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        Response of  Chlamydomonas reinhardtji  to the
         addition of  various  concentrations of Aroclor I242(
  I06H
5!
^
K
                                    O	 control
                                    O	 solvent control
                                    A-- .2 p. p.m.
                                    D- - 2 p.p.m.
                                    S-- 20 p.p.m.
-
,1

\ /'

' 1 1 , - 	 	 -1 	 1 	 1 	 1 	 1 	 1 	 I .....
0 2 4 6 8 10 12 14 16 18 20 2
  10
Fig. 31
                           DAYS
           Effect of the addition of O.2,  2, and
           20  pprn Aroclor 1242  on the growth of
           Ch 1 amydomona s reinhardt ii . Each point
           represents  the mean  of two obser-
           vations whose individual values are
           indicated as  the range.
                       46
-------
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         .5        360       3000 B,G,D,R, in
                                      dark

      71-4         .5        500       30OO B,G,D,R, in
                                      dark

      71-5         5        3OO       3000 B,G,D,R, in
                                      dark

      71-7         .5        5OO       3OOO all  flasks
                                      in  light

      71-9         .5-5      30O       30OO all  flasks
                                      in  light

Major deviations in experimental conditions.

BM-1
Experiment initialized at 3OOO ftc changed to 10OO ftc
on day 13.  Flow rate high and variable(  1OOO ml-40 ml)
average  5OO ml until day 22 changed to 14O ml/day.
Day 27 1O ml of culture in Flask B was  inoculated into
Flask D.  Day 13 protozoans re-inoculated to density of
.1 x 1O  analyzed day 35.

BM-3
Light intensity initialized at 1000 ftc changed to 3OOO
ftc on day 7 temp fluctuation on day 8  22.5    26.8 C
for 4-5 hours.  Cultures contaminated on  day 2O.
Analyzed day 24.

BM-4
    Operation per design.  Flow fluctuation, 340-62O.
Analyzed day 19.

                            47
-------
Table 1:  continued

BM-5
Initialized on Med 63 changed to Med 66 on day 15.
Initialized flow at 400-6OO 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
-------
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
B
G
C
D
R
BM 71-3 A
B
G
C
D
R
BM 71-4 A
B
G
C
D
R
4.33 140
4.95
3.15
4.54 148
1.15 .477
1.03 .269
3.31 332
4.75
4.23
3.78 330
1.56 .330
.76 .211
1.69 505
2.66
1.67
2.22 496
1.98 .066
1.26 0.37
BM 71-5 A
B
G
C
D
R
BM 71-7 A
B
G
C
D
R
BM 71-9 A
B
G
C
D
R
3.92
2.67
3.74
3.21
1.94
1.99
1.45
1.81
1.60
.74
1.30
1.61
1.70
2.60
5.00
2.75
4.32
6.84
257


251
1.103
.836
505


520
.OO69
.0075
347


316
.0515
.0635
-------
Table  3A:   BM-1   Chemical  Analysis
    Conditlf
BIOLOt   \L MODELS 70-1

 February 24. 1971
o
22 C
Flow rate 140 ml/day
0^140 ^.35
400
Cell Density
Algae Protoz.
& i. °°

vol.
raj.
485 A
Ul
O 395 B
G yield
465 C
410 D
R yield
xlO /ml


3.98

4.6
2.65
4.05
1.14
1.05
xlO /ml 16mM


.47

.33
.26
.38
*0£ .21
^,(J3 .19
(Condition 1)
Whole Whole
Culture culture
"Biomass"
ash free
dry wt Protein %
MK/ml


.33

.27
.25
.25
.20
.29
rag/ml


.06

.04
.05
.05
.04
.07
Protein


17.4

15.6
19.7
21.5
19.9
23.0
Liquid
only
mM

'
.005

.0003
.006
.0001
.0001
0.0000
Liquid
only
mM


-

.0008
.0016
.001
.0018
.0025
Whole
Cul ture
Total
NH£ N re~ .
covered
mM


.6

.5
.6
.6
.5
.8
mM


.605

.5003
.606
.6001
.5001
.8000,
14
C CPM

per ml


194.1

343.9
590.9
561.7
152.8
224.3
,
per 10 cells


48.8

74.8
223.0
138.7
134.0
213.6
Conclusions:
  Not Light Limited after 2nd flask.

  N limited in  the 1st flask .
  Algal "mortality" between B and yield (to be further explored).
      Consumption of algae occurred by protozoa;757. drop in algal cell density,presumably eaten in Flask D,slight further consimp-

       Protozoan "mortality" between D and yield  (to be further explored).                        ln yleld bottl««
       No indication of increased algal production with cropping.
       Slight if any increased NH4 free as a result of cropping; could be due  to increased algal uptake.
-------
 Table 3B:  BM-1  Pigment Analysis
                                           PIGMENTS
                                                                   BM 71-1
                                                                   3-31-71
         chla
                    P & S Analysis
                        mg/1
             chl b
carotenoids
                                                      Pheo pigment Analysis
                                                              mg/1
Chl a
Pheo a
A
B
 .562
 .204
G Yield  .330
D
RY
        1.431
2.492
0.552
.229
.118
.176
0.550
0.514
0.165
.139
.116
.134
0.330
.471
0.090
.478
.247
.388
1.256
.731
0.294
.148
.160
.080
0.312
2.951
0.391
-------
Table 4A:   BM-3   Chemical Analysis
Sherer unit Continuous
Condition:
3OOO fc. 12 hrs on, 12 hrs dark
20* c (B, G, D, Red-Dark)
Flow rate: 360 ML/Day
Culture: * Sterility
Cell Pcnsitv
Volume
455 A
455 B
360^3
507 uC
565 t)
•36**
Algae
x!06/ml
3.29
3.66
4.39
3.43
1.72
. 685
Protoz. OD
xlCT/ml 16raM
i ':
1 . 250 !'
! I'
! . 275 j.
j . 252 |
I . 244 i;
. 189 i . 210 i;
. 182 | . 145 I
Culture
Whole Whole
Culture culture
"Bionass"
ash free
dry wt Protein
M
-------
(Jl
CO
    Table 4 B:  BM-3  Pigment Analysis
                 P & S Analysis
                    mg/1
                                            PIGMENTS

A
B
G
C
D
R
chl a
0.96
1.12
1.06
1.02
1.12
0.83
chl b
0.34
0.41
0.41
0.36
0.36
0.24
Carotenoids
0.28
0.31
0.33
0.27
0.34
0.27
                  BM-71-3
                  4-26-71
Pheopigment Analysis
        mg/1
                                                              Chi  a
                                                              0.829
                                                              0.94
                                                              0.93
                                                              0.84
                                                              0.73
                                                              0.37
               Pheo a
               0.34
               0.27
               0.46
               0.30
               0.32
               0.77
-------
Table 5A:  BM-4  Chemical Analysis
Sherer Unit CC
Condition:
3OQO ft. 12 hrs light B
20 C 12 hrs dk. D
Flow rate 500 /Day
+ Contaminated
Cell Donritv
xX06/ml
Protoz. OD
xlO /ml 16
Vol
425 A, 2.53
£> 445 B 2.35
i
StKJf'G 2.10
425 d+ 2.52
43O D + 3.08
48O'/R4- 1.80
.026
.039
.G. (Dark)
.R. (Dark)
Whole
Culture
"Bicr.iass"
ash free
dry w£
:r:M Mf,/rol
26o! .2094
290 ; .2137
220 ! .1983
270 . .2072
220 ! .1883 I
215 j .2139 i
BM 71-4
5-19-71
VSiols Liquid Liquid Whole
culture only only Culture
KO Total ^
'ro „-. jjjjt N re- C CPM
Protein % ' 2 '"'4 covered
m^/t-,1 Protein c£I mH reJ-1 mM per ml per 10 cell
.0387 18.5 .140 O.OOO2 , ' 2604
i
.0385 18.0 .0288 " i ; ' ; 2551
.0472 ', 23.8 .0270' " ; ; '• 3128 '
.0401 ; 19.4 .0992, : . j 3104
.0474 j 25.2 .0002 " : ! 2918
.0456 ; 21.3 .0078! " ; ; ' 2681
-------
     Table 5B  BM-4
                                     PIGMENTS
             BM-71-4,  5-19-71
     P & S Analysis
        mg/1
Chi a      Chi b      Carotenoids
      Pheopigment Analysis
            mg/1
Chi a             Pheo a
A
B
GY
C
D
RY
.72
1.00
.86
.84
.95
.93
.27
.40
.41
.32
.34
.37
.16
.26
.20
.19
.24
.22
                                                    .64
                                                    .87
                                                    .70
                                                    .73
                                                    .82
                                                     766
                    .17
                    .24
                     .29
                    .19
                    .294
-------
         Table  6A:   BM-5   Chemical  Analysis
t_n
cn
Condition:  Sherer unit CC
3000 fc.
120hrs
-------


Table 6B BM-5
' Flask Chi a
A 4.64
B 3.01
*-,fW— r. _ 	 .».-.*,..... --
GY " 4.23 	
C 3.66
D 5.13
RY ' 	 "~ 	 5.61 •-••


P & S Analysis of Pigments
mg/1
Chi b
1.97
1.20
" 	 ~ 	 "'"•• " 	 2.13 - 	 	 ' "
	 :i;77 ~"~ 	 " '" 	
	 	 - 	 1.99 - ' •"" 	 ""
. 2.00'" ~"
BM 71-5
7-6-71

Carotenoids
0.90
0.59
" ' ' '1.04' ~
--•"- - +,820" 	
••" ""' "1.3 "~
1.22
-------
 Table  7A:   BM-7   Chemical  Analysis
                 9-20-1971

BM-7-71
12 hours  light - 12 hrs dark
Scherer unit - continuous culture
3OOO fc
Med #63
I.. -A
• -••I
Ln A
00
B
Green
C
D
Red
Temp. 22°C
Coll Densitv
:cl06/ml :d.OA/ml
Vol
427 .42x10
390 1.47x10
520 1.06xlO
427 .56x;106
Whole Whole
Culture culture
"BioTMss"
OD dry w£ Protein
I&TDM K"/:i\l ns/rl
.070
; .22OO
.300 .2850
.4OO
.140
445 .82x'l06.0056xlD4.340
540 1.47xl06.O044x|l04.328
: .2979
• .2039
! .2511
i .3217
1
.O419 :
.0457 •
.0364 i
.0380 j
.0433 |
.0480 '
Liquid Liquid Whole
only o31y Culture
KO Total u
\. ... ' + N re- C CPM
% "2 'N 4- J ' covered ,
Protein s£! iri-1 nsM nM per ml per 10 cell,
! : i
18.6 0^240 ^.0002| ! j 2269
1 . {
16. 0 O.O084^.OOO!2 \ [ 2847
12.2 0.003d <. 0002 J i' 3079
18.2 0.166': s. 0002 j 1.1796
i ^ . !' ;
' ••-•--•; 	 j !; ,
17.3 ;0. 0088 ^.0002 i , 3124 :
14.9 O.OO68COOOJ2 | ', 323O
   Sterile Culture
-------
           Table 7B:   BM-7
                        PIGMENTS

                        BM 71-7

                        Sampled 9-20
                          P + S Analysis
                              mg/1
un
*£>
              A
B
             GY
              D
             RY
                    chlor a
      0.700
1.137
      1.046
                    0.922
      0.841
      0.775
                   chlor b
             0.242
0.440
             0.387
                   0.337
             0.408
             0.372
                           carotenoids
              0.193
0.281
              0.263
                           0.2O6
              0.220
              0.159
Pheo Analysis
    mg/1
Pheopigments*


0.121

0.042

0.310

0.134

O.O71

O.419
             *  Variation between duplicates was larger than usual.
-------
      Table 8A:  BM-9   Chemical Analysis
     BM-71-9   10/28/71
     Chly #9O (reinhardtii)
     12 hrs. Dark,  12 hrs light  Q
     Shere unit.  3OOO fc.   Ts22 C
     Med #66                     Whole
Whole
Liquid Liquid  Whole

A1"
VolxlO
A 485
B 450
340
reen
C 475
D 455
Red 300
Coll Densitv


2.63
2.54 :
5.47 j
2.81 !
3.06 '• .135
5.55 ' ''.16B


OD
16rnM
.520
.71OO
1.450
.640
.900
1.52
i^uii-ure
Bicrsass'
ash free
dry wt
Mi /ml
'
i .5067
' .5356
; .6933
: .5467
! .5844
; .7383
cuicura cr»j.y oniy culture
.^°3
Protein % ™2 W14 m*
c^/nl - Protein siM mM mM
" |
.O878 ; 17.3 3.04i<-.005;
.1153 • 21.5 2.08K.005J
; . : i
.2699 j 38.9 ; 1.40J^.OO5j
.1085 j 19.9 , 2. 99 |<. 005;
.1646 ' 28.2 [ L.6o'<.005:
.3318 44.9 . 0.94;<.005[

local J^A
N re- C CPM
covered ,
mM per ml per 10 cell
i.
i
j 687
; 973
; 1 286
\ ; 643
:603
i: 217
Contaminat^ on 1O/6   Sterility showed +  on  1O/28 -  It was negative  on  1O/2O
-------
   Table 8B.  BM-9  PIGMENT ANALYSIS

            P & S Analysis
               mg/1
B
chl a
3.99
5.56
11.16
5.75
7.34
13.39
chl b
2.02
2.51
5.10
2.25
2.71
7.10
Carotenoids
1.58
1.44
3.02
1.41
1.82
4.00
       No Pheo Analysis conducted
-------
(Ti
                                               TABLE 9
             Summary of PCB toxicity studies.  +++—many survivors; ++ ~ most survivors;
                                + ^. few survivors;  0 =: no survivors.
Test Table
Situation # age dose of PCB (ppm)

Daphnia
fed PCB-containing
cells*
direct addition of
PCB-alcohol
non-fed
fed
PCB-containing
algal cultures


Ostracods
PCB-containing
algal cultures
Guppies
direct addition
of PCB-alcohol



2


3
3

4
5
5


6

7
8
* Daphnia in PCB-free
O.OOO2 O.OO2 O.O2 O.2 2


young 	 	 	 +++ +


young +++ +++ + 00
young +++ +++ ++ 00

arln 1 f- 	 — D

adult 	 +++ +++ ++ 0
adult 	 +++ +++ 0 O


adult 	 +++ +++ +++ ++

young 	 	 	 	 0
young 	 +++ +++ ++ 0
media were fed washed Chlamydomonas cells
20


O


O
0

O
o
o


0

o
0
grown
obser-
vation
day


4


4
4

3
2
9


3

7
1
in PCB
        suspensions of the  indicated doses.
-------
                    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, O.5 mM NO    As  can  be seen  from
the isopleth marked 3  (3x10  cells/ml) this density of
cells can be maintained as the steady state population
over the light intensity range of 10OO 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  not 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, 3.5x10  cells/ml) are considered.   With predation,
the maximal densities can be maintained only with flow
rates greater than 4OO ml and light intensities greater
than 3OOO 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  O.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 - .7x10  cells/ml.
The effect of the protozoan predation is  to sharply
reduce the range of conditions which  would  support even
the 0.5x10  cells/ml, but the O.3 and O.lxlO  cells/m.1
densities would occur under the same  environmental con-
                                                        4
ditions.  Protozoan population is extremely low,  .8 x 1O at the
                         63
-------
most, and would be ineffective over most of the environ-
mental range.

Increasing the nutrient concentration to S.OrnM 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.O 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 1O fold.

The examination of the actual experimental runs is com-
plicated by the high variability in daily flow rates
(usually ± 1O%, occasionally ± 2O%).  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
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. - f  (algal cone., substrate cone.)
     Algae protein cone. - f (substrate cone.)
     Algae growth rates - f (substrate cone., 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.

At - time lag  for algae response to changing conditions
threshold level of nitrate substrate  needed for algal
growth
protozoan growth rates - f  (algal density, algal
protein cone.)
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-1  (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 3O 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
-------
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  (O.2, 2, and 2O ppm) of Aroclor 1242-alcohol on
the density of Ch1amydomonas 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 Ch1amydomonas
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
-------
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 Ch1amydomonas 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.

                              14
In order to determine whether   C 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.OOO2, O.O2, O.2,2, and 2O
ppm), incubating NaH  CO ,  with samples taken from the
cultures at three different points in the  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   C uptake per cell
were all markedly reduced and showed no dose-related
response.  These results were undoubtedly  influenced
by the varying amounts of nutrients still  available.
The   C 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
-------
factors in the delayed growth and subsequent recovery
of the Ch1amydomonas 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 Ch1amydomon a s 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 2O ppm (mg/1) PCB had
been added, O.52 mg were detected in the algae, and
only O.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 lost 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.

                                    14
The effect of DDT on the growth and   C uptake of
Chiamydomonas cultures was studied.  In the tested
concentration range of O.2 ppm to 2O ppm DDT, the
algal growth curves did not differ appreciably from
those of the controls. It is thus possible that
Chiamydomonas, like Dunaliella, is relatively DDT
resistant, and yet may be effected by polychlorinated
biphenyls which may restrict   C 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  (O.Ol 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
-------
toxic to Daphnia pulex, however.  Young Daphnia died
at levels as low as O.O2 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
revealed 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 1OO% 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 (O.O8 g/1 and
4 g/1 Nutrient Borth); DDT  (10 ppm) and an algicide
(1.2 ppm, 2,3-Dichloro-l,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
-------
     2.  Community Metabolism

The 0  exchange of yields from some of the continuous
culture have been studied by C  uptake, continuous 0
and pH recording, or photosynthetic respirometry-  It
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.O 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 N0~ 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
-------
                     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
-------
                    SECTION VIII

                LIST OF PUBLICATIONS

1.  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.

2.  Taub, Frieda B., "A Biological Model Of A Freshwater
      Community:  A Gnotobiotic Ecosystem," Limnology
      and Oceanography.. Vol. 14, No. 1,  pp 136-142
      (1969a).

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

4.  Taub, Frieda B., "A Continuous Gnotobiotic (Species
      Defined) Ecosystem", pp 101-12O.  In John Cairns,
      Jr, ed., The Structure and Function of Fresh-Water
      Microbial Communities, American Microscopical
      Society,  (1969c).

5.  Morgan, Janet R, 1972.  "Effects of Aroclor 1242R
      (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.

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

7.  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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 SELECTED WATER
 RESOURCES ABSTRACTS
 INPUT TRANSACTION FORM
                                               I. Report No.
                                                                  w
 4.  Title
           BIOLOGICAL MODELS OF FRESHWATER COMMUNITIES
 7.  Aathor(s)
           TAUB, Frieda B.
         Washington University,  Seattle, College of Fisheries



12. Sponsoring Organization  u. S.  Environmental Protection Agency
25. Supplementary Notes

        Environmental Protection Agency report number,
        EPA-660/3-73-008, August 1973.
                                                                    S. PerfoTtaitJ!' Organization
                                                                      RejfoitNo.
                                                                  :0. Pfojecttio.
                                                                       16050 DXM
                                                                   11,  Conicsctl Grunt Wo
                                                                        EPA Grant 16050  DXM
                                                                   13.  Type of Repoz- and
                                                                       Period Covered
                                                                         Final Report
 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.  (Ta'ui-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
 17c. COWKR Field & Gioup  05C
 18, Availability
                        19.  S,' rarity C 'fisg.
                            (Report)

                        20.  Secun iy Class.
                            (Page)
21. No. of
   Pages

22. Price
Send To:
                                                       WATER RESOURCES SCIENTIFIC INFORMATION CENTER
                                                       U.S. DEPARTMENT OF THE INTERIOR
                                                       WASHINGTON, D. C. 2O240
 Abstractor  Frieda B.  Taub
                                      Ia-:.Hu:tion  University of Washington
R S i C ' O 2 (R £ V i i IN £•:
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