components and 1/10 of the organic salts, was used for
the Tetrahymena studies. Coarsely-filtered pond water
was used in the guppy toxicity tests. Disposable glass-
ware was used to prevent residue effects. At the start
of an experiment all culture vessels and media were
autoclaved at 250°C. for 25 minutes to insure sterility
(except the guppy experiments in,which pond water was
used). One hundred percent, p,p'-DDT, designated as
the "ESA pesticide reference standard" was used in all
DDT experiments and the Aroclor 1242 used was obtained
directly from Monsanto in St. Louis, Mjssouri. The
test chemical was generally added to the growth media
prior to the introduction of the organisms. Acetone
was used as a vehicle for DDT, and 95% ethyl alcohol
was used for the PCB at a concentration of 0.2 ml sol-
vent/liter media. Initial algal concentrations were
®.1 x 10° cells/ml. Experimental cultures containing
algae and/or Daphnia were grown at 28.5 C.+ 1°c. ‘under
a bank of fluorescent lights at 900 foot-candles.
Tetrahymena vorax cultures, grown in test tubes, were
placed on a roller tube apparatus which rotated the
tubes at 1/5 rpm. Protozoan, ostracod, and guppy
bioassays were conducted under the conditions of 23.5
ct#1°c, and 75 footcandles of light.

oO

The Chlamydomonas cells were killed and stained by adding
a drop of I,-KI solution, then counted using a haema-
cytometer at 100X. The Tetrahymena were stained in the
same manner, then ten 0.01 ml drops of the culture were
distributed on a Falcon counting plate and the cells in
each counted at 50X. Carbon-14 uptake by algal cells
subjected to various concentrations of both DDT an¢, PCB
was determined by incubating 5 ml samples with NaH’ “co
and using a standard filtration-planchet method. Gas 3
chromatography analysis was performed by Dr. James Saddler
of the Fisheries Research Institute on five algal cul-
tures--a control, a solvent control, and three cultures

to which 20, 2, and 0.2 ppm Aroclor had been added. The
algal pellets and supernatant samples were extracted

with acetonitrilepetroleum ether; purified by elution on

a Florisil column; and injected into a gas chromatograph
equipped with an electron capture detector.

12


Experimental algal standing
crop in experiment BM-4

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A second study (Taub, 1969b), showed that in batch com-
munities, even the presence of a larger herbivore, the
rotifer Philodina, resulted in only a temporary reduc-
tion in algal abundance. The major effect of the roti-
fer was to reduce the abundance of the protozoan either

by competition for food or by predation. The absence of

3 of the 6 kinds of bacteria had no effect. A third
report, (Taub, 1969 c), demonstrated that a two-stage
continuous culture of the alga, protozoan and 2 bacteria
could be maintained and that they responded to changes in
flow rate and light intensity. At high dilution rates
(1.0 and 1.4 per day) and high light intensities algal
populations and Pseudomonas were low in the upstream com-
munity, and the protozoa and E. coli became extinct,
whereas at lower dilution rates (0.57 and 0.73) all of

the organisms were able to maintain steady-state populations.
At reduced light intensities and the lower dilution rates,
the algal densities were markedly reduced and the protozoan
population was able to persist at a dilution rate of 0.5
but not 0.75 per day. An assumption of a Mechalis-Menten
relationship between the growth rate of the protozoan and
the abundance of the algaé as the substrate, accurately
predicted protozoan extinction, but not steady state
populations. The downstream and yield communities were
progressively denser and more stable to the environmental
changes. The continuous culture technique provided more
sensitive communities as well as being more amenable to
analyses. These formed much of the basis for the new work.

The comparative toxicities of DDT and Aroclor 1242, and to
a more limited degree, (2,3-Dichloro-1,4 naphtoquinone)
have been investigated. Various other exploratory studies
are reported here.

-6-


The severity of the response was dose related. After
day 5, the growth rate of these inhibited cultures
increased so that by day 22 they had cell densities
similar to the controls. By day 22, the standing crop
of Chlamydomonas in all of the cultures appeared to be
about the same. This experiment was repeated several
times with similar results, although the degree of
inhibition from equal additions of PCB varied somewhat.
This variation may be due to heterogeneity of the PCB
globular suspensions, or to differences in other
physical factors from experiment to experiment.

In order to determine whether 14, uptake by Chlamy-
domonas cells was also affected by the addition of
Aroclor 1242, the experiment was repeated using five
different doses of PCB(O.0002, 0.02, 0.2,2, and 20
ppm), incubating NaH “CO,, with samples taken from the
cultures at three differént points in t growth period.
On day 4, the control had the greatest C uptake and
the PCB containing cultures had uptakes per cell and
perml in inverse relationship to their PCB concen-
tration. By days 11 and 15, the Cc uptake per cell
were all markedly reduced and showed no dose-related
response. These results were undoubtedly influenced
by t varying amounts of nutrients still available.
The Cc uptake per ml tended to increase during the
duration of the experiment, the values generally in
inverse relation to the dose.

A possible reason for the apparent recovery of
Chlamydomonas from the initial effects of PCB-containing
media is that the PCB was degraded or otherwise made
less effective during the course of the experiment. The
possibility that the fluorescent lights used in growing
the algae cultures were causing degradation of the

added PCB was investigated. Mason jars of Media 63
containing 2 ppm Aroclor 1242 were placed under the bank
of fluorescent lights for 7,5,2 or O days prior to

their inoculation with Chlamydomonas. All of the PCB-
containing cultures exhibited delayed algal growth and
there did not appear to be any correlation between
degree of inhibition and the length of time the media
was under the lights after the PCB was added. This
seemed to indicate that neither degradation by light

nor adsorption of PCB on the glass were significant

67


60

Table 2:

Experimental Conditions and Results at Steady State

Average conditions (averaged over period considered stable)

Exp. flask algae prot flow Exp. flask algae prot flow
BM 71-1 A 4.33 140 BM 71-5 A 3.92 257
4.95 B 2.67
G 3.15 G 3.74
c 4.54 148 c 3.21 251
D 1.15 2477 D 1.94 1.103
R 1.03 ~269 R 1.99 836
BM 71-3 A 3.31 332 BM 71-7 A 1.45 505
B 4.75 B 1.81
G 4.23 G 1.60
Cc 3.78 330 Cc 74 520
D 1.56 330 D 1.30 .0069
R 76 211 R 1.61 ~0075
BM 71-4 A 1.69 505 BM 71-9 A 1.70 347
B 2.66 B 2.60
G 1.67 G 5.00
c 2.22 496 Cc 2.75 316
D 1.98 066 D 4.32 0515
R 1.26 0.37 R 6.84 -0635


Table 1: TABLE OF EXPERIMENTAL CONDITIONS

Experiment Media Flow Light
mM. ml/day ftc
BM 71-1 5 140 1000 all flasks
in light
71-3 25 360 3000 B,G,D,R, in
dark
71-4 5 500 3000 B,G,D,R, in
dark
71-5 5 300 3000 B,G,D,R, in
dark
71-7 5 500 3000 all flasks
in light
71-9 ~5-5 300 3000 all flasks
in light

Major deviations in experimental conditions.

BM-1

Experiment initialized at 3000 ftc changed to 1000 ftc

on day 13. Flow rate high and variable( 1000 ml-40 ml)
average 500 ml until day 22 changed to 140 ml/day.

Day 27 10 ml of culture in Flask B was inoculated into

Flask D Day 13 protozoans re-inoculated to density of
-l x lO” analyzed day 35.

BM-3

Light intensity initialized at 1000 ftc changed to 3000
ftc on day 7 temp fluctuation on day 8 22.5 26.8°C
for 4-5 hours. Cultures contaminated on day 20.
Analyzed day 24.

BM-4
Operation per design. Flow fluctuation, 340-620.
Analyzed day 19.

a7


€s

Table 4 B: BM-3 Pigment Analysis BM-71-3

PIGMENTS 4-26-71
P & S Analysis Pheopigment Analysis
mg/L mg/1

chl_a chl b Carotenoids Chl_a Pheo a
A 0.96 0.34 0.28 0.829 0.34
B 1.12 0.41 0.31 0.94 0.27
G 1.06 0.41 0.33 0.93 0.46
c 1.02 0.36 0.27 0.84 0.30
D 1,12 0.36 0.34 0.73 0.32

R 0.83 0.24 0.27 0.37 0.77



BM 71-5
7-6-71

Table 6B BM-5 P & S Analysis of Pigments
mg/L
Flask .. Chi_b Carotenoids

A 4.64 1.97 0.90

3.01 1.20" 0.59

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45


LIGHT

2.0
3.0

MAX 4.0

100

Figure 2.

300
FLOW

Response surface (isopleths) of
algal standing crop in downstream
community (flask B) at nutrient con-
centration of 0.5 mM NO, -

17


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Model algal standing crop

Figure 20,

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35


Also the model fails to demonstrate how the algal
density could be higher in the presence of predation
than in its absence as shown in this experiment. The
model predicted the timing of steady state quite well
with a somewhat too rapid a response to the change

in environmental conditions.

More experimental data is necessary to establish the
amount of experimental variation, random noise in the
system, that can be expected and the degree to which
the model forecasts the results. We can obtain some
insight into the amount of variation within an
experiment by examining the variation that occurred
between flasks A and C. The variation between experi-
ments of similar experimental conditions, BM-3, BM-5,
and BM-9, needs to be examined further.

Plotting the assumed steady state results on the
isopleths indicates that the exact position of the
curves may be questioned. However, the general shape
and relative numbers appear to be reasonable. Thus,
the model can be used as an integral part of designing
experiments and in understanding the system.

The chemical data and the activity data have not yet
been adequately incorporated into the model.

Toxicity Studies

Figure 31 illustrates the effect of three different
doses (0.2, 2, and 20 ppm) of Aroclor 1242-alcohol on
the density of Chlamydomonas over a period of 22 days.
Both the control and the solvent control followed the
typical parabolic growth curve with no initial lag
period. There was a period of rapid growth (the log
phase) followed by a leveling off of the population
size as the culture approached the stationary phase.
Aroclor-containing cultures did not follow this pat-
tern, however, but took a much longer time to approach
the cell density of the controls. In the Chlamydomonas
cultures to which the two highest dosages of Aroclor
were added there appeared to be not only a delay
before exponential growth began, but an initial drop
in the algal density below the original inoculum size.

66


ABSTRACT

Data from continuous cultures of an alga (Chlamydomonas
reinhardtii) and protozoan (Tetrahymena vorax)have been
used to construct a model of algal standing crop over
ranges of light intensity, dilution rate, and nutrient
concentration both in the absence and presence of pre-
dation by the protozoa.

The model has been used to demonstrate response surfaces
for the steady-state standing crop values for the algae
and protozoa over the ranges used in the laboratory
experiments (shown as isopleths). The physical variables
had a marked effect on the algal standing crop which
influenced the growth rate of the protozoan. The stand-
ing crop of the protozoan was determined by growth and
dilution rates. The effect of the predation was depen-
dent on the protozoan standing crop. The response
surfaces indicate that predation can reduce algal stand-
ing crop only within certain ranges of the variables
considered.

The experimental density results and the model pro-
jections, adjusted for the daily varying flow rates,
are shown. The chemical analyses for steady-state cul-
tures are reported but not entirely integrated into the
model.

The comparative toxicities of Aroclor 1242, a poly-
chlorinated biphenyl and DDT, were tested on the alga
and protozoan, and also on daphnids, ostracods, and

guppies.
This report was submitted in fulfillment of Project

Number 16050 DXM, under sponsorship of the Environmental
Protection Agency.

aii


toxic to Daphnia pulex, however. Young Daphnia died
at levels as low as 0.02 ppm. Mortality may be
linked with other stresses as well, however, for
when the Daphnia were supplied with algal culture,
the death rate was cut to one-third. A comparison of
the relative toxicity of PCB and DDT to ostracods
reveled a greater mortality of Cypridopsis vidua at
20 ppm PCB than at the same concentration of DDT.

At 20 ppm there were no survivors out of 20 ostracods
in the Aroclor-containing cultures while there were
13 survivors in the DDT cultures. At lower concen-
trations, however, DDT appeared to have more of an
effect than the PCB. The guppy Poecilia reticulata
had 100% mortelity at 20 and 2 ppm, but only 25%
mortality at 0.2 ppm. The behavior of the fish just
prior to death in PCB-containing cultures was quite
different from the normal symptoms of hydrocarbon
pesticide poisoning. In some cases behavior included
rapid pivoting of the body with the nose pressed close
to the bottom of the jar; fungus-like growths were
also often associated with dying or recently-dead fish.
This study has been published, Morgan (1972).
Exploratory Experiments

1. Pollution demonstration

To serve both as a class demonstration and as an
exploratory study of stress conditions, an experiment
was conducted on the effects on complex communities of
low and high levels of organic pollution (0.08 g/l and
4 g/l Nutrient Borth); DDT (10 ppm) and an algicide
(1.2 ppm, 2,3-Dichloro-1,4 naphtoguinone). The results
were obivous even to the most casual observation. With
low levels of organic enrichment, density of the algae
and zooplankton were enhanced, whereas at high levels,
the community was murky yellow green, objectionably
odoriferous and the zooplankton had died from lack of
oxygen. The DDT killed the zooplankton and an algal
bloom had occurred in the absence of predation. The
algicide killed the algae, and while not killing the
zooplankton, they were obviously starved and had had
no offspring.

69


factors in the delayed growth and subsequent recovery
of the Chlamydomonas cultures. Another possibility

is that the PCB may have been metabolized either by

the algae or by contaminating bacteria. No difference,
however, was noted in the chromatograms between con-
taminated and axenic cultures, nor in degree of algal
growth inhibition, hence bacterial degradation of
Aroclor 1242 was probably not a significant factor.
(The EPA laboratory at Athens has suggested that this
result might have resulted from surface effects on

the transfer of co, from the atmosphere).

Data from the gas chromatography analysis seem to
indicate that the Chlamydomonas was concentrating

the PCB, for a much higher concentration was found

in the algal pellet (which took up about 0.05 ml in
volume) than in the one liter of supernatant. For
example, in cultures to which 20 ppm (mg/l) ‘PCB had
been added, 0.52 mg were detected in the algae, and
only 0.04 mg in the supernatant. A large percentage

of PCB that was added was not recovered from either

the media or the algae. This may have been last because
of adherence to the glass sides of the mason jars,
sinking of small PCB globules to the bottom, vaporization,
codistillation with water, metabolism by the algae, or
loss in the extraction, Florisil clean-up, or injection
processes.

The effect of DDT on the growth and 14, uptake of
Chlamydomonas cultures was studied. In the tested
concentration range of 0.2 ppm to 20 ppm DDT, the
algal growth curves did not differ appreciably from
those of the controls. It is thus possible that
Chlamydomonas, like Dunaliella, is relatively DDT
resistant, and yet may be effe ted by polychlorinated
biphenyls which may restrict Cc uptake by a different
mechanism.

The toxicity of PCB and DDT to organisms other than
algae was then investigated. The growth curves of the
protozoan Tetrahymena vorax grown in the presence of
Aroclor 1242 (0.02 to 20 ppm) and in the presence of
DDT (0.01 to 100 ppm) seem to indicate that neither
chemical has any effect upon the growth rate or
population size. Aroclor 1242 appeared to be fairly

68


SECTION VII
ACKNOWLEDGMENTS

The efforts of Dr. Walter M. Sanders III, current Grant
project Officer and his predecessors, Dr. Frank Wilkes
and Mr. Joel Fisher are acknowledged with sincere thanks.

Mr. Daniel McKenzie has been responsible for the mathe-
matical modeling, as well as all computer services.

The culture work and chemical analyses have been con-
ducted by Ms. Ruth Hung and Mr. Fred Palmer with the
assistance of Ms. Kathleen Hamel. The pesticide studies
were conducted by Ms. Janet Morgan and are being con-
tinued by Mr. Joseph Cummins. Community metabolic studies
were conducted by Mr. Jonathan Heller. Exploratory pol-
lution and self-supporting communities were studied by

Mr. Benedict Satia. Ms. Liesel Rombouts has provided
secretarial support.

71


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28


Exploratory Experiments

1. Pollution demonstration.
2. Community Metabolism.
3. Multi-trophic level, self-sustained communities.

Methods are described briefly with the discussion.

13


MAX .67

FLOW

Figure 5. Response surface (isopleths) of algal
standing crop in upstream community
(flask A or C) at nutrient concen-

tration of 0.05 mM No, -

20


100 200 300
FLOW

Figure 1. Response surface (isopleths) of algal
standing crop in upstream community
(flask A or C) at nutrient concen-

tration of 0.5 mM NO.

16


eguvts.

a flask B

pest

© «flask C

in experiment BM-1.

Model algal standing crop

Figure 14,

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29


where wad Maximal growth rate of algae

L - light intensity at mid-depth of the culture
S - substrate concentration
K, K) constants (Michaelis-Menton constant)

The yield coefficient of the algae, Y, has been represen-
ted as a function of available nitrate substrate.

A linear regression equation was fit to the experimental

data in order to predict protein concentration from sub-

strate levels.

The optical density in the model is calculated from

a multiple regression equation that includes algal den-
sity and substrate concentration. The light intensity
at mid-depth of the culture is computed using Beer's Law.

_ -dh
Ta Te (6)

where I,- incident light intensity

A extinction coefficient as measured by optical
density
dad depth of interest

It is appropriate to recognize that the growth of the
protozoan predator should be similarly modified so that
He in equation (2) is replaced by:

Pp
[= Ww A
pmax 2A (7)
a
where uP is the maximum growth rate of protozoans

max
which is proposed to be a function of algae protein con-
centration and K, is a constant.

10


2. Community Metabolism

The O, exchange of yields from, gome of the continuous
cultufe have been studied by C “uptake, continuous O
and pH recording, or photosynthetic respirometry. ré
has been shown that the patterns are different in the
alga-only system as compared to the alga-protozoan
system. In general, the systems subjected to predation
tended to be the more productive per algal cell and per
volume (ml of culture). Algal cultures at higher
dilution rates also demonstrated greater O, production
per cell than cells grown at lower dilution rates.
These data are only an index of what might be occurring
in the growth flasks, since these tests must be con-
ducted on external samples under altered conditions.

3. Multi-trophic level, self sustained communities.

Aquaria of various sizes and with various nutrient con-
centrations were inoculated with a complex community

to explore the nutrient budget which would be required
for an alga-zooplankton-fish (guppy) community where
primary productivity could serve as the initial energy
source (i.e. without dependence on an external source

of fish food. It became obvious that at least 18 liters
with an initial NO, concentration of 5.0 mM NO, would be
necessary to even marginally supply enough N for guppy
growth, Without shelters, the zooplankton population
can be eliminated by the fish predation. If containers
of 18 liters (i.e. 5 gal. carboys) or larger can be
used, and if higher concentrations of NO. are used, and
if shelters for the zooplankton can be provided, the
feasibility of self sustained communities seems
reasonable. The non-homogeneous nature of such com-
munities would require that community be assessed either
by community 0, exchange or by total analysis of
replicate samples.

70


GS

Table 5B BM-4

P & S Analysis

mg/L
Chia Chi b Carotenoids
A 72 27 16
B 1.00 -40 26
GY 86 41 -20
Cc 284 32 19
D 95 34 24
RY 93 37 222

PIGMENTS

BM-71-4, 5-19-71

Pheopigment Analysis

mg/l
Chia Pheo a
64 17
87 24
70 229
73 19
~82 286
766 294



No.

3A

4A

5A

6A

7A

8A

TAB

LES

Experimental Conditions - Design

Experimental Conditions and Results
at Steady State

BM-1

BM-3

BM-4

BM-4

BM-9

Chemical Analysis
Pigment Analysis
Chemical Analysis
Pigment Analysis
Chemical Analysis
Pigment Analysis
Chemical Analysis
Pigment Analysis
Pigment Analysis
Pigment Analysis
Pigment Analysis

Pigment Analysis

Summary of PCB Toxicity Studies

ix

49

50

51

52

53

54

55

56

57

58

59

60

6l

62


40

LIGHT
f

% 10 ao 30 Co) 30
FLOW

Figure ll. Effect of predation on the response
surface (isopleths) of algal standing
crop in downstream community (flask D)
at nutrient concentration of 5.0 mm

NO 3°

26


rae)

TABLE 9

Summary of PCB toxicity studies. +4+++-=many survivors; ++ = most survivors;
+ = few survivors; 0 = no survivors.

Test Table obser-
Situation at age dose of PCB (ppm) vation
0.0002 0.002 0.02 0.2 2 20 day
Daphnia
fed PCB-containing
cells* 2 young --- --- --- ++ + ° 4

direct addition of
PCB-alcohol

non-fed 3 young +++ +++ + ° ° ie) 4
fed 3 young ttt tet +t ie) te) te) 4
PCB-containing
algal cultures 4 adult --- --- =o fe) fo) 3
5 adult ttt +44 Ab ° fe) 2
5 adult oo- t+ ttt ° ° ° 9
Ostracods
PCB-containing
algal cultures 6 adult --- ttt tte FE t+ fe) 3
Guppies
direct addition 7 young --- --- --- --- fe) fo) 7
of PCB-alcohol 8 young --- ttt te ++ fe) {e) 1

* Daphnia in PCB-free media were fed washed Chlamydomonas cells grown in PCB
suspensions of the indicated doses.


most, and would be ineffective over most of the environ-
mental range.

Increasing the nutrient concentration to 5.0.mM sharply
increases the maximal standing crop, 14 x 10 cells/ml
(darkly stippled area) and in the upstream flask, the
maximum occurs along the left edge, i.e. almost irres-
pective of light, but only at low flows. In the down
stream community, the range of conditions contributing
to the maximal conditions are reduced. The effect of
the predation is marked and prevents algal densities
greater than 6.0 x 10 cells/ml. The maximal protozoan
population is high, .9 x 10° cells/ml but can be main-
tained only under a limited range of conditions. Their
density is very sensitive to flow, but rather insensi-
tive to light intensity. Note that the protozoan den-
sity is only 50% increased, whereas the nutrient con-
centration was increased 10 fold.

The examination of the actual experimental runs is com-
plicated by the high variability in daily flow rates
(usually + 10%, occasionally + 20%). The seriousness

of this problem is demonstrated by the great sensitivity
of cell density to flow rate under some conditions, e.g.
Fig. 1, where a flow rate of 300 + 30 ml/day would be 6
expected to result in changes from almost .5 to 3 x 10
cell/ml densities.

As with any simulation model there are several problems
involved in determining the validity or predictability
of the model. One method of testing the model is to
provide two independent sets of data, one for estimating
the parameters and one for testing the prediction of the
model. Experiments BM-1 through BM-7 were used to
estimate parameters for the following relationships:

O.D. - £ (algal cone., substrate conc.)

Algae protein conc. - f (substrate conc.)

Algae growth rates - f£ (substrate conc., light)

Coefficient of predation - f (algal density,
protozoan density)

Algal natural mortalities

64


The following list of parameters and functions were
estimated by making reasonable assumptions about values
and by experimenting with the model to produce accep-
table results. At most these functions are based on
one or two data points.

4t - time lag for algae response tochanging conditions
threshold level of nitrate substrate needed for algal
growth

protozoan growth rates - f (algal density, algal
protein conc.)

protozoan mortality rates - f (protozoan density,
light)

parameters for dissolved organic components

parameters for recycling of nitrate substrate.

To demonstrate the models ability to predict the den-
sities of the experimental runs, the daily flow rates
were used in the model. Generally, the agreement
between the experimental and model algal densities was
good, but there are some troublesome disagreements,
only some of which have satisfactory explanations.
Differences in initial responses are probably due to
differences in initial conditions; the model assumed
that the initial densities in all flasks was 1 x 10
cells/ml, whereas it was not in fact uniform. This
effect is especially obvious in BM-4 (Fig. 19 & 20).

Of greater concern are the occurrances in BM-5 (Fig.22
and 23) where two major peaks in algal density

occurred in the experimental data, but not in the
model, The experimental and model protozoan populations
also generally agree, but there are some inadequately
explained disagreements. In BM-l (Fig. 15) and BM-5
(Fig. 24), the experimental population showed agreement
for some period of time, but then increased and were
highly variable. The model was then used to predict
the results of BM-9. Figures 28, 29 and 30 show the
experimental and model results. The experiment was
designed to be a near repeat to experiments BM-3 and
BM-5, and to give a measure of the response time to

a change in environmental conditions. It is difficult
to identify the causes of the lack of agreement between
the two results. As expected the model predicted
results that were much like the two earlier experiments,
however, the experimental results were quite different.

65


LIGHT

MAX .09

(.

gt

% 20 Ey 00 B00 00
FLOW
Figure 8. Response surface (isopleths) of proto-

zoan standing crop in downstream com-
munity (flask D) at nutrient concen-

tration of 0.05 mM No, .

23


lo

FIGURES

Response surface (isopleths) of algal standing
crop in upstream community (flask A or C) at

nutrient concentration of 0.5 mTM NO,-
Response surface (isopleths) of algal standing
crop in downstream community (flask_B) at
nutrient concentration of 0.5 mM NO...
Effect of predation on the response surface ,
(isopleths) of algal standing crop in downstream
community (flask D) at nutrient concentration of
o.5 mM NO..

3
Response surface (isopleths) of protozoan standing
crop in downstream community (flask_D) at
nutrient concentration of 0.5 mM NO.
Response surface (isopleths) of algal standing
crop in upstream community (flask A or C) at
nutrient concentration of 0.05 mM NO.
Response surface (isopleths) of algal standing
crop in downstream community (flask B) at
nutrient concentration of 0.05 mM NO,-
Effect of predation on the response surface
(isopleths) of algal standing crop in downstream
community (flask D) at nutrient concentration of
0.05 mM NO, -
Response surface (isopleths) of protozoan standing
crop in downstream community (flask D) at
nutrient concentration of 0.05 mM NO.
Response surface (isopleths) of algal standing
crop in upstream community (flask A_or c) at
nutrient concentration of 5.0 mTM NO,-
Response surface (isopleths) of algal standing
crop in downstream community (flask B) at nutrient
concentration of 5.0 mTMM NO,-

vi

PAGE

16

17

18

19

20

21

22

23

24

25


Section

Ir

Ir

TIr

Iv

VI

VII

VIII

CONTENTS

Conclusions
Recommendations
Introduction

Methods and Materials
Experimental Phase
Discussion
Acknowledgments

List of Publications

Page

15
63
71

73


One more additional consideration is necessary to make the
model more realistic. It is unlikely that growth rates
adjust instantaneously and to allow for this possibility

a time delay factor is included in the algae growth
equation. Thus, equation (5) can be written:

Heh DS) ag (5')

L(K + S)e_ att Se atk

This says that the growth rate depends on the substrate
concentration not at present time but at a previous time,
At units earlier. A time lag of .1 days has been deter-
mined by best fit.

The model has been used to calculate the response surface
for a range of flow and light conditions. To generate
each point on the isopleth grid, the model was run for

a sufficiently long time period that the populations
reached a steady state. Contours of equal population
density were then drawn on the grid. The-algal isopleths
represent cell density in millions/ml (10 /m}) and pro-
tozoan isopleths are in ten thousands/ml (10 /ml).

The model is programmed in MIMIC, a digital computer
simulation language packaged by Control Data Corporation
and run on the CDC 6400 computer. MIMIC performs
integration of systems of differential equations by

a centralized integration routine, using a fourth order
variable step Runge-Kutta algorithm. The language pro-
vides routines for the time sequencing of events and
output of the results in both tabular and plotted form.
Although the models will be subject to further refine-
ments, both the biological and mathematical models are
now functional tools.

Toxicity Studies

Five different organisms were used in the present inves-
tigation: the alga Chlamydomonas reinhardtii, the ciliate
protozoan Tetrahymena vorax, the cladoceran Daphnia pulex,
the ostracod Cypridopsis vidua, and the guppy Poecilia
reticulata. Media 63 was the experimental medium in which
the effects of the toxicants upon algae, cladocerans, and
ostracods were studied. Bioassay media, modified from
Fernell and Rosen to contain 1/100 of the organic

ll


25

26

27

28

29

30

31

FIGURES

Experimental algal standing crop in

experiment BM-7.

Model algal standing crop in experiment BM-7.

Experimental and model protozoan standing

crop in experiment BM-7.

Experimental algal standing crop in

experiment BM-9,.

Model algal standing crop in experiment BM-9.

Experimental and model protozoan standing crop

in experiment BM-9.

Response of Chlamydomonas reinhardtii to the

addition of various concentrations of Aroclor

1242,

viii

PAGE

40

41

42

43

44

45

46


2.0

MAX 3.99

30

LIGHT
4

300
FLGW

Figure 3. Effect of predation on the response
surface (isopleths) of algal standing
crop in downstream community (flask D)
at nutrient concentration of 0.5 mM
NO}.

18


Ecological Research Series



Model algal standing crop

in experiment BM-7,

vPigure 26,

_ 90030075, 90+ 300" vorsoore, yuesvore vor a
‘906 300°5, vor 300" ‘yueavo"r we avore Sue 40uF
9003007 90-300" 0840076 woe vate wus vorrt
906 s00°6 wor s0ry yor avant wus swore Soe uur

41


SECTION II
RECOMMENDATIONS

The adequacies of the model to explain experimental
results should be further explored. The community
metabolism and chemical composition data should be
integrated into the model.

The model should be explored for its adequacy to
explain natural communities. Specifically, the
usefulness of the analogy of washout and predation
mortality should be explored.

Effects of toxicity and other stresses should be
evaluated on these alga-predator systems.

More complex communities should be studied, both for
modeling and for use of toxicities and other stress
effects. Community metabolism or total system
analyses may have to be used, since these systems will
have to be non-homogeneous and aliquot sampling will
not be feasible.

Community metabolism and stresses of carbon limitation
should be studied with continuous cultures, with in
situ O, and pH probes, and with automatic and recor-
ding pa equipment.


SECTION I
CONCLUSIONS

The model demonstrates the range of experimental con-
ditions over which predation can reduce algal standing
crop. Under these conditions, the reduction in algal
population is marked and the range of conditions sup-
porting high algal populations is reduced. Predation
control cannot occur under either of two conditions:
(1) if the algal standing crop is very low, even if
the washout due to dilution is low, and (2) even if
algal growth rate and production is high and the
standing crop moderate, if the washout due to dilution
is high. The model provides a means of analyzing
standing crop as a function of experimental conditions
in spite of variabilities in flow rates (largely due to
the inadequacies of the pump).

Aroclor 1242 appeared to temporarily inhibit the
growth rate,gf Chlamydomonas as evidenced by cell
counts and Cc uptake, while DDT did not appear to
have any effect. A greater concentration of PCB was
found in the algal cells than in the media, indicating
that concentration and passage of the biphenyl com-
pounds through the food chain may take place, Neither
Aroclor 1242 nor DDT in the amounts tested appeared to
have any effect on the growth or cell density of
Tetrahymena vorax cultures. Daphnia pulex were quite
sensitive to additions of Aroclor 1242 as low as 0.02
ppm. The toxicity of PCB and DDT to the ostracod
Cypridopsis vidua appeared to be approximately the same.
Young guppies died in concentrations of 2 ppm Aroclor
1242. Aroclor 1242 appears to have a much lower
toxicity to cladocerans and guppies than p,p'-DDT.
Although the effect of PCB on algal growth appeared to
be temporary, in view of its evident capability to be
passed through the food chain and its selective
toxicity to certain zooplankton, it would be unwise to
attempt to predict its effect on the ecosystem without
further study.

The alga-protozoan model here is of limited use as a PCB
or DDT test system, but an excellent test system for
algicides. The PCB-DDT effects can be tested on our
alga-daphnids, alga-ostracods, or alga-daphnids-ostracod
guppy system.


vs

Table 5A: BM-4 Chemical Analysis

Sherer Unit cc

Condition: BM 71-4
30go ft, 12 hrs light B.G. (Dark) 5-19-71
20°¢ 12 hrs dk. -D.R. (Dark)
Flow rate 500/Day
+ Contaminated Waele Whole Liauid Liquid Whole
Cell Dencity Culture culture oaly only Culture
—— “Bienaes" Nt 5 Total a
Alec 7 . ash free = aoe - N re- Cc CPM
‘see Proves op dry wt Protein % Xo) OG Me Covered
x10°/ot x10" fmt Ugnt_a/ob mul Protein ot! mi mk mM per al. per 10%celis
Vol | : j i
425 &, 2.53 | 260! .2094 0387 «18.5 .140.0.0002 | 2604
i i : i :
445 B 2.35 | .290: 2137 0385 18.0 .o2zs8 " ! ot 2551
500-6 2.10 | 220, .1983 10472, 23.8 .0270 "| : 3128
425 & 2.52 | .270| «2072 19.4 10992 a i 7 3104
T i : |
430 D+ 3.08 .026 | .220/ 1883 25.2 0002" | 2918
i A H i
4807+ 1.80 .o39 =| zis} .2139 «=| .0456: 21.3 .0078 ' 2681



~genenenensgescensenseessensnsacoestsnesecanescecessecsenasseneacscasenssearonecorenereeeeseeseses QQ00" By ~

tect ec ceregecceccsecetpereoseagees oeeeeeege cer ee er ene ee ree eee e eR ere ETT ETS CETTE EY OE SEWED EEE EE One

*0000%29—

7 2 daes : :
> ast. 2 - ghey Seeewere ga0grae—
ai oder
a
i
> a5
—T is
> is
as
:
7 : :
z UOUUTET
7g :

aBuRyo eTPAH

2 carrera ree Bere VER ETI ENS 0 9999°3T

ween ence e eee eg ene REEL EN EE EFEY EEETESIEENES 644. 9999¢5—

90+300°S ~ 90030079 90+300°2
99+300°S 9003007 * 9o+300°2
90¢300°S “9043007 ~ 90+300%C 90630072
99+300°S 90300" 90°300°E 90+300°2 90+300°t 19

37


LIGHT

MAX 19.04

8.0
14.0
14.0

4 n 1 rn n

o

300
FLOW

Figure 10. Response surface (isopleths) of algal
standing crop in downstream community
(flask B) at nutrient concentration

of 5.0 mM NO, :

25




LIGHT

z g MAX 18.95)

2.0
100 200 300 400 500 600
FLOW
Figure 9. Response surface (isopleths) of algal

standing crop in upstream community
(flask A or C) at nutrient concen-

tration of 5.0 mM NO, -


19

Table 8B. BM-9 PIGMENT ANALYSIS

P & S Analysis

mg/1
chla chil b Carotenoids
3.99 2.02 1.58
5.56 2.51 1.44
11.16 5.10 3.02
5.75 2.25 1.41
7.34 2.71 1.82
13.39 7.10 4.00

No Pheo Analysis conducted


11

12

13

14

15

16

17

18

19

20

21

22

23

24

FIGURES

Effect of predation on the response surface
(isopleths) of algal standing crop in down-
stream community (flask_D) at nutrient con-

centration of 5.0 mTM NO.

Response surface (isopleths) of protozoan
standing crop in downstream community
(flask D) at nutrient concentration of

5.0 mM NO,-

Experimental algal standing crop in
experiment BM-1l.

Model algal standing crop in experiment BM-l.

Experimental and model protozoan standing crop
in experiment BM-l.

Experimental algal standing crop in experiment
Bm-3.

Model algal standing crop in experiment BM-3.

Experimental and model protozoan standing
crop in experiment BM-3.

Experimental algal standing crop in experiment
BM-4.

Model algal standing crop in experiment BM-4,.

Experimental and model protozoan standing crop
in experiment BM-4.

Experimental algal standing crop in experiment
BM-5.

Model algal standing crop in experiment BM-5.

Experimental and model protozoan standing crop
in experiment BM-5.

vii

PAGE

26

27

28

29

30

31

32

33

34

35

36

37

38

39


SECTION V

EXPERIMENTAL PHASE

Results

The current state of the model is most clearly demon-
strated by the response surfaces for algal and
protozoan standing crops calculated over the range of
variables tested, Fig. 1-12. The standing crop in
the actual experiment, and the model calculations
(based on environmental conditions including actual
daily flow rates) are shown in Fig. 13-30. The
nominal and actual experimental conditions are listed
in Tables 1 and 2. The chemical data collected on
the experimental runs at steady state is shown in
Tables 3-8.

Toxicity effects are reported in Table 9 and Fig. 31.

Results of the exploratory experiments are briefly
stated in the discussion.

15


es

Table 4A: BM~3 Chemical Analysis

360d

4.39

Sherer unit Continuous Culture BM-71-3
Condition: 4/26/71
30g0 fc. 12 hrs on, 12 hrs dark
20° © (B, G, D, Red-Dark)
Flow rate: 360 ML/Day
Culture: * sterility Whole Whole Liquid Liquid Whole
atts Culture culture only only Culture
Cell Density "Biomass" 0, Total wa
‘ we . ash free a - N re- c cpM
é tesk Alsse Prorezs op dry wt Protein % xo) NH wt Covered 6
o x10°/m) x10"/ml__ Léa’ Me fn mg/al Protein nl mt mM mf __per ml. per 10°cel1s
| \ : i
1 h : H
455 A_ 3,29 |. 250! .. 2500 i ' 1803
' i - : :
455 B 3.66 1. 275) . 3160 . 0423 13.4 0.0002<0.0002 | 1638
: a seen .
T

507 3.43

« 2432 + 0378

565 D 1.72 « 2439 5 . 0577 23.6 'qome | 1372

360°R. 685

. 182

. 145

- 2633 « 0471 17.9 +0042 . | 734

* cells analyzed on 4 - 26 - indicated negative

Sterility checked 5 - 18 "
However, Red yields held until 6/28 showed pasitive starting 4/22, 23, 24, 25 and 26.
Green yields hela ' 6/24 is negative.


6S

Table 7B: BM-7 PIGMENTS
BM 71-7
Sampled 9-20

P+ S Analysis

mg/l
chlor a chlor b carotenoids
A 0.700 0.242 0.193
B 1.137 0.440 0.281
GY 1.046 0.387 0.263
Cc 0.922 0.337 0.206
D 0.841 0.408 0.220
RY 0.775 0.372 0.159

Pheo Analysis
mg/L
Pheopigments*

0.121
0.042
0.310
0.134
0.071

0.419

* Variation between duplicates was larger than usual.


o
. &
&
: e
Es
= BE
a8
. ra
aa
: ge
24
: * aoa : : : : : Dew
a £EEE
we OS sees weeks vegveyee*** 000%
£ gaag TeOw
§ any 2eow
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=
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90+300°5 90-300" yor3007€ 90240072 40+300°1 +0
—sora00 yo 3007 ets “90*300- S0>300-T v
90+300°S 904300" yoe360°C 4o+300%2 30+300°T +0

sor s0T oF 300-T OF TOT TeIVOT T

32


SECTION VIII
LIST OF PUBLICATIONS

Taub, Frieda B. and Alexander M. Dollar 1968. "The
Nutritional Inadequacy of Chlorella and Chlamydo-
monas as Food for Daphnia Pulex", Limnology and
Oceanography, Vol. 13, No.4. pp. 607-617.

Taub, Frieda B., "A Biological Model Of A Freshwater
Community: A Gnotobiotic Ecosystem," Limnology

and Oceanography. Vol. 14, No. 1, pp 136-142
(1969a).

Taub, Frieda B., "Gnotobiotic models of freshwater
communities", Verh. Internat. Verein. Limnol.,
Vol. 17, pp 485-496 (1969b).

Taub, Frieda B., "A Continuous Gnotobiotic (Species
Defined) Ecosystem", pp 101-120. In John Cairns,
Jr, ed., The Structure and Function of Fresh-Water
Microbial Communities, American Microscopical
Society, (1969c).

Morgan, Janet R, 1972. "Effects of Aroclor 1242
(a Polychlorinated Biphenyl and DDT on Cultures of
an Alga, Protozoan, Daphnid, Ostracod and Guppy".
Bulletin of Environmental Contamination & Toxicology,
Vol. 8, No. 3, pp 129-137.

Taub, Frieda B., "Insights Into Stability Of Bio-
logical Systems as demonstrated by a Chemostat" (in
manuscript), (1972)

Taub, Frieda B., "Continuous cultures of an alga and
its predator”, (in manuscript, accepted for presen-
tation at the symposium "Modern Methods in the Study
of Microbial Ecology", International Biological Pro-
gramme, Sweden, June 1972).

-73-


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an “0000+
7 OE
aS
TO
ca
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g
oe
» SET
age
- FRe
Bz $
7 38 : : : : : : : we
Eee =
7 REE " i" Y T T : va
Rae >
Se
7 : TYE
Ht
X ve woes y tpooov
os
7 : TST

tos ioore ty
—- = eos qoere las ieee

u0"y 40° 400%
som == E08 400e——

33


Table 1: continued

BM-5

Initialized on Med 63 changed to Med 66 on day 15.
Initialized flow at 400-600 ml reduced on day 33 to
250-300 ml. Suspected low CO, flow on days 29-33.
Gross fungal contamination day 39. Analyzed day 46.

BM-7
Flow on day 21, 150 ml day 22 O ml. Analyzed day
56.

BM-9

Initialized with media 63,stabilized at day 20.
Switched to media 66, day 21. Changed glassware
day 29 because of excessive wall growth. Analyzed
day 38.

48


e 4 MAX .86

¢ ‘200 300 0 ‘300 ‘600
FLOW

Figure 6. Response surface (isopleths) of algal
standing crop in downstream community
(flask B) at nutrient concentration
of 0.05 mM NO 3.

21


TS

Table 3B: BM-l Pigment Analysis BM 71-1

3-31-71
PIGMENTS
P & S Analysis Pheo pigment Analysis
mg/1 mg/L

chla chl b carotenoids Chl a Pheo a
A 562 2229 139 -478 148
B ~204 118 .116 247 160
G Yield .330 .176 134 .388 -080
Cc 1.431 0.550 0.330 1.256 0.312
D 2.492 0.514 2471 -731 2.951

RY 0.552 0.165 0.090 0.294 0.391


Table 8A: BM-9 Chemical Analysis

BM-71-9 10/28/71

Chly #90 (reinhardtii)

12 hrs. Dark, 12 hrs light
Shere unit. 3000 fc. Ts 22°C

Med #66 Whole Whole Liquid Liquid Whole
Coll Density Culture culture only only Culture
sensi “Bicrass" 130, Total uw
Alga toze ach free e * .- N re- c CPM
vol 6, Profoze op éxy wt Protein % Ko, mig Eo vered
107 /mb 3x10" /m2 L6nM , Ma/md ma/ml Protein mM mf mM mM__.__ per. ml. | per 10°ceL
1 1 t H
H i . ! |
A 485 | 2.63 +520 f, .5067 | .0878 ; 17.3 . 3.04) ¢.005; ! i 687
eo : rr
B 450 (2.54 .7100! .5356 | 41153; 221.5  2.08!¢.005! i : 973
i | : : ' ! E
i i ay. t
Green 1.2699 [38.9 1.40} <-005) : | 286

643

7
i
u

340 15.47} j1.450 | 6933
i
i
'
i

7 t i = 7
ce 475 |2.81; ~640 | .5467 | .1085 | 19.9; 2.99!<.005: H
oe nnn ioe he i
7 i i it : ‘ y :
Dp 455 (3.06: .135 +900 | +5844 tL vre4e | | 28,2! 1.60! ¢.008! i 603
en ; fr A i H H _ a
Red 300 |5.55/ ‘168 1.52 |! .7383 i «3318 44.9 | 0.94'<.005| : . 217

Contaminatg¥ on 10/6 Sterility showed + on 10/28 - It was negative on 10/20


EPA Review Notice

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

ii


erereree 000695

verse qoa0' dy

OFFSFEEEYEEES 666 50°ET

40


o000+ae

Model algal etanding crop in

experiment BM-5,

Figure 23,

90+ 400°s, 90+ 40079 yos3007 90+ 40072 +0
90+300°S, = = 90rd00% “90rg00% =~ 90 4007. a ioe”
90+ 30075, 90+300°% 40+ 300°€ 90° 4007 0
= 99°300°S ~ =~ 909400" ~ s= 9oe300%C ~~ ~~~ ~~ 908900"? v

38


SECTION VI
DISCUSSION

The response surfaces of the model provide a pictorial
demonstration of the current model which incorporates
our present understanding. The interactions between
light intensity and flow (also dilution) rates can be
seen by examining the isopleths at any one concen-
tration, e.g. Fig 1, 0.5 mM.NO,. As can be seen from
the isopleth marked 3 (3x10 célls/ml) this density of
cells can be maintained as the steady state population
over the light intensity range of 1000 to 6000 ftc. if
the flow is relatively low, but can also be maintained
at progessively higher flow rate at progressively
higher light intensities. The downstream community
without predation, Fig. 2, shows that the potential for
further growth is rather limited. The effects of pre-
dation will be marked over most, but na&all of the con-
ditions (cf Fig. 3 with Fig.2). The effects of the
predation are obvious when the range of conditions which
will support,the maximal standing crop (light stippled
area, 3.5xlO cells/ml) are considered. With predation,
the maximal densities can be maintained only with flow
rates greater than 400 ml and light intensities greater
than 3000 ftc. The diagrams demonstrate that the algal
standing crop is not altered by the potential presence
of predators at either very favorable or very unfavorable
conditions.

The protozoan standing crop which results from the algal
cells consumed are shown in Fig. 4. They are highest

at the conditions which have demonstrated major effects
on algal crop.

A reduction in nutrient concentration to 0.05 mM NO,,not
only sharply reduces the maximal standing crop, but also
shifts the pattern of the isopleths so that the highest
algal standing crops (moderately stippled area) occur
along the left border, i.e. maximal density can occur at
any light intensity but only at very low flows. The
maximum algal densities are only .5 - .7xlO cells/ml.
The effect of the protozoan predation is to sharply
reduce the.range of conditions which would sypport even
the 0.5x10° cells/ml, but the 0.3 and O.1x10 cells/ml
densities would occur under the same envirgnmental con-
ditions. Protozoan population is extremely low, .8 x lo at

63

the


SECTION IV
MATERIALS AND METHODS

Biological Culture Model

The biological/computer simulation model systems have been
used to study the control of algal standing crop by herbi-
vores over a range of environmental conditions of nutrient
concentration, light intensity and flow rates. The test
organims are an alga (Chlamydomonas reinhardtii) and
herbivorous protozoan (Tetrahymena vorax v.58) -

The biological culture model consists of a two-flask con-
tinuous culture with only algal cells and a parallel
system with the addition of protozoa in the second growth
flask. The alga only system flasks are designated:

Reservoir —> A > B => G
(upstream) (downstream) ("green" yield)

The alga-protozoan system flasks are designated:

Reservoir > Cc > D > R
(upstream) (downstream) (“red" yield)
alga pop- alga and alga and protozoan
ulation protozoan populations
populations

This arrangement permits the second growth flasks and yield
reservoirs to be maintained in the dark for determining
algal death and predation rates, or in the light for
determining algal regrowth-predation-light interactions.
The variables of nutrient concentration, flow rate, and
light intensity have all been shown to be major deter-
minants of the community behavior. In general, the
physical conditions are set, the culture monitored by
optical density (OD) and cell count until a steady state
is reached (one week with less than 10% change), and then
the culture is harvested for chemical data and cell
enumerations. In some of the latter experiments, con-
ditions have been changed during the states to examine
response times. In most experiments the flow rates could
not be maintained at adequate constancy throughout the
course of the experiment.


os

Table 3A: BM-l Chemical Analysis

Conditic BIOL AL MODELS 74 - 1
1090 ft, 12 hre, light (all flasks in light) February 24, 1971
22°C
Flow rate 140 ml/day (Condition 1)
BY 140_ +235
400
Whole Whole Liquid Liquid Whole
Culture culture only only Culture
Gell Density "Biomase" No, Total ‘
ash free . . R N re- Cc UPM
Algse Protez op dry wt Protein 7% No, Mit mie ered 7
x10°/m1 x10'/ml___16mM ¥g/ml, mg/ml ___ Protein mM __ aM mM mM per ml per 10°celle
vol.
mL ‘
485 A 3.98 oA7 233 +06 17.4 1005 - 6 605 194.1 48.8
395 Bo 46 033 +27 204 15.6 20003 0008.5 5003 343.9 74.8
G yield 2.65 +26 225 205 19.7 .006 0016.6 606 590.9 223.0
465 C 4.05 38 225 +05 21.5 0001 .001 6 6001 561.7 138.7
410 D 1.4 »OB 21 +20 204 19.9 0001 .0018 45 5001 152.8 134.0
R yield 1.05 For) +19 229 07 23.0 0.0000 .0025 .8 8000. 224.3 213.6
Conclusions:

Not Light Limited after 2nd flask.

W limited in the Ist flask .
Algal "mortality" between B and yield (to be further explored).
Consumption of algae occurred by protozoa;75% drop in algal cell density,presumably eaten in Flask D,slight further consump-
Protozoan “mortality” between D and yield (to be further explored). in yield bottle.
No indication of increased algal production with cropping.
Slight if any increased wah free as a result of cropping; could be due to increased algal uptake.


Table 6A: BM-5 Chemical Analysis

Condition: Sherer unit cc BM - 71-5
3000 fc. 7/6/71
12,hrs lite - 12 hrs dark

20 ¢ Bi G; D, R% in the Dark

Flow rate 300/Day

* Sterility Whole hole Liquid Liquid Whole
Coll Density Culture culture ro only Culture total “
Alsse Projoze oD a eeee Protein % ‘ro, ‘wig vad covered eer
210° fmt x10"/mi___16rtt Me/ml ng/ml Protein oM __ mit mM {per ml per 10°cells
Flask vor i ] i : : |
| 3.97 | -680 25089! 41214 | 23.9 2.23 .0005_ , 2577
2.47 : 465 | 4456 17.5 | 3.02 .0010 | 2661 _
| +680 i “5383 i 28.0 “2.78 0042 29 :
| .680 | .5206 | .1352 | 26.0 2.88 oolo0 : | 2598 7
1.33 1.035 -540 || 5189 _waa3a | 25.7 272 0047 ‘ ‘2939 _
‘Red ¥| 300 J 1.63 .720 I +540 I 1.246 + .1569 £ 12,6% 2.50 0042 ! 13068

* Both Red and Green units showed gross contamination on 6/28/71
appears to be fungal contamination.
BM-71-5 started on 5/26 w/Med 63.
attached Med #66 on 6/4/71


Increase = inflow-outflow+production-utilization

do
=do_- -
om 07 dO, +k Ay k,05P (4)

where A algae population, A

first flask, Ay second
flask (numbers/ml)

1

P-protozoan population (numbers/ml)
S-nitrogen substrate (mg/ml)

O-organic dissolved substances (mg/ml of nitrogen
content)

D,,D

yy algal and protozoan death rates (daily

rates)
Harty algal and protozoan growth rates (daily
rates)
d dilution rate (daily rates)
Y- yield coefficient of algae

R nitrogen substrate available for algae growth
from recycling

k, kjk, rate coefficients
In the model the growth rate of the algal
population is based on the Mechalis-Mentén equation modi
fied to include the effects of both light and substrate
concentration. The following equation fits the experi-
mental results at the .05 level of significance:
uoeu? LS

a max ————
L (K.+8) +SK)


EPA-660/3-73-008
August 1973

BIOLOGICAL MODELS OF FRESHWATER COMMUNITIES

By

Dr. Frieda B. Taub
College of Fisheries
University of Washington
Seattle, Washington 98195

Project 16050 DXM

Project Officer

Dr. Walter M. Sanders III
Southeast Environmental Research Laboratory
Environmental Protection Agency
Athens, Georgia 30601

Prepared for

OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460

For sale by the Superintendent of Documents, U.8. Government Printing Office, Washington, D.C, 20402 - Price $1.05


Experimental algal standing
crop in experiment BM-3

1 $

5

: 2

z
“9Or200°S 300: 0+ 300°T ow 300" 2 ros z00* Tt a
Soe3o0's Soca00"> Seeder sors00e2 Sosa0rt cH
90°300°S “m= == 90090087 ~ 10° 200°C 19¢300* 19+ 300°T — T
90*300°S 90+300°% 90*300°E 90+300°2 90+300°T “0

31


SELECTED WATER 2, Report No.) 2.
RESOURCES ABSTRACTS

INPUT TRANSACTION FORM

4. Title 5. Renort Dace
BIOLOGICAL MODELS OF FRESHWATER COMMUNITIES 6
- 8 Performino Organiiation
7. Author(s) Report No.
TAUB, Frieda B. 19. Project Ma,
16050 DXM

2% Organization

Washington University, Seattle, College of Fisheries |!!. Coaicvet/ Grant Mo
EPA Grant 16050 DXM

13. Type of Repor: and
Period Covered

12, Sponsoring Organization Y, §, Environmental Protection Agency Final Report

18. Supplementary Notes

Environmental Protection Agency report number,
EPA-660/3-73-008, August 1973.

16. Abstract
Data from continuous cultures of an alga (Chlamydomonas reinhardtii) and
protozoan (Tetrahymena vorax) have been used to construct a model of
algal standing crop over ranges of light intensity, dilution rate, and
nutrient concentration both in the absence and presence of predation by
the protozoa, ‘The model predicts that predation can reduce algal standing
crop only within certain ranges of the environmental variables.

The comparative toxicities of Aroclor 1242, a polychlorinated biphenyl,
and DDT, were tested on the alga and protozoan, and also on daphnids,
ostracods, and guppies. (Taub-University of Washington)

17a. Descriptors
*Model studies, *Algae, *Protozoa, Chlamydomonas, Ciliates, Bacteria,
Nitrates, Light intensity, Primary productivity, Secondary productivity,
cultures, Pesticide toxicity, DDT

17b. Identifiers
*Chemostats, *Polychlorinated biphenyls

7c. COWRR Field & Group $5C

18, Availsbility 19. Security Class. 21, Bo. of Send To:
(Report) Pages
; WATER RESOURCES SCIENTIFIC INFORMATION CEN
20. Securnty Class. | 22. Price US) DEPARTMENT OF THE INTERION TER
(Page) WASHINGTON, D. C. 20240

Abstrector_ Frieda B, Taub [evasion University of Washington

Wasic 12 (Rey NINE Hare


LIGHT

2

9 MAX .689

2000+

Figure 4.

FLOW

Response surface (isopleths) of pro-
tozoan standing crop in downstream
community (flask D) at nutrient con-

centration of 0.5 mM NO,.

19


RESEARCH REPORTING SERIES

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

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

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

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


0000" 8

: > aoe + * + a
rrr rere ee reeeesens cee cenrnqese crn erreeessserserernag:
: : * a + “8 .

Experimental algal atanding

|

1

i
Figure 28,

? : . 4
—900300"s ~~ - —--90+300%% Yor300ce oez00%2 19¢300° t= —— 0
90°300°S 90+300°% 9o+3007€. 90+300°2 90+300°C 20
- 90+300°S 90°300°7 ~yoes00%E = «~~ 90430022 = > - ~-~ 906300" © 9
90°300°S 9o+300"% 90-300" 90°300°2 90¢300°1 +o

43


LIGHT

3
6
i)
oe]
6
3
ol

MAX .995

‘300
FLOW

Figure 12. Response surface (isopleths) of pro-
tozoan standing crop in downstream
community (flask D) at nutrient con-

centration of 5.0 mM NO,-

27


Experimental and model pro-
tozoan standing crop in

experiment BM-4.

. . . -e ove .
— Wa epee ere rene ee eee EEN Cree nee get eee REE EEEE BOE EF OGUEET—
rs . 5

Figure 21.

0630078 foe300"9 €o+300%% — Co+3002
Co+a00%8 Co+a00%s C0+z00% == Oe g0082 ee.

36


LIGHT

MAX .57

FLOW

Effect of predation on the response
surface (isopleths) of algal standing
crop in downstream community (flask D)
at nutrient concentration of 0.05 mM

Ne
CO 3-

22


SECTION IIT
INTRODUCTION

One of the consistent effects of urbanization is the eutro-
phication of water bodies. To the extent that the associated
increased primary productivity channels to invertebrates and
ultimately to fish production, and provided that the algal
standing crop does not become a nuisance, eutrophication is
beneficial. Complex aquatic communities are thought to have
compensatory mechanisms which damp responses over some
undefined range of stress, e.g., added nutrients. When these
compensatory mechanisms are stressed beyond their capacity,
the established communities rapidly shift to less desirable
structures. The most notable undesirable attribute is
usually a high algal standing crop. The failure of herbi-
vores to effectively crop down high algal standing crops
(provided the greens are not replaced by blue-greens) is not
currently well understood. In these conditions, herbivore
populations must be limited by something other than food
availability.

The complimentary use of a biological culture system and

a mathematical simulation model has provided the opportunity
for rapid exploration of hypotheses on the mathematical
model, and a check of their validity with the laboratory
biological model. This study has provided an intermediate
test system between pure cultures and the complexities of
real lake systems. In situations where nutrient input cannot
be eliminated, many of the noxious conditions of decaying
algal blooms could be prevented if the conditions favoring
consumption could be enhanced.

This report emphasizes the yet unpublished results of the
study. The model described here has been developed in part
from the earlier published work of this grant. The first
study (Taub, 1969a) demonstrated that batch gnotobiotic com-
munities could be maintained for periods as long as 170 days
provided that new growth medium was occasionally added to
make up for the volume lost by sampling and evaporation.
Communities of an axenic alga Chlamydomonas were compared
with communities of Chlamydomonas with the herbivorous pro-
tozoan Tetrahymena and the bacteria Pseudomonas, Cytophaga
and Aerobacter. The algal density was not reduced by the
presence of the other organisms in these batch communities.



8s

Table 7A: BM-7 Chemical Analysis

9-20-1971

BM-7-71
12 hours light - 12 hrs dark
Scherer unit - continuous culture

3000 fc
Med #63 |.
Temp. 22°C Whole Whole Liquid Liquid Whole
Cell Density Culture culture oaly ofly Culture
ass" NO, Total “Me
ea 4 _ cage - N ree c “crx
Alsce Protoze op dry ve Protein % BO, RNG we evened 6
210° /mL 32.0 "/m1 160 ; Metal ma/rd Protein mf mM mM mM .__ per ml_._ per 10°cell.
vol 6 | |
A 427 .42x10 | .070 I ~2200 1 20419 : 18.6 0.4240 /,0002, !
i | oe : ee
B 390 1,47x10° ; +300 | .2850 | .0457: 18.0 0.0084 ¢.0002 |
Green 520 1.06x10° 1.400 ; 22979 | .0364, 12.2 0.0030 ¢, 0002 :
c 427 .56x10° | 2140 | .2039 70380 | ;
! : mete Ee '
D 445 .82x10°.o0s6x1p*.340 | 2511 | .0433 |’. 17.3 '0.0088 0002 :
j > * 7 ; -
Red 540 1,47x10° .0044x10*. 328} .3217 | .0480' 14.9 0.0068 0002 :

Sterile Culture


Gout

outa,

aguytn,

poorer

Model algal standing crop in

experiment BM-9.

teseeeeee ue

” Figure 29.

_90+300°S

90 40

Yor su07E Yor au Jor qoord

yoe300"%

44


ALGAL DENSITY IN CELLS/ML

Response of Chlamydomonas reinhardtii to the
addition of various concentrations of Aroclor 1242@

i
1
!
S.
0 i @— control
1 O— solvent control
! A-- 2 ppm.
i O-- 2ppm.
fn 8-- 20 ppm.
'
i
‘
\ott}— ~ ——
(e} 2 4 6 8 10 1l2 14 6 18 20 22

DAYS
Fig.3l. Effect of the addition of 0.2, 2, and
20 ppm Aroclor 1242 on the growth of
Chlamydomonas reinhardtii. Each point
represents the mean of two obser-
vations wnose individual values are
indicated as the range.

46


The nominal environmental conditions were, therefore,
only an approximation of the actual experimental con-
ditions, and it was necessary to use an average flow
rate and average cell densities for the period of
"steady state" conditions when the culture was har-
vested for chemical analyses; the temperature was
20°+ 2c; light was provided by cool white fluorescent
and incandescent (except at 1000 ftc, when only the
former were used); the approximate culture volume was
500 ml per flask; the nutrient solution was Medium 63
(Taub and Dollar, 1968), except when noted that NO,
was altered (PO, concentrations were also altered to
maintain the same N:P ratio).

Computer Simulation Model

The computer simulation model computes the population
densities of the algal and protozoan populations and
the concentrations of dissolved organic and inorganic
substrates, given the physical conditions, by the pro-
cedure outlined below- Each of these four modeled
components is represented by a differential equation,
which expresses the rates of change of the component
with respect to time. The model starts with the basic
Lotka-Voltera equations for a predator-prey system.
The differential equations are then modified to include
the effects of inflow and washout to represent the
chemostat environment.

Second flask equations:
Increase = growth-mortality-outflow+inflow-predation

dA
=u_A
2 We

x -DA,-dA,+dA, -k,A,P (1)

2

Increase = growth-mortality-outflow

aP = u_P-aP-D,P (2)
at

Increase = inflow-outflow-consumption+recycling

2 = aS,-dS,-W A+R (3)
at Y