UPTAKE AND EFFECTS OF KEPO~ ON GROWTH, " RESPIRATION AND PHOTOSYNTHESIS OF

CHI.ORELLA SOROKINIANA AND CHLOROCOCCUM HYPNOSPORUM/

by

Gary Robert Young u ~ ;,

Thesis submitted to the Graduate Faculty of the

Virginia Polytechnic Institute and State University

in partial fulfillment of the requirements for the degree of

Master of Science

in

Plant Physiology

Approved:

s. W. BinghKDl, Co~Chairman ~ S~ade?, Co-Chairman

C. R. Drake

November 1978

Blacksburg, Virginia

ACKNOWLEDGEMENTS

The author wishes to express a preserving gratitude to his graduate

coDDllittee, and , for their

suggestions and cooperation in the preparation of this thesis and to

for supplying a culture of algae.

Thanks go to for his technical assistance and

to , a colleague, for our discussions on the

problems associated with Kepone(B in these investigations. 14 The C-Kepone and technical grade Kepone was supplied gratis by

Allied Chemical Corporation.

The author thanks the faculty and staff of the Department of

Plant Pathology and Physiology for the education and friendships

acquired throughout his graduate tenure.

Finally, the author wishes to express the greatest appreciation

to his wife, • for her moral support and for the typing of this

thesis.

ii

TABLE OF CONTENTS

TITLE PAGE ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• i

ACKN'OWI..El)(;mf.ENTS •••••••••••••••••••••••••••••••••••••••••••••••••••• ii

TABLE OF CONTENTS •••••••••••••••••••••••••••••••••••••••••••••••••• iii

LIST OF TABLES •••••••••••••••••••••••••••••••••••••••••••••••••••••• iv

LIST OF FIGURES •••••••••••••••••••••••••••••••••••••••••••••••••••••• v

INTRODUCTION ••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 1

LITERA'l'IJR.E REVIEW' •••••••••••••••••••••••••••••••••••••••••••••••••••• 4

MA.TERIAI.,S .AND ~THODS ••••••••••••••••••••••••••••••••••••••••••••••• 12 Kepone ••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 12 Algae culture •••••••••••••••••••••••••••••••••••••••••••••••••• 12 Effect of Kepone on respiration •••••••••••••••••••••••••••••••• 14 Effect of Kepone on photosynthesis ••••••••••••••••••••••••••••• 14 Removal of Kepone from solution •••••••••••••••••••••••••••••••• 16 Efflux of Kepone from Chlorella and Chlorococcum ••••••••••••••• 17

RESULTS .AND DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••• 19 Influence of Kepone on growth •••••••••••••••••••••••••••••••••• 19 Effect of Kepone on respiration •••••••••••••••••••••••••••••••• 19 Effect of Kepone on photosynthesis ••••••••••••••••••••••••••••• 23 Amount of 14c-Kepone removed from solution ••••••••••••••••••••• 25 Efflux of Kepone ••••••••••••••••••••••••••••••••••••••••••••••• 30

S~Y ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 33

LITERATU'RE CITED •••••••••••••••••••••••••••••••••••••••••••••••••••• 34

VITA •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 3 7

iii

LIST OF TABLES

TABLE

I. Effect of Kepone on growth of Chlorella •••••••••••••••• , ••••••• 20

II. Effect of Kepone on growth of Chlorococcum ••••••••••••••••••••• 21

III. Effect of Kepone on respiration •••••••••••••••••••••••••••••••• 22

IV. Effect of Kepone on photosynthesis ••••••••••••••••••••••••••••• 24

iv

LIST OF FIGURES

Fig.

1. Absorbance of Chlorella at 520nm as a function of cell concentration •••••••••••••••••••••••••••••••••••••••• 15

2. Absorbance of Chlorococcum at 520nm as a function of cell concentration ••••.••.•••••••.•...•.•..••.•...•.••• 15

3. 14 Amount of C-Kepone remaining in solution after uptake by Chlorella over a 48 hr. sampling period •••••••••••••••• 26

4. 14 .

Amount of C-Kepone remaining in solution after uptake by Chlorococcum over a 48 hr. sampling period ••••••••••••• 28

5. Percent increase of Chlorella cell number during uptake study over 48 hr •••••••••••••••.•••••••.••••.•••••••••..•• 29

6. Percent increase of Chlorococcum cell number during uptake study over 48 hr •••••••••••••••.•••••.••••••••.•••• 29

7. 14 Efflux of C-Kepone after 48 hr. from Chlorella cells ••••••••• 31

8. 14 Efflux of C-Kepone after 48 hr. from Chlorococcum cells •••••• 32

v

INTRODUCTION

The effect of pesticides on water quality, pesticide pollution,

has become increasingly important as a determining factor of the qual-

ity of the world's environment. The increase in human population has

resulted in greater use of water for industrial, municipal, and recre-

ational activities. Often, without adequate monitoring, pollution

problems develop to quite high levels before being realized. The

growth of human population has led to increased industrial productivity

and the creation of new industries. The pesticide industry is one of

the most recent industries to develop during the last 30 years. Many

pesticides have been produced and development is continuing, under

stringent regulations, with the release of several new pesticides

annually. Early growth of the pesticide industry resulted in the de-

velopment of a number of various pesticides which were very effective

for their particular use, but little was known about the persistence

and long term effect of the pesticides on the environment. Scientific

investigations have proven that a few of the early pesticides were not

restricted from use unless an environmental hazard was predicted or

actually occurred. Kepone, along with other pesticides, exists in

aquatic environments and accumulates in aquatic organisms. In order

for a pesticide pollutant to be removed from the environment it must be

degraded; however, degradation is a slow process. Persistent pesti-

cides which are accumulated can be distributed over areas away from the

source of deposition. This accumulation of pesticides by aquatic

organisms may alter the organisms' metabolism, result in degradation of

the pesticide, cause death of the organisms, or a combination of all

1

2

these. Reports have indicated resistance and survival of some organisms

(i.e. algae) to particular pesticides and that these organisms accumu-

late pesticides to levels much higher than that in the external environ-

ment (Menzel et al. 1970, Vance and Drummond 1969, and Wurster 1968).

Algae are common inhabitants of water, soil and surfaces exposed

to air and light. Algae are important in regulating water quality by

modifying pH, color, turbidity and quantity of inorganic and organic

compounds. Although algae may be nuisances, they can be used to im-

prove water quality. Algal populations vary and are dependent upon

the amounts and kinds of dissolved and suspended materials which serve

as nutrients. In recent years, attention has been devoted to algae

because they accumulate and produce toxic organic substances which

affect many kinds of wild and domestic animals (Palmer 1962).

Our society is becoming increasingly dependent upon water for

domestic purposes; therefore, the effects of pesticides on non-target

organisms in the aquatic food chain is being considered. Upon entry

into the food chain, some pesticides are passed from one organism to

another resulting in concentration of a toxic substance in the food

chain. Algae are more resistant to certain organic compounds such as

chlorinated insecticides and consequently, upon accumulation, cause

severe effects on organisms higher in the food chain (Vance and Drummond

1969). Algae are, therefore, important in removing or redistributing

residues from pesticide laden aquatic environments because they are in

intimate contact with water, reproduce rapidly if the nutrient supply

is sufficient, form a large surface to volume ratio, and accumulate

pesticides.

3

The objectives of this research were to evaluate the level of

uptake and accumulation of Kepone by two species of green algae and to

determine the effect of this chemical on growth, photosynthesis and

respiration of the algae.

LITERATURE REVIEW

® Kepone is the registered trade name for decachlorooctahydro 1,3,

4-metheno-2H-cyclobuta cd pentalen-2-one, cODDnon name chlordecone.

It is a member of the cyclodiene family of insecticides, others of which

are chlordane, heptachlor, aldrin, dieldrin, endrin, Kelevan and Pentac

(Kenaga and Allison 1971). Depending upon impurities, crystalline

Kepone appears white to light gray or tan. The structure of the Kepone

molecule appears cage-like, much like Mirex, except for a keto-oxygen

replacing two chlorine atoms as shown below. Kepone is the ketone

Cl Cl

Anhydrous Kepone mw 490.68

analog and degradation product of Mirex {USEPA 1978, Andrade and Wheeler

1975, Carson et al. 1976). It is made by reacting hexachlorocyclopen-

tadiene with sulfur trioxide, sodium hydroxide and sulfuric acid in the

presence of the catalyst antimony pentachloride. The above reaction

yields approximately 81 percent Kepone {USEPA 1978). Kepone is soluble

in acetic acid, acetone, lower aliphatic alcohols, other organic sol-

vents and aqueous sodium hydroxide {Merck Index 1973, USEPA 1978).

4

5

Kepone is more water soluble than Mirex due to the oxygen atom forming

hydrogen bonds. In water Kepone is soluble from 1.5 to 2.0 mg/liter

over a 4.0 to 7.0 pH range. Solubility increases from 5 mg to 70

mg/liter when the pH is increased from 9 to 10. Anhydrous Kepone ex-

posed to normal temperatures and humidities forms hydrates with water.

This water of hydration has been specified as being 3.5 to 6.0% of the

technical grade Kepone (USEPA 1978).

The insecticide was first formulated in 1952 by Allied Chemical

Corporation. It was primarily manufactured in the United States to

control ants and cockroaches. Much of the Kepone produced was exported

to Central America and Africa to use on insect pests on bananas and

potatoes and to Europe to be converted to other products. In the

United States, use was limited to ant and roach traps with maximum

levels set at 0.125% (USEPA 1978). Kepone also was recommended for use

as a stomach poison for grasshoppers and housefly larvae (Metcalf et

al. 1962).

The James River is the largest river in Virginia flowing more than

680 km from its source in West Virginia to the Chesapeake Bay. The

lower portion of the James River, 180 km from Richmond to the Chesapeake

Bay, is subject to diurnal tides. The river bas very high turbidity

throughout most of its length, with maximum turbidity occurring be-

tween Dancing Point, 129 km, and Hog Island, 56 km, from the Chesapeake

Bay. The Dancing Point and Hog Island area is the saline-fresh water

interface and where the greatest sedimentation occurs. Kepone was

manufactured by Allied Chemical Corporation intermittenly from 1966 to

1974 at their plant in Hopewell, Virginia. In November, 1973, the

•

6

Life Science Products Company, Inc. was contracted to produce Kepone.

The city of Hopewell allowed discharge of its wastewater into the city

sewerage system. In October, 1974, a Virginia State Water Control

Board survey revealed that Kepone was being discharged into the city

of Hopewell's sewerage system due to improper digestion equipment oper-

ation. By late July, 1975, the Life Science Products Company, Inc.

agreed to terminate production due to evidence of air, soil, water and

building contamination by Kepone and the compounds used for its manu-

facture. The insecticide was found in the blood of employees and their

families and caused a neurological disorder. Subsequent investigation

in the James River revealed high levels of Kepone in economically im-

portant shellfish and f inf ish. The result of these investigations

caused the Food and Drug Administration to close the James River to

coDDDercial and sport fishing in December 1975 (USEPA 1978).

Sources of Kepone contaminated discharge were in the process of

drying and bagging, atmospheric releases, water discharges, wastewater

spills and bad batch dumping of solid and liquid wastes on terrestrial

sites. The total amount of Kepone discharges were impossible to quan-

tify due to the lack or unavailability of daily records. Estimations

of total discharge were based on the material balance of raw hexachloro-

pentadiene supplied by Allied Chemical to Life Science Products, the

theoretical yield and sales to Allied Chemical. The total production,

total sales to Allied Chemical gave an estimate of more than 91,000 kg

potential loss from operations by Life Science Products. Between 1966

and 1974, the total production by Allied Chemical was 763,000 kg.

Losses also occurred from Allied Chemical's plant; however, these

7

losses were even more difficult to estimate. Environmental samples

taken in May, 1967, revealed Kepone levels from 0.02 to 0.04 µg/g in

sediment samples at the Dancing Point-Jamestown area. Preserved sam-

plea of oysters and fish further confirmed the analysis (USEPA 1978).

In 1976, Kepone levels in the top 9 cm of bottom sediment ranged from

0.02 to 1.0 µg/g throughout the James River from Hopewell to Newport

News, Virginia. Bottom sediment levels in Bailey Creek were as high

as 10 µg/g (USEPA 1978). Assays and estimates conducted by the Virginia

Institute of Marine Science (VIMS) in 1977 revealed the total amount

of Kepone to be from 11,000 to 18,000 kg in the bottom sediment of the

James River. Surface waters in 1975 were found to contain 0.3 µg/l

in the James River near Hopewell and from 1 to 4 µg/l in Bailey Creek. ·

Recent studies have found that most of the levels of Kepone in the

James River water are below detectable limits of 0.01 µg/l (Huggett et

al. 1978); however, samples were found to contain 0.042 µg/l (VIMS

1977).

Much concern was emphasized on the movement of Kepone in the James

River. Possible pathways of movement were through volatilization,

sorption-desorption, bioconcentration and physical movement of Kepone

·laden suspended solids (USEPA 1978). Theoretical calculations sug--5 2 gested that Kepone volatilizes from 1.34 to 4.92 x 10 g/hr/m of

water surface. 6 2 In terms of Bailey Bay which comprises 3.2 x 10 m

(800 acres), volatilization losses would be in the order of 80 g/hr.

Garnas et al. (1977) found that Kepone volatilization did not occur at

detectable levels in static and continous flow experiments. Studies

conducted at the Battelle Memorial Institute also confirmed that Kepone

8

did not volatilize when dry Kepone-contaminated sediments and sediments

standing in water were exposed for 12 weeks to direct sunlight. Vol-

atilization was probably insignificant due to the strong particulate

affinity of Kepone. Water was considered as a sink for Kepone; however,

due to the strong sorption of Kepone onto particulate matter, concentra-

tions of Kepone dissolved in water were found below detectable limits.

Sorption-desorption and laboratory elutriate tests found that Kepone

partition coefficients (water to sediment concentrations) were in the -4 order of 10 , indicating a small fraction of Kepone would be present

in water with high particulate levels (USEPA 1978).

Kepone sorption is similar to partitioning in an immiscible two

solvent system. Non-polar to slightly polar solutes partition between

the two solvent systems through their comparative affinity for each

solvent. A slightly polar compound like Kepone accumulates in organic

material because it has a higher affinity for organic material than

water (USEPA 1978). According to Huggett et al. (1977), desorption is

influenced by water quality parameters such as salinity, temperature,

aeration and pH. Solution pH is the only parameter known to affect

Kepone desorption. Leachate content of Kepone is increased from 0.03

to 6.84 µg/l when pH is increased from 7.2 to 12.0 (USEPA 1978 and

Garnas et al. 1978).

Migration of Kepone is known to be a result of runoff and leachate

solubilizing Kepone from contaminated sediments. Assays of runoff and

leachates are estimated as being from a few grams per day during drought

periods to 64 g per day during average rainfall from Hopewell to

the James River. Mathematical modeling estimates Kepone movement

9

seaward at a rate of 76 to 170 kg/yr past Burwell Bay, 28 km from the

Chesapeake Bay. As much as 65% of this movement is due to desorption

of Kepone from sediments into the dissolved state. Transport of Kepone

out of the James River by anadromous fish is estimated at 10 to 100

kg/yr (Huggett et al. 1977). Movement of Kepone into the Chesapeake

Bay is of great concern. However, the low rates of movement and sorp-

tion onto uncontaminated sediments are believed too small to cause

adverse affects on the Bay's environment. The above low rates of move-

ment indicate that environmental and economical impacts will continue

long into the future. Furthermore, laboratory information reveals that

the effects of Kepone on aquatic life will continue until levels are

reduced to 0.008 µg/l in water and 0.015 µg/g in sediment and food for

biota (USEPA 1978).

The food chain begins with algae at the primary trophic level and

ends with the tertiary consumers such as birds and mammals. Bioconcen-

tration and bioaccumulation are two terms which are important in

describing Kepone entry into the food chain. Bioconcentration is the

process by which organisms of the same trophic level concentrate a given

substance directly from water and/or sediments. Bioaccumulation is the

process by which organisms acquire a given substance through water, sed-

iments or food and continually increase the concentration within the

body throughout the organism's active life (Kneip and Lauer 1973).

Kepone contamination of the James River is of great concern to environ-

mentalists because it poses a threat to aquatic and terrestrial biota.

The concern is based on the toxicity, bioconcentration and bioaccumula-

tion of Kepone in estuarine organisms found in laboratory studies

10

(Hansen et al. 1976).

The fate and accumulation of inorganic and organic compounds,

especially the organochlorines, are important because these compounds

degrade slowly in nature, and cause long-lasting effects on the environ-

ment. Aquatic organisms are important in the translocation and

metabolic transformation of compounds which enter and exit bodies of

water. Accumulation of organochlorine compounds altering metabolic

processes could cause interference in competition among organisms;

therefore, possibly causing the reduction of desirable organisms and

an increase in less desirable organisms (Bourquin 1977).

Concentrations of Kepone approaching those in the James River

sediment inhibit growth and oxygen uptake of aerobically grown micro-

organisms. Nontoxic effects of Kepone on anaerobic bacteria have

suggested involvement of Kepone with electron transport and respiration

(Bourquin 1977). Butler (1963) demonstrated that 1 mg Kepone/l reduced

carbon fixation by 95%. Growth of four marine unicellular algae,

Chlorococcum, sp., Dunaliella tertiolecta, Nitzchia sp. and Thalassio-

~ pseudonana was inhibited by Kepone (Walsh et al. 1977). In studies

conducted by Bahner et al. (1977), Chlorococcum concentrated Kepone

to near 6000 times that in solution within 48 hours in static tests.

Laboratory studies have shown that Kepone is taken up by algae

(Walsh et al. 1977 and Bahner et al. 1977). According to Huggett et

al. (1977), the average bioconcentration levels of Kepone in James

River phytoplankton were 1.3 µg/g (wet weight or dry weight was not

disclosed). Algae are located at the primary trophic level and pass

Kepone to other organisms in the food chain. Bahner et al. (1977)

11

conducted food chain studies using Kepone contaminated algae,

Chlorococcum, sp. fed to eastern oysters (Crassastrea virginica) and

Kepone contaminated plankton, brine shrimp (Artemia salina) fed to

mysids (Mysidopsis bahia) which were in turn fed to spot (Leiostomus

xanthruus). Bioconcentration of Kepone was demonstrated by the algae-

oyster food chain and the plankton-mysid-f ish food chain. Oysters were

found to depurate the bioconcentrated Kepone within 10 days when fed

Kepone-free algae. Kepone depuration by crustaceans and fish was slow

with concentrations in tissues decreasing to 50% in 28 days.

According to Lowe et al. (1971), bluecrabs (Callinectes sapidas)

fed Kepone contaminated eastern oysters bioaccumulated Kepone. When

fed Kepone-free oysters, the bluecrabs contained detectable concentra-

tions of Kepone after 90 days. The above food chain studies demonstrate

Kepone is transferred. However, the major route of uptake in fish is

from Kepone dissolved in water while the route of uptake by bluecrabs

is from food material. Furthermore, Kepone detection in various bird

species such as ducks, herons, gulls, geese and eagles reveals that

Kepone is having an extensive impact on the environment of eastern

Virginia (USEPA 1978).

MATERIALS AND METHODS

Kepone. Growth, respiration and photosynthesis studies were conducted

with technical grade Kepone (89%). Uptake and efflux evaluations were 14 conducted with uniformly labeled C-Kepone (specific activity 35.71 µCi/

mg).

Algae culture. Chlorella sorokiniana Shihira and Kraµs and Chlorococcum

hyPnosporum Starr were obtained from Dr. J. A. Swader and Dr. D. M.

Orcutt, respectively, Department of Plant Pathology and Physiology,

Virginia Polytechnic Institute and State University, Blacksburg, VA.

The algae were grown under aseptic conditions for four days in 250 ml

Erlenmyer flasks containing 100 ml of ~ strength Bold's Basic Medium

(James 1974), adjusted to a pH 7 with 1 N NaOH, hereafter referred to

as nutrient solution. Each flask was aerated with humidified compressed

air. Aliquots from the stock cultures were sonicated (Bransonic Model

12 ultrasonicator, Branson Instruments) for 60 seconds and centrifuged

at 3000 x g for 2 minutes. The algal pellets were resuspended in

sterile nutrient solution by sonication. The centrifugation and re-

suspension procedures were repeated 12 times (James 1974). Washed

algal cells were then dispersed with an atomizer using filtered com-

pressed air onto the following agar (15 g/l) nutrient media: Nutrient

solution with 0.1 g protease peptone/100 ml and nutrient solution with

0.3 g yeast extract/100 ml, each containing 30 µg of Penicillin "G"

(46.8 units) and 50 µg of Streptomycin sulfate/100 ml. The antibiotic

stock solution was filter sterilized with a 220 nm pore size milipore

filter before addition to autoclaved media.

12

13

The algae were grown for several days over Westinghouse Cool

White flourescent lamps which provided a light intensity of 85 µE/m2/sec

at the surface of the petri plates. Algal cononies were transferred

aseptically to the two groups of media described above with antibiotics

deleted. Axenic algal cells, after several days of growth, were trans-

£erred aseptically to 250 ml Erlenmyer flasks containing 100 ml of

sterilized nutrient solution per flask. The algae were grown under

aseptic conditions, aerated with filtered, humidified, compressed air

at room temperature under a light intensity of 90 µE/m2/sec provided

by flourescent lamps. After four days, the cells were harvested by

centrifugation, resuspended in sterile nutrient solution, pH 8.0, and

counted with an improved Neubrauer counting chamber. The cells were

transferred aseptically to a 6 liter Erlemnyer flask containing 3 liters

of sterile nutrient solution, pH 8.0. Initial cell concentrations were

1 x 105 Chlorella cells/ml and 4 x 104 Chlorococcum cells/ml. Algae

were treated with O.O, 0.5, 1.0, 1.5 and 2.0 mg Kepone/l and 50 ml

aliquots dispensed aseptically into four 125 ml Erlenmyer flasks. Ke-

pone stock solutions were prepared with acetone and 50 µ1/1 of solution

was added to the treatments and the same amount acetone was added to ·

the controls. The incubation flasks were placed on a rotary shaker set 2 at 80 excursions/min., under flourescent lamps providing 85 µE/m /sec

at the surf ace of the treatment solution. The algae were incubated un-

der aseptic conditions at 25°C and aerated with 200 cm3/min humidified,

compressed air. Cell populations were measured spectrophotometrically

at 520 nm at 12 hr., 24 hr., 48 hr., and 96 hr. time intervals. The

absorbance of the algal cells ploted as a function of cell density was

14

calibrated with the counting chamber (Fig. I and II). Averages of

four replications were tabulated as cells/ml for each treatment at each

time interval. The data was tested for significance at the 5% level

of confidence according to the Duncan's Multiple Range Test (Sokal and

Rohlf 1969). The experiments were repeated with results similar to

those recorded.

Effect of Kepone on respiration. Chlorella and Chlorococcum grown for

four days in the growth studies were used to study the effect of Kepone

on respiration. Algal cell concentrations were 1 x 108 cells/ml and

6 x 106 cells/ml for Cblorella and Chlorococcum, respectively. Respir-

ation was measured by oxygen uptake from solution with an oxygraph

(Model K-ICC, Gilson Medical Electronics) equipped with a Clark elec-

trode and a 2 ml reaction chamber. Five replicates of the control,

control plus acetone and 1.0 mg/l Kepone treatment were examined under

dark conditions for five minutes at 25°c. Averages of five replications were tabulated as µmol o2 consumed/mg dry wt/hr. The data was tested for significance at the 5% level of confidence according to the Duncan's

Multiple Range Test (Sokal and Rohlf 1969). The experiments were

repeated with results similar to those reported.

Effect of Kepone on Photosynthesis. Photosynthesis was measured with

aliquots of Chlorella and Chlorococcum from the growth study. The cells

were centrifuged and diluted with fresh nutrient solution to obtain 7 6 1 x 10 cells/ml and 6 x 10 cells/ml for Chlorella and Chlorococcum,

respectively. The 2 ml reaction vessel was illuminated with two 300W,

120v Ken-Rad reflector lamps placed on each side of the reaction cell,

1.0

.8

.6

.4

.2

0

15

10 20 30 40 50 Cells/al x 10-6

Fig. 1. Abaorbance of Chlorella at 520nm as a function of cell concentration

1.0

.8

~ .6 0 N 11"1

< .4

.2

0

0.6 1.2 1.8 2.4 3.0 Cells/ml x 10-6

Fig. 2. Absorbauce of Chlorococcua at 520nm a1 a £unction of cell concentration.

16

one lamp per side. 2 Each lamp provided 2000 µE/m /sec at the surface of

the reaction vessel. Tap water flowing through a Pryex container be-

tween each lamp and the reaction cell served as a heat filter. Photo-

synthesis was measured by oxygen evolution with the oxygraph. Averages

of five replications were tabulated as µmol o2 evolved/mg chlorophyll/ • hr. Amount of chlorophyll was determined according to the method

described by Amon (1949). The data was tested for significance at

the 5% level of confidence according to the Duncan's Multiple Range

Test (Sokal and Rohlf 1969). The experiments were repeated with

results similar to those reported.

Removal of Kepone from solution. Chlorella and Chlorococcum were grown

in nutrient solution for four days, after which the cells were harvested

by centrifugation and resuspended in fresh nutrient solution. The algae

suspension was then transferred to a 6 liter Erlenmyer flask containing 3 3 liters of sterile nutrient solution, aerated with 500 cm /minute of

cotton filtered, humidified air. After 10 days in a growth chamber at

25°c and 85 µE/m2/sec of light, the algae were counted and volume de-

termined to obtain 8 x 109 cells of Chlorella and 8 x 108 cells of

Chlorococcum. After centrifugation, each of the following alga concen-

trations, 1x107, 5 x 106 and 1x106 cells/ml for Chlorella and

1 x 106, 5 x 105, and 1 x 105 cells/ml for Chlorococcum, were suspended

in 500 ml of radiolabeled nutrient solution, pH 7.0. Chlorella and

Chlorococcum were exposed to 14c-Kepone concentrations of 60.9 µg/l

(2.17 µCi/ml) and 59.2 µg/l (2.12 µCi/ml), respectively. Each cell

concentration was replicated five times in 125 ml Erlenmyer flasks

17

containing 100 ml of algae suspension. The cells were incubated at 0 2 25 C at a light intensity of 15 µE/m /sec on a reciprocal shaker set

at 80 excursions per minute. The incubation period was for 48 hours.

A complete set of flasks without algae were included to serve as a con-

trol. At each sampling interval, the cells were shaken by hand and

4 ml samples were centrifuged at 3000 x g for two minutes. At the

termination of study, a portion of the algal cells were counted and

percent increase in cell number was calculated. Aliquots of 0.5 ml

of the supernatant were added to 3 ml of Aquasol (New England Nuclear)

and radioactivity measured with a liquid scintillation counter (Beckman

Model LS-250). All samples were corrected for quenching. The data was

expressed as disintegrations per minute (DPM)/ml remaining in solution

for each cell concentration at each sampling interval and tested for

significance at the 5% level of confidence according to the Duncan's

Multiple Range Test (Sokal and Rohlf 1969).

Efflux of Kepone from Chlorella and Chlorococcum. Algal cells were ob-

tained after the 48 hour incubation period of the uptake study and

utilized to measure efflux of 14c-Kepone. Four milliliters of incuba-

tion medium for each of five replicates of each alga cell concentration

were centrifuged at 3000 x g for 2 minutes. The algal cells were

resuspended in fresh sterile medium without labeled material. After

three washings the cells were suspended in 2 ml of nutrient solution,

allowed to incubate for 20 minutes under the same conditions as de-

scribed for the uptake study, centrifuged and radioactivity of 0.5 ml

of supernatant was measured by liquid scintillation counting. All

18

samples were corrected for quenching. The data was expressed as DPM/ml

for each alga cell concentration and was tested for significance at the

5% level of confidence according to the Duncan's Multiple Range Test

(Sokal and Rohlf 1969).

RESULTS AND DISCUSSION

Influence of Kepone on growth. The growth of Chlorella and Chlorococcum

was inhibited by 1.5 and 2.0 mg/l of Kepone after 12 hr. of incubation

(Tables I and II). All treatments of Kepone after 48 hr. inhibited the

growth of Chlorococcum, whereas, Chlorella was inhibited by Kepone

concentrations greater than 0.5 mg/l. No effect on growth was evident

on both algal species by Kepone concentrations up to 1.5 mg/l 96 hr.

after treatment, indicating recovery from the effect of Kepone.

Chlorococcum appeared to be more sensitive to Kepone than Chlorella,

but the initial cell concentration of Chlorella was 2.5 times higher

than Chlorococcum.

These results show that Kepone inhibited algal growth, but not

completely. The concentrations of Kepone and algal cells used in these

experiments were greater than those in the James River (USEPA 1978).

Lower concentrations of various pesticides, than those used in

this study were toxic to algae (de la Cruz et al. 1973, Vance and

Drummond 1963, Wurster, 1968, Menzel et al. 1970 and Butler 1963),

however, Hollister et al. (1975) found that Mirex, a compound similar

to Kepone, did not affect growth when various species of algae were

exposed to 0.2 µg/l. Populations of algae may, therefore, be affected

depending upon the concentrations of Kepone to which they are exposed

in the natural environment.

Effect of Kepone on respiration. Respiration of Chlorella and Chloro-

coccum was reduced by 47 and 34%, respectively when exposed to 1 mg/1

of Kepone for 96 hr. (Table III). These results support observations

19

TABL

E I

Eff

ect

of K

epon

e on

Gro

wth

of

Chl

orel

la

-5

a/

Kep

one

Cel

ls x

10

/ml-

Hou

rs o

f in

cuba

tion

(m

g/l)

12

24

48

96

o.o

2.02

a

5.75

a

38.1

.a

357

a 0.

0 +

Ace

tonJ

!./

1.89

a

5.35

ab

35.4

ab

356

a 0.

5 1.

89 a

5.

75 a

29

.8 b

e 32

4 ab

1.

0 2.

02 a

4.

24 a

b 27

.4 c

25

4 be

1.

5 1.

89 a

3.

84 b

e 29

.2 b

e 25

4 be

2.

0 1.

89 a

2.

65 c

23

.1 c

23

2 be

a/V

alue

s in

eac

h co

lum

n ar

e th

e m

eans

of

four

rep

lica

tes

and

thos

e fo

llow

ed b

y th

e sa

me

lett

er w

ithi

n ea

ch c

olum

n ar

e no

t si

gnif

ican

tly

diff

eren

t at

the

5%

leve

l of

con

fide

nce

acco

rdin

g to

the

Dun

can'

s M

ulti

ple

Ran

ge T

est.

bf

Stoc

k so

luti

ons

of K

epon

e w

ere

prep

ared

wit

h A

ceto

ne a

nd

the

sam

e vo

lum

e of

Ace

tone

, 0.

5 µ

l/m

l, w

as a

dded

as

in

the

Kep

one

trea

tmen

ts.

N

0

TABL

E II

Eff

ect

of K

epon

e on

gro

wth

of

Chl

oroc

occu

m

-5

a/

Kep

one

Cel

ls x

10

/ml-

Hou

rs o

f in

cuba

tion

(m

g/l)

12

24

48

96

o.o

0.62

5 a

1.01

6 a

2.60

0 a

13.3

8 a

0.0

+ A

ceto

nJ!-

1 0.

673

a 0.

858

b 2.

520

a 12

.79

a 0.

5 0.

600

a 0.

800

b 1.

830

b 12

.36

a 1.

0 0.

653

a 0.

754

b l .

620

b 12

.26

a 1.

5 0.

600

a 0.

600

c 1.

070

c 8.

97 a

2.

0 0.

600

a 0.

600

c 0.

650

c 2.

55 b

a/V

alue

s in

eac

h co

lum

n ar

e th

e m

eans

of

four

rep

lica

tes

and

thos

e fo

llow

ed b

y th

e sa

me

lett

er w

ithi

n ea

ch c

olum

n ar

e no

t si

gnif

ican

tly

diff

eren

t at

the

5%

leve

l of

con

fide

nce

acco

rdin

g to

the

Dun

can'

s M

ulti

ple

Ran

ge T

est.

b/

Stoc

k so

luti

ons

of K

epon

e w

ere

prep

ared

wit

h A

ceto

ne a

nd

the

sam

e vo

lum

e of

Ace

tone

, 0.

5 µ

l/m

l, w

as a

dded

as

in

the

Kep

one

trea

tmen

ts.

N .....

TABL

E II

I

Eff

ect

of K

epon

e on

Res

pira

tion

Kep

one

(mg/

l)

o.o

0.0

+ A

ceto

nJ!-

1

1.0

µmol

o2

Cons

umed

(mg

dry

wt)

-1/h

r x

lo!-

/ C

hlor

ella

C

hlor

ococ

cum

3.16

a

9.92

a

3.58

a

9.22

a

1.89

b

5.83

b

a7V

alue

s in

eac

h co

lum

n ar

e th

e m

eans

of

five

re

pli

cate

s an

d th

ose

foll

owed

by

the

sam

e le

tter

w

ithi

n ea

ch c

olum

n ar

e no

t si

gn

ific

antl

y d

iffe

r-en

t at

the

5%

leve

l of

con

fide

nce

acco

rdin

g to

th

e D

unca

n's

Mul

tipl

e R

ange

Tes

t.

b/S

tock

sol

utio

ns o

f K

epon

e w

ere

prep

ared

wit

h A

ceto

ne a

nd t

he s

ame

volu

me

of A

ceto

ne,

0.5

µl/

ml,

was

add

ed a

s in

the

Kep

one

trea

tmen

ts.

N

N

23

of de la Cruz et al. (1973) that 1 ppm of Mirex inhibited respiration

of Chlamydomonas.

Mitochondria have many important roles in plant and animal cells,

such as a source of energy for metabolic functions. Acute, or chronic

influences of various substances may cause interference with cellular

functions (Pardini et al. 1971). Mitochondrial membranes, as well as

other cellular membranes, have a high content of phospholipids. Since

organochlorine pesticides are lipid soluble, it is expected that cell

membranes would contain high concentrations of the pesticides which

might interfere with mitochondrial function (Pardini et al. 1971). In-

secticides which inhibit respiration are heptachlor, chlordane, toxa-

phene, rotenene, Mirex and Kepone (Pardini et al. 1971, Byard et al.

1975, Anderson et al. 1977, Anderson et al. 1978, Desaiah et al. 1975

and Desaiah et al. 1977). 2+ Kepone inhibits the animal enzymes Mg stimulated ATPase, and

lactate, malate and glutamate dehydrogenases in vitro (Desaiah et al.

1976, Anderson et al. 1978, Anderson et al. 1977 and Freedland and

McFarland 1965). Kepone probably affects these same enzymes in plant

cells.

Effect of Kepone on photosynthesis. Kepone at 1.0 mg/l reduced rates

of photosynthesis of Chlorella and Chlorococcum 17 and 10%, respec-

tively, after 96 hr. of incubation (Table IV). Effects of chlorinated

hydrocarbons on photosynthesis of algae have been documented; however,

the mechanism is not understood (Menzel et al. 1970 and de la Cruz et

al. 1973). Some reports suggest that chlorinated hydrocarbons inhibit

TABL

E IV

Eff

ect

of K

epon

e on

Pho

tosy

nthe

sis

Kep

one

(mg/

l)

o.o

0. 0

+ A

ceto

nJ!-

1 1

.0

µmol

o2

Evol

ved

-1

a/

(mg

chlo

roph

yll)

/h

r-C

hlor

ella

C

hlor

ococ

cum

66.0

b

76.4

a

63.4

b

33.8

ab

34.1

a

30.8

b

a/V

alue

s in

eac

h co

lum

n ar

e th

e m

eans

of

five

re

plic

ates

and

tho

se f

ollo

wed

by

the

sam

e le

tter

w

ithi

n ea

ch c

olum

n ar

e no

t si

gnif

ican

tly

dif

fer-

ent

at t

he 5

% le

vel

of c

onfi

denc

e ac

cord

ing

to

the

Dun

can'

s M

ulti

ple

Ran

ge T

est.

b/

Stoc

k so

luti

o.ns

of

Kep

one

wer

e pr

epar

ed w

ith

Ace

tone

and

the

sam

e vo

lum

e of

Ace

tone

, 0.

5 µ

l/m

l, w

as a

dded

as

in t

he K

epon

e tr

eatm

ents

.

N ~

25

the Hill reaction (Butler 1963, Wurster 1968 and Sodergren 1968). 7 Chlorella at a concentration of 2.54 x 10 cells/ml exposed to 1

I -5 mg Kepone 1 in these experiments gave a ratio of 4 x 10 µg Kepone/

cell. While no actual information was found before completion of this

study on populations of algae throughout the James River, information

was available on cell populations in the Hampton Roads area-and chloro-

phyll a concentrations in the James River. Marshall (1967) found that

populations of algae in the Hampton Roads area were approximately 200

cells/ml during the SUDDller months. Chlorophyll a content in the James

River ranged from less than 10 µg/l near Hampton Roads to more than

40 µg/l near Hopewell, Virginia (USEPA 1978). Assuming cell concentra-

tion as a direct function of chlorophyll content, cell populations

ranged from 200 to 800 cells/ml. Sampling of the James River water

revealed Kepone concentrations from as high as 0.042 µg/l to less than

0.006 µg/l (USEPA 1978). Calculations of the above information re-

vealed the amount of Kepone/cell ranged -6 10 µg Kepone/cell in the James River.

-4 from 2.1 x 10 µg to 7.5 x -5 The 4 x 10 µg Kepone/cell is

within the range found under natural conditions and it appears Kepone

may affect photosynthesis of algae in the James River.

14 Amount of C-Kepone removed from solution. Chlorella, at concentra-

tions of 1 x 106, 5 x 106 and 1 x 107 cells/ml removed 25, 40 and 57%

of Kepone from solution, respectively, after 30 min. of incubation

(Fig. 3), indicating the removal of Kepone was dependent upon cell con-

centration. Chlorella cells showed a significant efflux of Kepone

during longer incubations. The rates of efflux peaked at 1 and 3 hr.

incubations at the lower cell concentrations but continued during the

...... a -C""I 5 a

4 .

Control~/

ab ab ab ~- .!ho .!2.

1o 3 ...... '

...... a -C""I I 0 ...... >I ~ p.. Q

·,

1 ·,

0

5

4

. 25 . 5 1 3 18 24 48 Time (hours)

5 x 106 Cells/ml

o .......... ~1---..... ---t--+--+--+-~ . 25 . 5 1 3 18 24 48

26

1 x 106 Cells/ml

5

4 e ....... a -C""I I 0 3 ......

><

~ 2 Q

1

0 .25 .5 1 3 18 24 48

Time (hours)

1 x 107 Cells/ml

5

e e e ...... a -

27

entire 48 hr. period at the highest cell concentration. The lower cell

concentrations of Chlorella removed Kepone from solution at a steady

rate after 3 hr. of incubation until the experiments were terminated,

which may have been the result of higher cell populations (Fig. 5).

The amount of Kepone removed in 48 hr. by each of the cell concentra-6 6 7 tions, 1 x 10 , 5 x 10 and 1 x 10 cells/ml was 16.6, 17.3 and 9.9%,

respectively.

Chlorococcum cells did not demonstrate the efflux of Kepone ob-

served with Chlorella except at the 1 x 106 cells/ml (Fig. 4). It

appears that an equilibrium of Kepone in solution and with the algal

cells was reached rapidly. A trend in an increased removal of Kepone

was evident in the later sampling interval, however, this may have been

due to increased cell number (Fig. 6). Chlorococcum at,l x 105, 5 x 105

6 and 1 x 10 cells/ml removed 7.8, 40.4 and 56.8%, respectively, of the

Kepone from solution.

The reason for Kepone efflux was not completely resolved, however,

equilibriums between adsorption/desorption and absorption are probably

involved. Many organochlorine insecticides have adsorptive properties

(Acree 1963) and Kepone has a high affinity for organic material (USEPA

1978). Adsorbed Kepone may be desorbed when the pH of the medium be-

comes more alkaline (USEPA 1978). It is not known if the pH of the

medium changed during the incubations; however, algal cells increase

the pH of the medium during photosynthesis (Swader 1978).

Absorption may occur actively or passively and is a physical and

complex physiological process in which photosynthesis and respiration

play an important role (Hill et al. 1967). Lipid soluble materials

Control!/ 5 a~

I b -

4 •

3,

2 ~ .

1,

0

5

4

.-4 s

- !?£. d l?£. des_ -

• 25 • 5 1 3 18 24 48 Time (hours}

5 x 105 Cells/ml

M- 3 ~LI r-...::.:=- i k lm

1 0 .-4

H 2 !: i:::i

1

0 • 25 • 5 1 3 18 24 48

28

5

4 .-4 s -M 3 I 0 .-4

H

!: 2 i:::i

1

0

5

4

:! 7 3 0 .-4

1

1 x 10 5 Cells/ml ..

~ef ef ef d . _f&_ R -. . .

• 25 • 5 1 3 18 24 48 Time (hours}

1 x 106 Cells/ml

0 ......... -+--1--+--+--+--I--~25 .5 1 3 18 24 48

Time (hours} Time (hours) 14 Fig. 4. Amount of C-Kepone remaining in solution after uptake by

Chlorococcum over a 48 hr. sampling period • .!,/All bars with the same letter are not significantly different at the 5% level of confidence according to the Duncan's Multiple Range test.

29

Chlorella 100. Cells/ml

A. 1 x 106

80' B. 5 x 106 .... c. 1 x 107 s -Cll .... .... 60 . ..

c.J GI -Cll ~ ~

40. - -CJ i::

1-1

H 20.

0 A B C

Cells/ml Fig. 5. Percent increase of Chlorella cell number during uptake study over 48 hr.

200.

'i1 160 . -Cl) .... G1 120 c.J G) Cll Id Q) 80. ~

~ H 40,

0

Chlorococcum Cells/ml

A. 1 x 105

B. 5 x 10~ c. 1 x 10 -

- -A B C

Cells/ml Fig. 6. Percent increase of Chlorococcum cell number dur-ing uptake study over 48 hr.

30

move through lipid containing cell walls and membranes. This penetra-

tion is due to lipid and water solubility of the substance with the

lipid solubility determining the extent of penetration. A substance

which has a higher lipid than water solubility will move from the water

phase to the lipid phase (S8dergren 1968). The solubility of Kepone

in water is approximately 1.5 to 2.0 mg/liter at pH 7. Its solubility

in lipids is quite high, but the actual quantity is not known (USEPA

1978). Algae are approximately 20% or less lipid on a dry weight basis

(Orcutt 1978), therefore, the rapid initial uptake plus the high lipid

solubility of Kepone suggests absorption was the primary means of

Kepone uptake. Kepone inhibits photosynthesis, respiration and various

enzymes and an alteration of the metabolism of Chlorella and Chlorococcum

by Kepone may have caused the efflux of Kepone.

Efflux of Kepone. The data (Fig. 7 and Fig. 8) illustrate a portion of

the activity associated with algal cells was not bound and was released

into solution, indicating that Kepone was both adsorbed and absorbed by

algal cells. If absorption was the only mechanism of uptake, the data

would have indicated more Kepone returning to solution. Adsorption was

probably the primary mechanism of Kepone uptake, however, absorption

did occur. Algae can accumulate Kepone to toxic levels from non-toxic

concentrations (Walsh 1977); however, the data reported here indicate

that desorption occurred. Movement of Kepone contaminated algae by -····,.-··-·-.. ·-···.,..""'""'_.,~,.~·'•'•'~0''' -- "••••,.-......... ,.,o••~·. M·~ .. -. .- .... ~~-·

31

5

4,

i - 3 • N I 0 ..s.. .....

>4 2 . ~ A

1,

0 A B c

Cell Concentratio~/ 14 Fig. 7. Efflux of C-Kepone after

48. hr. from Chlorella cells. Cell concentrations,-A,B,C, re5~r to fi-nal 'ell counts, A(6 x ~O ), B(2.85 x 10 ), and C(5.69 x 10) centrifuged from 4 ml of treatment solution and placed in 2 ml of nutrient solution without labeled mate.rial:. · a/Bars with the same letter are not significantly different at the 5% level of confidence.

32

10.

8,

r-4 ,..!.. SI - 6, N I 0 r-4

~ 4 . ~ ,:::i

2 •

0 A B c

eell Concentrations 14 Fig. 8. Efflux of C-Kepone after

48 hr. from Chlorococcum cells. Cell concentrations, A,B,C, refer to final cell counts, A~9.9 x 105), B(4.0 x 106), and C(6.8 x 10 ) centrifuged from 4 ml of treatment solution and placed in 2 ml of nutrient solution without labeled material. a/Bars with the same letter are not sig-~ificantly different at the 5% level of confidence.

SUMMARY

Kepone affected the growth of Chlorella and Chlorococcum in studies

with concentrations of Kepone approaching the solubility limit in water.

Growth of Chlorella was more tolerant than the growth of Chlorococcum

to 2 mg/l of Kepone. Effects on growth were significant after 24 hours

of incubation.

Photosynthesis and respiration were inhibited in both algae by 1 mg

Kepone/l. Photosynthesis of Chlorella and Chlorococcum was inhibited

by 17 and 10%, respectively. Respiration of Chlorella and Chlorococcum

was inhibited by 47 and 34%, respectively.

The initial uptake of Kepone was proportional to the cell concen-

trations of both algae. Kepone was released back into the medium by

algal cells after the initial uptake, which may have been the result of

Kepone inhibiting the algal metabolism. Uptake of Kepone was propor-

tional to Chlorococcum cell concentration at the end of the 48 hours

of study, but not for Chlorella cells. Both algae at all cellular

concentrations showed an efflux of Kepone when washed, suspended in

untreated nutrient solution and incubated for 20 minutes.

33

LITERATURE CITED

Acree, F., Jr. 1963. Insecticide volatibility, codistillation of DDT with water. J. Agric. Food Chem. 11:278-280.

Anderson, B.M., S.T. Kohler and R.W. Young. 1978. Interactions of Kepone with rabbit muscle lactate dehydrogenase. J. Agric. Food Chem. 26:130-133.

Anderson, B.M., C. Noble, Jr. and E.M. Gregory. 1977. Kepone in-hibition of malate dehydrogenases. J. Agric. Food Chem. 25:485-489.

Andrade, P., Jr. and W.B. Wheeler. 1975. Identification of a Mirex metabolite. Bull. Environ. Contam. Toxicol. 14:473-479.

Arnon, D.I. 1949. Cooper enzymes in isolated chloroplasts. Poly-phenol oxidase in Beta vulgaris. Plant Physiol. 24:1-15.

Bahner, L.H., A.J. Wilson, Jr., J.M. Sheppard, J.M. Patrick, Jr., L.R. Goodman and G,E, Walsh. 1977. Kepone bioconcentration, accum-ulation, loss and transfer through estuarine food chains. Chesapeake Sci. 18:299-308.

Bourquin, A.W., P.H. Pritchard and W.R. Mahaffey. 1977. Effects of Kepone on estuarine microorganisms. Developments in Industrial Microbiology. 19. ·

Butler, P.A. 1963. Pesticide wildlife studies: a review of fish and wildlife investigations during 1961 and 1962. Commercial fish-eries investigation pp.11-25. J.L. George(ed). Fish and Wildlife Service Circ. 167, U.S. Dept. Int., Washington, D.C., 109 P•

Byard, J.L., v.c. Koepke, L. Abraham, L. Golberg and F. Couston. 1975. Biochemical changes in the liv~s of mice fed Mirex. Toxicol. Appl. Pharmacol. 33:70-77.

Carlson, D.A., K.D. Konyha, W.B. Whyeler, G.P. Marshall and R.G. Zaylskie. 1976. Mirex in the environment: Its degradation to Kepone and related compounds. Science 194:939-941.

de la Cruz, A.A. and S.M. Naqvi. 1973. Mirex incorporation in the environment: Uptake in aquatic organisms and effects on the rates of photosynthesis and respiration. Arch. Environ. Contam. Toxicol. 1:255-264.

Desaiah, D. and R.B. Koch. 1975. Inhibition of ATPases activity in channel catfish brain by Kepone and its reduction product. Bull. Environ. Contam. Toxicol. 13:153-158.

34

35

Desaiah, D., I.K. Ho and H.M. Mehendale. 1977• Effects of Kepone and Mirex on mitochondrial Mg2+ ATPase activity in rat liver. Toxicol. Appl. Pharmacol. 39:219-228.

Desaiah, D., I.K. Ho and H.M. Mehendale. 1977. Inhibition of mito-chondrial Mg2+ ATPase activity in isolated perfused rat liver by Kepone. Biochem. Pharmacol. 26:1155-1159.

Freedland, R.A. and L.Z. McFarland. 1965. The effect of various pesti-cides on purified glutamate dehydrogenase. Life Sci. 4:1735-1739.

Garnas~ R.L. and A.W. Bourquin and P.H. Pritchard. 1978. The fate of 1 C-Kepone in Estuarine Microcosms. In: Appendix C to EPA Mitigation Project Report, USEPA, Environmental Research Labora-tory, Gulf Breeze, FL.

Hansen, D.J., D.R. Nimmo, S~C. Schimmel, G.E. Walsh and A.J. Wilson, Jr. 1976. Kepone: Hazard to aquatic organisms. Science 193:528.

Hansen, D.J., D.R. Nimmo, S.C. Schimmel, G.E. Walsh and A.J. Wilson, Jr. 1977. Effects of Kepone on estuarine organisms. In: Recent Advances in Fish Toxicology: A Symposium. EPA publication 600/3-77-085. pp.20-30.

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The vita has been removed from the scanned document

UPTAKE AND EFFECTS OF KEPON/J ON GROWl'H,

RESPIRATION AND PHOTOSYNTHESIS OF

CHI.ORELLA SOROKINIANA AND CHLOROCOCCUM BYPNOSPORUM

by

Gary R. Young

(ABSTRACT}

KeponefJ, the registered trade name for decachlorooctahydro-1,3,

4-metheno-2H-cyclobuta cd pentalen-2-one, inllibited growth and reduced

rates of photosynthesis and respiration of Chlorella sorokiniana Shihira

and Kraus and Chlorocbccum hypnosporum Starr.

rates of respiration more than photosynthesis.

The insecticide reduced 14 Uptake of C-Kepone

by the algae was proportional to cellular concentration. A net efflux

of Kepone was exhibited by Chlorella cells after 30 minutes of incuba-

tion, whereas, equilibrium occurred within 15 minutes of incubation for

Chlorococcum. Desorption of Kepone was evident when both algal species

were removed from Kepone treated solutions and incubated in untreated

nutrient solutions.

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