Effect of DDT On Growth Rate and Photosynthesis in Pediastrum boryanum (Chlorococcales)

EFFECT OF DDT ON GROWTH RATE

AND PHOTOSYNTHESIS IN PEDIASTRUM ptt) Sehictte eae

Robert Alan Michaels

EFFECT OF DDT ON GROWTH RATE AND PHOTOSYNTHESIS

IN PEDIASTRUM BORYANUM (CHLOROCOCCALES )

by

ROBERT ALAN MICHAELS

B.S., The City College of New York, 1967

A Thesis Submitted to the Graduate Faculty of the University of Georgia in Partial Fulfillment of the

Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

EFFECT OF DDT ON GROWTH RATE AND PHOTOSYNTHESIS

IN PEDIASTRUM BORYANUM (CHLOROCOCCALES)

by

ROBERT ALAN MICHAELS

roved:

J raeeaMlegaaal” /4 >/

a. Mp Led Date Lagecad 2 19) Chairman, Reading Committee 4 /

Approved: ;

Dean, Graduate School

Cagust 4/92

Date

ACKNOWLEDGMENT

This traditional space shall serve to express my appreciation of and respect for my major professor, Dr. James Schindler, for his guidance and encouragement, and above all for his being a fine, sensitive, far from traditional human being.

My sincere thanks are extended to Dr. Donald Scott and Dr. Eugene Odum for reading my thesis and making helpful (and copious) suggestions for its improvement.

Dr. James Schindler and Mr. Ken Honick are thanked for their help in operating the gas analyzer, which may well explain its longevity. For the rest, I have only myself to blame, although my friends and fellow students, with whom I shared many disconnected experiences,

contributed to my rather healthy delinquency.

iii

TABLE OF CONTENTS

Page LIST OF TABLES 2. 1 ww ee ee ee ee ee v LIST OF FIGURES . 1 1 6 ee we ee ee ee ee ee vi

INTRODUCTION © 6 we ee ee ee ee he 1

“ON THE BIOLOGY OF PEDIASTRUM BORYANUM AND NAVICULA

PELLICULOSA . 1 1 1 ee ee ee ee 4 METHODS AND MATERIALS . 2 1 we we ee ee ee ee ee 8 STATISTICAL ANALYSIS «ow 1 ww ee ee ee ee ee 16 RESULTS £6 ee ee ee ee 22 DISCUSSION OF RESULTS . 2 1. we ee ee ee ee ee ee ee 39 ADDITIONAL RESEARCH CONSIDERATIONS . 2. 1 ee ee ee ee ee ee 49 SUMMARY AND CONCLUSIONS 2 6 6 1 ee ee ee ee ee ee 52 LITERATURE CITED 2 ww ww ee ee ee ee ee ee ee 54

APPENDIX 21 we ee ee ee ee ee 56

iv

10.

LIST OF TABLES

Defined medium for P. boryanum ..... 1... 2.24 Relationship between optical density and cell density

for P. boryanum. 2. 1 ee ee ee ee ee ee ee

Regression equations relating time (X) and difference in cell density (Y) from controls up to 160 hours of growth, at three DDT concentrations (X = hours,

Y = cells/ml.) for P. boryanum and N. pelliculosa. .

14¢ experiment data... 2. ee ee ee Yotal inorganic carbon data... ... 1... ee eee P. boryanum growth experiment data .........--.

P. boryanum growth experiment data transformation to

cell density vs. time. . 2... . ee eee ee ee N. pelliculosa growth experiment data. ........

Cumulative effect of DDT on P. boryanum cell density as a function of time. .......

Cumulative effect of DDT on N. pelliculosa optical

density as a function of time... . se eee eee

Page

Figure

4-5.

6-7.

10-11.

12.

13.

14,

1S.

16.

17.

18.

LIST OF FIGURES

Young P. boryanum colonies (x 2,000) (after Smith,

1916) Growth chamber with aeration

Relationship between optical

density: P. boryanum . Effect of DDT on P. boryanum

sions. Growth experiments

Effect of DDT on P. boryanun sions. Growth experiments

Effect of DDT on P. boryanum sions. Growth experiments

Effect of DDT on P. boryanum sions. Growth experiments

Effect of DDT on N. pelliculosa cell density:

regressions .

Effect of two DDT concentrations on light !4C-uptake

in P. boryanum

befect of 1.0 ppm DDT on net P. boryanum.......

Effect of DDT on P. boryanum experiment la . oe .

Effect of DDT on experiment Ib .

|~

. boryanun

and sampling apparatus density and cell

cell density: regresla andb .

cell density: regreslc and 2a .....

cell density: regres2b andc....

cell density: regreslabe and 2abc, means

light !c- “uptake in

cell density: growth

cell density: growth

Effect of DDT on P. boryanum cell density: growth experiment Ic. .

Effect of DDT on P. boryanun cell density: growth experiment 2a.......

vi

Page

ll

‘igure Page

19. Effect of DDT on P. boryanum cell density: growth experiment 2b... 6 1 we ee ee ee ee ee ee 83

20. Effect of DDT on P. boryanum cell density: growth experiment 2c... ee ee eee ee ee ee 84

21. Effect of DDT on P. boryanum cell density: growth experiments labc, mean effect... .. 2... ee eee 85

22. Effect of DDT on P. boryanum cell density: growth

experiments 2abc, mean effect. .........08. 86 23, Effect of DDT on N. pelliculosa cell density ...... 87

INTRODUCTION This study investigated the effect of DDT on photosynthesis, respiration and population growth rate in two algae by comparing the rates of fixation of '4C-labeled bicarbonate, total inorganic carbon and optical density changes of DDT-treated and control cultures. The

algae were Pediastrum boryanum (Chlorococcales) and the pennate diatom

Navicula pelliculosa. They were chosen because they were available,

easily cultured, fresh water representatives of different algal divisions.

Little research has been reported on the effects of DDT on algae, and still less on fresh water algae (see discussion). Existing research indicates a great variability in response to DDT of algal population growth rate and photosynthesis. Further investigation into the different conditions under which DDT may be expected to affect these processes is needed to answer such questions as: (1) Do colonial forms differ from unicellular forms in their sensitivity to DDT?

(2) Do skeletal structures affect the uptake process? (3) Are increasing levels of DDT increasingly toxic, or is there a threshold and a saturation level between which toxicity is independent of concentration (is a dose-response curve demonstrable)? (4) What are the ecological implications of the DDT concentrations found in the fresh water bodies of the world? (5) By what mechanism is the uptake of DDT achieved?

The present study does not answer these questions. A separate

discussion is presented of a few problems suggested by the present research.

A number of hypotheses have been advanced to characterize the disruption in cellular systems caused by DDT. Most of this work dealt with animal, especially nerve, cells (Matsumura and Patil, 1969; Hilton and O'Brien, 1970; Matsumura, 1970; Koch, Cutkomp and Do, 1968; Koch, 1969). DDT inhibits the Na*,K*,Mg2*-adenosine triphosphatase system in rat brain nerve endings (Matsumura and Patil, 1969). These authors state that the Na*,k*,Mg@+-adenosine triphosphatases are thought to contribute to the process of active transport in many biological membranes. Perhaps disruption of the membrane systems of algal cells occurs as well, with the formation of an "ion gate" (Hilton and O'Brien, 1970), altering the normal permeability of the membranes.

The nature of such disruption is hypothesized by Hilton and O'Brien (1970), who report that the K* conductance induced by 1o-& molar valinomycin in lecithin-decane membranes is reversed by 3 x 10-6 molar DDT.

Wurster (personal communication) states that DDT and DDE are inhibitors of electron transport in both chloroplasts and mitochondria, and that this was obviously at the root of the photosynthetic inhibition reported in his experiments (Wurster, 1968).

Chloroplasts isolated from some DDT-treated varieties of barley showed reduced Hill reaction activity, as measured by the rate of reduction of the blue dye 2,6-dichlorophenolindophenol to its leuco (colorless) form (Lawler and Rogers, 1967).

The exact mechanism of action of DDT in algae is not known; nor is

it known why effects observable in some algae are not observable in others. The present study demonstrates reduction of the population growth rates in P. boryanum and N. pelliculosa, with no effect on the rate of photosynthesis. Respiration rate is apparently increased by

addition of DDT.

ON THE BIOLOGY OF PEDIASTRUM BORYANUM AND NAVICULA PELLICULOSA

Pediastrum boryanum (Turpin), Meneghini

Division: Chlorophyta Class: Chlorophyceae Order: Chlorococcales Family: Hydrodictyaceae

P. boryanum occurs as an irregularly shaped colony consisting of 8, 16, 32, or 64 multinucleate cells (Smith, 1916), though occasionally 128 celled colonies cccur (Fritsch, 1965}. In young cultures, larger colonies predominate, while 8 and 16 celled colonies predominate in cultures over a month old. The colonies are not invested with a gelatinous sheath, as are many members of the Chlorococcales (Smith, 1916).

According to Smith (1916), each cell contains 1 or 2 (in young colonies) to 4, 8, or 16 nuclei (in mature colonies). The number of pyrenoids may vary. Starch bodies produced in photosynthesis are visible around them, and are used for storage.

Colony formation can be by asexual or sexual means. In asexual reproduction, according to the description of Smith (1916), each cell in the parent colony eventually divides, though not simultaneously, into 16 (biflagellate (Fritsch, 1965)) zoospores. These are discharged through a split in the mother-cell wall, the innermost layer of which

contains them. The empty "ghosts" of the parent colonies are left

behind. Rapid movement occurs within the vesicle, lasting for about 15 minutes, by which time the irregularly shaped mass of cells forms a flat plate. After four hours, by gradual stages of emargination, the cells have developed the horns characteristic of the species (Fig. 1). By 24 hours the intercellular spaces have been eliminated, and the cells have become closely applied to one another. Growth in size follows.

In sexual reproduction (Fritsch, 1965) biflagellate isogametes are produced in the same way as the zoospores. They are smaller and in larger numbers, and liberated as individual swarmers through a hole in the parent-cell membrane. Following fusion of the isogametes,

reduction division and coleny formation occurs.

Navicula pelliculosa

Division: Pyrrophyta Class: Bacillariophyceae Order: Pennales Family: Naviculae

Sexual reproduction in N. pelliculosa has never been reported. According to the description of Darley (1969 and personal communication), vegetative reproduction begins with a light-dependent increase in size in the pervalvar direction, accommodated by slippage of the silica girdle bands past one another. A mitotic nuclear division ’ occurs, followed by cytokinesis. The resulting biprotoplastic cell consists of two daughter protoplasts enclosed within the parent

frustule. Silica deposition begins within the daughter protoplasts in

Figure 1. Young P. boryanum colonies (x 2,000) (after Smith,

1916)

Young P. boryanum Colonies (X 2,000) (after Smith, 1916)

the median position corresponding with the raphe, and proceeds toward both girdle bands. By an unknown mechanism, new cell walls are laid down within the new valves, and the old cell walls between the new valves disappear.

The result is two complete cells which at first adhere to one another, but soon separate into cells of equal size. No diminution of size as a result of vegetative reproduction, characteristic of many diatoms, has been reported. The whole division cycle takes about

10 hours.

METHODS AND MATERIALS

Growth Experiments

The effect of three concentrations of DDT on growth of P. boryanum (Chlorococcales) and N. pelliculosa (Bacillariophyceae) was measured. Periodic readings of optical density, shown to be linearly related to cell density, were taken to monitor growth of cultures from inoculation to senescence. Cultures containing one of three concentrations of DDT (0.01, 0.1, 1.0 ppm) applied in acetone solution as a carrier were compared with two controls containing no DDT, one being an acetone control to measure the effect of acetone on growth. (All DDTcontaining cultures, regardless of concentration, received the same amount of acetone as did the acetone control.) Optical density was measured with a Klett-Summerson Photoelectric Colorimeter (model 800-3).

A defined medium was employed to culture P. boryanum (Table 1). The diatom N. pelliculosa was cultured on the same medium with the addition of 100 mg/1 Na,Si0,-9H,0 as a silicon source. Ail media were autoclaved for at least 20 minutes before use.

Stock cultures were maintained in test tubes for one month. Fresh media were inoculated from these at the end of each month, and kept in the laboratory at room temperature under fluorescent and incandescent light 24 hours per day, in addition to ambient sunlight from the

windows. Larger aliquots of culture medium were inoculated to yield

Table 1. Defined medium for P. boryanum

Ca(NO,) *4H,0 100 mg/1 vitamin B,,

K, HPO, *3H,0 135 mg/1 biotin

MgSO, 7H,0 25 mg/1 thiamin-HICl Na,CO, 20 mg/1 trace elements

glass distilled water to 1.0 liter

pH 8.2 + 0.1, adjusted with NaOH and HCl

1.0 ug/1 2.0 ug/1 0.5 mg/1

1.0 ml1/1

*/H5BO, 568 mg/1 ZnCl, 624 mg/1 CuC1, +2H,0 268 mg/1 NaMo0,, +2H,0 252 mg/1 MnC1, + 4H1,0 360 mg/1 CaCl, -6H,0 420 mg/1 FeSO, +7H,0 2500 mg/1 Na, tartrate +2H,0 1760 mg/1

glass distilled water to 1.0 liter

10 cultures of appropriate size and density for experiments.

Ten empty acid (HC1 and H,SO,) bottles, and five one-gallon vinegar jugs, sufficed as growth chambers (Fig. 2). They contained from 2000 to 2500 milliliters of culture medium. These were arranged in three horizontal groups of five similar bottles suspended before a battery of four fluorescent lights such that each bottle within a group received equal light intensity. A 250 watt incandescent light was placed opposite the fluorescent lights, with the 15 growth chambers between the two light sources. These and presumably all other incidental sources of light illuminated equally the members of each group. Light was applied continuously during experiments (intensity 4000 foot candles), and the cultures were at 27 to 28°C.

Aeration, and consequent agitation, of growth chambers was accomplished by introducing a cotton filter between the laboratory air supply and a series of 15 manifolds, connected in parallel by rubber tubing. Each manifold controlled air flow (which was vigorous) to one growth chamber equipped with a rubber stopper bearing three glass tubes with the following functions: one inlet tube (with a secondary cotton filter) for delivery of air to the bottom of the chamber, one short exit tube (with a cotton filter to prevent possible airborne contamination), and one sampling tube drawing from the bottom of the chamber, operated by closing off the exit tube to increase atmospheric pressure over the culture sufficiently to force out the desired sample. Each sampling tube contained a short length of Tygon plastic tubing which was sealed off by a clamp except during sampling.

Sampling was preceded by vigorous shaking of the chambers for

Figure 2.

Growth chamber with aeration and sampling apparatus

Growth Chamber with Acration and Sampling Apparatus

Sampling

Tygon 2. we foTT Air supply tubing-->, ; yw Clamp i Rubber \ Sample “me | Flask D>

Algal

~~ cnleure

12 about 10 seconds to insure homogeneity of the samples. This was especially important when sedimentation of algae occurred. A 10 milliliter initial aliquot was discarded to empty the sampling tubes of unmixed material before samples were accepted.

Sequential 10 milliliter samples were taken approximately three times daily from each chamber. They were drawn directly into a Klett tube and optical densities were recorded immediately. Sampling times were chosen for convenience and/or (visually) apparent change in color of the cultures. Experiments lasted 5 to 10 days.

One growth experiment was performed using N. pelliculosa in which

only five growth chambers were used, and these were placed in a 23°C

water bath with overhead fluorescent and incandescent light sources.

l4carbon Experiments

The effect of two concentrations of DDT (1.0 and 10.0 ppm) on the

rate of !4carbon-labeled bicarbonate fixation by P. boryanum was

measured at various stages in the growth curve, at constant temperature (23°C) and light intensity (1500 foot candles). DDT was introduced in acetone solution into two biological oxygen demand (BOD) bottles, while two untreated BOD bottles received equal amounts of pure acetone, which was allowed to evaporate from all bottles. A second set of four BOD bottles was treated similarly to provide replication of each experiment.

Algae were obtained from a stock culture of known optical density, previously prepared for the purpose, and were introduced into the BOD

bottles in approximately equal amounts (120 milliliters). In some

13 cases the cultures were incubated for 24 hours before addition of the isotope.

l4c was introduced as liquid bicarbonate prepared from solid Ba*CO, (New England Nuclear Corporation, Boston, Mass.) by SteemannNielsen's (1952) method. Ampoules containing fixed amounts (approximately 10 pCi per ampoule) of the isotope were broken open and the contents removed with a 10 milliliter syringe. The ampoule, followed by the syringe, was washed once with culture material to ensure complete transfer. The culture was then capped and shaken vigorously to achieve homogeneity. Dark controls were covered with aluminum foil immediately, and all bottles were then incubated in a water bath.

After five hours the BOD bottles were removed and the contents filtered under reduced pressure through Millipore filters (HA 0.45 yu) (Millipore Filter Corporation, Bedford, Mass.). The filters holding the algae were washed with 20 milliliters of 0.001 N HCl to drive off adsorbed !4c, and then with 30 milliliters of distilled water. Placed in labeled two-inch diameter planchets, the filters were wetted with liquid soap to keep them flat, and desiccated in a carbon dioxide-free atmosphere to prevent loss of fixed !4C through exchange with atmospheric carbon dioxide. (The desiccant was silica gel, and absorption of atmospheric carbon dioxide was accomplished by the presence of soda lime in the desiccator.)

Following desiccation the filters were counted by use of a Spectro/Shield Scaler (model 8703) coupled with a Spectro/Shield Automatic Low Background Planchet Sample Changer (model 110), and

Printing Timer (model 008437) (Nuclear Chicago, Des Plaines, I1l.).

14 The machine was programmed to count beta emissions through a thin window for a pre-set total of twenty-thousand counts, recording the

number of minutes for this to be achieved.

Inorganic Carbon Experiments

Inorganic carbon was converted to carbon dioxide and measured with a Lira Infrared Analyzer (model 200, Mine Safety Appliances Co., Pittsburgh, Pa.). Thirty 10 milliliter ampoules received 5-milliliter aliquots of culture medium of known optical density. Half of these ampoules received 1.0 ppm DDT from a prepared stock solution in which the acetone fraction of an acetone solution of DDT was allowed to evaporate before addition of the algae.

The 15 treated and 15 untreated ampoules were each divided into three groups of five: initial, final-light, and final-dark. Initial total inorganic carbon was determined from the initial five ampoules of both groups. Five treated and five untreated ampoules were wrapped in aluminum foil, while the remaining 10 ampoules were left exposed.

The final groups were then incubated in a water bath at 24°C and exposed to fluorescent light of intensity 1500 foot candles for 24 hours. Total inorganic carbon was then measured with the infrared analyzer. From the three inorganic carbon values (those for the initial, final-light and final-dark groups) from the treated and untreated ampoules, net and gross photosynthesis and respiration were determined by use of the following formulas:

Gross photosynthesis = Net photosynthesis - Respiration,

Net photosynthesis = Light total inorganic carbon (TIC) -

15 Initial TIC, Respiration = Dark TIC - Initial TIC, and

Gross photosynthesis = Light TIC - Dark TIC.

Determination of Relationship between Optical and Cell Density

A culture of P. boryanum was prepared for the purpose of relating optical density to cell density. At 10 different optical densities arrived at during the maturation of the culture, ranging from 12 to 116, samples of the algae were counted by use of a haemacytometer.

Each cell count estimate (in cells per milliliter) was derived from totals from four entire fields (25 squares per field) of the haemacytometer.

The number of groups of cells and cells per group were also counted to determine whether cells of P. boryanum occurred in groups of

different sizes at different optical densities.

STATISTICAL ANALYSIS

Relationship between Optical and Cell Density

A linear relationship between optical density and cell density was established (Fig. 3), defined by the linear regression equation

Eq (1) Y = 45,578 X - 683,008, where X equals optical density in Klett units and Y equals cell density in cells per milliliter.

This equation was derived from data (Table 2) obtained from haemacytometer counts. The strength of the linear relationship was measured by the correlation coefficient (r) which was equal to 0.96, 100 r? being equal to 92.9, indicating that 92.9 percent of the variation in cell density could be attributed to differences in optical

density.

Growth Experiments

A linear transformation of data from Klett units to cells per milliliter (Tables 6 and 7, Appendix) was made for growth experiments with P. boryanun, using equation (1). Since the relationship between optical and cell densities was not determined for N. pelliculosa, no transformation of data was possible (Table 8, Appendix).

For each time, the change in cell density since the preceding time was calculated. Each resulting value was then added to all those that

preceded it, producing a cumulative change in cell density for each

Figure 3.

P. boryanum

Relationship between optical density and cell density:

Relationship Between Optical Density and Cell Density: P. boryanum

Py

Cell Density (cells/ml X 10

oot ES TE, MELT ee a as el a wl)

CLT SR PIETY SANE OA MERE TNRIE ACO UA RABE

0 25 50 75 1c0 125

Optical Density (klett units)

Table 2. Relationship between optical density and cell density for P. boryanum

moe: se —

Optical density Cell density Round to (Klett units) (cells per milliliter) (« 108)

0 0 0.0 12 184,000 0.2 17 132,500 0.1 25 612,500 0.6 32 356,666 0.4 37 500,000 0.5 43 857,500 0.9 63 2,040,000 2.0 75 2,090,000 2.1 101 4,530,000 4.5

116 4,930,000 4.9

total amount of time clapsed in the growth experiment. The cumulative changes in cell density occurring in the acetone control were subtracted from those for each DNT concentration, thereby isolating the cumulative effect of the DDT factor alone on cell density at each DDT concentration (Tables 9 and 10, Appendix).

For N. pelliculosa, the cumulative effects of DDT were expressed as optical rather than cell density changes, but this difference does not change the shape of the curves (Fig. 15 through 23, Appendix).

In one set of P, boryanum growth experiments, no acetone control was run for one of the three simultaneous replicates. In order to make use of the data from this replicate, the following method was used to generate values that probably would have occurred had an acetone control been available. At each time, the ratio of the mean of the two acetone controls to that of the two respective non-acetone controls was multiplied by the corresponding value of the non-acetone contrel of the replicate lacking an acetone control. The justification for this procedure lies in the assumption that the effect of acetone is the same for all three replicates.

P, boryanum data were pooled into two groups each composed of the mean values of three simultaneous replicates, for purposes of visual comparison.

For each of the 24 curves for P. boryanum and three N. pelliculosa eurves, linear regression equations were generated, and the strengths ef the linear relationships (r) computed. The regression equations were computed using data to only 160 hours of growth, since the systems

become more complex (see discussion of results) beyond this time, and

the results begin to reflect "bottle effects" more than treatment

effects.

luc Experiments

A transformation of data from Klett units to logarithms (base 10) of cells per milliliter was made, using the linear regression equation

Eq (2) log Y = 5.17 + 0.015 X, where X is the optical density in Klett units and Y is the cell density in cells per milliliter, Equation (2) is derived from equation (1), and has a correlation coefficient of r = 0.95. r* = 0.90, indicating that 90 percent of the variation in cell density can be accounted for (but not necessarily caused) by changes in optical density.

This transformation was used because the resulting regression equation has a positive Y-intercept, making it unnecessary to consider "negative'' populations of algae at low optical densities.

At each cell density, two experiments were performed, with either of two DDT concentrations: 1.0 ppm or 10.0 ppm. For each experiment, dark fixation (counts per minute minus background per milliliter) was subtracted from light fixation of radioactive bicarbonate. Net fixa-

tion of treated groups was then expressed as a percentage of controls.

Total Inorganic Carbon Experiment

The treated (1.0 ppm DDT) and untreated total inorganic carbon (TIC) data were of three types: initial, final-light, and final-dark. F-tests were used to compare the variances of the data from the treated and control groups of all three types. The variances of the data from

the treated groups were found to be equal to those of the untreated

21 groups at the 5 percent level (i.e., it was not 95 percent certain that the variances were unequal).

Net primary production (NPP) equals final-light TIC minus initial TIC. In order to arrive at a value for NPP, the mean of the initial TIC data was subtracted from that of the final-light TIC for both the treated and untreated groups. The NPP for the control (untreated group) was subtracted from that of the treated group. A confidence interval for this difference was erected using the t-statistic, at the 5 percent level. If the confidence interval included zero, the NPP's were considered to be equal for the treated and control groups.

The gross primary production (GPP) and respiration values were compared analogously. The GPP and respiration of the control group were subtracted from those of the DDT-treated group, and confidence belts erected within which it was 95 percent certain that the true differences lay. If the confidence intervals included zero, the values

were considered equal.

RESULTS

Growth Experiments

In 21 of 24 P. boryanum, and all 3 N. pelliculosa regressions (Fig. 4 through 12), the correlation coefficients were high enough (for the number of values considered) to conclude at the 2 percent level of

~significance that the linear relationship did not result from chance alone (Table 3).

In all cases the slopes of the regression lines were different from zero. (A slope of zero indicates no effect of DDT on growth.) Confidence intervals for the slopes at the 5S percent level did not, generally, include zero. For P. boryanum this was true of all experimental units treated with 1.0 ppm DDT, all but two treated with 0.1 ppm

DDT and of ali but three treated with 0.01 ppm DDT. For N. pelliculosa,

all three confidence intervals for the slopes did not include zero at the 5 percent level.

An analysis of variance of the slopes of the regression lines for the P. boryanum experiments was performed. Four slopes were compared for the four DDT concentrations: 0.0, 0.01, 0.1 and 1.0 ppm. The hypothesis that the means of the slopes of all four regression lines were equal was tested.

The variance ratio, F, was high enough to indicate at the 1 percent level that the greater variation between samples than within

samples did not occur by chance, but could be attributed to a real

Figures 4 and 5.

Effect of DDT on P. boryanum cell density:

regressions. Growth experiments la and b.

P)

Cell Density Difference from Control (cells/ml X 10

Effect of DDT on P. boryanum Cell Density:

———- 0.01 ppm

rousssesas 1.0 ppm

Time (hours)

Regressions. Growth Exp’ts. la and b

Time (hours)

Figures 6 and 7.

Effect of DDT on P. boryanum cell density:

regressions. Growth experiments le and 2a.

Cell Density Difference from Control (cells/ml X 10°)

|. Je |

Effect of DDT on P. boryanum Cell Density:

———- 0.01 ppm soonsnseens - 0.1 ppm

aumzme 1.0 ppm

70 190 130 160

ES (oo)

Time (hours)

Regressions. Growth Exp’ts. Ic and 2a

40 70 400 130

Time (hours)

Figures 8 and 9. Effect of DDT on P. boryanum cell density:

regressions.

Growth experiments 2b and c.

Effect of DDT on P. boryanum CellDensity: Regressions. Growth Exp’ts. 2b and c

_

a <) fs

1 ray EIN AL REIT ARES a EN OT BNE TE LTS SY AGES MPR SA REID

' i)

——--- 0.01 ppm

1 w

mast 1.0 ppm

Cell Density Difference from Control (cells/ml x 10°)

1 >

70 100 130 160 40 70 100 130 160

» fon)

Time (hours) ; Time (hours)

Figures 10 and 1l.

Effect of DDT on P. boryanum cell density:

regressions. Growth experiments labe and 2abc, means.

Cell Density Difference from Control (cel!s/m! X 10°)

Effect of DDT on P. boryanum Cell! Density:

Time (hours)

Regressions. Growth Exp’ts. labe and 2abc, Means lg

Time (hours)

Figure 12.

sions.

Effect of DDT on N. pelliculosa cell density:

regres-

Effect of DDT on_N- pelliculoss Cell Density: Regressions.

Cptical Density Difference from Control (Klett units)

0 40 70 100 130 160 Time (hours)

reat NK aT KEN

0.01 ppm

ASECIMEDATAMAMAN 1.0 ppm

Table 3. Regression equations relating time (X) and difference in cell density (Y) from controls up to 160 hours of growth, at three DDT concentrations (X = hours, Y = cells/ml.) for P. boryanum

a = =: =

Growth DDT No. of Correlation Experiment Concentrations Readings Coefficient No. (ppm) Regression Equations (N) (r)

0.01 Y= 0.1659 - 0.0034 X 10 -0.88 1 0.1 Y = 0.3507 - 0.9071 X 10 -0.97 1.0 Y= €.9310 - 0.0156 X 10 -0.96 0.01 Y = -0.1301 - 0.0001 X 9 -0.05* 2 0.1 Y= 0.0997 - 0.0027 X 9 -0.77 1.0 Y= 0.3305 - 0.0061 X 9 -0.90 0.01 Y= 0.3664 - 0.0054 X 10 -0.93 3 0.1 Y= 0.3147 - 0.0049 X 10 -0.92 1.0 Y= 0.8730 - 0.9125 X 10 -0.96 0.01 Y = 0.4148 - 0.0056 X 9 -0.77 4 0.1 Y= 0.0445 - 0.0013 X 9 -0.58* 1.0 Y= 0.2705 - 0.9074 X 9 -0.99 0.01 Y = -0.2057 + 0.0012 X 9 0.62 5 0.1 Y = -0.4366 + 0.0052 X 9 0.89* 1.0 Y = 0.1705 - 0.0072 X 9 -0.99 0.01 Y= 0.3131 - 0.0068 X 9 -0.98 6 0.1 Y= 0.4032 - 0.0078 X 9 -0.97 1.0 Y= 1.0996 - 0.9187 X 9 -0.97

Table 3 (continued)

Growth DDT No. of Correlation Experiment Concentrations Readings Coefficient No. (ppm) Regression Equations ay) (r) (r?) 0.01 Y= 0.1322 - 0.0029 X 10 -0.94 0.88 Mean 0.1 Y= 0.2530 - 0.0049 X 10 -0.97 0.95 123 1.0 Y= 0.7102 - 0.0114 X 10 -0.97 0.93 0.01 Y= 0.1741 - 0.0037 X 9 -0.86 0.74 Mean 0.1 Y = 0.0050 - 0.0013 X 9 -0.91 0.83 456 1.0 Y= 0.5137 - 0.0111 X 9 -0.99 0.98 0.01 Y= 0.1509 - 0.0033 X 19 -0.57 0.32 Mean 0.1 Y= 0.2459 - 0.0043 X 19 -0.56 0.31 1-6 1 Y= 0.6053 - 0.0112. X 19 -0.82 0.67 0.01 Y= 0.14 - 0.20 xX 18 -0.96 0.92 For N. pelliculosa** 0.1 Y=-2.10 -0.16 X 18 -0.93 0.86 1.0 Y=-1.22 -0.14 X 18 -0.95 0.90

*Signifies lack of 98 percent certainty that the population correlation coefficient is not zero.

**X = hours, Y = Klett units.

30 difference in the actual slopes (betas) of the regression lines for the

four DDT concentrations.

IMC Experiments:

From the graph below (Fig. 13) it can be seen that net !4C bicarbonate fixation was variable, and that no effect of DDT on net fixation was apparent at either DDT concentration. There was insufficient justification for assuming linear relationship (r = 0.23).

A t-test was made of the hypothesis that the mean of all values of 14 uptake as a percent of contrcls equalled 100 (i.e., that net fixation among treated groups is 100 percent of that of controls).

This hypothesis was accepted at the 5 percent level, indicating that it was less than 95 percent certain that there was a difference between treated and untreated groups.

Because there was a loss of degrees of freedom in expressing net 14¢ bicarbonate fixation by treated units as a percentage of controls, the absolute values were compared (Fig. 14 and Table 4). An F-test for equality of variances was performed in which equality of variances was verified at the 5 percent level. Analysis of variance was used to test the hypothesis that the mean of all net 14¢ fixation values of the treated groups was equal to that of the untreated groups. The means

were equal for both 1.0 ppm and 10.0 ppm DDT, at the S percent level.

Total Inorganic Carbon Experiment

The net primary production (NPP) values differed significantly (Table 5). The range between which it was 95 percent certain that the

true difference ir NPP's lay was 0.059 to 0.341 mg C/1/day. Because

Figure 13. Effect of two DDT concentrations on light 14C-uptake

in P. boryanum

14 Effect of Two DDT Concentrations on Light C-Uptake in

P. boryanum

160 140) - 120 5 0) S fo) w ° l i CPE LEAT eink CQ Oma) SLA 2& [so 5.2 54s CSG 5.8 6.0 a O Log Cells per Milliliter ‘ ) mm 980) fe) a or x? |

Net Nn oO

Ee

O denotes 1.0 ppm DDT X denotes 10.0 ppm DDT

Figure 14, Effect of 1.0 ppm DDT on net light !4C-uptake in

P. boryanun

C-Uptake in P boryanum

Effect of 1.0 Ppm DDT onNet Light

8.04

N“ , ne mre 3 ey re o oé ° NN "tee, NN tee, ~~ Meee, NN rey, NN td <>< N o =< NP Xx NN NX Ss. N N ‘2 Oy 0 aASoOapg og

(0 X e3nuru z3d

sjunod) Jaary iad a, 307

5.6 5.8 6.0

5.4 Log Cells per Milliliter

x denotes DDT-treated

5.2 0 denotes Control

5.0

Table 4. !"C experiment data

Experiment No. Light- Counts per min. 14¢-uptake (log cells/ml) Dark Counts per min. Vol. minus background as a percent (ppm DDT) (L-D) DDT-Control minus background (mi) per ml of control

1 L Control 45,452 126 361 (5.36) L DDT 54,051 132 499 114.4

(1.0 ppm) D Control 2,519 128 20

D DDT 2,401 125 190

2 L Control 49,997 128 Bel (5.36) L DDT 47,616 152 313 76.2

(1.0 ppm) D Control 2,217 130 170

D DDT 4,402 154 29

3 L Control SU ,S06 126 721

(5.60) L DDT 105 ,260 132 797 S$i-8 Gas

(1.0 ppm) D Control 10,360 128 81 4Z-7

D DDT 24,688 125 198

4 L Control 99,997 128 781 (5.60) L DUT 64,513 152 424 58.8

(1.0 ppm) D Control 31,247 136 240

D DDT 16,390 154 106

5 L Control 666 ,664 126 5,291 (5.89) L DDT 999 ,997 132 7,576 150.9

(1.0 ppm) D Control 68,963 131 526

D DDT 48,777 127 384

Table 4 (continued)

Sa

=

Experiment No. Light- Counts per min. 14¢-uptake (log cells/ml) Dark Counts per min. Vol. minus background as a percent (ppm DDT) (L-D) DDT-Control minus background (m1) per ml of control

6 L Control 666 ,664 128 5,208 (5.89) L DDT 666,664 152 4,336 81.0

(1.0 ppm) D Contrel 83,330 131 636

D DDT 86,954 127 685

7 L Control 58,821 120 490 (5.29) L DDT 60 ,603 120 505 90.6

(1.0 ppm) D Control 1,405 120 12

D DDT 8,581 120 72

8 L Control 76,920 129 641 (5.29) L DDT 71,429 120 595 88.6

(1.0 ppm) D Control 870 120 7

D DDT 4,013 120 33

9 L Control 399 ,994 120 3,333 (5.35) L DDT 399,994 120 3,333 100.6

(1.0 ppm) D Control 7,214 120 60

D DDT 4,848 120 40

10 L Control 399 ,994 120 3,333 (5.35) L DDT 399,994 120 3,333 100.8

(1.0 ppm) D Control 6,129 120 51

D DbT 6,036 120 50

ve

Table 4 (continued)

—== —

SS ES

Experiment No. Light- Counts per min. 14c-uptake (log cells/ml) Dark Counts per min. Vol. minus background as a percent (ppm DDT) (L-D) DDT-Control minus background (ml) per ml of control ll L Control 166,661 120 1,389 (5. 80) L DDT 166,661 120 1,389 100.0 (10 ppm) D Control 2,126 120 18 D DDT 2,138 120 18 12 L Control 181,812 120 1,515 (5.80) L DDT 181,812 120 1,515 100.1 (10 ppm) D Control 2,650 120 22 D DDT 2,460 120 21 13 L Control 499 ,994 120 4,167 (5.51) L DDT 499 ,994 120 4,167 100.4 (10 ppm) D Control 25,968 120 216 D DDT 24,090 120 201 14 L Control 499 ,994 120 4,167 (5.51) L DDT 499 ,994 120 4,167 102.2 (10 ppm) D Control 39,984 120 333 D DDT 29,845 120 249

s¢

Table 5. Total inorganic carbon data

Initial Final-Light T.1.C. Final-Dark T.I.C. T.I.C. (mg C/1) (mg C/1) No. (mg C/1) No. 1.0 ppm DDT No. 1.0 ppm DOT 1 3.4 6 1.9 11 3.6 2 3.4 7 2.2 12 3.5 3 3.4 8 2.1 13 3.7 4 3.3 9 1.8 14 5 3.1 10 2.0 15 3.7 Final-Light Final-Dark Initial (mg C/1) (mg C/1) No. (mg C/1) No. Untreated No. Untreated 16 3.1 21 1.3 26 3.2 17 3.1 22 1.9 27 3.4 18 3.2 23 1.2 28 3.1 19 3.0 24 2.2 29 2.9 20 3.1 25 1.6 30 3.2 Xx = 3.3 xX = 2.0 xX = 3.6 (1-5) (6-10) (11-15) Xx = 3. xX = 1.6 xX = 3.2 Xc16-20)" 3° X (21-25) (26-30) ~

1.0 ppm DDT

tt

Net Primary Production (NPP) = L - I = -1.3 mg C/1

Respiration = D - I = 0.3 mg C/1

Gross Primary Production (GPP) = L - D = -1.6 mg C/1 0.0 ppm DDT

NPP = -1.5 mg C/1

Table 5S (continued)

E.

F,

0.0 ppm DDT (continued) Resp. = 0.1 mg C/1

GPP = -1.6 mg C/1

38 this range is entirely above zero, the inorganic carbon present after Olin day was probably higher for the treated algae. The NPP was therefore lower for the DDT-treated algae, since less of the inorganic Carbon was converted to organic carbon in the process of photosynthesis and/or more was utilized in the process of respiration.

The gross primary production values did not differ significantly between the control and DDT-treated groups. The confidence interval for the difference included zero, and the GPP's were considered equal.

Respiration of the control group, when subtracted from that of the treated group, was not equal to zero, and the confidence interval erected also did not include zero. Respiration was higher for the

Dbl-treated group than for the control group, the range between which

it was $S percent certain that the true difference Jay being from

9,95 to 0.35 mg C/1/day.

DISCUSSION OF RESULTS

Growth Experiments

Population growth rate (incrcase in biomass) is a function of the difference between photosynthetic production and respiratory consumption, and it varies with the maturity of the algal culture. Juvenile cultures display a large excess of photosynthetic activity over respiration. At maturity, photosynthetic production equals respiratory consumption, In closed systems and laboratory cultures, a sequence from juvenile to mature to senescent stages occurs with cultures eventually beconing increasingly limited by the nutrient content of the mediun, the metabolites and growth inhibitors present in it, as well as simple crovding and competition for light resulting from one cell shading another (Fogg, 1965). These effects, to the extent that they are more important in the growth chambers than in nature, fall into the general category of "bottle effects," and become increasingly important as the growth experiment progresses. The data obtained in the experiments here reported indicate 160 hours to be an appropriate cut-off point, up to which treatment effects predoninate and after which bottle effects predominate. In addition to the self-limiting characteristics ef a competing population of algae, bottle effects also encompass the increasing contribution of bacterial browth to the dynamics of the system (in part due to the substrate provided them by the growth

chambers).

According to Gromov (1967), the weight of bacteria in open air algal cultures may reach 60 percent of the algal biomass, with the most active bacterial multiplication usually occurring only in the first stages of cultivation. Gromov reports that when the density of algal cells became high, many bacteria] forms died partially or completely, and that some algae exerted inhibitory action on the phages of the coli-typhoid bacterial group. He states that the cause of bacterial death in algal eultures is as yet unknown, but that some authors believe it to depend largely upon pll changes in the medium. Depression of bacterial growth in some Chlorella cultures cultivated in organic glucose media was connected with pl! fall, according to Gromov. In ‘purely mineral] media, Gromov reports insignificant bacterial contamination, but where contamination did occur it sometimes resulted in inhibition of algal population growth rate, or death. Organic compounds exereted by algae were found to be available to some bacteria.

In the growth chambers of the present study, excretion and nonbiological processes like solution and oxidation of pigments and organic compounds from dead algae could contribute significant organic nutrients to the medium and cause an increase in bacterial population. This could account for the tendency of optical density to increase even after the cultures were discolored, sedimented and obviously unhealthy. At this stage, both light and nutricnt limitation are indicated.

After 160 hours of growth, significant degradation of the DDT could have occurred as a result of the limited ability of some algae to degrade it (Christie, 1969) as well as the possibility of bacterial

degradation by such species as Hydrogenomonas (Focht and Alexander,

41 1970) and hai: ..n-induced photolysis of DDT (Miller and Narang, 1970).

If degracation of DDT does occur it may introduce an additional source of organic carbon into the treated cultures, which would result in greater bacterial growth in treated than control chambers. The additional bacterial contribution to optical density could explain why the optical density of the treated cultures sometimes surpasses that of the controls.

Clearly the effects of DDT on growth rate cannot be nicasured when this stage arrives, since bottle effects predominate over treatment effects. Natural systems are characterized by a diversity of algae and the presence of other trophic levels. Increases in population of one alga give way not to wholesale collapse of the natural system, bui to a succession of dominance of other species of algae. According to Wurster (1968), selective toxic stress by DD? on certain algae may alter the species composition of a natural phytoplankton community. Menzel, Andersen and Randtke (1970) state that chlorinated hydrocarbons, while not toxic to all species, may exert a dramatic influence on the succession and dominance of individual forms. The natural system containing DDT is likely to have a relatively constant DDT concentration, since there is an exchange between the dissolved and suspended fractions and the sediments (Cope, 1966), or a constant input from industrial sources. In contrast, the growth chambers received a single input of DDT which was probably taken up by the algae at once. Sédergren (1968) reports that Chlorella cultures with a density of 1 x 10? cells per milliliter can accumulate 1.6 milligrams of DDT per

liter, and that 1.9 x 108 cells can absorb more than 0.38 micrograms

42 of 14C-DDT.

At inoculation, the effective DDT dose per algal cell in the experimental chambers is relatively close to the applied dose. After numerous cell divisions, the fixed DDT content of the chambers is distributed over a larger number of cells, with a consequent reduction in the effective dose per cell. Experimental evidence of this phenomeNon was reported by Wurster (1968). The magnitude of the DDT-induced

reduction of photosynthesis in Skeletonema costatum decreased with

increasing cell concentration.

Rapid bubbling of air into experimental chambers probably resulted in significant reduction in DDT content of treated growth chambers by the process of codistillation. Codistillation of DDT in aquecus systems is higher than the theoretically derived value because of the tendency of DDT to concentrate at the air-water interface (Acree, 1963; Cope, 1966) and enter the atmosphere with water molecules. Another source of loss of DDT from the experimental systems was through sampling.

In comparing treated and control chambers, the division of the data into a two-component system is helpful. After 160 hours of growth, there is a tendency of the optical density of the DDT-treated cultures to catch up to and sometimes surpass that of the controls. This is not surprising. Since DDT reduces population growth rate up to 160 hours of growth, it also reduces the rate of utilization of the nutrients present in the medium and the rate of output by the algae of growth inhibitors and metabolites. Therefore the control cultures would

becone limited by bottle effects earlier than would the PDT-treated

43 cultures. Eventually the treated cultures would contain as meny algae as the control cultures, since the upper limit of population is still determined by the nutrients available in the inedium, even though the rate of their utilization may be slowed by DDT.

The present study shows that DDT, in all three experimental doses, reduces population growth rate in most cultures of P. boryanum and all

of N. pelliculosa. Similar research demonstrates great variability of

effect of DDT on the growth rates of different algae. Using material from a waste stabilization pond, Christie (1969) found less than a 3 percent difference between mixed DDT-treated (100 ppm) and untreated

cultures containing Scenedesmus, Chlorella, Ankistrodesmus and Euglena,

after four days of growth. Measuring cells per milliliter in uniaigal

cultures of Chlorella pyrencidosa at pH 6.0 and 9.0, Christie also

found less than a 10 percent difference between DDT-treated (100 ppm) cultures and controls. A similar result is reported by Palmer and Maloney (1955) using

Cylindrospermum licheniforme, Microcystis aeruginosa, Scenedesmus

obliquus, Chlorella variegata, Gomphonema parvulum and Nitzschia palea.

2.0 ppm DDT was toxic to only Microcystis and Gomphonema during part of

their growth curves. Ukeles (1962) reports lack of effect of 0.6 ppm

DDT on growth of Dunaliella euchlora, Chlorella sp., Protococcus sp.

and Phacodactylum tricornatum, but inhibition of growth in Monochrysis

lutheri. Christie (1969) theorizes that lack of effect is a result of ‘the low solubility of DDT in water (1.2 ppb (Babers, 1955)) and some ability of the algae to degrade it.

Menzel, Andersen and Randtke (1970) report no significant effect

of DDT up to 1 ppm on Dunaliella tertiolecta and Coccolithus huxleyi

cell division, but significant blockage of cel] division in Skeletonema

costatum and Cyclotella nana.

lic Experiments

Because population growth rate (biomass increase) is a function of the rates of photosynthesis and respiration, agents like DDT which influence population growth rate may interfere with or enhance these processes. The reduction of population growth rate by all three DDT doses suggests a possible effect on photosynthesis and respiration.

The !*C-bicarbonate fixation experiments provide a measure of relative photosynthetic rates tor treated and control algae. While it is possible to derive approximate net or gross primary productivity values from specific net !4C bicarbonate fixation values (Doty and Oguri, 1959; Ruttner, 1966; Steemann-Nielsen, 1952; Strickland, 1960; Strickland and Parsons, 1968), relative valves are adequate for most primary productivity studies (Vollenweider, 1969).

Sddergren (1968) studied !4C-DDT uptake in Chlorella, and reported uptake at the end of one minute to be not less than the amount retained after 24 hours of exposure. Fifty-two to 77 percent of the added M4C-pDY was accumulated in Chlorella, and uptake was completed in under 1S seconds. Evidence was presented indicating the involvement of physical, rather than biological, processes in the 14c¢_DDT uptake mechanism in Chlorella. Assimilation processes were precluded by the

discovery that the rate of uptake was equally rapid after killing of

the cells. This suggested that the rate of penctration through the

45 cell wall is a measure of the diffusion rate of !*C-DDT molecules in aqueous solution. Absorption rather than adsorption was the dominant process, since contamination of fresh media when exposed algac were transferred to them did not occur. Had adsorption been the dominant uptake process, an equilibrium with the fresh medium would have been established.

Considering the rapid uptake of 14C_ppT in Chlorella, it was

considered prudent in the present study to add !4C-bicarbonate to the BOD bottles immediately after mixing of the DDT and algac, allowing 5 hours of incubation thereafter. No significant difference between

treated and control cultures was observed.

s suggested th

o igges

t DDT uptake in P. boryanur

that observed by Sddergren (1968) in Chlorella. The colonial form of P. boryanum, and its skeletal structure, would support this possibility.

DDT uptake in P. boryanum may be primarily an adsorption process, unlike that of Chlorella, a naked unicell with relatively great surface area for absorption. P. boryanum occurs in groups of cells, often complete colonies, which reduces the cell's surface area for absorption. Skeletal protrusions increase its total surface area for adsorption. That DDT tends to concentrate at surfaces and interfaces (Acree, 1963; Cope, 1966), supports the hypothesis of adsorption as the major uptake process, followed by absorption into the cell.

For the !4C-bicarbonate fixation experiments, the implication of this possibility was that more time was required for penetration

through the cell wall to occur. Twenty-four hours were allowed for

incubation of all cultures, including those exposed to DDT. 14C was

then added, and again no difference between treated and control cultures was observed. Perhaps penetration of DDT through the cell wall was totally prevented in P. boryanum. At least it was felt that an equilibrium existed between the cell walls and the interior of the cells which greatly favored the cell walls, reducing the effective DDT dose present inside the algae.

In order to test this hypothesis, the applied dose was increased to 10.0 ppm DDT in four trials, in hopes of increasing the effective dose by an order of magnitude. No difference between treated and control cultures was observed in two trials in which !4C was added simultaneously with DDT, and no effect was observed in two trials which included a 24 hour incubation period before !4C addition.

Higher concentrations of DDT were considered to be ecologically meaningless, and were not employed. From the data (Table 4) no conclusion can be drawn about the extent or mechanism of DDT uptake in P. boryanun, nor can a prediction be made of whether net 14C_bicarbonate fixation would be affected by DDT if the DDT could enter the cells.

However, the data do indicate that there is no significant difference in net !"C-bicarbonate fixation between control and treated cultures of P. boryanum. Since net 14¢-bicarbonate fixation is influenced by photosynthesis but not respiration, the data indicate that interference with the photosynthetic machinery is not the cause of the observed reduction in population growth rate.

In contrast to these findings, Wurster (1968) reports significant difference in net !4¢-bicarbonate fixation in the marine diatom

Skeletonema costatum, the coccolithophore Coccolithus huxleyi, the

green alga Pyra:imonas sp., and the neritic dinoflagellate Peridineun

trochoideum, exposed to DDT doses up to 0.5 ppm. The results obtained by Menzel, Andersen and Randtke (1970) show no effect on net !4C-bicarbonate fixation in Dunaliella tertiolecta,

but significant reduction in Skeletonema costatum and Coccolithus,

huxleyi, exposed to 9.1 ppm DDT. Cyclotella nana showed reduced uptake

at concentrations above 0.001 ppm. MacFarlane (1970) reports reduced net !4C-bicarbonate fixation per milligram of chlorophyll ain the

diatom Nitzschia delicatissum at DDT concentrations up to 1.0 ppm, with

maximun effect occurring in the range of 0.1 to 1.0 ppm.

Inorganic Carbon Experiments

The inorganic carbon experiments, which have no counterparts in the literature, verify that photosynthesis is not affected by DDT in the test algae, as gross primary production (GPP) was the same for treated and control algae. Net primary production (NPP), however, was reduced by DDT. These facts imply that respiration was increased by DDT. An examination of the valies obtained for respiration rates reveals that the difference in NPP could have been due to increased respiration of the DDT-treated algae. However, in one of the organic carbon experiments (not reported here) in which TIC data were taken, the TIC data for respiration showed higher respiration for the control. Because of the relative reliability and replication of the TIC experiments, and the lack of statistical significance in the organic carbon experiment, it is probably safest to ignore the aberrant result.

The great variation in results probably reflects the great

48 variation in the experimental methods (use of ampoules in the TIC experiments, growth chambers in the two unreported organic carbon experiments), as well as the variability of the algae at different

stages in their growth curve (initial optical densities of 17, 34, and

13 Klett units).

ADDITIONAL RESEARCH CONSIDERATIONS

Artificial Selection by DDT

Perhaps genetic adaptation by artificial selection could explain the tendency of DDT-treated cultures to catch up to the controls. Fogg (1965) notes that "algae evidently possess considerable powers of adaptation to antimetabolites and antibiotics." Whether DDT is a significant artificial selection factor over a 10 day period (corresponding to the maximum length of a growth experiment) is open to question. This wouid depend, among other things, upon how strongly selection favored those algae which were genetically resistant to DDT. If in fact selection is an important process in the present growth experiments, as it has been hypothesized to be in nature, the effect of DDT observed in the algal populations might be far less than its effect on only those algae not possessing factors proffering resistance to DDT. It would be illuminating to compare the effect of DDT on algal populations with a long history of exposure to DDT with pcepulations never exposed to DDT. Also, the possibility of non-genetic adaptation to DDT should not be ignored.

Possible Additional Mechanisms of DDT Action

Algae like P. boryanum and N. pelliculosa, which are equipped with

skeletons, could have the ability to absorb large quantities of DDT to

their surface. If DDT is observed to inhibit photosynthesis and/or

50 growth rate, as it has been shown to do in some studies, perhaps the mechanism of action of DDT is dependent upon entry into the cell during and immediately following cell division when the skeletal structures of the daughter ceils have not yet been completely formed. This could be especially applicable to algae whose storage product is oil rather than starch, since DDT is oil-scluble. A preliminary review of the literature indicates no correlation between algal storage product and effect of DDT on population growth rate and/or photosynthesis. However, Keil and Priester (1969) hypothesize the existence of natural detoxifier organisms which remove oil-soluble pesticides like DDT from the water. These organisms include diatoms, which store oil and leucosin rather than starch. Whether such organisms would truly be effective detoxifiers is highly questionable, since diatoms are major contributors to the next trophic level, the zooplankton, and therefore represent possible vehicles of toxicity as well as detoxifiers.

The absence of a second trophic level (with the exception of bacteria) in the unialgal growth chambers leaves open the question of whether storage product is correlated with effect. Although a literature search did not demonstrate this correlation, it still remains possible that organisms placed in nutrient-limited systems like the growth chambers presently used would eventually have to utilize these reserves. Upon depletion, the DDT dissolved in the food reserve would be mobilized, and in the case of oil reserves, this amount could conceivably be a lethal dose. More research could be done on this

subject.

Cell Structure Sddergren (1968) and MacFarlane (1969) report cell deformation in

Chlorella and Nitzschia delicatissum, respectively, after exposure to

DDT. Cell deformation in P. boryanum and N. peiliculosa was observed

not to occur under the conditions of the experiment, whenever it was looked for. However, it must be added that the search for this effect was not systematic and persistent, and it could have been accidentally ignored. The possibility for observing deformation due to addition of DDT was diminished by the deformation of colonies which occurred in both treated and control cultures equally, and which was probably due to the rapid bubbling of air into the cultures. The breaking apart of colonies which was observed would tend to obscure similar effects

caused by DDT.

SUMMARY AND CONCLUSIONS

Three experimental approaches were used to measure the effects of DDT on photosynthesis, respiration and population growth rate. Population growth rate was measured by the rate of increase of optical density of treated and control cultures. Photosynthesis was estimated by measurement of the fixation of !"C labeled bicarbonate. Photosynthesis and respiration was also measured using a gas analyzer to measure inorganic carbon as carbon dioxide. An attempt to measure dissolved, particulate and total organic carbon with the carbon analyzer was unsuccessful due to shortcomings in the method.

All three concentrations of DDT (0.01, 0.1, 1.0 ppm) were shown to reduce population growth rate in two fresh water algae, the diatom

Navicula pelliculosa and the green alga Pediastrum boryanum (Chloro-

coccales). A DDT dose of 1.0 ppm increased respiration in P. boryanum,

but neither 1.0 nor 10.0 ppm DDT appeared to affect photosynthesis. Reports of other research reveal great variability of response to DDT of algal population growth rate and photosynthesis. In some cases there is no effect, in others, marked interference.

The role of bacteria in the experiinental growth chanbers was significant in the latter part of the growth experiments. For this reason, results of ecological significance are obtainable only from the first 160 hours of growth, since beyond this time the growth chambers

become increasingly unlike natural systems.

A possible mechanism of uptake is presented, exclusively involving non-biological processes, probably a mixture of adsorption and absorption. Reference to the uptake mechanism of !4¢ DDT by Chlorella is discussed. Hypotheses of the exact mechanisms of interference by DDT are cited, primarily including the disruption of cell membrane permeability properties, and inhibition of electron transport.

Cell deformation associated with addition of DDT was observed not to occur at the DDT levels employed. Lack of correlation was noted between effect of DDT on algae and algal storage product (oil or starch). Possible genetic adaptation to DDT was hypothesized to explain the accelerated growth rate of DDT-treated cultures toward the latter part of the growth experiments. Tho possibility of increased affect of DDT during and immediately following cell division was also

hypothesized.

LITERATURE CITED

Acree, F., Jr., M. Beroza, and M. Bowman. 1963. Codistillation of DDT with water. J. Agr. Food Chem. 11:278.

Christie, A. E. 1969. Effects of insecticides on algae. Water Sewage Works 116(5):172-176.

Cope, Oliver B. 1966. Contamination of the fresh water ecosystem by pesticides. J. App. Ecol. 3(Suppl.):33-44.

Darley, W. Marshall. 1969. Silicon and the division cycle of the diatoms Navicula pelliculosa and Cylindrotheca fusiformis. Proc. N. Amer. Paleontological Cony., Sept. 1969, Part G. 994-1009.

Doty, Maxwell S, and Mikihiko Oguri. 1959. The carvon-14 technique for determining primary plankton productivity. Pubbl. Staz. Zool. Napoli Suppl. 31:70-94.

Focht, D. D. and M. Alexander. 1970. DLT metabolites and analogs: Ring fission by Ilydrogenomonas. Science 170:91-92.

Fogg, GS. E. 1965. Algal Cultures and Phytoplankton Ecology. University of Wisconsin Press, Madison, Wisc.

Fritsch, F. E. 1965. The Structure and Reproduction of the Algae. Vol. 1. Cambridge University Press, London, England.

Gromov, B. V. 1967. Main trends in experimental work with algal cultures in the U.S.S.R. In Algae, Man, and Environwent. D. F. Jackson, Ed. Syracuse University Press, Syracuse, N.Y.

Hilton, B. D. and R. D. O'Brien. 1970. Antagonism by DDT of the effect of valinomycin on a synthetic membrane. Science 168:841843.

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APPENDIX

Table 6. P. boryanum growth experiment data

Optical Density (Klett Units)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT August 18, 1970

Growth Experiment la 26 1 1 2 3 3 47.5 3 3 4 3 3 71.5 6 5 5 5 5 79 6 7 6 4 3 84.5 10 9 7 S 3 93 7 12 9 8 4 108.5 10 17 14 11 6 120 15 21 17 13 6 131 17 27 22 17 6 141.5 24 35 29 22 6 156 34 45 38 28 9 162 35 48 42 28 9 168 40 53 47 34 9 178.5 44 58 54 40 ll 190 56 65 59 47 11 204.5 70 76 75 67 16 211 74 82 80 68 16 226 87 90 85 72 19 237.5 94 99 96 85 21 248 102 104 106 93 26 264 114 114 113 97 34 278.5 124 125 124 113 42 288.5 134 133 135 124 50 301 155 145 144 136 60 310 165 150 152 142 65 324.5 178 160 161 15] 71 330.5 184 164 166 156 75 347 195 171 177 164 84 355 204 174 181 166 86 371 223 186 194 182 96 379 230 188 202 184 97 401 255 202 220 195 103 425 288 217 234 206 115 452 320 231 262 213 120

Table 6 (continued)

Optica] Density (Klett Units)

Elapsed Tine No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 9 ppm DDT DDT DDT DDT DDT

August 18, 1970 Growth Experiment ib

26 3 2 3 4

47.5 5 3 4 5

71.5 7 4 5 7

79 6 3 5 5

84.5 10 4 7 7

93 10 6 8 7 108.5 14 11 14 15 120 15 13 14 12 131 19 17 18 14 141.5 22 22 22 15 156 31 i 28 20 162 35 33 32 23 168 41 42 35 23 178.5 50 47 42 28 190 53 55 46 30 204.5 70 72 60 42 21) 74 77 63 44 226 84 87 71 $3 237.5 93 102 77 58 248 102 112 84 68 264 110 123 93 80 278.5 126 141 100 91 288.5 138 155 110 103 301 151 174 121 12i 310 156 179 124 131 324.5 172 193 135 158 330.5 177 201 139 169 347 190 216 146 190 555 195 222 148 201 371 215 240 158 229 379 222 244 163 241 401 242 256 166 274 425 270 305 184 325

452 304 346 19] 393

s9

Table 6 (continued)

Optical Density (Klett Units)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT

August 18, 1970 Growth Experiment lc

26 5 5 3 3 3 47.5 5 6 5 4 6 71.5 8 8 7 6 6 79 5 6 4 4 4 84.5 9 9 5 4 3 93 9 12 9 9 6

108.5 20 20 19 18 il

120 22 23 21 20 8

131 26 26 24 24 i0

141.5 33 34 31 28 12

156 43 45 2 40 16

162 45 46 44 44 18

168 53 55 52 48 22

178.5 56 71 62 53 25

190 66 78 67 59 29

204.5 76 93 &8 75 49

211 7 938 96 80 43

226 92 11s 115 95 50

237.5 97 120 122 107 57

248 108 137 143 111 71

264 116 144 154 127 78

278.5 146 158 198 146 94

288.5 156 165 213 161 115

301 173 188 252 179 136

310 180 202 274 191 147

324.5 188 217 324 211 178

330.5 196 222 352 224 188

347 208 242 435 240 218

355 219 254 510 248 232

371 230

379 240

Table 6 (continued)

Optical Density (Klett Units)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppa 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT

November 20, 1970 Growth Experiment 2a

22 2 2 3 2 2 47 5 5 6 4 3 69 10 12 10 ll 6 79 14 16 16 14 8 92 19 21 23 20 13 99 21 23 26 23 14 117 26 33 31 28 20 123 27 36 33 35 22 139 36 45 35 41 7 152 43 $2 39 49 33 167 53 61 52 56 42 174 53 62 55 55 43 189 61 70 63 64 $2 198 63 72 65 65 54 214 75 82 79 74 64 225 77 86 81 76 69 238 85 93 88 82 76 260 90 7 92 87 83

286 100 108 100 95 96

Table 6 (ccntinued)

Optical Density (Klett Units)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT November 20, 1970 Growth Experiment 2b 22 3 3 4 4 4 47 6 6 5 5 4 69 13 13 11 12 6 79 18 15 14 16 7 92 24 21 19 22 1J 99 26 25 24 24 14 117 26 31 31 38 19 123 26 38 37 44 23 139 21 47 4G $3 29 152 19 52 55 63 33 167 17 61 63 72 41 174 17 64 66 74 45 18S 15 72 76 85 53 198 15 75 79 88 57 214 16 83 90 101 64 225 15 gs 97 107 70 238 17 94 105 116 77 260 20 161 111 124 87 286 22 169 124 138 101

Table 6 (continued)

sevesesser ens: — —

Optical Density (Klett Units)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT

November 20, 1970 Growth Experiment 2c

22 3 3 3 3 3 47 5 5 3 4 4 69 10 10 8 7 6 79 13 15 10 10 7 92 2 22 16 17 12 99 23 26 19 20 13 117 35 39 28 2 18 123 40 5 32 31 19 13S £2 56 42 41 21 152 61 68 2 50 28 167 72 8] 65 él 32 174 75 85 68 65 36 189 88 78 81 ? 42 198 $3 104 87 82 46 214 105 139 102 93 85 225 111 127 112 98 62 238 125 137 123 110 67 260 129 150 137 128 73

286 187 i71 160 140 83

Table 7. P. boryanum growth experiment data transformation to cell density vs. time

Cells per Milliliter (x 10°)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT

August 18, 1970 Growth Experiment la

26 -0.64 -0.64 -0.59 -0.55 -0.55 47.5 -0.55 -0.55 -0.50 -0.55 -0.55 71.5 -0.41 -0.46 -0.46 -0.46 -0.46 79 -0.41 -0.36 -0.41 -0.50 -0.55 84.5 -0.23 -0.27 -0.56 -0.46 -0.55 93 -0.36 -0.13 -C.27 -0.32 -0.50 108.5 -0.23 0.09 -0.04 -0.18 -0.41 120 0.00 0.27 0.08 -C.09 -G6.41 131 0.09 0.55 0.32 0.09 -0.4) 141.5 0.41 0.91 0.64 0.32 -0.41 156 0.87 1.37 1.05 0.59 -0.27 162 0.91 1.59 1.23 0.59 -0.27 168 1.14 1.73 1.46 0.87 -0.27 178.5 1.32 1.96 1.78 1.14 -0.18 190 1.87 2.28 2.01 1.46 -0.18 204.5 2.51 2.78 2.74 2.37 0.05 211 2.69 3.05 2.96 2.42 0.05 226 3.28 3.42 3.33 2.60 0.18 237.5 3.60 3.83 3.69 3.19 0.27 248 3.97 4.06 4.15 3.56 0.50 264 4.51 4.51 4.47 3.74 0.87 278.5 4.97 5.01 4.97 4.47 1.23 288.5 5.42 5.38 5.47 4.97 1.60 301 6.38 5.93 5.88 5.52 2.05 310 6.84 6.15 6.24 5.79 2.28 324.5 7.43 6.61 6.66 6.20 2.55 330.5 7.70 6.79 6.88 6.43 2.74 347 8.20 7.11 7.38 6.79 3.15 355 8.61 7.25 7.57 6.88 3.24 371 9.48 7.79 8.16 7.61 3.69 379 9.80 7.89 8.52 7.70 3.74 401 10.98 8.52 9.34 8.20 4.01 425 12.44 9.21 9.98 8.71 4.56 452 13.90 9.85 11.26 9.03 4.79

Table 7 (continued)

Cells per Milliliter (x 10°)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT August 18, 1970 Growth Experiment 1b

26 -0.55 -0.55 -0.59 -0.55 -0.50

47.5 -0.46 -0.44 -0.55 -0.50 -0.46

71.5 -0.36 -0.38 -0.50 -0.46 -0.36

79 -0.41 -0.36 -0.55 -0.46 -0.46

84.5 -0.23 -0.25 -0.50 -0.36 -0.36

93 -0.23 -0.10 -0.41 -0.32 -0.36 108.5 -0.04 -0.18 -0.04 0.00 120 0.00 0.00 -0.09 -0.04 -0.14 131 0.18 0.46 0.0 0.14 -0.04 141.5 0.32 0.46 0.32 0.27 0.00 156 0.73 0.93 0.73 0.59 0.23 162 0.91 1.16 0.82 0.78 0.37 168 1.19 1.47 1.23 0.91 0.37 178.5 1.60 2.26 1.46 1.23 0.59 190 1.73 2.12 1.82 1.41 0.68 204.5 2.51 3.01 2.60 2.05 1.23 211 2.69 3.30 2.83 2.19 1.32 226 3.15 3.70 3.28 2.55 1.73 237.5 3.56 4.18 3.92 2.83 1.96 248 3.97 4.42 3.15 2.42 264 4.33 4.92 3.56 2.96 278.5 5.06 5.74 3.87 3.46 288.5 5.61 6.38 4.33 4.01 301 6.20 7.25 4.83 4.83 310 6.43 7.48 4.97 5.29 324.5 7.16 8.11 5.47 6.52 330.5 7.38 8.48 5.65 7.02 347 7.98 9.16 5.97 7.98 355 8.20 9.44 6.06 8.48 371 9.12 10.26 6.52 9.75 379 9.44 10.44 6.75 10.30 401 10.35 10.98 6.88 11.81 425 11.62 13.22 7.70 14.13 452 13.17 15.09 8,02 17.23

Table 7 (continued)

Cells per Milliliter (x 10°)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours ) 0 ppm DDT DDT DDT DDT DDT

August 18, 1970 Growth Experiment lc

26 -0.46 -0.46 -0.55 -0.55 -0.55 47.5 -0.46 -0.41 -0.46 -0.50 -0.41 71.5 -0.32 -0.32 -0.36 -0.41 -0.41 79 -0.46 -0.41 -0.50 -0.50 -0.50 84.5 -0.27 -0.27 -0.46 -0.50 -0.55 93 -0.27 -0.14 -0.27 -0.27 -0.41 108.5 0.23 0.23 0.18 0.14 -0.18 120 0.32 0.37 0.27 0.23 -0.32 131 9.50 0.50 C.41 0.41 -0.23 141.5 0.82 0.87 0.73 0.59 -0.14 156 1.28 1.37 1.23 1.14 0.05 162 1.37 1.41 1.32 1.32 0.14 168 1.73 1.82 1.69 1.50 0.32 178.5 1.87 2.55 2.14 1.73 0.46 190 2.33 2.87 2.37 2.01 0.64 204.5 2.78 3.56 3.33 2.74 1.14 211 2.87 3.78 3.69 2.96 1.2 226 3.51 4.56 4.56 3.65 1.60 237.5 3.74 4.79 4.88 4.19 1.91 248 4.24 5.56 5.83 4.38 2.55 264 4.60 5.88 6.34 5.11 2.87 278.5 5.97 6.52 8.34 5.97 3.60 288.5 6.43 6.84 9.03 6.66 4.56 301 7.20 7.89 10.80 7.48 $.51 310 7.52 8.52 11.81 8.02 6.02 324.5 7.89 9.21 14.08 8.93 7.43 330.5 8.25 9.44 15.36 9.53 7.89 347 8.80 10.35 19.14 10.23 9.25 355 9.30 10.89 22.56 10.62 9.89 371 9.80

379 10.26

Table 7 (continued)

Cells per Milliliter (« 10°)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DpY DDT DDT November 20, 1970 Growth Experiment 2a

22 -0.59 -0.59 -0.55 -0.59 -0.59 47 -0.46 -0.46 -0.41 -0.50 -0.55 69 -0.23 -0.14 -0.23 -0.18 -0.41 79 -0.04 0.05 0.05 -0.04 -0.32 92 0.18 0.27 0.37 0.23 -0.09 99 0.27 0.37 0.50 0.37 -0.04 117 0.50 0.82 0.73 0.59 0.23 123 0.55 0.96 0.82 0.91 0.32 139 0.96 1.37 0.91 1.19 0.55 152 1.28 1.69 1.09 1.55 0.82 167 1.73 2.10 1.69 1.87 1.23 174 1.73 2.14 1.82 1.87 1.28 189 2.10 2.51 2.19 2.23 1.69 198 2.19 2.60 2.28 2.28 1.78 214 2.74 3.05 2.92 2.69 2.23 225 2.83 3.24 3.01 2.78 2.46 238 3.19 3.56 3.33 3.05 2.78 260 3.42 3.74 3.51 3.28 3.10 286 3.87 4.24 3.87 3.65 3.69

Table 7 (continued)

Cells per Milliliter (x 10°)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours) 0 ppm DDT DDT DDT DDT DDT November 20, 1970 Growth Experiment 2b 22 -0.55 -0.55 -0.50 -0.50 -0.50 47 -0.41 -0.41 -0.46 -0.46 -0.50 69 -0.09 -0.09 -0.18 -0.14 -0.41 79 0.14 0.00 -0.04 0.05 -0.36 92 0.41 0.27 0.18 0.32 -0.18 99 0.50 0.46 0.41 0.41 -0.04 117 0.50 0.73 0.73 1.05 0.18 123 0.50 1.05 1.00 1,32 0.37 139 C.27 1.46 1.41 i.73 0.64 152 0.18 1.69 1.82 2.19 0.82 167 0.09 2.10 2.19 2.60 1.19 174 0.09 2.23 2.33 2.69 1.37 189 0.00 2.60 2.78 3.19 1.73 198 0.00 2.74 2.92 3.33 1.91 214 0.05 3.10 3.42 3.92 2.23 225 0.00 3.33 3.74 4.19 2.51 238 0.09 3.60 4.10 4.60 2.83 260 0.23 3.92 4.38 4.97 3.28 286 0.32 4.28 4.97 5.61 3.92

Table 7 (continued)

Cells per Milliliter (x 106)

Elapsed Time No Acetone O ppm 0.01 ppm O.1 ppm i.C ppm (hours ) 0 ppm DDT DDT DDT DDT DDT

November 20, 1970 Growth Experiment 2c

22 -0.55 -0.55 -0.55 -0.55 -0.55 47 -0.46 -0.456 -0.55 -0.50 -0.50 69 -0.23 -0.23 -0.32 -0.36 -0.41 79 -0.09 C.00 -0.23 -0.2 -0.35 92 0.23 0.32 0.05 0.09 -0.14 99 0.37 9.50 0.18 0.23 -0.09 117 0.92 1.05 0.59 9.59 0.14 iz3 1.14 1.37 0.78 0.75 0.18 139 1.69 1.8? 1 1.15 0,27 152 2.10 2.42 1 1.60 9.46 1€7 2.60 5.01 2 2.10 0.78 174 2.74 3.33 2 2.28 0.96 189 3.33 3.78 3 2.74 1.24 198 3.56 4.06 3. 3.08 1.41 214 4.10 4.74 3.97 3.56 1,82 225 4.38 5.11 4.42 3.78 2.14 235 5.01 5.56 4.92 4.33 2.37 260 5.20 6.15 5.56 5.18 2.64 286 6.47 7.1 6.61 5.70 3.15

| | i { 1 | 1

Table 8. N. pelliculosa growth experiment data

Optical Density (Klett Units)

Elapsed Time No Acetone 0 ppm 0.01 ppm 0.1 ppm 1.0 ppm (hours ) 0 ppm DDT DDT DDT DDT DDT 0 4 4 4 4 4 3.5 7 7 7 7 7 9.25 4 6 4 6 4 18.5 2 3 0 1 1 26 0 1 0 0 0 32 2 2 1 0 0 44.5 3 3 1 1.5 2 56.5 3 4.5 1.5 2.5 2 68 3 6 1.5 3 2 79 3 8 2 4 3.5 gl 3 11 5 9 8 98 7 13 2.5 5 4 103.5 7 14 4 4.5 7 115.5 9 21 9 10 10 122 15 28 10 13.5 12 128 20 32 10 13.5 16.5 136 20 37 14 18 23 144 17 39.5 18 25.5 23 153 36 45 18 25 25.5 163.5 40 58 18.5 47 26 169.5 40 64 22 37 32 176 55 60 31 45 39.5 187.5 47 62 37 54 45

193.5 $4 83 69 60 67

Table 9.

Cumulative effect of DDT on P. boryanun cell density as a function of time

Elapsed Time

Cells per Milliliter (x 10°)

(hours) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT Growth Experiment la

47.5 0.00 -0.09 -0.09

71.5 -0.05 -0.09 -0.09

79 -0.10 -0.23 -0.28

84.5 -0.14 -0.28 -0.37 93 -0.19 -0.28 -0.46 108.5 -0.18 -0.36 -0.59 120 -0.23 -0.45 -0.77 131 -0.28 -0.55 ~1.05 141.5 -0.32 -0.68 -1.41 156 -0.37 -0.87 -1.73 162 -0.32 -1.00 -1.86 168 -0.32 -0.95 -2.09 178.5 -0.23 -0.91 -2.23 130 -0.32 -0.91 -2.55 204.5 -0.09 -0.50 -2.82 211 -0.14 -0.72 -3.09 226 -0.14 -0.91 -3.33 237.5 -0.19 -0.73 -3.65 248 0.04 -0.59 -3.65 264 -0.09 -0.86 -3.73 278.5 -0.09 -0.63 -3.87 288.5 0.04 -0.50 -3.87 301 -0.10 -0.50 -3.97 310 0.04 -0.45 -3.96 324.5 0.00 -0.50 -4.15 330.5 0.04 -0.45 -4.14 347 0.22 -0.41 -4.05 355 0.27 -0.46 -4.10 371 0.32 -0.27 -4.19 379 0.58 -0.28 -4.24 401 0.77 -0.41 -4.60 425 0.72 -0.59 -4.74 452 1.36 -0.91 -5.15

Table 9 (continued)

Elapsed Time

Cells per Milliliter (x 10°)

(hours) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT Growth Experiment lb

47.5 -0.07 -0.06 -0.07

71.5 -0.08 -0.08 -0.03

79 -0.15 -0.10 -0.15

84.5 -0.21 -0.11 -0.16

93 -0.27 -0.22 -0.31 108.5

120 -0.05 -0.08 -0.19 131 -0.28 -0.36 -0.55 141.5 -0.05 -0.23 -0.51 156 -0.13 -9.38 -0.75 162 -0.27 -0.42 -0.84 168 -0.37 -0.60 -1.15 178.5 -0.93 -1.07 -1.72 190 -0.43 -0.75 -1.49 204.5 -0.54 -1.00 -1.83 211 -0.60 -1.15 -2.03 226 -0.55 -1.19 -2.02 237.5 -0.39 -1.39 -2.27

Table 9 (continued)

Cells per Milliliter (x 10)

Elapsed Time (hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT

Growth Experiment Ic

47.5 0.04 0.00 0.09 71.5 0.05 0.00 0.00 79 0.00 0.00 0.00 84.5 -0.10 -0.14 -0.19 93 -0.04 -0.04 -0.18 108.5 -0.32 -0.28 -0.32 120 -0.37 -0.33 -0.60 131 -0.36 -0.28 ~0.64 141.5 -0.41 -0.47 -0.92 15¢ -0.41 -0.42 -1.23 162 -0.36 -0.28 -1.18 168 -0.40 -0.51 -1.41 178.5 -0.68 -1.11 -2.00 190 -0.77 -1.15 -2.14 204.5 -0.50 -1.11 -2.33 211 -0.36 -1.11 -2.41 226 -0.27 -1.20 ~2.87 237.5 -0.18 -0.89 -2.78 248 0.00 -1.49 -2.91 264 0.19 -1.08 -2.91 278.5 1.55 -0.86 -2.82 288.5 1.92 -0.49 -2.18 301 2.64 -0.72 -2.28 310 3.02 -0.81 -2.40 324.5 4.60 -0.59 -1.78 330.5 5.65 -0.22 -1.55 347 8.52 -0.43 -1.10

355 11.40 -0.58 -1.00

Table 9 (continued)

Celis per Milliliter (x 10°)

Elapsed Time (hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT

Growth Experiment 2a

22 47 0.01 -0.04 -0.69 69 -0.13 -0.04 -0.27 79 -0.04 -0.09 -0.37 92 0.06 -0.04 -0.36 99 0.09 0.00 -0.41 117 -0.13 -0.23 -0.59 123 -0.18 -0.05 -0.64 139 -0.50 -0.18 -0.82 152 -0.64 -0.14 -0.87 167 -0.45 -0.23 -0.87 174 -0.36 -0.27 -0.86 189 -0.36 -0.28 -0.87 198 -0.36 -0.32 -0.82 214 -0.17 -0.36 -0.82 225 -0.27 -0.46 -0.78 238 -0.27 -0.51 -0.78 260 -0.27 -0.46 -0.64

286 -0.41 -0.59 -0.55

Table 9 (continued}

Elapsed Time

Cells per Milliliter (x 106)

(hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT Growth Experiment 2b

47 -0.10 -0.10 -0.14 69 -0.14 -0.10 -0.37

79 -0.09 0.00 -0.41

92 -0.14 0.00 -0.50

99 -0.10 -0.10 -0.55 117 -0.05 0.27 -0.60 123 -0.10 0.22 -0.73 139 -0.10 0.22 -0.87 152 0.08 0.45 -0.92 167 0.04 0.45 -0.96 174 0.05 0.41 -0.91 189 0.12 0.54 -0.92 198 0.12 0.54 -0.88 214 0.26 0.77 -0.92 225 0.35 0.81 -0.87 238 0.44 0.95 -0.92 260 0.40 1.00 -0.79 286 0.63 1.28 -0.51

Table 9 (continued)

Cells per Milliliter (x 10°)

Elapsed Time (hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT

Growth Experiment 2c

47 -0.09 -0.04 -0.04 69 -0.09 -0.13 -0.18 79 -0.23 -0.23 -0.36 92 -0.27 -0.23 -0.46 99 -0.32 -0.27 -0.59 117 -0.50 -0.50 -0.95 123 -0.59 -0.64 -1.19 139 -0.64 -0.68 -1.60 152 -0.73 -0.82 -1.96 167 -0.73 -o.9 -2.23 174 -0.91 -1.05 -2.37 189 -0.77 -1.04 -2.55 198 -0.78 -1.01 -2.65 214 -0.77 -1.18 -2.92 225 -0.69 -1.33 -2.97 238 -0.64 -1.23 -3.19 260 -0.59 -1.00 -3.51

286 -0.50 -1.41 -4.01

Table 9 (continued)

Elapsed Time

Cells per Milliliter (x 10°)

(hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT Growth Experiments labc, Mean 47.5 -0.01 -0.05 -0.023 71.5 -0.027 -0.057 -0.04 79 -0.083 -0.11 -0.143 84.5 -0.150 -0.177 -0.24 93 -0.167 -0.18 -0.317 108.5 -0.25 -0.32 -0.455 120 -0.217 -0.287 -0.52 131 -0.307 -0.397 -0.747 141.5 -0.26 -0.46 -0.947 156 -0.303 -0.557 -1.237 162 -0.317 -0.567 -1,.293 168 -0.363 -0.687 -1.55 178.5 -0.613 -1.03 -1.983 190 -0.507 -0.937 -2.06 204.5 -0.377 -0.87 -2.327 211 -0.367 -0.993 -2.51 226 -0.52 -1.10 -2.74 237.5 -0.25 -1.003 -2.90

Table 9 (continued)

Cells per Milliliter (x 105)

Elapsed Time (hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT

Growth Experiments 2abc, Mean

47 -0.06 -0.06 -0.09 69 -0.12 -0.09 -0.273 79 -0.12 -0.107 -0.38 92 -0.116 -0.09 -0.44 99 -0.11 -0.123 -0.517 117 -0.227 -0.18 -0.713 123 -0.29 -0.157 -0.853 139 -0.413 -0.213 -1.097 152 -0.43 -0.17 -1.25 167 -0.38 -0.23 -1.353 174 -0.407 -0.303 -1.38 189 -0.337 -0.26 -1.447 198 -0.34 -0.263 -1.45 214 -0.227 -0.257 -1.553 225 -0.203 -0.327 -1.54

238 -0.157 -0.263 -1.63

Table 10.

Cumulative effect of DDT on N. pelliculosa optical density as a function of time

Cells per Milliliter (x 10§)

Elapsed Time

(hours ) 0.01 ppm DDT 0.1 ppm DDT 1.0 ppm DDT 3.5 0 0 0 9.25 -2 -2 -2 18.5 -9 -10 -8

26 -7 -9 -7 32 -7 -10 -8 44.5 -8 -9.5 -7 56.5 -10 -10 -8.5 68 -11.5 -11 -10 79 -13 -12 -10.5 91 -13 -10 -9 98 -17.5 -16 -15 103.5 -17 -17.5 -13 115.5 -19 -19 -17 122 -25 -22.5 -22 128 -29 -26.5 -21.5 136 -30 -27 -20 144 -28.5 -22 -22.5 183 -34 -28 -25.5 163.5 -46.5 -19 -38 169.5 -51 -37 -40 176 -38 -25 -28.5 187.5 -34 -18 -25 193.5 -23 ~33 -24

Figure 15.

experiment la

Effect of DDT on P. boryanum cell density:

growth

(Sanoy) awty

tal

Ove

[osha SE RR OR DOLOD TL OOS

6 Gell Density Difference from Control (cells/ml! X 10 )

wid |"g

|

Ayisuag 1j99 wnuehiog gy vo Jgg yo 490449

Figure 16.

experiment 1b

Effect of DDT on P. boryanum cell density:

growth

"80

Effect of DDT on P. boryanum Cell Density:

STE 0D

1h

= = t h2 8 SALE IN Ts LS ee SR — = i at : ai (OL X [W/8}429) Jouquog wesy aouaday sig Ayisuag j[9g

129 169 200 240 Time (hours)

Figure 17. Effect of DDT on P. boryanum cell density: growth

experiment Ic

Effest of DDT on P. boryanum Ceil &

I Growth Exa’t. Ie

bhi

6 ty Difference from Control (celis/mi X 10 )

Ceti Dd

APIA TOES ST ONE HRN VSR RNOSST 2

id i20 160 200 240 Time (hours)

Figure 18.

experiment 2a

Effect of DDT on P. boryanum cell density:

growth

Effect of DDT on P. boryanum Coll Density:

1 Growth Exp’t. 2a

.

Cell Density Sifference from Control {cells/mi X 10

“3 ALR ST SET EL ER PETES BIT A A TE EE OIE 4p 3a i2d 163 20% 240 Time (hours)

Figure 19.

experiment 2b

Effect of DDT on P. boryanum cell density:

growth

Effect of ODT on P. boryanam Geii Baasity-

1s firowth Exp’t. 2h

f)

Se Re > > : o > - s = s 3 5 ¢ § : = 2 :

i.Qapm

Density Difference from Control (cells/ml X 10

‘ it

Se

SES CCS OE SRE NS

there aes

1 a a,

AEN EEL PO ATRIA EEA TR TAS RSS TMC Se eT

40 a) 129 160 90 246

Time (hours)

Figure 20.

experiment 2c

Effect of DDT on P. boryanum cell density:

growth

(sanay)

|

dall Gensity Difference from Gontrai weils/mi X 10 }

é | |

Ayishag [9d WaUeAdGg gy uO fgg yo y8assq

2 71,dxz yp morg

onmee eee en ennes a

ET LOSER 2 TE EET SS) RN LE UE A ee

Figure 21. Effect of DDT on P. boryznum cell density:

experiments labc, mean effect

growth

Effect of DOT on P. horyanum Cell Density:

| Growth Exp’t. labe, Mean Effect

— 2 om H}

=) i om

- i =

—

on” ¥ a

<2 —

— a g oH

oa

iJ

i—J

o

IRE EEG MLA

piriraa ah:

4 nr

RAPES TES BLS

Geil Density Difference from

O29 ODF AAI, TSR, A TT TT

te 169 Time (hours)

Figure 22. Effect of DDT on P. boryanum cel] density:

experiments 2abc, mean effect

growth

Effect of NOT on P. boryanum Cell Density:

1 Growth Exp’t. abe, Mean Effect

0.01 ppm a an

' i)

6 Cell Density Difference from Gontrol (cells/ml! X10 )

a E I

eR STASIS OTT i EAS PMI TAT OY

a@ 4g 80 i20 160 200 240 Time (hours)

Figure 23.

Ettect of DDT on N. pelliculosa cell density

Optical Density Density’ Difference from Control(Klett units)

! i) mn

“75

Effect of DDT on N.pelliculosa Cell Density

0.01 ppm

0.1 ppm

iseanargarcciaveapss == | () ppm

40 80 120 160 ~=———200 Time (hours)

Publication citation

Michaels, Robert Alan. Effect of DDT On Growth Rate and Photosynthesis in Pediastrum boryanum (Chlorococcales). Athens, Georgia, USA; University of Georgia, Institute of Ecology, Department of Zoology; Master of Science (MS) Degree, James E. Schindler, Major Professor; Donald C. Scott, Reading Committee Chairperson; Eugene P. Odum, Distinguished Reading Committee member; 89 pages plus Appendices, August 1971.

Robert A. Michaels on ResearchGate