 g/100 ml
.7 g/10 ml
12 g/100 ml
1 g/100 ml
71 mg/ml
12.8 mg/ml
3.075 mg/ml
1 mg/ml
. 1 mg/ml
contains .5 yg/ml
.5 yg/ml
Per Liter
100 ml
10 ml
100 ml
25 ml
1 ml
1 ml
10 ml
.1 ml
10. Trace Metals (TM-Metals)'
11.
12.
13.
1 ml of stock solution contains the following:
.0098 mg (0.0025 mg Cu)
.022 mg (0.005 mg Zn)
.010 mg (0.0025 mg Co)
.180 mg (0.05 mg Mn)
.0063 mg (0.0025 mg Mo)
1. 140 mg (0.2 mg B)
Cu S04-5H20
Zn SOk'7U20
Co C12-6H20
Mn C12-4H20
Na2Mo Cv2H20
H3B03
Adjust Ph
TRIS (pH 7.8, 5%
Autoclave
solution)
7.8
10 ml
*pH - 7.8
**Guillard, et al., 1962.
215
-------
RESULTS
Temperature and Illumination
Plants of Codium fragile grew most rapidly at 300 to 600 ft. C at
15-22°C. At other temperatures plants grew slower but at these tempera-
tures, growth was still most rapid at 350-600 ft. C (Figure 1). When
illumination was held constant at 600 ft. C and temperature was varied,
best growth occurred at 16-20°C. (Figure 2). Above and below this range,
growth was reduced. Plants of Enteromorpha compressa grew most rapidly at
300-500 ft. C when the temperature was held constant at every temperature
regime tested (Figure 3). However, when the light intensity was held con-
stant at 300 ft. C most rapid growth occurred at 20-25°C (Figure 4).
Salinity
Growth of C_. fragile and E. compress a were much different at the
range of salinities tested, i.e. 0-60 °/°° E. compressa, on the other hand,
showed new growth nearly equal at 5 °/°° to 25 7°° salinity, with a gradual
decrease at higher salinities (Figure 5).
The nutrients phosphorus and nitrogen were varied over a wide range
for both C_. fragile and E_. compressa. Nutrient requirements differ quite
markedly for the two species. Growth of Codium was not appreciably in-
creased by increasing the amounts of phosphorus and nitrate. One-tenth the
amount of phosphorus and nitrogen used in TM-6 produced growth equal to
the amount normally added (Figures 6 § 7). Conversely, E. compressa was
definitely stimulated by higher amounts of both phosphorus and nitrogen
(Figures 8 £ 9).
216
-------
10,-
X
rt
O
CM
<1>
Bl
,_,
O
p
c
(D
-1.U
9
8
7
6
5
4
3
2
1
-
-
u
-XO~~°"^.
/ \
0 °v
/ \
0X X
1 1 1 1_. 1 1 ,
Figure L.
200 400 600 800 1000 1200
Illumination (foot-candles)
Increase in length of Codium fragile tips at various intensities
of illumination (in ft. c) Temperature 18° C.
X
03
O
CM
J_,
P
M-l
03
O
X
4_>
c>o
-------
c/l
I*
O
CM
CD
oS
f ^
O
2
CD
i-J
20
18
16
14
12
10
8
6
4
2
-
_
x°\
- / A
/ OV>0 o
O ^^^^rt
- /
- /
0
1 1 1 1 1 1 1 1 1 1 1 1
Figure 3.
200 400 600 800 1000 1200
Illumination (foot-candles)
Increase in length of Enteromorpha compressa tips at various
intensities of illumination (in ft. c). Temperature 20°C.
Temperature (°C)
Figure 4. Increase in length of Enteromorpha compressa tips at various
temperature regimes from 5°C. to 30°C. Illumination 300 ft. c,
218
-------
Figure S.
18
in ,,
* 16
T\
14
H 12
S 10
20p» Enteromorpha compress a
o o Codium fragile
o
(N
O
0>
8
1 /'
D" 1 1 1 1 1
V
^°-0
II II
15 30 45
Salinity (°/oo)
60
Increase in length of tips of Enteromorpha ^Tessa ( ) and
Codium fragile (o-o) placed in sea water of vanous salinities
Temperature 20°C. Illumination 400 ft. C.
10
9
&0
0)
j
100 mg Nitrogen/L.
10 mg Nitrogen/L.
1 mg Nitrogen/L.
0 mg Nitrogen/L.
Days
Figure 6. Increase in length of tips of Codium fragile placed in media
containing increasing amounts of nitrogen. Temperature 20°C.
Illumination 400 ft. C.
219
-------
nr
o
N '
4->
bO
C
0)
J
10
9
8
7
6
5
4
3
2
1
- 100 mg Phosphorus/L.
_ 0 o 10 mg Phosphorus/L.
1 mg Phosphorus/L.
~ o o 0 mg Phosphorus/L.
S1
0x^
_^ ""*'"" o«***
^r* .-^
^^*****^ ~^
*^ ~ *
/o
^ -
,/ *mi!^ -" ^.. ^~
i i i i i i i i i i i I i i i I i
o
0
1
4 8 12 16 18
Days
Figure 7. Increase in length of tips of Codium fragile placed in media
containing increasing amounts of phosphorus. Temperature 20°C.
Illumination 400 ft. C.
40
35
30
6 25
o °
f.
+j
bo
20
15
10
5
100 mg
o o 10 mg Phosphorus/L.
1 mg Phosphorus/L.
o o 0 mg Phosphorus/L.
12
16
20
Days
Figure 8. Increase in length of tips of Enteromorpha compressa placed in
media containing increasing amounts of phosphorus. Temperature
20°C. Illumination 400 ft. C.
220
-------
CJ
30
25
20
c
-------
Figure 10.
10
10
£> Q
Cu J
o 8
fn 7
Q) '
oj 6
' > i-
S 5
o
3 4
bO ,
0
J 2
1
Increas
deficiei
Illumin.
20
18
l/l
fr 16
13
-------
MISCELLANEOUS OBSERVATIONS
During the month of August 1972, large, round, non-flagellated
spores were released from some tips of Codium and grew when placed in
unenriched sea water. However, the individual spores developed into un-
differentiated filamentous masses identical to tips grown in a complete,
highly enriched sea water. These plants were retained for three months
in culture but did not develop into mature Codium plants. Another
interesting observation is the ability of Codium to grow from either end
of an excised tip. This does not always occur; and the controlling factor
for this phenomenon is not yet known.
DISCUSSION
The two species of algae, C. fragile and E. compressa, both green
algae, both estuarine, and both abundant in nature, exhibit quite different
responses to nutrients when studied in the laboratory. Codium appears to
be a species with fairly limited salinity tolerances, and fairly limited
temperature tolerances. Whereas E. compressaappears to be a species in
which growth is definitely stimulated in highly enriched conditions, C_.
fragile does not appear to be stimulated by these conditions. It tends to
prefer adequate but lower levels of enrichments.
The most outstanding difference in growth requirements of these two
species is the requirement by C_. fragile of a very low level of heavy
metals. When heavy metal ions are abundant, growth is definitely depressed
or non-existent.
How then do these two species co-exist in nature? To some extent,
they are usually not found growing abundantly in the same places. Entero-
morpha compressa and other Ulvales are abundant in bays and harbors that
contain a high degree of organic pollution and raw sewage. Codium fragile
223
-------
is found in "salt ponds" or shallow embayments receiving a constant tidal
flux. Could this type of environment tie up or chelate the metal ions,
thereby allowing Codium to grow abundantly?
According to Fralick and Mathieson (1973) C^. fragile photosynthesizes
most efficiently at 20-25 C and at 900 ft. C. In our study however, growth,
or increase in tip length was more efficient at light intensities of from
200-600 ft. C. Their optimum temperature compares well with ours, however.
Studies by Borden and Stein (1969) on the Pacific coast of North America
indicate that this optimum temperature for C^. fragile from that coast is
much lower (8 C).
The problem of differentiation of tissue into the dichotomies character-
istic of Codium is not yet fully answered. Ramus (1972) obtained differentia-
tion of the tissue by shaking. Borden and Stein (1969) did not obtain
any differentiation of tissue. In my cultures, excized tips differentiate
only in unshaken media with low metals. Cultures obtained from spores did
not differentiate in this medium, however. Is it possible that growth
requirements change as the thallus differentiates from juvenile filaments
to the adult plant? Perhaps differentiation of all stages of Codium is
dependent not only on concentration of heavy metals in the medium but on
the osmotic pressures set up in the interstitial areas of the filaments
of the plants. In an uncompacted juvenile plant this would be less im-
portant than in a tightly compacted adult thallus.
Enteromorpha compressa, rather than developing into a useful bio-
assay tool, is a good indicator of highly enriched water. In some instances,
growth rates of 3-5 cm per day were obtained, and in a single low salinity
experiment where nutrients were never limited over a six week period, a
plant 1.5m was obtained. With growth rates of this order, it is not
difficult to understand how areas such as Boston Harbor and other highly
enriched areas become choked with ulvaceous growth.
224
-------
REFERENCES
Borden, C. and J. Stein. 1969. Mitosis and Mitotic Activity in
Codium-fragile (Suringar) Hariot. Phycologia 8(3): 149-156.
Davey, E., J. Gentile, S. Erickson and P. Betzer. 1970. Removal
of Trace Metals from Marine Culture Media. Limnol. and
Oceanogr. 15(3):486-438.
Fralick, R. and A. Mathieson. 1973. Ecological Studies of Codium
fragile in New England, USA. Marine Biology 19(2):127-132.
Guillard, R. and J. Ryther. 1962. Studies on Marine Planktonic
Diatoms. I. Cyclotella nana Hustedt and Detonula confervaceae
(Cleve) Gran. Can. J. of Micro. 8:229-239.
Levring, T., H. Hoppe, and 0. Schmid. 1969. Marine Algae: A Survey
of Research and Utilization. Botanica Marina Handbooks, Vol. 1.
Cram, De Gruyter & Co., Hamburg.
Lewin, J. 1966. Silicon Metabolism in Diatoms. V. Germanium Dioxide,
a Specific Inhibitor of Diatom Growth. Phycologia 6(1):1-12.
Moeller, H. 1969. Ecology and Life History of Codium fragile subsp.
tomentosoides. Ph.D. Thesis. Rutgers University, New Brunswick,
N. J. 76 p.
Perkins, E. and 0. Abbot. 1972. Nutrient Enrichment and Sand Flat
Fauna. Marine Pollution Bull. 3(5):70-72.
Ramus, J. 1971. Codium: The Invader. Discovery (New Haven, USA)
6:59-68.
1972. Differentiation of the Green Alga Codium fragile.
Amer. J. Bot. 59:478-482.
225
-------
GREAT LAKES NUTRIENT ASSESSMENT
Nelson A. Thomas, Katharine Hartwell,
and William E. Miller
INTRODUCTION
The large lakes program of the Grosse lie Laboratory has both extramural
and inhouse programs concerned with biostimulation. The extramural program deals
with three different areas of algal growth studies. The first study is centered
around the delineation of the nutrient requirement for the green alga Cladophora.
This alga often causes nuisances around the Great Lakes during late summer when
it breaks loose and washes up in windrows along swimming beaches. A basic
knowledge of the nutrition and key growth factors is required in developing a
sound basis for control of this alga. The first phase of this project is to
develop culture techniques and synthetic culture medium. Secondly, the organic
and inorganic nutritional requirements will be determined. A major part of the
experiment will be to determine the critical concentration for each of the
essential inorganic nutrients, thus hoping to determine the limiting nutrient
concentration in the lake for Cladophora. Another phase of this project will be
to sample the Cladophora growths in the lake and determine which element is
restricting its growth.
Two types of procedures will be employed to determine the critical limiting
nutrients for controlling Cladophora growths. The first procedure involves the
comparison of the inorganic analysis of the alga with the critical growth con-
centration of elements as obtained in laboratory studies. The second procedure
involves the assay of Cladophora samples for nitrogen and phosphorus availability
through measurements of phosphorus releases upon boiling and ammonia uptake in
the dark.
226
-------
The second extramural project is the testing for the long-term availability
of the various forms of nitrogen and phosphorus present in water samples
collected at selected seasons in the Lake Ontario Basin. This includes both
tributary and main lake sampling. The study will include the determination of
those fractions of the nutrients that stimulate or control alga nutrient growths
through a combined program of chemical analysis and nutrient assays. The chemical
composition of the water during alga growth can be monitored, therefore, deleting
those nutrients that are available for long-term alga growth. The second phase
of this project is to determine the effect of reducing the phosphorus content in
a particular water sample by using alum or iron salts. During this study it was
found that the results could not be reproduced when the phosphorus was readded.
When phosphorus was readded some growth element evidently, had been removed
with the alum or iron salts. When phosphorus was readded in its pure form, the
resultant growth was not equal to that of the untreated samples.
The third extramural project is the study of algal assay procedures that
might be appropriate for the Great Lakes. These tests will include both
Selenastrum capricornutum and natural populations of algae. Another phase is to
determine the effect of various nutrient levels on the level of standing crop and
which, if any, nutrients are limiting. This procedure includes the measurement of
growth with nutrient additions both as a spike and as a constant concentration.
This experiment will utilize the technique of chemically analyzing each test
chamber daily and then adding the amount of nutrients to its initial level. The
rationale for such experimental design is based on nutrient studies in the large
lakes, which indicate that the nutrient concentration reductions are very long
term. To perform experiments that have nutrient reductions over a ten day
227
-------
period is, therefore, unrealistic.
The inhouse program at the Grosse lie Laboratory was designed to provide
answers to the questions of the importance of phosphorus in limiting phytoplankton
growth in Lake Michigan, as well as to attempt to define a phosphorus standard
for Lake Michigan,
The subject of algal nutrient stimulation on the phytoplankton of Lake
Michigan has been discussed in many reports including the Lake Michigan
Enforcement Conference and the scientific literature. There has been
strong evidence presented by Schelske and Roth (1973) that phosphorus has
the greatest nutrient limiting effect on phytoplankton growth. The study
on the relationship of the growth of phytoplankton to individual nutrients
in relationship to Lake Michigan water is very incomplete. However, nutrient
growth data are required to formulate criteria for the establishment of
phosphorus limits in large bodies of water.
Algal assays were conducted on Lake Michigan water in connection with
the Phosphorus Technical Committee to the Lake Michigan Enforcement Confer-
ence. Water samples were collected from a 3 meter depth along a transect
from Milwaukee, Wisconsin, to Ludington, Michigan, during January, May, and Aug.,
1972 (Figure 1). A similar transect was sampled between Benton Harbor, Michigan,
and Chicago, Illinois. Samples were collected at the 6 meter depth during
the months of April and July, 1972. A total of 25 samples were analyzed.
The January, April and May samples analyzed by the National Eutrophication
Research Program were assayed for growth potential utilizing the standard
algal assay procedure with a Coulter Counter. Samples analyzed at the
Grosse lie Laboratory were assayed according to standard procedures with a
fluorometer. In addition, the growth response of the natural population was
also determined on selected split samples. The natural population growth was
228
-------
MILWAUKEE
CHICAGO
LUDINGTON
BENTON HARBOR
Fig. 1. Algal assay sampling stations, Lake Michigan, January-August, 1972.
229
-------
estimated by the amount of chlorophyll £ present in the samples. Raw water
chemistry is presented in Table 1.
The samples analyzed according to the standard algal assay procedure
received the following treatment:
Prior to assay each sample was pretreated in two ways: (1) one-half
was filtered through a 0.45 micron membrane filter to remove indigenous
biomass and (2) the other half was autoclaved to solubilize any nutrients
contained within the indigenous biomassequilibrated with a mixture of
1 percent CO,, in air until the original pH was attainedand then passed
through a 0.45 micron membrane filter. The assays were carried out in
500 ml Erlenmeyer flasks containing 100 ml of total sample. The following
additions of nitrogen (as NaNOo), phosphorus (as K^HPO,) , iron (as FeCL^),
and trace elements contained in the Algal Assay Procedure medium singly
or in combination were made to the Lake Michigan water samples: 1.00 mg N/l,
.005 to .020 mg P/l, .033 mg Fe/1, and complete AAP medium trace element
mixture. The nutrient combinations assayed are listed in Table 2.
Growth of the natural population was estimated by measuring the
concentration of chlorophyll after 22 days of incubation. Samples were
filtered through GF/C filters which were dissolved in acetone. Filters
were ground and allowed to steep for one hour then centrifuged. The optical
density of the extract was determined spectrophotometrically.
RESULTS
To determine the suitability of Selenastrum capricornutum as a test alga for
Lake Michigan, split samples were run with the natural population as well as
Selenastrum capricornutum (Tables 3, 4 and 5). The algal response to nutrient addi-
tions were similar except for the 5ppb phosphorus spike (Figure 2). The natural
230
-------
TABLE 1 Lake Michigan chemistry when collected mq/1 - 18' all sample
Station
Number
1
2
3
4
5
1
ro
CO
- 2
3
4
5
6
7
8
9
Sample No.
8647
8648
8649
8650
8651
8908
8909
8910
8911
8912
220
223
224
225
Date
Collected
5/72
5/72
5/72
5/72
5/72
8/72
8/72
8/72
8/72
8/72
7/72
7/72
7/72
7/72
Ammonia
Nitrogen
.011
.014
A. 0015
.01
<.01
.01
.01
.02
.03
.01
.02
.02
.02
.02
Nitrite
Nitrate
Nitroaen
.201
.211
.21
.21
.21
.15
.14
.15
.15
.16
.17
.15
.11
.11
Total
P
.008
.005
.003
< .003
.004
.007
.004
.005
.004
.004
.011
.007
.006
.005
Ortho-
P
.001
.002
<.001
.003
< .003
.006
.003
.003
.003
.003
.003
.003
.003
.003
Alka-
1 inity
105
A86
no
78
127
119
118
116
116
115
98
114
114
114
Total
dis P
^.001
.003
\.0025
.001
Temp
22.2
23.0
23.8
T9.'5
Depth
18*
18'
181
18'
pH
8.6
8.7
8.6
8-5
-------
TABLE 1 Lake Michigan Chemistry when collected mg/1 - 18',all sample (Con't)
Station
Number
10
6
7
8
9
10
tN, 1
CO
rv>
2
3
4
5
Sample No.
228
8627
8626
8625
8624
8623
8482
8483
8484
8485
8486
Date
Collected
7/72
4/23/72
4/23/72
4/23/72
4/23/72
4/23/72
1/11/72
1/11/72
1/11/72
1/12/72
1/12/72
Ammonia
Nitroge
.02
<0.01
<0.01
<0.01
<0.01
<0.01
.03
.02
.02
.02
.04
Nitrite
'Nitrate
Nitrogen
.13
.23
.22
.21
.21
.19
.28
.26
.26
.25
.28
Total
P
.003
.006
<.003
.004
.005
.010
.014
.008
.009
.008
.012
Ortho-
P
.003
<.003
< .003
.003
.003
.008
.003
.003
.007
.004
.004
Alka-
linity
118
131
160
133
129
129
136
144
138
141
138
Tottl
dts-.P
Temp
16°
Depth
18'
pH
8.4
-------
TABLE 2. EXPERIMENTAL DESIGN TO STUDY ALGAL GROWTH POTENTIAL OF
LAKE MICHIGAN WATERS
Treatment
1. Lake water control
2. Lake water + .020 mg P/l
3. Lake water + 1.000 mg N/l
4. Lake water + 1.000 mg N/l + .020 mg P/l
5. Lake water + 1.000 mg N/l + .020 mg P/l + .033 mg Fe/1
6. Lake water + .020 mg P/l + .033 mg Fe/1
7. Lake water + .005 mg P/l + 1.000 mg N/l + .033 mg Fe/1 + T.E,
8. Lake water + .010 mg P/l + 1.000 mg N/l + .033 mg Fe/1 + T.E.
9. Lake water + .015 mg P/l + 1,000 mg N/l + .033 mg Fe/1 + T.E,
10. Lake water + .020 mg P.I + 1.000 mg N/l + .033 mg Fe/1 + T.E.
11. Full strength AAP medium
All samples were pretreated by (1) filtration, and (2) selected samples
by autoclaving followed by filtration
233
-------
TABLE 3 Corvallis Results. Maximum Standing Crop. Mg/1. Coulter Counter.
Station
Number
1
2
3
4
5
10
ro
% 8
9
7
6
1
2
3
4
Sample
Number
8482 a & f
M - 3
8483 a & f
M - h
8484 a & f
M - %
8485 a & f
M - 3/4
8486 a & f
L - 3
8623 a & f
8625 a & f
8624 a & f
8626 a & f
8627 a & f
8647 a & f
8648 a & f
8649 a & f
8650 a & f
Date
Collected
1/72
1/72
1/72
1/72
1/72
4/72
4/72
4/72
4/72
4/72
5/72
5/72
5/72
5/72
Control
.066
.079
.087
.133
.103
.08
.040
.189
.040
.050
.067
.083
.086
.115
.02 P
.178
5.453
.505
.204
.097
5.60
1.926
6.257
5.069
5.764
4.642
2.389
3.234
3.72;
1.0 N
.065
.046
.107
.034
.028
.12
.060
.033
.046
.096
.06'
.05*
.06!
.05
.02 P
1.0 N
3.762
2.873
2.943
.151
1.379
8.88
4.422
8.167
6.921
5.887
6.51
1.121
4.296
i 4.894
.02 P
1.0 N
.033 Fe
.910
1.558
4.167
6.749
4.651
9.40
4.444
13.784
2.776
5.397
6.80
2.661
9.017
6.650
.02 P
. .033 F
7.32
5.181
5.887
5.784
5.191
5.55
1,569
5.244
5.6Q2
.005 P
. +all
.78
.381
.307
.300
.825
.34
.184
.283
.136
.01 P
-t-all
1.99
.722
.120
.865
.661
1.09
.37
.519
,276
.015 P
+a.l 1
3.59
1.520
.370
1.493
1.577
1.52
1.220
1.117
1.532
.02 P
4.32
2.085
.755
4.031
5.102
3.42
.845
1.355
2.745
-------
TAR! E 3 Corvallis Results. Maximum Standing Crop. Mq/1 . Coulter Counter. (Con't)
Station
Number
1
2
3
4
5
10
ro
CO
01 2
8
4
6
1
2
3
4
Sample
Number
M-3 filters
M-1/4 f
M-l/2 f
M-3/4 f
L-3 f
8623 f
8624 f
8625 f
8626 f
8627 f
8647 f
8648 f
8649 f
8650 f
Date
Collected
d 1/72
1/72
1/72
1/72
1/72
4/72
4/72
4/72
4/72
4/72
5/72
5/72
5/72
5/72
Control
.193
.075
.091
.139
.115
.04
.067
.036
.084
.077
.036
.085
.059
.036
.02 p
3.858
.059
.299
5.421
..038
5.52
1.996
.664
2.049
.216
..754
3.222
4.552
4.366
1.0 N
.115
.074
.115
.043
.073
.12
.038
.045
.049
.033
.077
.076
.046
.058
.02 p
1.0 H_
5.049
6.057
3.059
.541
1.992
8.23
1.893;
1.811
1.178
.193
3.505
5.390
6.050
5.060
.02 P
1.0 N
.033 Fe
8.605
5.324
3.531
6.544
5,369
8.86
8.635
1.646
.954
.357
4.716
5.715
4.194
8.850
.02 P
.033 E
5.65
6.614
.857
.573
.551
2.183
5.133
5.048
5.457
.005 P
' +all
.14
.067
.575
.275
.172
.838
.449
.717
.422
.01 P
+all
2.12
.076
.696
1.010
.802
1.527
1.162
.815
.991
.015 P
+all
3.13
1.539
1.596
2.903
1.011
1.734
2.116
1.380
2.035
.02 P
+ * 1 1
4.69
.149
1.220
1.611
.914
2.225
2.085
3.074
2.969
-------
TABLE 3 Corvallis Results. Maximum Standing Crop. Mg/1 . Coulter Counter. (Con't)
SC.'itJon
K umber
5
5
ro
Co
CTi
Sample
Number
8651 f
8651 a&f
Date
Collected
5/72
5/72
Control
.055
.067
.02 P
3.210
2.480
1.0 N
.063
.060
.02 P
1.0 N
4.939
3.171
.02 P
1.0 N
.033 Fe
6.446
5.129
.02 P
5.236
6.425
.005 P
.463
.178
.01 P
2.103
.978
.015 P
2.460
1.930
.02 P
+ a 1 1 .
4.648
2.330
-------
TABLE 4 Grosse He Calculated mg/m Chlorophyll a_ Natural Algae. Spectrophotometer.
Station
Number
6
10
1
5
ro
<-U
^i
Sample
Number
220
228
8908
8912
Date
Collected
7/23/72
7/23/72
8/16/72
8/16/72
Control
19
107.5
9.8
15,0
.02 P
58.5
76
7.6
1.1,8
1.0 N
19
91
7.2
13.0
.02 P
1.0 N
156
296
37.9
45.3
.02 p
1.0 N
.033 Fe
148
214
NA
NA
.02 7
,033.F
29.0
37
NA
NA
.005 P
>. +all ...
16.7
111.5
NA
NA
.01 P
+all
49.5
177
NA
NA
.015 P
+a.ll
NA
NA
NA
NA
.02 P
+ nl 1 _
119.5
304.5
NA
NA
-------
TABLE 5 Grosse lie. Day 14 Standing Crop. Mg/.l . Fluorometer
Station
Number
1
2
3
3
6
7
ro
OJ
oo 8
9
10
1
2
3
4
5
Sample
Number
8647a,f
8648a,f
8649a,f
8649f
220f
223f
224f
225f
228f
8908f
8909"
8910f
8911 f
8912f
Date
Collected
5/9/72
5/9/J2
5/9/72
5/9/72
7/23/72
7/23/72
7/23/72
7/23/72
7/23/72
8/16/72
8/16/72
8/16/72
8/16/72
8/16/72
Control
.002
.011
0
.005
.005
0
0
.03
0
.019
.006
.037
.028
0
.02 P
8.36
6.657
1.6
.47
1.57
.57
.73
1.05
4.61
1.17
1.08
1.41
1.91 .
.08
1.0 N
.007
.006
0
0
0
0
.011
0
.014
.01
.007
.018 '
.018
0
.02 P
1.0 N
4.72
2.738
.56
.19
1.23
.22
.99
.68
4.57
1.86
1.02
1.15
4.96
4.37
.02 P
1.0 N
.033 Fe
4.58
1.16
.49
.28
1.87
.18
2.24
.69
8.04
.98
.91
1.35
3.24
.22
.02 P
,033 I
5.93
1.34
3.4
.44
1.62
1.01
2.98
6.07
3.10
2.45
1.11
1.51
1.51
.26
.005 P
+all .
1.2
.187
1.6
1.9
1.31
3.64
1.90
2.35
3.01
.83
1.54
1.65
2.25
.64
.01 P
+all
6.38
6.17
6.5
6.8
6.17
7.14
7.04
6.45
7.52.
2.37
6.70
7.28
5.37
4.09
.015 P
+aU
11.65
9.26
11.6
8.0
8.94
9.26
10.92
12.38
10.19
9.88
8.11
8.94
13.8
8.32
.02 P
+ rill.
6.01
10.61
16.3
10.1
10.68
11.54
13.62
14.14
13.52
15.3
10.50
12.69
15.0
9.98
-------
to
on
15
10
o
a:
o
<
i
<
0
0
Benton Harbor-Chicago
July, 1972
,o 10 Natural population
10 S. capricornutum
6 S. capricornutum
6 Natural population
5 10 15 20
TOTAL PHOSPHORUS SPIKE, p/l
25
01
CD*
350
300
250
200^
150 §
3
100 5
50
0
Fig. 2. Growth of natural algae and Selenastrum capricornutum at
various phosphorus concentrations.
-------
population did not respond to the low level phosphorus spikes; the phosphorus
content of the water was less than the critical level necessary to support
growth of the natural population. The similarity of growth responses provides
validity for the use of Selenastrum capricornutum to determine the limiting
nutrients in Lake Michigan.
During January, all samples indicated phosphorus limitations by the
increasing growth with increases in phosphorus concentration. The additional
response to nitrogen indicates that nitrogen was secondarily limiting.
The April sampling of the transect from Benton Harbor to Chicago indicates
that algal nutrient limiting effect increased from Benton Harbor to Chicago.
The phytoplankton near Chicago (Station 10) had the greatest response to the
addition of phosphorus and nitrogen thus indicating the supply of other nutrients
and trace elements was sufficient. The autoclaved samples produced increased
growth over the filtered samples thus indicating that autoclaving released
additional nutrients. Phosphorus was also limiting in the autoclaved samples
from April.
The results of the May sampling of the transect from Milwaukee to Ludington
indicated the same phosphorus limitation. There was no response to the addition
of nitrogen. However, the combination of nitrogen and phosphorus did produce
a slightly higher growth than the single addition of phosphorus.
The response of the natural population to nutrient additions indicates
that during July the near-shore water off Benton Harbor (Station 6) was
limited primarily by phosphorus. Water samples collected near Chicago (Station
10) were primarily limited by phosphorus and secondarily by nitrogen. The
near-shore waters were more nutrient limited than the off-shore stations.
During August the near-shore waters off Milwaukee were limited by
240
-------
phosphorus, secondarily by nitrogen. The near-shore water near Ludington was
limited equally by phosphorus and nitrogen (Figure 3). The addition of
trace elements was necessary to stimulate maximum growth.
The growth rate curve resulting from the addition of phosphorus to either
Selenastrum capricornutum or to the natural population was linear. The increase
in the population was directly proportionate to the concentration of phosphorus.
The addition of other trace elements was requrired to stimulate the growth of
Selenastrum capricornutum. In a few tests nitrogen was required in addition
to phosphorus to cause a growth in the natural population.
The data was too limited to correlate the concentration of chlorophyll a_
and total phosphorus present at the time of the sampling. Likewise, the data
points were too limited to correlate the chl _a and dry weight (fluorometer)
produced without spiking.
During January, the total and orthophosphate averaged 10.2 and 4.2 ppb,
respectively. During May and August the same stations averaged 4.7 and
3.0 ppb for total and ortho phosphorus, respectively.
CONCLUSION
Algal nutrient limitations for Lake Michigan can be determined through
the use of test algae (Selenastrum capricornutum) or with natural population.
Phosphorus is the primary limiting nutrient during January, April, May,
July and August in the areas sampled. Nitrogen is secondarily limiting and
in one case it was equal to phosphorus in limiting algal growth. Auto-
claving of samples releases nutrients which results in additional growth.
Tests in the laboratory indicate that on spiked samples there was a 5.0
percent increase in algal dry weight per increase in each ppb of phosphorus.
In laboratory growth experiments with unspiked lake water, there is a 0.5
241
-------
Water Sample 8908
SPIKES, mg/l r
Day 22, Natural Algae Population
20 40 60 80
100
ir
T
T
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Control
0.005 P
0.01
0.02
0.005
0.01
0.2
0.005
0.01
0.02
0.005
0.01
0.02
0.25N
0.5
1.0
0.005
Water Sample 8912, August 16, 1972, 3 Miles off Ludington
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Control
0.005P
0.01
0.02
0.005
0.01
0.02
0.005
0.01
0.02
0.005
0.01
0.02
0.25N
0.5
1.0
0.25
0.25
0.25
1.0
20 40 60 80
CHLOROPHYLL a, mg/m3
100
Fig. 3. Resultant growth of natural populations at various levels of nitrogen and phosphorus.
242
-------
percent increase per ppb of total phosphorus. The difference in growth
rates probably was caused by the unavailability of the phosphorus in the
unspiked samples.
Lake Michigan, as indicated by the algal assay tests, is primarily
limited by phosphorus and secondarily by nitrogen. In that Lake Michigan
produces such large amounts of algae with small additions of phosphorus, a
program to assess the present levels and interactions of phosphorus and
phytoplankton must be implemented.
The results of these tests indicate that phosphorus limits the total
algal standing crop in Lake Michigan. The results also indicate that
changes in the ppb range substantially changes the standing crop; therefore,
to base a phosphorus standard on the tests thus far performed, is not
possible. Algal assay work is being continued in an effort to establish a
standard.
REFERENCE
Schelske and Roth. 1973. Limnological Survey of Lakes Michigan, Superior,
Huron and Erie. University of Michigan, Great Lakes Research
Division, Publication No. 17. pp 108.
243
-------
ASSESSING TREATMENT PROCESS EFFICIENCY WITH THE ALGAL ASSAY TEST
Richard E0 Thomas and Robert L0 Smith
INTRODUCTION
Wastewater discharges containing substantial quantities of
nitrogen and phosphorus have been identified as an important factor
in the eutrophication problem plaguing many lakes throughout the
United States. The development of a reliable algal assay procedure
for assessing the sensitivity of receiving waters to change and
the effects of changes in waste treatment processes on receiving
waters offers the hope of extending the essay procedure to other
applications.
Ongoing research studies on the treatment efficiency of the
spray-runoff (overland flow) method of wastewater treatment by
land application provided us an opportunity to make preliminary
evaluations for one such extension of the algal assay procedure.
One of these ongoing research studies involved spray-runoff treat-
ment of rainfall runoff from a beef cattle feedlot. The other
ongoing research study has the primary objective of determining
the feasibility of utilizing the spray-runoff soil treatment
process as a simple and economical system for complete treatment
of raw domestic wastewater. The algal assay procedure was
244
-------
included in the analytical program for these two studies to obtain
a measure of the capability of spray-runoff to reduce the potential
of a wastewater to stimulate algal growth. The primary objective
of including the algal assay procedure in the analytical program
was to observe the relative reduction in the capability to stimu-
late algal growth in relation to the reduction in nitrogen and
phosphorus concentrations achieved by the treatment process.
METHODS
All samples were collected in non-metallic containers and
either transported to the analytical laboratory in a few minutes
or packed in an ice chest for transport to the analytical lab-
oratory. Indigenous algae were killed by autoclaving rather than
removed by filtration since it was desired to determine the algal
biomass that could be grown from all nutrients in the water includ-
ing those solubilized by autoclaving. The standard bottle test
procedure was followed utilizing Selenastrum capricornutum as the
inoculum and measuring the development of the maximum standing
crop with fluorescence measurements and a gravimetric determination
at the end of the test period. Aliquots of each sample were
analyzed for solids, biochemical oxygen demand, total nitrogen,
and total phosphorus as well as many other parameters of interest
for evaluating treatment efficiency of the spray-runoff treatment
process.
245
-------
RESULTS
Two algal assay tests have been completed for each of the
ongoing research projects. Samples for the tests involving the
runoff from the beef cattle feedlot were taken from a storage
lagoon from which the wastewater was pumped to the spray-runoff
area and from the spillway of a small farm pond which was the final
step in the multi-unit treatment system. Samples for the tests
involving the municipal wastewater were taken from the discharge
line of the standard rate trickling filter plant used by the city
and from the overflow at a flow measuring flume at the spray-
runoff test area. The results of the algal assay tests and perti-
nent chemical quality data are summarized in Table 1.
Table 1. CHEMICAL QUALITY AND BIOMASS PRODUCTION
(mg/1)
H
4J 3
co cr
cu -i-i
H H-l
Storage lagoon
Farm Pond
H
cfl
13 CJ
CU -H
C5 CO CU C T3
T-H CU *"O Q CU pi
cfl O- vH O 60 nj
4-1 CO H O P"> B
O 3 O -H X 3
H 00 c/l pq O O
Feedlot Runoff
81 6
127 25
11 5
8 6
a
0)
60
.-H O
cfl )-i
4-1 4-1
0 -H
E~* £5
15.2
11.5
3.6
6.3
CO
M
O
i 1 ft
cd co
4-1 O
0 JT
H PM
3.7
4.6
0.2
0.5
T3 C
CU O
4J -H
cd co 4-1
H CO O
3 cd 3
a 0 -a
r-l O O
cd -H M
U « P-i
K
220
194
31
53
Municipal Wastewater
Sewage Plant Effluent
Spray-Runoff Plots
32 13
20 16
20 20
10 10
20.8
16.3
6.4
4.0
8.5
7.3
2.5
1.0
198
121
90
60
*Biomass calculated with factor derived between fluorescence
measurements and gravimetric measurements for feedlot tests.
246
-------
The treatment system consisting of the spray-runoff area and
the farm pond achieved substantial reductions in the concentration
of all four of the chemical parameters shown in Table 1 for the
studies on the feedlot runoff. Changes observed in biomass pro-
duction paralleled the changes in the total nitrogen and total
phosphorus concentrations but showed little relationship to the
total suspended solids or biochemical oxygen demand.
The spray-runoff treatment system for municipal wastewater
produced a product water of better chemical quality than the
standard rate trickling filter plant. As was the case for the
feedlot test, the biomass productivity also paralleled the changes
in total nitrogen and total phosphorus.
DISCUSSION
The results of two algal assay tests conducted on each of
two wastewater treatment systems support the hypothesis that the
algal assay test shows promise for evaluating the eutrophying
potential of product waters from different wastewater treatment
processes. Even though nitrogen and phosphorus concentrations of
the product waters were substantially greater than those in waters
for which the algal assay test was developed, biomass production
was directly related to the concentration of nitrogen and/or
phosphorus in the product water. The general relationships
observed in these two tests appear suitable for direct comparisons
247
-------
at a specific location. The different relationships observed
for the feedlot tests versus the municipal wastewater tests indi-
cate that attempts to extrapolate results between wastewaters and
to differing wastewater sources could lead to erroneous
interpretations.
SUMMARY
The algal assay test was included in the evaluation of
wastewater treatment processes for feedlot runoff and for munici-
pal wastewater. The biomass productivity was directly related
to the total nitrogen and/or phosphorus concentration in the
product water from the treatment process. The results of these
exploratory tests suggest that the algal assay test has promise
for comparing the eutrophying potential of the effluents from a
variety of wastewater treatment processes. This capability to
measure eutrophying potential could be an important factor in
determining cost effectiveness of treatment processes in many
localities.
248
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UTILIZATION OF ENERGY BY PRIMARY PRODUCERS
IN FOUR PONDS IN NORTHWESTERN FLORIDA
Gerald E. Walsh
INTRODUCTION
Amounts of solar energy incorporated by primary producers in
four small ponds in northwestern Florida were estimated by use of
field data collected during an annual cycle and current theory of
photosynthesis as discussed by Arnon et al. (1968), Kok (1965),
and Rabinowltch and Govindjee (1969). Several reports have des-
cribed photosynthetic rates in relation to light intensity (Ryther
1956, McAllister 1961, Steeman Nielsen and Hansen 1961, Goldman
et al. 1963, Strickland 1965, Kalff 1969), but none has given the
amount of solar energy incorporated by photosynthesis into organic
compounds in natural communities.
The research reported here was concerned with seasonal changes
in energy utilization. The objectives of the investigation were
to:
1) determine the amount of solar energy incorporated into the
first trophic level in each pond,
2) relate energy incorporated to the amount of solar radiation
impinging upon the water,
3) determine the efficiency of incorporation of energy absorbed
by primary producers into organic compounds,
4) study the role of C02 concentration in limiting the rate of
primary production, and
5) describe changes in phytoplankton over a seasonal cycle.
249
-------
DESCRIPTIONS OF THE PONDS
The ponds were located near Pensacola, Florida, at approximately
87°9' West Longitude and 30°20' North Latitude. Their bathymetric
contours are given in Figure 1; area, volume, and depth in Table 1;
and pH, nitrate-nitrogen, alkalinities, and conductivities in Table 2.
TABLE 1. AREA, VOLUME, AND DEPTH OF PONDS USED IN ENERGY STUDIES.
Pond
1
2
3
4
Area (ha)
1.95
2.30
0.91
0.15
Volume ( nr)
17,800
16,200
9,600
650
Avg. Depth
0.91
0.71
1.06
0.43
(m)
Each pond had a narrow band of emergent vegetation (Typha sp.)
around the margin, and each was surrounded by nearly bare sand. Sub-
strata were coarse sand that contained increasing amounts of organic
debris with depth. Water color was light brown, and a Secchi disc
could be seen resting on the bottom at maximum depth in each basin
on all sampling days. Thermal stratification was never observed.
The ponds appeared highly productive of vegetation. Potamogeton
pectinatus and Chara sp. grew in all ponds. The phytoplankton was
dominated by the blue-green algae Lyngbya sp., Anabaena sp., and
Oscillatoria sp., and the green alga Oedogonium sp. The green alga
250
-------
1X5
H-
OQ
c
f-i
CD
w
CU
rt
rt
i-i
H-
n
O
o
p
rt
O
i-i
cn
en
(D
H-
3
0
0>
i-i
OQ
C/3
rt
P^
H-
ro
en
POND I
N
METERS
50
POND 4
POK8D 2
-------
TABLE 2. AVERAGE pH, NITRATE-NITROGEN, ALKALINITY, AND CONDUCTIVITY IN PONDS, 1968.
en
ro
Pond
1
2
3
4
8
7
8
7
7
PH
AM
.35
.44
.77
.06
2
7
8
7
7
PM
.60
.62
.99
.82
8
0
0
0
0
N03,
AM
.107
.095
.122
.227
mg/1
2
0
0
0
0
PM
.099
.092
.116
.198
Alkalinity
mg CaC03/l
23.95
43.29
68.17
42.66
Conductivity
millimhos
1.
2.
1.
0.
10
71
22
85
-------
Spirogyra sp. was present between March and May, and Zygnema sp.
was found in small numbers between January and May in Ponds 1, 2,
and 3.
Unicellular algae, mostly diatoms, were never prominent in the
phytoplankton. The most numerous genera were Pinnularia, Tabellaria
Navicula, Pleurostigma. Stauroneus. Amphora t Synedra, Diatoma, and
Cymbella.
METHODS AND THEORY
Water samples were taken from each basin with a plastic Van Dorn
bottle at approximately weekly intervals from 22 January to 11 December
1968. Water was collected at approximately 0800 and 1300 hours at the
shore, and at the surface, mid-depth, and bottom in the basins of
Ponds 1, 2, and 3, and at the surface, mid-depth, and bottom of Pond
4 in both basins. Water was analyzed for dissolved oxygen by the
Winkler method (Strickland and Parsons 1965). Gross oxygen production
of the total pond was estimated by photosynthesis/respiration ratios
(photosynthetic quotient, Q_) for algal production. Photosynthetic
quotients were calculated by the light-and-dark bottle method (Strick-
land and Parsons 1965) , and bottles were incubated in situ at the
depth of collection. Although photosynthetic quotients derived from
algal productivity are not exactly those of the total pond, they are
more likely to be better approximations than the constant values of
about 1.0 used by some workers because they reflect seasonal changes.
Corrections were made for diffusion of oxygen across the surface of
the water (Welch 1968) and for oxygen stored in intracellular lacunae
of rooted plants (Hartman and Brown 1967).
253
-------
Total chlorophyll was determined by the method of Strickland and
Parsons (1965). Free C02 was calculated from total alkalinity and
pH values (Rainwater and Thatcher 1960). Uptake of C02 was calculated
from its diurnal changes in the water (Verduin 1956). The data between
0800 and 1300 hours were plotted and integrated in relation to length
of day to yield values for the total number of hours of sunlight.
Nitrate-nitrogen was measured by the method of Kahn and Brezenski
(1967). Phytoplankton were counted each week. Samples were collected
with a plankton net (155 y porosity) towed beneath the water surface
by a boat. The volume of water filtered was 7.2 cubic meters. Algae
were counted in a Sedgwick-Rafter counting chamber.
Several diel studies were made in which water samples were
taken at three-hour intervals from 0600 hours of one day to 0600
hours of the next. Chemical analyses were identical to those
described above.
On 3 April 1968 a wettable powder formulation of the herbicide
f Ry
dichlobenil (Casoronvy , 2,6-dichlorobenzonitrile) was applied at
an active ingredient concentration of 1.0 ppm to Pond 4 in order to
study fixation of solar energy by algae in the absence of rooted
plants. Dichlobenil is effective in control of rooted plants but has
no demonstrable effect upon unicellular or filamentous algae at the
concentration used.
According to current theory of photosynthesis, there are two
Registered trademark, Thompson-Hayward Chemical Company. References
to trade names in this publication do not imply endorsement of the
products by the Environmental Protection Agency.
254
-------
biochemical reactions that require light energy (the "light reactions")
and one that does not require light (the "dark reaction") . Molecular
oxygen is formed from the first light reaction:
light
4lT + 4e , (1)
and reduced NADP is formed in the second light reaction:
light
2NADP + 4e~ + 2H+ >2NADPH. (2)
In the dark reaction, energy from (1) and (2) is utilized to incor-
porate C02 into carbohydrate:
2H+ + 2NADPH + CC>2 > 2NADP + H20 + CH 0. (3)
The amount of energy required for evolution of any observed
amount of oxygen can be calculated. Eight to ten photons are re-
quired for evolution of one molecule of oxygen by the light reactions
(Clayton 1965). I used nine photons in the following calculations.
Light, as it impinges upon the pigment system, is converted to
red quanta at wavelengths between 680 and 700 nm (Kok 1965). The
00
energy content of one Einstein (6.02 X 10 quanta) of light at a
wavelength of 700 nm is 41 kcal.
If the number of oxygen molecules evolved over a period of time
is known, the rate of absorption of energy can be calculated. The
example below, taken from Pond 4 on 4 April, shows how energy re-
quired for evolution of oxygen was calculated:
255
-------
2 23
Oxygen evolved, molecules/M /day = 1.91 X 10
o 23
Number of photons required/M^/day = 17.19 X 10
2
Number of Einsteins/M /day
17.19 X 1023 = 2.86
6.02 X 1023
2
Total energy absorbed = 2.86 X 41 kcal = 117 kcal/M /day.
Energy incorporated by light reactions is not transferred with
100 percent efficiency to organic compounds in the dark reaction.
For the general reaction
light
H?0 + CO ^ Organic molecules + 0
approximately 180 kcal of light energy are required to evolve one mole
of oxygen (Kok 1965). Using the data from above, 0.317 moles of oxygen
2
were evolved/M /day. If 180 kcal were incorporated into organic
2
matter for each mole of oxygen evolved, then 57 kcal/M /day were
stored and available for respiration and as food for herbivores.
Hereafter, this energy, which is that potentially stored in organic
molecules, is referred to as e. Efficiency of e may be calculated
from the expression
0e = ! X 100.
insolation
Walsh and Heitmuller (1971) reported mean monthly insolation on
the ponds and those data were used to calculate 0e.
Expressions (1), (2), and (3) imply that when substances such
as inorganic phosphorus and nitrogen are limiting, solar energy can
256
-------
be utilized and oxygen evolved through conventional photosynthetic
pathways without formation of organic molecules.
The limiting effect of CO concentration upon primary production
and the efficiency of conversion of e to organic compounds was
estimated. Pond 2 contained very little dissolved CO , Ponds 1 and 3
contained larger amounts, and Pond 4 contained the greatest concen-
tration of free CO . Approximately 140 kcal of light energy are
required for incorporation of one mole of CO into organic molecules
and the efficiency of storage is 81% (Bassham 1965). The amount of
energy actually stored was estimated by assuming that all C0_
absorbed was used to form new organic molecules. The number of
moles of CO2 absorbed, multiplied by 113 (0.81 X 140 kcal), gave
an estimate of the amount of energy stored. This estimate,
hereafter called E, must necessarily be a minimum value for gross
primary production because utilization of endogenous C02 was not
considered.
There is another important factor to consider here. The values
of E are underestimations of productivity because the method used
estimated free CO- in a system where
C02 + H O^H2CO -^=i H+ + HC03~S=^H+ + C03 =
and excluded C02 in other forms, such as CaCO^. This underestimation
is especially significant for data from Pond 2, where the average
annual alkalinity was 43.3 mg CaC03/l but the pH was always high
(e.g., 9.34 on 10 September).
257
-------
The rate and efficiency with which light energy is used to form
new compounds with CO- are two basic ecological processes associated
with primary production. Efficiency was estimated from the expression
E' = | X 100
EFFECTS OF HERBICIDE
Dichlobenil at 1 part per million reduced the density of benthic
plants in Pond 4. After application of the herbicide on 3 April, P_.
pectinatus and Chara sp. declined in numbers. Effects of the herbi-
cide appeared greatest around 1 May, when all P_. pectinatus and
approximately 70 - 80% of the Chara sp. were eliminated. As benthic
plants died, a bloom of filamentous algae occurred. In Pond 4 at
the height of the bloom, dominant phytoplankters were Lyngbya sp.
(533,400 filaments/M3), Oedogonium sp. (327,150 filaments/M3), and
Oscillatoria sp. (35,520 filaments/M ). In the other ponds, algae
did not bloom at that time. Presumably, as benthic plants died,
the algae utilized nutrients released from them to sustain the bloom.
By 1 June, Chara sp. had returned to approximately the pretreatment
density, a few individuals of P_. pectinatus had appeared, and the
algal bloom had subsided. By 2 July, density of P_. pectinatus
appeared similar to that existing before treatment. Treatment had
no measured effect on energy incorporation.
ENERGY INCORPORATION AND UTILIZATION
Diel studies demonstrated differences between ponds in the amount
of solar energy incorporated by the light reactions of photosynthesis,
258
-------
efficiency of its incorporation, the rate of uptake of C02, and the
amount of light energy stored (Table 3). Pond 4 had the greatest
primary productivity and absorbed the greatest number of einsteins
and quantity of CC^ per square meter. In addition, 0e values and the
amount of energy stored (E) were much greater than in the other ponds.
During a phytoplankton bloom in May, when most benthic plants were
gone, total chlorophyll concentration in Pond 4 was 25.3 mg/£ and
2
energy stored was 23.4 kcal/M /day. In Pond 3 on the same day, total
chlorophyll content was only 3.9 mg/£ and energy stored was only 2.2
2
kcal/M /day.
Incorporation and utilization of energy in Pond 2 differed
greatly from those in the other ponds. During the diel study,
Pond 2 contained relatively small numbers of phytoplankters as
shown by total chlorophyll concentration, but its bottom was
covered by dense growth of P_. pectinatus. Concentration of free
CO. was 1.5 mg/£ at 0600 hours and 0.4 mg/£ at 1500 hours and was,
therefore, limiting to photosynthesis. Rate of incorporation of
solar energy by the light reactions of Pond 2 was relatively rapid
because of the dense growth of benthic plants. Rate of incorporation
into organic molecules was low however, probably due to the limiting
effect of low concentration of CO . Although rate of absorption of
energy in Pond 2 was more than double that in Pond 1, the estimated
rate of absorption of C02 was only l/6th that in Pond 1.
Values of E' are estimates of efficiency of incorporation of
259
-------
TABLE 3. ENERGY OF LIGHT REACTIONS, AMOUNT OF C02 TAKEN UP, AND MAXIMUM AMOUNT OF LIGHT ENERGY
STORED DURING DIEL STUDIES IN PONDS. (e = solar energy potentially stored in organic molecules,
cf>e = efficiency of e, E = estimate of amount of energy stored from C02 uptake data, E' = ratio
of E to £ X 100).
cr>
O
o
Einsteins/M /day
e, kcal/M2/day
0e, %
C02, mm/M2/day
E, kcal/M2/day
Chlorophyll, mg/1
E', %
-------
trapped solar energy into organic compounds. Table 1 shows that
Pond 4 was most efficient and Pond 2 was least efficient. Ponds 1
and 3 were approximately equal in their ability to transfer energy
to organic molecules during diel studies in December.
ANNUAL STUDIES
Annual variations in the amount of energy required for production
of the calculated amounts of oxygen are given in Figure 2. In all
ponds, the amount of energy used in the light reactions was greatest
o
in summer, and monthly average reached a peak of 11.1 Einsteins/M /
day in Pond 4 in June. The amount of energy that could be stored in
organic substances, assuming 100 per cent efficiency of transfer, is
estimated by the value of e. This varied between an average of 4.5
kcal/M^/day in Pond 1 in February and an average of 250.5 kcal/M /day
in Pond 4 in June. The primary producers in Pond 4 absorbed a
greater amount of energy than did those in the other ponds. Extremes
of average efficiency, expressed as 0e, were 0.1 per cent in Pond 3
in December and 7.2 per cent in Pond 4 in September. On an annual
basis, Pond 4 was most efficient in the absorption of light energy
(Table 4). The monthly averages of CO absorbed and energy required
for its fixation are given in Figure 3. In each pond, except Pond 2,
there was an increase in the rate of uptake of CO in summer. Cal-
culated CO concentrations in Pond 2 were very low throughout the
2
summer. Between April and November, highest concentration was 0.30
mg/£, compared to 16.9 mg/£ in Pond 4 in July (Table 5).
261
-------
POND 1
POND 2
POND 3
POND 4
CO
CO
N
Figure 2. Mean Monthly Solar Energy Utilized by Light
Reactions in Ponds, 1968.
262
-------
70
50
!\ .
i \ *
\
i \ i
I » *
i » »
! v
30
I
I
I
I
I
I
I
I
f
I
10
0
600
400
POND 1
POND 2
POND 3
POND 4
200
0
N
D
Figure 3. Mean Monthly Values of Rate of Uptake of C02 and
Energy Required for its Fixation, 1968.
263
-------
TABLE 4. MEAN ANNUAL EFFICIENCY OF ABSORPTION OF LIGHT
ENERGY IN PONDS, 1968.
Pond
1
2
3
4
0e,%
0.7
1.0
0.9
3.1
TABLE 5. CONCENTRATIONS OF FREE C02 IN PONDS, 1968.
Pond
1
2
3
4
co2,
Annual average
1.4
0.4
1.8
5.0
mg/1
Daily
0.1 -
0.0 -
0.2 -
range
3.3
1.7
5.3
0.1 -16.9
264
-------
Although measured concentrations of CO were low in Pond 2, it
should not be construed that productivity was low. From 4 June to
6 November, afternoon pH values were between 8.89 and 9.34 (average
9.11). Free CO was seldom found in the water between those dates,
whereas blue-green algae attained their highest numbers during that
time. Clearly a source of CO , other than CO as we measured it,
22
was utilized by primary producers in Pond 2. For discussions of
chemical equilibria that relate to CO in natural waters, see
Schindler (1967) and Lee and Hoadley (1967).
If all CO absorbed was incorporated into organic compounds (E),
2
the amounts of energy fixed were greatest in Pond 4, where an average
o
of 64.0 kcal/M /day was required in August. On a seasonal basis,
energy required for fixation of CO varied widely between ponds and
within any one of them, but was greatest in Pond 4 (Table 6).
Efficiency of transfer of absorbed light energy to organic com-
pounds was greatest in Pond 4 (Table 7). Pond 2 absorbed more light
energy than Ponds 1 and 3, probably because of dense growth of P_.
pectinatus, but was less efficient in formation of new compounds due
to low concentrations of free CO . At the concentrations of free CO
in Pond 2, photosynthesis was rate-limited because of its dependence
upon bicarbonate as a source of carbon (Wright and Mills 1967, Fogg
1968). Thus, it seems probable that rates at which the light re-
actions could proceed were not limited, but photosynthesis was
limited by the slow rate of supply of C02 to the dark reaction.
265
-------
TABLE 6. ENERGY REQUIRED FOR FIXATION OF C0? IN PONDS, 1968.
Pond
1
2
3
4
E, kcal/M2
Annual Mean
4.0
0.8
5.4
33.5
/day
Daily
1.1 -
0.5 -
0.1 -
2.8 -
range
10.4
5.8
14.9
89.9
TABLE 7. EFFICIENCY OF INCORPORATION OF ABSORBED ENERGY
CALCULATED FROM C02 DATA (Ef), 1968.
Pond
1
2
3
4
Annual average, %
15.3
3.2
17.8
26.8
Range, %
4.4 - 25.4
0.7 - 12.9
6.1 - 46.5
11.3 - 37.2
266
-------
Great limitation did not occur in the other ponds, where con-
centrations of free CC>2 were not consistently low. Primary pro-
ducers of Pond 4 absorbed the greatest amount of light energy and
utilized the greatest amount of free C09. They were also most
efficient in transfer of light energy to organic compounds, possibly
because concentrations of free CC>2 were seldom limiting.
There was no demonstrable effect of herbicide on absorption of
solar energy or uptake of CO in Pond 4. Rate of absorption of
solar energy increased in all ponds from March to June, and the
increased rate of uptake of CCU in Pond 4 when benthic plants were
almost gone was accompanied by increases in Ponds 1 and 3.
PHYTOPLANKTON RELATIONSHIPS
3
The average number of filamentous phytoplankters/M /day,
3
average number of blue-green algae/M /day, and the percentage of
blue-green algae in the total for each month are given in Tables
8, 9, 10, and 11. Blue-green algae bloomed in the summer to such
degree that green algae were almost excluded from the ponds.
The photosynthetic quotients also increased during the summer
and were as high as 2.7 during the blue-green algal blooms (Tables
8 - 11). Fogg (1968) stated that (1) the photosynthetic quotient
for young, growing plants and algal cultures is approximately 1.1,
such a value indicating that the primary product of photosynthesis
is carbohydrate, and (2) the photosynthetic quotient for plant tissue
or algal cells in a later phase of growth, where fat accumulation
267
-------
TABLE 8. AVERAGE TOTAL NUMBER OF FILAMENTOUS PHYTOPLANKTERS AND
AVERAGE NUMBER OF BLUE-GREEN ALGAE PER CUBIC METER ON EACH SAMPLING
DAY, AND PHOTOSYNTHETIC QUOTIENT (Qp) IN POND 1, 1968.
Month
J
F
M
A
M
J
J
A
S
0
N
D
Total Number
49,314
37,935
19,867
88,685
73,302
152,290
Lyngbya too
116,443
26,620
16,498
Blue-Green
3,473
2,060
1,867
4,060
15,754
149,447
numerous and matted
57,454
10,914
6,061
% Blue-Green
7.0
5.4
9.3
4.6
21.5
98.1
to count
49.3
40.9
36.7
QP
r
0.6
1.1
1.2
1.0
1.9
2.3
2.3
2.5
2.1
1.1
1.1
1.0
TABLE 9. AVERAGE TOTAL NUMBER OF FILAMENTOUS PHYTOPLANKTERS AND
AVERAGE NUMBER OF BLUE-GREEN ALGAE PER CUBIC METER ON EACH SAMPLING
DAY, AND PHOTOSYNTHETIC QUOTIENT (Q ) IN POND 2, 1968.
Month
J
F
M
A
M
J
J
A
S
0
N
D
Total Number
58,819
70,388
55,710
189,528
761,580
Lyngbya too
111,980
15,088
17,119
3,120
7,930
Blue-Green
17,381
7,524
5,309
17,600
213,532
numerous and matted
105,670
13,700
16,599
2,210
4,680
% Blue-Green
29.6
10.7
9.5
0.9
28.0
to count
94.4
90.8
97.0
70.8
59.0
0
0.9
0.6
0.5
0.3
0.9
1.2
1.4
1.8
1.2
1.1
1.0
0.4
268
-------
TABLE 10. AVERAGE TOTAL NUMBER OF FILAMENTOUS PHYTOPLANKTERS AND
AVERAGE NUMBER OF BLUE-GREEN ALGAE PER CUBIC METER ON EACH SAMPLING
DAY, AND PHOTOSYNTHETIC QUOTIENT (Q ) IN POND 3, 1968.
Month Total Number Blue-Green % Blue-Green
J
F
M
A
M
J
J
A
S
0
N
D
62,129
39,588
85,426
328,338
Lyngbya
48,341
54,379
42,075
28,425
8,525
56,426
36,179
45,526
155,582
too numerous and matted
47,315
52,830
39,865
27,645
8,005
90.8
91.4
53.3
47.4
to count
97.9
97.2
94.7
97.1
93.9
F
1.2
1.5
1.9
2.0
2.2
2.3
2.5
2.6
2.1
1.9
1.9
0.4
TABLE 11. AVERAGE TOTAL NUMBER OF FILAMENTOUS PHYTOPLANKTERS AND
AVERAGE NUMBER OF BLUE-GREEN ALGAE PER CUBIC METER ON EACH SAMPLING
DAY, AND PHOTOSYNTHETIC QUOTIENT (0 ) IN POND 4, 1968.
Month Total Number Blue-Green % Blue-Green Q
J 46,700 13,461 28.8 0.9
F 65,193 18,965 29.1 1.2
M 103,387 21,427 20.7 1.7
A 645,045 232,392 36.0 1.7
M 941,413 457,527 48.6 1.6
J 301,210 301,210 100.0 2.7
J 259,731 256,463 98.7 2.7
A 25,374 25,374 100.0 1.9
S 4,592 4,592 100.0 1.0
0 5,883 5,883 100.0 0.9
N 1,907 1,863 97.7 0.9
D 1,745 1,745 100.0 0.6
269
-------
occurs, can be as high as 3.3. Rabinowitch and Govindjee (1969)
and Fogg (1968) pointed out that as cells mature, the photosynthetic
product changes from protein and carbohydrate to highly reduced sub-
stances, such as fats and "lipidlike" substances. Bassham and Jensen
(1967) stated that as much as 30 per cent of the CO^ taken up by
algae could be incorporated into fats.
Fat production by mature cells is stimulated by light. Spoehr
and Milner (1949) demonstrated that Chlorella pyrenoidosa contained
greatest fat stores when grown under high light intensity, and Lund
(1969) suggested that, unlike other types of algae, blue-green algae
thrive under intense illumination and high temperatures. Walsh (1971)
showed that the amount of short-wave radiation that entered the ponds
was greatest during the blue-green algae blooms.
Fat contains more energy than protein or carbohydrate. According
to Morowitz (1968) their Gibbs free energy values are; protein: 5.50
kcal/g, carbohydrate: 4.10 kcal/g, and fat: 9 ,-30 kcal/g. Increased
photosynthetic quotients and quantities of light energy impinging upon
the ponds in summer suggest that fat production also increased at that
time. Increased production of fat may explain how the energy stored
during CQ~ incorporation increased in the summer, and suggests a
mechanism whereby relatively large amounts of energy can be in-
corporated into eutrophic systems.
270
-------
SUMMARY
The amounts of solar energy absorbed by photosynthetic pro-
cesses in four small ponds in northwestern Florida in 1968, esti-
mated from diel and annual oxygen data, were compared with energy
required for fixation of CO . On an annual basis, energy required
for oxygen evolution and for fixation of C02 varied widely between
ponds and within any one of them. Efficiencies of energy in-
corporation also varied greatly. For example, the mean annual
efficiencies of absorption of light, calculated from oxygen data,
were between 0.7 and 3.1% of incident solar radiation. Average
annual efficiencies of absorbed solar energy incorporation into
organic molecules, calculated from CO. data were between 3.2 and
26.8%.
Blue-green algae were the dominant phytoplankters in all ponds
during most of the year and attained greatest numbers between
April and October. Photosynthetic quotients were highest during
the blue-green algae blooms. It is hypothesized that increased
production of fat may explain why the energy required for CO 2
fixation increased during the summer.
271
-------
REFERENCES
Arnon, D. I., H. Y. Tsujimoto, B. H. McSwain, and R. K. Chain. 1968.
Separation of Two Photochemical Systems of Photosynthesis by
Fractionation of Chloroplasts. In: Comparative Biochemistry
and Biophysics of Photosynthesis (K. Shibata, A. Takamiya,
T. A. Jagendorf, and R. C. Fuller, eds.). University Park
Press, College Station, Pennsylvania, p. 113-132.
Bassham, J. A. 1965. Photosynthesis: The Path of Carbon. In: Plant
Biochemistry (J. Bonner and J. E. Varner, eds.). Academic
Press, New York. p. 875-902.
Bassham, J. A. and R. G. Jensen. 1967- Photosynthesis of Carbon
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Life (A. San Pietro, F. A. Greer, and T. J. Army, eds.). Academic
Press, New York. p. 79-110.
Clayton, R. K. 1965. Molecular Physics in Photosynthesis. Blaisdell
Pub. Co., New York. 205 p.
Fogg, G. E. 1968. Photosynthesis. American Elsevier Publishing
Company, Inc., New York. 116 p.
Goldman, C. R., D. T. Mason, and B. J. B. Wood. 1963. Light Injury
and Inhibition in Antarctic Freshwater Phytoplankton, Limnol.
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Hartman, R. T. and D. L. Brown. 1967. Changes in Internal Atmosphere
of Submersed Vascular Hydrophytes in Relation to Photosynthesis.
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Kahn, L. and F. T. Brezenski. 1967. Determination of Nitrate in
Estuarine Waters: Comparison of a Hydrazine Reduction with a
Brucine Procedure and Modification of a Brucine Procedure.
Environ. Sci. Technol. 1:488-491.
Kalff, J. 1969. A Diel Periodicity in the Optimum Light Intensity
for Maximum Photosynthesis in Natural Phytoplankton Populations.
J. Fish. Res. Bd. Canada 26:463-468.
Kok, B. 1965. Photosynthesis: The Path of Energy. In: Plant
Biochemistry (J. Bonner and J. E. Varner, eds.). Academic Press,
New York. p. 903-960.
Lee, G. F. and A. W. Hoadley. 1967. Biological Activity in Relation
to the Chemical Equilibrium Composition of Natural Waters. In:
Equilibrium Concepts in Natural Water Systems (R. F. Gould, ed.).
American Chemical Society, Washington, D. C. p. 319-338.
272
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Lund, J. W. G. 1969. Phytoplankton. In: Eutrophication: Causes,
Consequences, Correctives. U. S. Nat. Acad. Sci., Washington,
D. C.
McAllister, C. D. 1961. Observations on the Variation of Planktonic
Photosynthesis With Light Intensity, Using Both 02 and Cl4
Methods. Limnol. Oceanogr. 6:483-484.
Morowitz, H. J. 1968. Energy Flow in Biology. Academic Press,
New York. 179 p.
Rabinowitch, E. and Govindjee. 1969. Photosynthesis. John Wiley
and Sons, Inc., New York. 273 p.
Rainwater, F. H. and L. L. Thatcher. 1960. Methods for Collection
and Analysis of Water Samples. Geological Survey Water-Supply
Paper 1454. U. S. Government Printing Office, Washington, D. C.
301 p.
Ryther, J. H. 1956. Photosynthesis in the Ocean as a Function of
Light Intensity. Limnol. Oceanogr. 1:61-70.
Schindler, P. W. 1967. Heterogeneous Equilibria Involving Oxides,
Hydroxides, Carbonates, and Hydroxide Carbonates. In: Equilibrium
Concepts in Natural Water Systems (R. F. Gould, ed.). American
Chemical Society, Washington, D. C. p. 196-221.
Spoehr, H. A. and H. W. Milner. 1949. The Chemical Composition of
Chlorella: Effect of Environmental Conditions. Plant Physiol.
24:120-149.
Steemann Nielsen, E. and V. K. Hansen. 1961. Influence of Surface
Illumination on Plankton Photosynthesis in Danish Waters (56° N)
Throughout the Year. Physiol. Plant. 14:595-613.
Strickland, J. D. H. 1965. Production of Organic Matter in the
Primary Stages of the Marine Food Chain. In: Chemical
Oceanography, Vol. 1 (J. P- Riley and G. Skirrow, eds.).
Academic Press, New York. p. 478-610.
Strickland, J. D. H. and T. R. Parsons. 1965. A Manual of Sea Water
Analysis, 2nd Ed. Fish. Res. Board Canada Bull. No. 125. 203 p.
Verduin, J. 1956. Energy Fixation and Utilization by Natural Communities
in Western Lake Erie. Ecology 37:40-50.
Walsh, G. E. 1971. Energy Budgets in Four Ponds in Northwestern
Florida. Ecology 52:298-304.
273
-------
Walsh, G. E. and P- T. Heitmuller. 1971. Uptake and Effects of
Dichlobenil in a Small Pond. Bull. Environ. Contain. Toxicol.
6:279-288.
Welch, H. E. 1968. Use of Modified Diurnal Curves for the Measure-
ment of Metabolism in Standing Water. Limnol. Oceanogr. 13:
679-687.
Wright, J. C. and I. K. Mills. 1967- Productivity Studies on the
Madison River, Yellowstone National Park. Limnol. Oceanogr.
12:568-577.
274
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HETEROINHIBITION AS A FACTOR IN ANABAENA FLOS-AQUAE
WATERBLOOM PRODUCTION
Llewellyn R. Williams
INTRODUCTION
Late-summer, warm water conditions often give rise to waterblooms of
blue-green algae in New Jersey ponds and lakes. Anabaena flos-aquae is
frequently the major constituent of these blooms, often appearing, at
least briefly, in codominance with Microcystis aeruginosa. That such
blooms are an annual occurrence in some waters, while appearing only
occasionally in similar waters nearby, suggests that we are dealing not
with a single causative factor, but rather with an interaction of physical
and chemical conditions leading to bloom production. Attempts to generalize
the waterbloom phenomenon as a single cause-response relationship have been
unsuccessful.
I would like to suggest the following mechanism for the production
of waterblooms of Anabaena flos-aquae, based upon my studies in the
laboratory, as well as natural system studies carried out in Lake Nelson:
1. Trigger for initial growth is the response of Anabaena
to the advance of warm surface waters deeper into the
water column and/or the direct benthic warming from
penetrating solar radiation. A filament grows from
the akinete, or overwintering cell of Anabaena, lying
on the bottomj the early filament competes successfully
in late-summer waters.
2. A. flos-aquae secretes into the water a heteroinhibitory
compound; this active substance inhibits the growth of a
broad spectrum of algal species in the community.
275
-------
3. Upon death and lysis of the inhibited cells, inorganic
and organic compounds are liberated into the system,
providing stimulation to the advancing Anabaena popu-
lation through nutrient or growth-factor effects.
4. The resultant high concentration of Anabaena at the
surface, largely the result of extensive gas-filled
pseudovacuole formation, is recognized as a waterbloora;
it is composed of senescent Anabaena flos-aquae fila-
ments .
MATERIALS AND METHODS
Laboratory Studies
Anabaena flos-aquae, obtained as axenic culture No. 1444 (U. of Indiana),
was cultured in liter batches under constant light of 200-300 ft. candles and
constant agitation with magnetic stirring. A mineral medium high in available
nitrogen, phosphorus and sodium was used throughout:
Chemical Compound Concentration (mg/1)
NaN03 2000
MgS04.7H20 25
K2HP04 250
NH4C1 50
CaCl2.2H20 50
The one-liter cultures appeared to peak in density at about 21 days and
remained stable for several weeks before degenerating. When bubbled air
agitation was substituted for magnetic stirring, early population declines
were noted. Standard aseptic techniques were used throughout the study;
cultures which failed to produce contaminant growth on glucose-supplemented
or Bacto-Agar media were considered axenic.
276
-------
Mineral medium in which Anabaena has been grown and removed by glass-
fiber filtration is the basis for test growth media. These cell-free
"conditioned" media were derived from cultures in log growth phase, usually
7-10 days after inoculation. No microscopic evidence of cell lysis was
npted at this stage. Table 1 presents the test media and algae used in the
various laboratory studies, noting any treatments or extraction procedures
modifying the basic "conditioned" medium.
A simple CHClg-MeOH extraction procedure was used to remove lipid
elements from conditioned medium for subsequent GLC analysis. The remain-
ing lipid-free medium, cleared of residual solvent by flash evaporation,
is called "extracted conditioned" medium; it is subsequently handled the
same as the conditioned medium against which it is compared.
Scenedesmus dimorpha was the primary test organism used to evaluate
the toxicity of Anabaena excretion products, Scenedesmus spp. are strong
summer dominants in my study pond. Other test algae representative of pre-
bloom genera included Pandorina morum, Euglena gracilis, Platydorina
caudata and a Chlorococcum sp.
Inhibition tests were run in rotating culture apparatus which I designed
for this purpose (Figure 1). The rotating turntable holds sixteen 1" diameter
spectrophotometer tubes (Bausch and Lomb) which serve as culture tubes. The
position and rotation of the tubes assure continuous and uniform illumination
of approximately 300 ft. candles, depending upon the age of the circular
fluorescent tube. To each tube is added 15 ml of test culture medium, a
small magnetic stir-bar, and a cotton plug. The magnetic stir-bars provide
periodic agitation in each culture tube as it passes over a magnetic stirring
unit. The culture tubes with medium, stir-bar, and cottonplug in place are
autoclaved as a unit prior to inoculation with test algae.
277
-------
Table 1. CONDITIONS OF LABORATORY INHIBITION STUDIES
rv>
^i
oo
9a
9b
9c
9d
10
11
12
Test Organism
S . dirnorpha
S. dimorpha
S. dimorpha
S. dimorpha
S, dimorpha
S. dimorpha
S. dimorpha
S. dimorpha
E. gracilis
Chlorococcum sp.
P. morum
P. caudata
Lake Nelson'
phytoplankton
Lake Nelson
phytoplankton
S. dimorpha
A. Flos-aquae
Test Growth Medium
(a) CHClo-MeOH extracted conditioned (b) Conditioned
(a) P.M.M. (b) P.M.M. with CHCl-j-MeOH extract added
(a) CIICU-MeOH extracted conditioned (b) as (a) but with
extract returned
(a) CIlClo-MeOH extracted conditioned (b) MeOH treated conditioned
(c) conditioned
(a) CHCLo-MeOH extracted conditioned (b) Conditioned (Autoclaved)
(c) Conditioned (Sterile-filered)
(a) P.M.M. (b) P.M.M. plus Oleic acid (c) P.M.M. plus Linoleic acid
(d) P.M.M. plus Linolenic acid
(a) CHC1 extracted conditioned (b) Diethyl ether extracted conditioned
(c) Hexane extracted conditioned (d) Conditioned
(a) Hexane extracted conditioned (b) Hexane treated conditioned
(a) CHClo-MeOH extracted conditioned (b) Conditioned
(a) CHCl^-MeOH extracted conditioned plus early-bloom extract (b) CHC1.,-
MeOH extracted conditioned plus late-bloom extract
(a) CHCl,,-MeOH extracted conditioned (b) as (a) but with extract
returned
(a) Early-bloom lake water (Mineral supplemented) (b) Late-bloom lake
water (Mineral supplemented)
Note: P.M.M. = Pure mineral medium
-------
r\>
*>i
vo
Cotton Plug
Fluorescent Tube
Turntable
Magnetic Stir-Bar
Rubber Rim Drive
Motor
Culture Tube
Magnetic Stirrer
Figure 1. Rotating culture apparatus
-------
The length of an experimental run varied from three days to as long as a
week. At the termination of each growth study all culture tubes were vigor-
ously agitated. A 1 ml aliquot was removed from each, diluted to 10 ml with
distilled water and run through a 24 mm diameter Millipore filter (0.45u, HAWP)
and dried. The filters were cleared with cedar oil and the test organisms
enumerated by the method of McNabb (1960), utilizing an ocular grid as a
counting aid against the plain Millipore filters.
Natural System Studies
Lake Nelson, in Middlesex Co., New Jersey, which regularly exhibits
waterblooms of blue-green algae, was selected for my field study. This
eutrophic impoundment of about 15 acres surface area has a maximum depth
of about 2 meters.
Phytoplankton and zooplankton composition were monitored on a daily
basis through two consecutive waterblooms. Air and water temperatures
were similarly monitored and a weather log was maintained for this period.
Total plankton samples were preserved with acid Lugol's solution and
enumerated at 100X magnification in a counting chamber. Net plankton
samples were usually counted fresh. Plankton and filtered water samples
were extracted with CHClo-MeOH for fatty acid analysis. Gas chroraato-
graphic analysis of extracts was performed on a Micro-Tek MT-220 unit
equipped with flame ionization detectors, polar and non-polar columns.
Quantitative estimates were obtained with a Disc Integrator.
RESULTS
JLaboratory Studies
An initial comparison between Scenedesmus growth in conditioned
medium and CHCl3MeOH extracted conditioned medium showed highly signifi-
cant differences in growth between the two media; less growth is noted in
the conditioned medium (Table 2a). In an attempt to demonstrate inhibitory
280
-------
TABLE 2 - SUMMARY OF LABORATORY RESULTS
RATIONALE
Is the inhibitor lipid in nature?
Tested by lipid extraction of
conditioned medium, coinparis ion
with test organism growth in
unextracted conditioned medium.
2a.
Conditioned
CKCl3-MeOH
extracted
(1)
(2)
(3)
TEST RESULTS
Conditioned medium inhibi-
tory .
Lipid extraction removes
inhibition.
Excludes mineral nutrient
inequity effects.
P>.99
PO
CO
Lipid extraction removed inhibition.
Tested extract per se for activity.
(1) Added to pure mineral medium
(2) Returned to extracted condi-
tioned medium.
2c.
Minera 1
medium
Minera 1
mc-d ium
+ extract
Extracted
medium
Extract
returned
Extract does not retain potency,
No difference; suggests that
inhibitor is either unstable
lipid or nonlipid.
No difference
-------
TABLE 2 (CONTINUED). SUMMARY OF LABORATORY RESULTS
IX)
co
Condit toned
If not lipid, is it proteinaceous?
Tested by:
(1) jMethanolic treatment.
(2) Heat treatment (autoclaving)
of conditioned medium. 2e.
MeOH treated
cond itioned
CHC13-MeOH
extracted
CMCIT-MeOH
extracted
Conditioned
filtered
Conditioned
autoclaving
Both conditioned media equally
inhibitory.
Tested against extracted: P> .95
Protein ruled out as inhibitor
Both conditioned media equally
inhibitory.
Tested against extracted: P>.99
Is the inhibitor an unsaturated
fatty acid unstable to extraction
and/or subsequent handling?
2f.
Mineral
medium
Oleic Acid
added
No difference.
No activity
against test
alga by any
fatty acids
tested.
-------
TABLE 2 (CONTINUED).SUMMARY OF LABORATORY RESULTS
Tested by addition of dominant
unsaturated fatty acids at levels
occurring in Anabaena culture.
2f.
Linoloic Acid
added
Linolenic Acid
added
No difference
No activity
against test
alga by any
fatty acids
tested.
00
I'f the solvent extraction is
tivating the inhibitor, would
other organic solvents do the
iible key to nature of the
compound, assess growth in
conditioned media extracted
with solvents of different
polarity.
CIIC1 extracted
Ether extracted
Ilexane extracted
Conditioned
Growth im
CHCl.^ Ether) Hexane)> Conditioned
P>.95 P>.95 P>.95
Increased polarity increases
removal of inhibition.
-------
TABLE 2 (CONTINUED).SUMMARY OF LABORATORY RESULTS
It extraction per se inactivates
inhibitor, will "treatment" with
organic solvent prove as effec-
tive as physical separation of
the phases?
Tested by comparing hexane ex-
traction with hexane treatment.
2h.
Hexane
extracted
Hexane
treated
No difference.
Consistent with inactivation at
the water:organic solvent inter-
face.
oo
What is the response of other
test algae to conditioned medium?
Tested jguqJLena, Chlorococcum,
Pandorina and Platydorina for
growth in conditioned and ex-
tracted conditioned media.
CHCl3-MeOH
extracted
Conditioned
r
All inhibited by conditioned
medium.
Morphological and cytological
differences noted.
(Figure 2; Plate 1-4)
Do organic compounds in J
natural waters during Anabaena
water blooms affect phyto-
plankton growth?
Growth in the presence and
absence of early bloom extract
is compared.
Lake
Nelson
Phyto-
P lank ton
Early bloom
extract
added
Extracted
Lake water
r» ^nabaena the only alga sti-
mulated by early bloom extract
> Anacystis, Pandorina favored
without extract.
* No difference for seven other
genera.
(Figure 3)
-------
TABLE 2 (CONTINUED).SUMMARY OF LABORATORY RESULTS
Hov; do the same phytoplankters
respond to lab Anabaena ex-
tract?
Tested by returning extract to
extracted medium, assaying
activity.
2k.
Lake
Nelson
Phyto-
Plankton
CHC 13
Me OH
extracted
Extract
returned
1
Anabaena not stimulated by
its own extract.
Micractinium better without
extract .
Pandorina. Eudorina . Scene-
desmus
OD
en
ter with extract.
(Same do well at end of water
bloom)
(Figure 4)
What is effect of filtered lake
water from a waterbloom on the
growth of Anabaena and scene-
dc-STilUS?
Tested by co-growing in early bloom
and late bloom water (both mineral
supplemented),
21.
Early
bloom
water
Late
bloom
water
Growth in;
Early bloom^ for Anabaena
Late bloom) for Scenedesmus
Anabaena growth better in
early than late bloom water,
while Scenedesmus is favored
in late bloom water, consis-
tent with natural system find-
ings .
(Figure 5)
-------
activity believed to be contained in CHCl^-MeOH extract from the conditioned
medium, I added the extract to pure mineral medium, comparing Scenedesmus
growth to mineral medium without extract supplement (Table 2b). No
difference was noted in the growth in these two media. Upon returning
CHClo-MeOH extract to the conditioned medium from which it had been removed,
no difference in growth of Scenedesmus could be attributed to extract
activity when compared with growth in extracted conditioned medium (Table
2c). Removal of extract removed activity, but return did not restore the
inhibitory properties to either medium.
To screen proteinaceous materials from consideration as the source of
activity in the conditioned medium, I compared conditioned medium which was
autoclaved, with that which was sterile-filtered; autoclaving had no effect
upon the activity present in the conditioned medium (Table 2e). Both
conditioned media showed significant reduction in growth when compared to
the extracted control. In subsequent studies activity was retained with
up to 60 minutes of autoclaving, the longest period evaluated. Methanol
treatment had no effect upon activity (Table 2d). Linoleic, oleic, and
linolenic acids were tested for inhibition of Scenedesmus growth at levels
corresponding to their occurrence in conditioned medium. No activity was
noted (Table 2f).
The action of three different solvent extractions in effecting re-
moval of activity from conditioned medium are compared in Table 2g. All
solvents tested remove sufficient activity to allow growth significantly
higher than conditioned medium. The activity is removed more completely
in proceeding from less polar to more polar solvent extraction. While
substantial reduction in activity is provided by ether or chloroform
extractions, inhibition appears to be more completely removed with the
highly polar CHCl^-MeOII solvent system noted earlier.
286
-------
Table 2h shows results of a test comparing the effect of hexane ex-
traction with hexane treatment--hexane is added, then removed by evapor-
ation. No difference was noted between the abilities of these media to
support the growth of Scenedesmus, even though hexane extraction had been
shown to remove activity.
Representatives of 4 genera found in Lake Nelson in July-August 1970,
were substituted for Scenedesmus to serve as indicators of the inhibitory
activity of the conditioned medium. Pandorina and Platydorina were complete-
ly eliminated in tubes containing conditioned medium. Euglena and Chloro-
coccum were present in reduced numbers, but showed evidence of cytological
and morphological changes in-conditioned medium relative to the extracted
controls. A loss of flagella, vacuolate appearance and spherical shape were
noted in Euglena grown in conditioned medium. The absence of actively
dividing cells was seen"in Chlorococcum grown in conditioned medium. In
addition, individual cells showed increased density consistent with continued
"assimilation without division." (See Figure 2)
Two parallel laboratory studies were performed in an effort to determine
the response of natural Lake Nelson phytoplankton to CHCl~-MeOH extracts of
(1) early-bloom lake water, and (2) laboratory conditioned medium. In
comparing phytoplankton growth in the presence or absence of early bloom lake
water extract (Figure 3), note that only Anabaena grew better with addition
of the extract than without. The difference was highly significant (P>.99).
No difference in growth was noted for seven other genera. However, Anacystis
and Pandorina growth was greater without the lake water extract.
The extract from laboratory conditioned medium did not stimulate
Anabaena growth, Micractinium grew better without the added extract;
Pandorina, Eudorina, Scenedesmus and Chlorogonium grew better in the pres-
ence of extract (Figure 4). Interestingly, in the natural system, the last
287
-------
60-i
50 -4
ro
co
co
a>
u
c
o
20-^
t-9.L /
df=6
df=o
CONTROL
CONDITIONED
nnnr
<
i-
Z
u
<
1
Pandorina Platydorina Chlorococcum Euglena
morum caudata gracilis
Figure 2.
Inhibition Study 9Comparative growth of four representative genera of algae in
conditioned medium and extracted conditioned (control) medium. Values represent
the average of mean cell counts per field for 4 tubes in each treatment combination,
-------
Co
i-D
u
z
20-
E ARL Y- B LOOM E X T R A C T
Wl T HOUT EXTRACT
t=7.92**
df = 14
Figure 3. Study 10Comparison of growth of natural Lake Nelson phytoplankton in extracted
pond water with and without early bloom lipid extract. Values are 8-tube averages
of mean cell counts per field.
-------
ro
WD
O
10-
9-
8-
0 7-
_rf
t*t
6-
oc
u«
Q.
5-
t=2.496'
df = 13
t=5.158**
df = 14
~--2. 049
.95
t=1.858
df = 13
. QO
-------
four algae showed moderate to strong growth responses during decline of the
second Anabaena waterbloom in 1970 suggesting stimulation by lipid,materials
released from Anabaena.
To determine whether dissolved organics in the natural lake water would
mediate competition between major algal constituents, I ran co-growth studies
with Anabaena and Scendesmus using waters from early-bloom and late-bloom
lake conditions. The results of these experiments, followed on a daily
basis, are shown in Figure 5. Note that Scenedesmus grown in early-bloom
wate-r varies little from the initial inoculum concentration, while the
Anabaena is "favored" under these conditions. Mineral supplements to the
lake waters minimized any role of inorganic ion depletion in mediating the
differential response noted.
Natural System Observations
The growth and early decline of constituent phytoplankton populations
is illustrated in Figure 6 which depicts the mean cell counts of all other
species of algae contrasted with the growth curve of Anabaena flos-aquae
in Lake Nelson. Response of zooplankton during the bloom is shown in Figure 7,
Percent composition of all identified plankton populations, derived by direct
cell counts of daily net plankton samples, is included in the Appendix.
Comparison of individual phytoplankton population responses to the growth
of Anabaena is also included in the Appendix.
Two major weather disturbances - heavy, prolonged thunderstorms -
significantly altered the phytoplankton succession pattern during the
sampling period. The first of these, on August!, essentially removed what
remained of the first Anabaena bloom. Wind effects can be noted in the
daily counts; precipitous drops in Anabaena concentration followed periods
during which strong west wind activity piled surface algae at the opposite
291
-------
5 0
4 0
ANABAENA
EARLY BL OOM
LATE BLOOM
SCENEDES MUS
EARLY BLOOM
LATE BLOOM
3 0-
QC
LU
Q.
1 0-
Figure 5. Study 12Comparison of Anabaena flos-aquae and
Scenedesmus dimorpha grown together in early-bloom
(8 Aug.) and late-bloom (25 Aug.) filtered pond water
(Lake Nelson, 1970). Points represent 8-tube averages
of mean coenobium or filament counts per field. Counts
of membrane filter-trapped algal samples were made daily,
Scenedesmus
Anabaena
t=0.278
df=14
t=1.248
df=14
t=4.628*
df=14
t-1.136
df=13
t=4.588*
df=13
t=6.570*
df=12
292
-------
10,000
ANABAENA
OT HER PHYT 0 PLA NKT 0 N
10
10
20
25
AUGUST
30
5
SEPT
Figure 6. Mean variation of phytoplankton components
during an Anabaena flos-aquae waterbloom in Lake
Nelson, 197CK
293
-------
10,000-j
1,000-
100
Ana baena
F i I i n i a
Other Rotiferi
Immature Cope-
pod t
20
AUGUST
Figure 7. Variation of zooplankton components during an
Anabaena flos-aquae waterbloom in Lake Nelson. Counts
were made from plankton net concentrates (1970).
294
-------
end of the pond. High concentrations noted on July 19 followed several
days of easterly breezes piling algae against the dam at my sampling site.
Heavy concentrations spilling over the dam resulted in a fish-kill in the
receiving brook July 21.
Attempts to culture Anabaena or Microcystis from surface waters during
waterbloom formation were singularly unsuccessful. Growth was obtained only
with Anabaena from the shallow waters of the inlet brook to Lake Nelson.
In sampling these waters the bottom sediments were disturbed and silt
entered the samples.
Gas Chromatbgraphy
I analyzed the free fatty acids of daily cell extractions by gas
chromatography through the development of an Anabaena waterbloom. No
attempt was made to measure the actual quantity of fatty acids p$r unit
of raw sample; amounts are expressed as relative percentages of the fatty
acids analyzed. GLC analyses of extracts from filtered lake water are
included where available (Table 3). Odd-chain fatty acids are plotted
separately in Figure 8.
DISCUSSION
Let us examine the evidence in support of each step in the proposed
mechanism for Anabaena flos-aquae water-bloom formation.
Initiation of filament growth from akinetes deep in the
water column is triggered by the advance of warm surface
waters and/or the direct benthic warming from penetrating
solar radiation. The early filament competes successfully
in mid to late summer waters.
Healthy laboratory cultures of Anabaena regularly settled to the
bottom of the flask upon removal of agitation. Cultures which have
degenerated show some tendency to surface; this is more apparent with
295
-------
TABLE 3
% COMPOSITION OF FATTY ACIDS FROM LABORATORY AND LAKE SOURCES
DATE CARB.Q£L NUMBER
7/29
7/30
8/1
8/2
8/3
8/4
8/5
8/10
8/13
8/25
7/29
£^12
11.5
(8.0)
9.3
15.8
(1.8)
11.2
(3.7)
17.4
3.4
2.2
(4.8)
5.2
(11.0)
1.8
(5.4)
(11.4)
(2.1)
C-13.
LAKE
18.0
(5.3)
4.6
5.3
(1.8)
9.1
(9.1)
1.6
0.8
0.7
(20.5)
2.1
(9.0)
0.9
(5.3)
(15.3)
BUDD
(10.6)
C-14
NELSON
11.4
(11.3)
7.2
4.2
(4.2)
9.7
(4.3)
1.6
1.9
7.0
(16.9)
4.3
(9.5)
1.3
(5.5)
(9.3)
LAKE,
(8.8)
LABORATORY CULTURE OF
12.0
(7.0)
3.5
(1.8)
11.1
(6.1)
C--15
, 1970
9.8
(7.5)
2.0
0.5
(4.2)
1.5
(5.5)
0.8
1.1
2.4
(Tr)
1.8
(1.2)
1.3
(9.2)
(1.2)
1970
(8.8)
C-16
18.2
(54.7)
33.4
10.5
(28.0)
39.6
(18.8)
30.2
47.0
52.9
(22.9)
43.0
(31.6)
53.5
(32.0)
(20.3)
(34.2)
C-17
21.4
(10.8)
5.1
Tr
(22.9)
1.5
(14.4)
0.8
1.1
1.9
(8.2)
2.2
(10.6)
1.2
(18.9)
(7.6)
(7.4)
C-18
5.1
(3.0)
30.9
63.2
(36.4)
27.2
(43.2)
54.9
43.3
33.3
(26.7)
38.7
(27.0)
39.0
(23.8)
(17.4)
(27.9)
A. FLOS -AQUAE
3.2
(0.5)
36.5
(16.5)
3.5
(3.3)
29.1
(65.7)
Values represent gas chromatographic analysis of 12 - 18-
carbon fatty acid methyl esters from CKCl^-MeOH extracts
of cells (open) and cell-free (in brackets).
296
-------
70
C- 13
C- 15
C- 17
Z
o
o
a.
10
5-
r r
29
JULY
5
A!
G U ST
Figure 8. Variation in the percent composition of the total
fatty acids analyzed represented by the odd-chain
fatty acids C-13, C-15, and C-17. Values were obtained
by gas chromatographic analyses of daily cell extracts
taken through the onset of an Anabaena flos-aquae water-
bloom.
297
-------
pseudovacuole formation noted in natural populations. That surfacing
filaments are not healthy, but rather degenerate and incapable of normal
growth when placed in culture medium, is strongly suggested by culture
studies of waterbloom samples taken from seven lakes and ponds in New
Jersey from 1968 to 1970. The only filaments to grow successfully were
obtained at the inlet to Lake Nelson; no surface film of Anabaena was
present at this site. The sample contained some silt and benthic diatoms
as-well as viable Anabaena filaments.
I have found in my culture work that Anabaena flos-aquae is inhibited
by light in excess of 400 foot-candles; under these conditions the filaments
become chlorotic; rapid degeneration soon follows if the cultures are
not removed to lower light conditions. Supraoptimal light and temperature
in the surface waters may play a significant role in the senescence of
cells in the surface film (Hutchinson, 1967).
In the Northeast blue-green blooms occur at a time when warm surface
waters extend deep in the water column. In the summer of 1970 bloom
reports began to come in from central and northern New Jersey weeks prior
to their anticipated appearance. The surface water temperature of some
of these lakes had reached 35 C after a sustained period of high insolation
and high air temperatures. Optimum temperatures reported for the growth
of Anabaena are, with few exceptions, far below the surface temperature
noted for these lakes, but more closely approximate hypolimnetic temper-
atures. As warm summer waters approach benthic muds, large amounts of
phosphate and sometimes nitrate and ammonia may be released into the
water from the sediments (Hutchinson, 1967) .
It is not at all unusual for blue-green algal waterblooms to be first
"discovered" when surface filming has already begun. However, it is
hardly possible to conceive of this sudden appearance of surface film
298
-------
based upon the reproductive rate for surface-dwelling filaments, subjected
to unfavorable conditions of light and temperature. Accumulation at the
surface, of filaments rising through the water column, can account for
the rapidity of surface-film development as well as the "greening" of a
lake noted prior to surface-film development in carefully watched water-
blooms. If, as has been suggested, akinetes are the source of repopulation
of Anabaena for the subsequent year, why can they not be found in surface
waters prior to Anabaena waterbloom formation, if not for the fact that
they are indeed resting on or in the sediments of the lake bottom.
The heterocyst-containing Anabaena filament has* a competitive "edge"
in late-summer waters through the mechanism of nitrogen-fixation.
Fitzgerald(1970) found A. flos-aquae to fix N actively when other sources
2
of nitrogen were low. The waters classically hold relatively low nitrate
concentrations during this period, while providing phosphate in adequate
amounts to support phytoplankton growth. Dependence upon the oxidized
forms of nitrogen may limit many phytoplankters under these conditions
The autumnal pulse, provided by convection-borne nutrients when surface
waters cool, is largely a result of nitrate replenishment in the upper
waters. In studies of Anabaena cylindrica, Fogg (1949). found heterocyst
production to be greater at low light intensities. He found also that
some free sugars and organic acids stimulate the formation of heterocysts
in this species. The presence of nitrate, ammonia or dissolved amina-
acid-N tended to retard or inhibit heterocyst production.
Luxury uptake of phosphorus in blue-green algae is well-documented
and a reproductive explosion can continue for many divisions after
depletion of the phosphorus from surrounding waters. The dynamics of
sediment-water phosphorus exchange would further tend to .favor the growth
of early Anabaena populations low in the water coulumn, or early filament
299
-------
production from akinetes on the sediment surface. Fitzgerald (1970)
reports that the presence of iron can alter phosphorus solubilities
shifting the equilibrium to favor algal growth at pH 9-9.5.
Anabaena flos-aquae secretes into the water a heteroinhibitory
compound; this active substance inhibits the growth of a broad
spectrum of algal species in the community.
It was important initially, to establish that activity against
other algal forms was a result of Anabjiena-produced compounds excreted
during active growth of the culture. Periodic spot-checks on bulk liquid
cultures of the blue-green never revealed more than low-level background
contamination when healthy growing cultures were examined under oil
immersion. Upon degeneration of some cultures, I have noted strong
increases in bacterial flora. Laboratory observations indicated that
healthy A. flos-aquae cultures tend to keep bacterial contaminants at
very low levels.
The conditioned medium, taken from the logarithmic growth phase with
essentially no cell lysis, is biologically active, as the results of ray
inhibition studies consistently show, suggesting that release of active
materials by cell breakdown is unnecessary. Evidence from co-growth
experiments in early- and late-bloom water further suggests that much of
the biological activity is lost late in the bloom when cell death and
decomposition is high. Natural system studies in Lake Nelson show
dramatic fall-off of several phytoplankton species prior to maximum
concentration of Anabaena in the surface waters, but late-bloom recovery,
in some instances, was just as dramatic, even though high numbers of
Anabaena filaments remained. The long-terra toxicity reported by Proctor
(1957a) was not evident, although there was a shift noted in phytoplankton
dominants, in comparing pre-bloom and post-bloom communities. Fogg (1952)
300
-------
found extracellular products of Anabaena to be liberated not by autolysis,
but as an "invariable concomitant of growth". It was noted that Anabaena -
conditioned medium was consistently inhibitory to all representative forms
tested,relative to growth in extracted control medium. Microscopic
examination of the algal forms suggested that division had been inhibited
in Chlorococcum and Euglena gracilis.
In studies with natural phytoplankton, I found A. 'flos-aquae -to be
stimulated by the return of early-bloom lipid extract; no other algae
were stimulated significantly.
The extract per se, when reintroduced to the extracted medium or to
pure mineral medium, did not result in restoration of inhibitory activity.
This has been a consistent finding, spurring attempts to explain the
inactivation of the biologically active compound upon extraction. A
comparison was made between hexane extraction, shown to remove a signif-
icant amount of biological activity and hexane "treatment", in which
hexane was added, shaken up with the conditioned medium, and volatized
rather than physically separated. That no difference was noted in the
inhibitory properties of these media strongly suggests that the inactivation
is accomplished at the interface of the aqueous-organic solvent compartments,
rather than in subsequent handling of the extract. The strong possibility
of an active chemical complex between hydrophyllic and lipophyllic
moieties which separate into inactive substituents at the slovent-water
interface was suggested by Litchfield (personal communication).
The inverse relationship of zooplankton response to Anabaena growth,
suggested by my data, may reflect actual damage to the zooplankton
population, or perhaps an avoidance behavior on the part of these motile
forms to the products of surface concentrations of Anabaena. Such
behavior has been suggested in studies by Hardy (1936) and Vance (1965).
301
-------
If the inverse relationship of zooplankton to Anabaena should be a
consistent finding, it could be recognized as a further competitive
advantage in the ascent of Anabaena to dominance.
Upon the death and lysis of inhibited cells, stimulation is
provided to Anabaena flos-aquae by the nutrients and/or growth
factors so liberated.
Co-growth studies showed enhanced growth of A. flos-aquae in lake
water from early-bloom conditions, when compared with late-bloom water.
The early-bloom water, obtained on August 8, represents a period of strong
Anabaena ascendency, while most other species were declining. August 25,
the date of the late-bloom water, represents a period of Anabaena decline,
coupled with the recovery of other algal populations. The possibility
of the relative successes of Anabaena in the co-growth studies being
related to autoinhibition by self-produced compounds in the late-bloom
waters was not overlooked. That Anabaena is stimulated by the presence
of compounds in the lipid extract of early-bloom water was confirmed
by comparing growth in extracted early-bloom lake water to that occurring
in the same waters with the return of the lipid extract. In the absence
of extractable lipid, Anabaena growth was modest at best. Self-produced
lipid materials failed to stimulate Anabaena growth; the compound(s)
stimulatory to Anabaena are produced from other sources.
Anabaena can use external organic compounds in synthesis, utilizing
fructose and sucrose with high efficiency in formation of extracellular
polysaccharides (Moore and Tischer, 1965). These authors demonstrated
the presence of the enzymes, fructose diphosphate phosphatase, and fructose
diphosphate aldolase. Anabaena readily photoassimilates acetate (Hoare,
Hoare, and Moore, 1967), fatty acids--palmitic, stearic, and oleic--
to be incorporated into lipids (Nichols, Harris, and James, 1965).
Nichols, _et al., found acetate to be largely incorporated into phosphatidyl
302
-------
glycerol, the major phospholipid of blue-green algae. In each of the
studies mentioned uptake and destination of C "*- labeled sugars and organic
acids were determined by radioassay of chromatographically separated
products. Fogg (1952) found that Anabaena cylindrica continued to
assimilate organic compounds for some time in the dark, finding also
that organic acids and sugars stimulated N fixation. Kuenzler (1970)
found C -fructose to be taken up by blue-green algae at low CO tension.
Lange (1970) found a three-fold increase in Anabaena circinalis growth
with the addition of sucrose to the mineral medium. These algal cultures
were not axenic, and Lange suggests the role of bacteria in metabolizing
the sucrose to CO , the latter representing the primary algal nutrient.
2
He does not rule out direct utilization of sucrose by the Anabaena.
Jakob (1961) found greatly increased growth of Nostoe in Chu medium with
addition of glucose; "muscarine" activity persisted at a reduced level.
That Anabaena can utilize extracellular organic compounds is rather
well-established; whether or not the algal community provides such a
carbon source is another question. While it can be argued that there
are sources for dissolved organic compounds in natural waters other than
phytoplankton production, it is assumed by some researchers that the bulk
of dissolved organics are phytoplankton-prpduced (Domogalla, et al.,
1925; Hellebust, 1967). Fogg (1962) suggested that only a small per^-
centage of these organics are liberated by active, growing cells, the
major part arising from the decomposition of phytoplankton. Lefevre, et al.,
(1952) reports rapid degeneration of some algal species subjected to
blue-green algae-produced inhibitors with cell lysis and consequent
303
-------
release of organic compounds. Fitzgerald (1970), working with A.
flos-aquae (Ind. 1444), found the best source of nutrients in his study
to be killed algal cells, live algae tending to hold onto their nutrients
and providing less growth than the control mineral medium. Meeuse (1962)
reports the reserve products of green algae to be true starches, sucrose
and glucose. As most of the pre-bloom phytoplankton represents this
group, sudden release of such reserves might certainly be expected to
enhance Anabaena growth.
In view of the high efficiency with which some simple sugars and
organic acids are reportedly assimilated, their role as potential carbon
sources for Anabaena must not be underestimated, particularly under
conditions of low CCL availability. That such conditions do occur in
nature has been strongly stated by Kuentzel (1969). He has suggested
that a mutualistic relationship exists between -waterbloom-producing
blue-green algae and bacteria, whereby the bacteria obtain oxygen and
organic compounds (mucilages) in return for CO- utilized by the algae.
Kuentzel further suggests that high BOD in natural waters may stimulate
blue-green waterblooms by boosting the bacterial populations. Analyses
of GLC peaks from cell extracts through the onset of an Anabaena
bloom do not suggest high bacterial activity.
The presence of a high percentage of odd-chain fatty acids
was noted in samples from the late degenerative phase of the first of two
Anabaena waterblooms. Similar high odd-chain percentages were noted
several weeks into a major A. flos-aquae bloom on Budd Lake. That such
odd-chain fatty acids are largely bacterial in origin has been documented
304
-------
(Parker, Van Baalen and Maurer, 1967). This suggests that the late-
bloom degeneration is accompanied by high bacterial populations (not at
all unexpected). However, if we extend the use of odd-chain fatty acids
as a rough index of bacterial populations, we do not note evidence of
bacterial blooms preceding the second Anabaena bloom, nor paralleling
the bloom in significant concentration. Levels of odd-chain acids
during the initiation of the waterbloom do not exceed levels noted in
healthy Anabaena cultures with low background contamination. Kuentzel
(1969) cites studies which suggest the importance of anticedent bacterial
populations in their role as C02-providing symbionts in blue-green algal
blooms. The fatty-acid findings would tend to ascribe a maintenance
and "mopping-up" role to the bacteria, rather than an active role during
initiation of the waterbloom. This is not meant to infer that the maintenance
of a major bloom, lasting for weeks at a time, is an unimportant bacterial
contribution. Additional subjective evidence of bacterial population
increase after peak Anabaena concentrations comes from filtration of water
samples. Following the peak there was a daily increase in the rate of
filter-clogging although the planktonic elements were either stable or
declining in numbers. As the glass-fiber filter (0.1 y pore size) is an
effective bacterial filter, the increased clogging was ascribed to
increased bacterial concentration. Potaenko and Mikheeva (1969) noted
inverse relationships between blue-green algal populations and bacteria,
citing evidence from their own studies and those of other workers by
Byelorussian lakes. His findings indicated, however, that increased
305
-------
eutrophication appeared to modify, and occasionally reverse, this
relationship.
I exposed sterile conditioned medium and sterile extracted, con-
ditioned medium (50 ml of medium in 125 ml Ehrlenmeyer flasks) to air-
borne contamination for several minutes, then refitted the cotton plugs
and set the flasks aside. Within days, fungal and bacterial contamination
began to overgrow the extracted medium. No appearance of contamination
was noted in the conditioned medium after four months. This simple
experiment has been repeated several times; the results are the same.
The resulting high concentration of A. flos-aquae
at the surface, largely the result of extensive
pseudovacuole formation, is recognized as a waterbloom;
it is composed chiefly of senescent Anabaena filaments.
In natural waterblooms, the color of A. flos-aquae filaments
ranges from brown to purple as a result of the light scattering properties
of the gas filled pseudovacuoles. Upon centrifugation, the filaments
displayed positive buoyancy, remaining at the top of the waters; a
concentration of heterocysts was found at the bottom. The heterocysts
did not appear to contain pseudovacuoles. Waaland and Waaland (1970)
report light induction of pseudovacuole formation in Nostoc muscorum - gas
vacuoles were formed only when illumination exceeded 400 foot candles,
and formation ceased when culture density lowered the mean illumination
sufficiently. Light scattering properties of the gas vacuoles reduced
light absorption according to the authors. While I did not observe
pseudovacuole formation in laboratory Anabaena, the vacuole-inducing
light value represents a level sufficient to induce degenerative
changes.
306
-------
Two additional factors may play a role in the rapid surface
concentration of Anabaena. Hutchinson (1967) suggests a dying surge of
multiplication under the supra-optimal conditions of light and tempera-
ture in surface waters. A second factor, noted consistently in the
laboratory, is a positive phototactic response. If pseudovacuole forma-
tion in Anabaena should prove to be induced by high light'conditions,
then the observed phototaxis may indeed play a greater role in bringing
Anabaena filaments to the surface than has heretofore been suspected.
Attempts to culture A. flos-aquae from surface waters met with
consistent failure. As in old laboratory cultures, a senile degeneration
appears to occur which is not reversible by transfer into fresh medium.
CONCLUSIONS
Anabaena flos-aquae can secrete a powerful broad-spectrum hetero-
inhibitor, which appears to prevent cell division in algae. A dramatic
decline in phytoplankton populations accompanies the ascent of Anabaena
to bloom concentrations; representative genera respond similarly in
laboratory studies in medium conditioned by log-phase Anabaena. Growth
of bacteria was suppressed in healthy cultures of Anabaena; data suggest
such suppression exists in the natural system, also.
Lipid extracts of early bloom lake water containing fatty acids,
readily assimilable by Anabaena, were stimulatory to the growth of the
blue-green in laboratory studies. The stimulatory products are presumably
released as other phytoplankton populations decomposed. Further studies
are necessary to confirm the precise origin of the products in lake water
extracts.
307
-------
Phototaxis in Anabaena has been demonstrated and its role in surface
concentration of a waterbloom has been suggested. As filaments from
surface waters were incapable of further growth in all culture media tested,
concentration by proliferation of filaments in surface waters is indeed
doubtful. Buoyancy of pseudovacuole-filled Anabaena filaments was noted.
A simple bioassay procedure has been suggested for testing the nature
and effect of Anabaena heteroinhibition. The next step must be an
approach to extraction without inactivating the inhibitor-. Bioassay with
the active fractions can then aid in the purification and ultimate identi-
fication of these fractions.
Much remains unsolved. The precise conditions governing filament
growth from akinetes, heterocyst production, phototactic behavior, and the
formation of gas-filled pseudovacuoles are not known. However, a major
role for heteroinhibition in the formation of Anabaena flos-aquae water-
blooms is supported by my observations in the laboratory and in the
natural system.
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311
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APPENDIX A - % Composition of Net Plankton
Lake Nelson - August 1970
CO
ro
Genera
Anabaena
Scenedesraus
Pediastrum
Dactylococ-
copsis
Pleodorina
Platydorina
Eudorina
Micractiniui
Golenkinia
Chlorococcum 0.9
Pandorina
Tetraedron
Chlorogoni'
Phacus
8/8
8.4
3.9
2.3
0.2
0.7
0.1
0.6
TOO
1-i.* £
1.1
OQ
. y
0.1
8/10
88.6
tr
tr
2.1
tr
0.8
00
. o
tr
tr
4- v
tr
tr
8/11
91.0
0.3
0.1
0.2
2.5
0.1
0.5
00
. £
0.2
0.1
IT
. X
8/12
84.3
1.4
n A
\J * *T
0.9
0.4
2.7
0.2
2.7
0.2
0.2
0.4
2-1
. o
8/15
68.5
0.7
0.4
0.2
1.3
6.1
3.8
0.4
0.2
OA
. t
0.2
8/17
48.0
0.2
0.2
9.0
2.9
0.2
0.5
0.5
0.7
8/18 8/19 8/20 8/21
37.6 57.8 50.1 25.1
0.8 3.0 1.5 0.7
0.4
0.4
1.1
1.6 1.7 0.6 3.8
1.6 0.9
0.4 0.4 -- 0.7
0.4 0.1 0.4
0.4 --
1.0 0.4
8/24
64.6
5.8
OQ
. «/
0.5
12.1
1.4
0.5
8/25 8/26
57.9 21.8
26.6 48.8
1.1
0.9 1.1
OC -11
. J J. . JL
3.5 2.5
0.5 ~
0.4
_ __
8/27
10.9
50.5
1.2
0.3
6.1
0.8
0-3
. 3
0.8
0.3
On
. J.
8/28
6.6
12.5
0.2
3.7
60.5
0.9
0.5
0.2
8/29
3.2
13.7
0.2
4.4
56.9
0.9
1.2
0.2
_
-------
Genera
APPENDIX A (continued) - % Composition of Net Plankton
Lake Nelson - August 1970
8/8 8/10 8/11 8/12 8/15 8/17 8/18 8/19 8/20 8/21 8/24 8/25 8/26 8/27 8/28 8/29
Anacystis 0. 1
Oscillatoria 0.9
Po ly edr iops is 0.4
Filinia 1.8
Other
.....«, D 4* .3
Rotifers
Immature .
Copes
OJ
CO
- .
1.
0.
0.
1.
0.
o
1
_- .
3 2.6 4.5
1 0.2 0.4
T
7 1.0 1.4
4 tr 0.5
6__ __,
0.4
2.7
0.2
8.1
3.4
2.7
1.1 16
1.8 2
2
22.2 17
10.9 20
1.9 0
.2 9.2
.0 5.7
.0 ~
.7 7.5
.3 13.0
.4
01
10.1 13.0
2.2 2.5
0.2 --
26.0 30.3
7.5 20.9
0.4
0-7
4.5 0.9
1.8 0.9
1.4
0.9
7.2 1,9
_ r\ c.
u. D
2 3
? 7
1.4
1.8
1.4
2.2
5.0
0.4
10
. o
01
, I
Q P
1.5
1.8
4.5
1.1
0.7
1C
. y
?? 7
2.3
1.1
0.4
1.6
0.5
Q 7
3.5
3.2
2.0
8.0
0.2
1 7
tr = 0.05% or less
-------
10000
1000
cc
in
a.
CO
oi
o
cc.
o
CO
LU
100
10-
ANABAENA
EUGLENA
PANDORIN A
ii
10
20
25
30 SE'PT
APPENDIX B - Variation of phytoplankton components during
an Anabaena flos-aquae waterbloom in Lake Nelson, 1970.
314
-------
IOOOO
IOOO
oc
UJ
Q.
CO
UJ
O
CC
O
CO
H
Z
UJ
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-'
\/
ANABAE NA
SCE NEDESMUS
OSCI LL ATORI A
58LY
2o
SEPT
APPENDIX C - Variation of phytoplankton components during
an Anabaena flos-aquae waterbloom in Lake Nelson, 1970.
315
-------
IOOOO
IOOO
cc
UJ
Q.
UJ
o
cc
o
CO
t-
IU
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ANJABAEN A
CHfLOROGONIUM
PLlATYDORI NA
10
APPENDIX D - Variation of phytoplankton components during
an Anabaena flos-aquae waterbloom in Lake Nelson, 1970.
316
-------
CO
I
Plate 1.
Plate 2.
Plate 3.
Plate 4.
APPENDIX E
Euglena gracilis grown in extracted conditioned medium. (lOOx)
Euglena gracilis grown in Anabaena-conditioned medium. (lOOx)
Euglena gracilis grown in extracted conditioned medium. (750x)
Euglena gracilis grown in Anabaena-conditioned medium. (750x)
-------
CONTRIBUTORS*
MICHAEL B. BARRON
Southeast Environmental Research
Laboratory
Athens, Georgia
KENNETH E. BIESINGER*
National Water Quality Laboratory
Duluth, Minnesota
DONALD L. BROCKWAY*
Southeast Environmental Research
Laboratory
Athens, Georgia
GARY B. COLLINS*
Methods Development and Quality
Assurance Research Laboratory
Cincinnati, Ohio
WILLIAM R. DUFFER*
Robert S. Kerr Environmental
Research Laboratory
Ada, Oklahoma
WILLIAM J. DUNLAP
Robert S. Kerr Environmental
Research Laboratory
Ada, Oklahoma
CRAIG W. DYE*
Department of Pollution Control
State of Florida
Tallahassee, Florida
JAMES W. FALCO*
U.S. Army Corps of Engineers
Waterways Experiment Station
Vicksburg, Mississippi
JOSEPH C. GREENE*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
KATHERINE HARTWELL
Grosse lie Laboratory
Grosse lie, Michigan
NORBERT A. JAWORSKI*
Pacific Northwest Environmental
Research Laboratory
Con/all is, Oregon
ALBERT KATKO*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
PAT C. KERR
U.S. Army Corps of Engineers
Waterways Experiment Station
Vicksburg, Mississippi
RAY R. LASSITER*
Southeast Environmental Research
Laboratory
Athens, Georgia
THOMAS E. MALONEY*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
JAMES McNABB
Robert S. Kerr Environmental
Research Laboratory
Ada, Oklahoma
ELLEN MERWIN
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
WILLIAM E. MILLER*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
WALTER M. SANDERS, in*
Southeast Environmental Research
Laboratory
Athens, Georgia
TAMOTSU SHIROYAMA*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
^'Asterisk denotes participants in the conference
318
-------
PAUL D. SMITH*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
ROBERT L. SMITH
Robert S. Kerr Environmental
Research Laboratory
Ada, Oklahoma
DAVID T. SPECHT*
Pacific Northwest Environmental
Research Laboratory
Corvallis, Oregon
R. MARIE STEAD
Southeast Environmental Research
Laboratory
Athens, Georgia
RICHARD L. STEELE*
National Water Quality Laboratory
Narragansett, Rhode Island
NELSON A. THOMAS*
Grosse He Laboratory
Grosse lie, Michigan
RICHARD E. THOMAS*
Robert S. Kerr Environmental Research
Laboratory
Ada, Oklahoma
GERALD E. WALSH*
Gulf Breeze Environmental Research
Laboratory
Gulf Breeze, Florida
LLEWELLYN R. WILLIAMS*
National Environmental Research
Center
Las Vegas, Nevada
DAVID J. YOUNT*
Watershed Ecosystems Branch, EPA
Washington, D. C.
*Asterisk denotes participants in the conference
319
-------
TECHNICAL REPORT DATA
f "lease read Instructions on the reverse before completing)
' REPORT NO.
EPA-66CV3-75-034
4. TITLE AND SUBTITLE
3. RECIPIENT'S ACCESSI ON> NO.
5. REPORT DATE
Proceedings: Biostimulation - Nutrient
Assessment Workshop
6. PERFORMING ORGANIZATION CODE
7. AUTHORlS!
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Pacific Northwest Environmental Research
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200 SW 35th St., Corvallis, OR 97330
10. PROGRAM ELEMENT NO.
IBA031
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Pacific Northwest Environmental Research
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200 SW 35th St., Corvallis, OR 97330
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16. ABSTRACT
The workshop was held to bring together those investigators in the
Environmental Protection Agency who are actively engaged in research relating
to biostimulation and nutrient assessment to present the results of their studies.
The papers presented were concerned with the results of algal assays conducted
on various waters and wastes to determine their biostimulatory effects as well
as the results of other research relating to the assessment of nutrients and
their effects on the aquatic ecosystem.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
bioassay, algae, nutrients,
eutrophication
biostimulation
nutrient assessment
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and Medical
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