• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Marine algae -- the seaweeds
 Scope of the problem
 Literature review
 Experimental procedure
 Results and discussion
 Summary and conclusions
 Appendix
 Bibliography
 Biographical sketch
 Copyright














Title: Ralationship of certain macroscopic marine algae to ZN65.
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Title: Ralationship of certain macroscopic marine algae to ZN65.
Series Title: Ralationship of certain macroscopic marine algae to ZN65.
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Creator: Bedrosian, Paul Harry,
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
        Page v
    List of Figures
        Page vi
        Page vii
    Introduction
        Page 1
        Page 2
        Page 3
    Marine algae -- the seaweeds
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
    Scope of the problem
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Literature review
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
    Experimental procedure
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
    Results and discussion
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
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        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
    Summary and conclusions
        Page 102
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
    Appendix
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
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        Page 154
        Page 155
        Page 156
        Page 157
    Bibliography
        Page 158
        Page 159
        Page 160
        Page 161
        Page 162
        Page 163
        Page 164
    Biographical sketch
        Page 165
        Page 166
    Copyright
        Copyright
Full Text












RELATIONSHIP OF CERTAIN MACROSCOPIC

MARINE ALGAE TO ZN65












By

PAUL HARRY BEDROSIAN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
June, 1961















ACKNOWLEDGMENTS


The author is sincerely grateful to Doctors A. P. Black, George

K. Davis, Alan D. Conger, and to Professors John E. Kiker, and George

B. Morgan for serving as members of the supervisory committee and for

giving freely of their time and knowledge towards the completion of

the research studies undertaken. Grateful acknowledgment is made to

Dr. James B. Lackey who not only served as Chairman of the committee

but also guided the author through the biologic phase of the research.

The author wishes to thank Miss Myrtice E. Smith and Aubrey

I. Covington for assistance in the radiological laboratory and to

student assistants Miss Carrie E. Bennett, William Carr and Robert A.

Menzies for assistance in marine biology. Appreciation is extended

also to the Curator's staff at the Marineland Studios, Marineland,

Florida for its cooperation in providing sea water for this study.

Much credit is due to his wife for her understanding during

this period of academic hardship and for her helpful comments and

kind assistance in the drafting of the first manuscript copy.

Appreciation is extended to Mrs. Marjorie A. DuMez for her patience

and understanding in typing the final manuscript.

This research was supported in part by U. S. Public Health

Service Traineeship Number 60-218.
















TABLE OF CONTENTS


ACKNOWLEDGMENTS . . . . . .

LIST OF TABLES . . . . . .

LIST OF FIGURES . . . . . .

CHAPTER

I. INTRODUCTION . . . .

II. MARINE ALGAE -- THE SEAWEEDS

III. SCOPE OF PROBLEM . . .

IV. LITERATURE REVIEW.. . .

V. EXPERIMENTAL PROCEDURE . .

VI. RESULTS AND DISCUSSION .

VII. SUMMARY AND CONCLUSIONS .

APPENDICES . . . . . . .


BIBLIOGRAPHY . . . . .

BIOGRAPHICAL SKETCH . . . .


iii


Page

ii

iv


. 1I

S 4

S 12

S 21

S 40

. 54

. 102


. . . . . . .


. . . . . .I

. . . . . .


. . .















LIST OF TABLES


Table Page

1. SPECIFIC ACTIVITY OF ZN65 CONCENTRATED BY SPHACELARIA . 55

2. SELF-ABSORPTION CORRECTION FACTORS FOR ZN65 IN SEA WATER 56

3. THE APPROXIMATE MAXIMUM CONCENTRATION FACTORS FOR ZN65
TAKEN UP BY THREE MARINE ALGAE UNDER VARIOUS CONDITIONS
OF LIGHT AND TEMPERATURE . . . . . . . ... 68

4. COMPARISONS OF THE APPROXIMATE MAXIMUM CONCENTRATION
FACTORS OF THE EQUIVALENT ALGA TO THOSE OF THE ACTUAL ALGA 86

5. COMPARISONS OF THE APPROXIMATE MAXIMUM CONCENTRATION
FACTORS AS DERIVED FROM ORIGINAL AND REPLICATED UPTAKE
EXPERIMENTS .. . . . . . . . . . . 87

6. COMPARISONS OF THE APPROXIMATE DILUTION FACTORS OF THE
MARINE ALGAE AS DETERMINED BY THE CONCENTRATION FACTOR
EQUIVALENT VOLUME AND BY THE RATIO OF INITIAL AND FINAL
WATER ACTIVITY . . . . . . . . .. . . 91

7. COMPARISONS OF ASSAYED ALGAL CONCENTRATION FACTORS IN
MEDIUM OF HIGHER ACTIVITY AS COMPARED TO REFINED
CONCENTRATION FACTORS IN MEDIUM OF ABOUT 25 COUNTS PER
MINUTE PER MILLILITER ...... . . . . .... 96

8. ACTIVITY OF SPECIFIC PORTIONS OF GRACILARIA FOLIIFERA USED
IN THE PREPARATION OF THE AUTORADIOGRAPHS . . . .. 101

9. GRACILARIA UPTAKE DATA AT 25C . . . ..... 117

10. GRACILARIA UPTAKE DATA AT 180C . . . . . ... .120

11. GRACILARIA UPTAKE DATA AT 5C . . . . . ... 124

12. ENTEROMORPHA UPTAKE DATA AT 250C . . . . . .. ..128

13. ENTEROMORPHA UPTAKE DATA AT 180C . . . . ... .131

14. ENTEROMORPHA UPTAKE DATA AT 50C . . . . . .... 134

15. SPHACELARIA UPTAKE DATA AT 250C . . . . . . .. 138












Table Page

16. SPHACELARIA UPTAKE DATA AT 180C . . . . . . . 142

17. SPHACELARIA UPTAKE DATA AT 5C . . . . . . .146

18. GROWTH RATE DATA FOR ENTEROMORPHA . . . . . . 151

19. GROWTH RATE DATA FOR SPHACELARIA . . . . .... 152

20. RETENTION DATA FOR SPHACELARIA AT 180C IN PLAIN SEA WATER 156

21. RETENTION DATA FOR SPHACELARIA AT 18 C IN ZINC ENRICHED
SEA WATER . . . . . . . . . . . 157















LIST OF FIGURES


Figure Page

1. SCHEMATIC FOR THE ARRANGEMENT OF AQUARIA, LIGHT SOURCE,
AND ALGAE FOR A GIVEN UPTAKE EXPERIMENT . . . .. 43

2. ARRANGEMENT OF GLASS AQUARIA AND APPURTENANCES IN THE
WATER RECIRCULATION SYSTEM FOR THE UPTAKE EXPERIMENTS . 44

3. ARRANGEMENT OF BEAKERS AND ALGA IN TRANSLOCATION
EXPERIMENT . . . . . . . ...... 51

4. THE SELF-ABSORPTION EFFECT AND CORRECTION CURVES FOR SEA
WATER OF APPROXIMATELY 35 %/oo SALINITY FOR THREE
CONCENTRATIONS OF ZN65 . . . . . ... . . .. 57

5. CONCENTRATION FACTORS FOR GRACILARIA FOLIIFERA IN ZN65-
DOSED SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 25C 64

6. CONCENTRATION FACTORS FOR GRACILARIA FOLIIFERA IN ZN65-
DOSED SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 180C 65

7. CONCENTRATION FACTORS FOR GRACILARIA FOLIIFERA IN ZN65-
DOSED SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 50C 66

8. THE APPROXIMATE MAXIMUM CONCENTRATION FACTORS OF
GRACILARIA FOLIIFERA FOR ZN65 IN DOSED\SEA WATER UNDER
DIFFERENT LIGHTING AND TEMPERATURE CONDITIONS . . . 67

9. CONCENTRATION FACTORS FOR ENTEROMORPHA PROLIFERA IN ZN65-
DOSED SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 250C 70

10. CONCENTRATION FACTORS FOR ENTEROMORPHA PROLIFERA IN ZN65-
DOSED SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 18C 71

11. CONCENTRATION FACTORS FOR ENTEROMORPHA PROLIFERA IN ZN65-
DOSED SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 5C 72

12. THE APPROXIMATE MAXIMUM CONCENTRATION FACTORS OF
ENTEROMORPHA PROLIFERA FOR ZN65 IN DOSED SEA WATER UNDER
DIFFERENT LIGHTING AND TEMPERATURE CONDITIONS ...... 73

13. CONCENTRATION FACTORS FOR SPHACELARIA SP. IN ZN65-DOSED
SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 25C . 75

14. CONCENTRATION FACTORS FOR SPHACELARIA SP. IN ZN65-DOSED
SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 180C . . 76














15. CONCENTRATION FACTORS FOR SPHACELARIA SP. IN ZN65-DOSED
SEA WATER UNDER VARIOUS LIGHTING CONDITIONS AT 5 . . 77

16. THE APPROXIMATE MAXIMUM CONCENTRATION FACTORS OF
SPHACELARIA SP. FOR ZN65 IN DOSED SEA WATER UNDER DIFFERENT
LIGHTING AND TEMPERATURE CONDITIONS . . . . .. 78

17. GROWTH RATE CURVES FOR SPHACELARIA SP. AND ENTEROMORPHA
PROLIFERA UNDER VARIOUS CONDITIONS OF LIGHTING, TEMPERATURE,
AND MEDIUM ENRICHMENT . . . . . . . .. 79

18. THE CONCENTRATION FACTORS FOR SPHACELARIA SP. AT 25C IN
ZN -DOSED SEA WATER ENRICHED WITH KCL . . . . . 83

19. TYPICAL WORKING GRAPHS DEPICTING THE DETERMINATION OF THE
CORRECTED-MEDIUM CURVE . . . . .. . . . 89

20. RETENTION OF ZN65 BY SPHACELARIA SP. AT 18C, UNDER 500
FOOT-CANDLES OF LIGHT, IN PLAIN SEA WATER AND IN SEA WATER
ENRICHED WITH STABLE ZINC . . . . . . ... 93

21. RESOLUTION OF THE RETENTION CURVE FOR SPHACELARIA SP. AT
180C, UNDER 500 FOOT-CANDLES OF LIGHT, AND IN PLAIN SEA
WATER . . . . . . . .. . . . . 94

22. RESOLUTION OF THE RETENTION CURVE FOR SPHACELARIA SP. AT
18C, UNDER 500 FOOT-CANDLES OF LIGHT, AND IN SEA WATER
ENRICHED WITH STABLE ZINC . . . . . . .. 97

23. AUTORADIOGRAPHS OF CONCENTRATION SITES FOR ZN65 TAKEN UP
BY GRACILARIA FOLIIFERA UNDER 500 FOOT-CANDLES AT 180C.
LEFT ORGANISM WITH FRUITIN(. BODIES, RIGHT WITHOUT FRUITING
BODIES . . . .... . . . . ..... . 99


Figure


Page















CHAPTER I


INTRODUCTION


The discharge of industrial and domestic waste to a receiving

body of water has long been the keen interest of sanitary engineers

throughout the world. Deep concern over proper waste discharge

practices is evidenced because water is of vital importance to the well

being and comfort of man and, therefore, its conservation and its

protection against waste contamination are highly desirable.

In some areas the desire for maintenance of clean water arises

principally out of the need for the conservation of this invaluable

resource, in other areas the aesthetic appearance and recreational uses

of the receiving water are the motivating forces which protect it

against man's pollution. It is only in a few instances, such as the

discharge of highly poisonous cyanide wastes, where these receiving

waters are protected for the sake of man's health.

Most streams and other bodies of water can adequately handle

reasonable amounts of untreated wastes with no detrimental effect to

the water. The process of self-purification in a stream, for example,

is sufficient to prevent the occurrence of a polluted situation. This

process is essentially one of a biological nature operating under

optimum conditions for its successful completion. In self-purification,

potential organic pollutants are reduced by the biologic activity to

harmless, non-pollutant mineral matter. There are, however, wastes












such as radioactive ones which can be reduced to simple mineral matter

by this process and yet be considered as highly undesirable pollutants.

Man's abhorrence of these radioactive pollutants stems not from the

usual reasons associated with the dislike of pollution in general, but

primarily from the high potential health hazard offered by these wastes.

This is the main reason for man's great concern in the treatment of

radioactive wastes and in their proper discharge to the environment.

Radioactivity is a complex phenomenon which is generally harmful

to the well being of most living entities. Its ionizing radiations

react with matter and alter the natural life functions of living

creatures thereby causing radioactive wastes to be undesired in the

environment. Biologic activity, which has proved to be successful

in reducing most other wastes to innocuous mineral matter, cannot

alter the nature of radioactivity, nor has any other method been found

to be effective in this regard. Basically, the only remedy to protect

man from the deleterious effects of radioactivity is to remove the

radioactive substances from his environment and to make certain that

they will not return.

Increasing usage of radioactive isotopes in industry, research

and hospitals, coupled with production of power by atomic energy, has

augmented the quantity of radioactive wastes in the environment. Atomic

power at present is not confined just to land installations, but is now

operative on a few ocean-going ships and submarines. It will only be a

question of time before both land and marine atomic installations will

grow in number. The reality is present that waste discharges from

atomic plants located on land near the ocean, together with the waste












discharges from marine power plants, can create in the coastal waters a

potential health hazard to man (34, 37, 60).

It would not be wholly justified for man to set aside atomic

energy and sacrifice all the potential benefits that are within his

reach just because radioactive wastes might constitute a menace to his

well being. This, of course, would be the most positive method in

protecting the environment from radioactive sources, but it is considered

that adequate protection can be achieved by careful, deliberate control

in the discharge of radioactive wastes. Accordingly, it is the purpose

of this study to ascertain the merits of one small aspect of this

control as.it pertains to man's closest marine environment -- the

coastal waters (52).

The fact has long been known that plants, algae, and many

microorganisms can take up and concentrate both radioactive and non-

radioactive elements from their immediate aquatic environment thereby

reducing the concentration of such elements in their surrounding water (72).

Is it not possible then to apply the same principle to the ocean? Why

cannot some organisms, for example macroscopic marine algae, take up

radionuclides from the ambient water and thereby reduce the aquatic

concentration of radioactivity to allowable limits? Heretofore, very

few investigations of this potential control mechanism have ever been

made. It is felt that there is some merit in the decontaminating

potentialities of these macroscopic marine alage which could possibly

not only afford protection to the coastal environment, but also increase

the efficiency of the ocean as a radioactive waste disposal medium.














CHAPTER II


MARINE ALGAE -- THE SEAWEEDS


Marine algae are photosynthetic plants, some of which grow

completely submersed in water, while others thrive with only partial

submersion. The size of these plants varies widely from one species to

another ranging from one-celled microscopic flagellates to giant multi-

cellular kelp. Like terrestrial vegetation, the algae possess chlorophyll

by which they are able to utilize the sun's energy to synthesize their

food. The myriad chemical compounds ever-present in the oceans, are

used by the algae in this process of photosynthesis. However, unlike

spermatophytes the algae have no flowers, leaves, seeds, or roots (80, 36).

The plant kingdom, although highly ramified, consists of four

primary divisions: Thallophyta, Bryophyta, Pteridophyta, and

Spermatophyta. Only the Thallophyta and Spermatophyta divisions are

represented in the sea with the algae belonging to the former (75).

Although most algae usually require in their environment similar physical

factors such as air, light, and moisture for existence, they differ in

cell structure, reproductive processes, size, shape, color and habitat.

Therefore, it is possible for many algae to look alike and yet belong

to different genera, families, and even orders (28).

Marine algae have been generally classified, according to the

color manifestations of their chromataphores, into four principal groups

which are Chlorophyceae (green), Myxophyceae (blue-green), Rhodophyceae












(red), and Phaeophyceae (brown). There is also a nebulous fifth classi-

fication comprising the yellow-green algae which are a heterogeneous

group. The yellow-green algae for the most part are floating or plank-

tonic forms whereas the others, except for some of the blue-green algae,

are primarily attached forms. The macroscopic forms of these attached

algae, which bear a close physical resemblance to terrestrial plants,

are collectively known as seaweeds and species of these were used in

this study.

The growth of algae occurs chiefly in the littoral region of the

ocean. The boundaries of this region extend continuously from the high

tide level onshore to approximately the 200 meter depth offshore. This

particular depth is generally accepted by scientists to approximate the

position of the outer edge of the continental shelf. They also consider

200 meters to be the depth which separates the lighted portion of the

sea from the dark regions. It therefore follows that this depth could

conceivably represent the limiting extent to which photosynthetic

organisms can survive. However, the depth through which sufficient

light can penetrate in order to sustain the growth of attached plants

(seaweeds) is approximately the 50 meter depth offshore. The stretch

of ocean between this depth and the high tide level is known as the

eulittoral or upper littoral zone (75). Since seaweeds often are both

sessile and photosynthetic, they can only grow in a region which will

afford both light and attachment. Such conditions are provided in the

eulittoral regions and, only under special circumstances, on the high

sea (49, 62).











As algae are photosynthetic organisms, the complete deletion of

light at great ocean depths restricts the growth of algae to the shallow

waters of the littoral regions which comprise about 2 per cent of the

ocean floor (75). The depth to which sunlight can penetrate so as to

be used effectively in plant photosynthesis usually varies from one

oceanic area to another and also varies with the latitude. The lighted

depth is greater in the low latitudes and less in the higher latitudes.

In the northern seas the lower limit of the lighted depth is approxi-

mately 40 to 50 meters (28). In the warm seas, where there is usually

less sediment in suspension, this depth ranges between 100 to 130 meters.

In any event, the maximum depth at which algae seem able to live has

been arbitrarily set at 200 meters (67).

The growth of seaweed in the littoral region exhibits a very

general tendency towards a zonal distribution along the ocean floor.

The sun-forms (primarily Chlorophyceae), grow in the upper portions of

the eulittoral region, commonly known as the intertidal belt, and

utilize strong light for their photosynthesis, while much of the shade-

forms of seaweeds (mainly Rhodophyceae and Phaeophyceae), grow in the

lower reaches of the littoral section where they utilize the weaker

light in their photosynthesis.

High up on the intertidal belt, seaweeds grow in very shallow

water or in no water at all except for water sprays they might receive

by the action of waves or wind. Here, the green and blue-green algae

thrive abundantly as all spectral light is available to them. As the

sun's light penetrates into the deeper water, portions of its spectrum,

the red, orange and yellow components, are filtered out by the water.











At this depth, the resultant light is efficiently utilized in photo-

synthesis by the brown algae and it is in this zone where the browns

grow luxuriously. When after penetrating into still deeper water where

all that is left of the sun's light is the blue component, the red algae

appear in greater abundance with the obvious reduction of other forms.

The filtering of the sunlight is a function of the turbulence of the

surface water and also of the amount of materials in suspension. It is

not wholly a function of depth. In other words, it is possible for red

algae to thrive in turbid water only a few meters deep as well as in

clear water many meters deep. In both situations, the resultant light

reaching the red algae would be about the same.

The zonation of seaweeds is not clean cut but exists with much

overlapping. This unique type of distribution is generally attributed

to the principal accessory pigment contained in the algal chromataphores.

There is, however, no absolute correlation between the color of the algae

and the depths at which they live or of the intensity of the illumination

they receive (75).

Members of the Chlorophyceae are found mainly in the warmer

waters of the upper littoral zone, the depth of which extends from high

tide level to approximately the 10 meter depth offshore. These algae

thrive best in well lighted habitats and of all the marine algae, they

are most closely related to the fresh water algae. The green algae vary

in size from microscopic to large broad fronded (comparable to terrestrial

leaves) specimens. The blue-green algae consist solely of small, poorly

organized plants of both single and multicellular variety. The blue-

greens are found in both fresh and brackish warmer waters and are the

organisms which usually cause sliming of these waters.











Red algae vary in size from single-celled microscopic plants to

filamentous, branching forms of broad fronds one or two meters long.

The red seaweeds are widely distributed but are most abundant in

temperate climates. Generally they occur in water ranging in depth from

the intertidal zone to depths of 130 meters or more. The brown seaweeds

are structurally more complex than the red and grow at shallower depths

than the red. The browns are also widely distributed, being more

abundant in the temperate and even cooler waters and vary in size from

small filamentous forms to giant kelps. Both the browns and reds are

found predominantly in the marine environment with only a few occurring

in fresh water (75, 26).

In appearance, large marine algae grow very much like flowering

plants. The marine organisms seem to grow out of the ground or out of

whatever substratum to which they are attached. This illusion is created

by the algae's root-like appendage -- the holdfast. It is by means of

the holdfast that seaweeds attach themselves to rocks, sand, shells, and

other substrata. The substratum is not exclusively inorganic since

some algae grow while attached to animals, wood, and even to other algae.

Different species of algae are found on different substrata but this is,

for the most part, due to the variations in roughness, hardness, and

other physical differences of the substrata and not to their chemical

characteristics. The holdfast merely holds the seaweed to the substratum

and is not an organ used to take in nourishment for the plant (67, 48).

Nourishment is taken in by the seaweeds directly through their cell

walls from the ambient waters. Algae attached to substrata do not move

except to sway with currents, but a constant change in water provides











nutrients. Floating algal forms may not receive as much food because

they move along with their food and do not have too much opportunity to

make contact with other potential food as do the attached algae. It is

for this reason that large size is attained only by the attached forms.

The ocean currents also tend to stabilize the temperature, salinity, and

pH of the ambient water and thereby tend to create a more favorable

growth environment for the seaweed. As seaweeds can withstand large

changes in these physical factors, it is not acutely essential that they

be controlled within narrow limits (49).

The physical factors affecting sea water exhibit but small

variations out in the open sea. Organisms which inhabit the open sea

are so very much adapted to their surroundings, which for all practical

purposes can be considered fairly constant, that they cannot adapt

themselves to an ever changing environment. These organisms are, there-

fore, very nonresistant to their environment and readily succumb to

significant changes. There is, however, a zone of the sea which

experiences very large variations in its physical state and thereby

gives rise to the propagation of the more highly resistant marine forms.

This zone, previously mentioned, is the intertidal belt and is bounded

by the extreme high and low tide levels.

The shore in the intertidal belt is alternately covered and

uncovered by the ocean tides. Not only are the seaweeds which grow on

this shore subjected to the tidal effects, but also to pooling effects.

Along the shore and inlets algae may become isolated in pools of water

which eventually could become highly saline by the evaporation of the

water or become very dilute by rain or extremely warm by the sun and






-10-


perhaps even become very basic or acidic by the photosynthetic action

of the algae. The effects of the tide upon the seaweeds is, therefore,

to expose the plants to desiccation, to severe temperature change, and

to wide variation in both salinity and pH. The seaweeds that thrive on

this shore, therefore, must be very resistant to withstand desiccation,

and extreme sudden changes of temperature, salinity, and hydrogen-ion

concentration (28, 63).

Some species of Fucus, for example, can survive out of water

for periods of 24-48 hours (49). Other seaweeds are known to live out

of water for more than 15 consecutive days (38) and still others have

been discovered which were able to withstand desiccation for such a

long time that they were still alive even after becoming brittle and

easily pulverizable. Such desiccation is tolerated only by the resistant

littoral forms. As a matter of fact, some of these forms require

desiccation in order to live, otherwise they will die (67, 2).

In conjunction with the temperature of the ocean water, it is

fairly well established that the thermal variations are much greater

for shallow water, i.e. the intertidal zone, than for the deeper water

of the open sea. Temperature variation is, therefore, a function of

depth. Not only does the temperature vary with depth, but it also

varies from one geographical location to another. These temperature

variations govern the distribution of algae both to species located in

a certain geographical site and to vertical location in the ocean depths.

Some algae are known to inhabit the higher levels of the ocean in the

winter and migrate to lower depths in the summer when the surface and

near surface water becomes warmer (67). The Fucales as well as many






-11-


other seaweeds common to the intertidal region have been known to

withstand temperatures as low as -200C and as high as 340C (25).

Species of seaweeds thriving in the high tide level and which

become exposed when the water is at low tide can tolerate salinity

concentrations of 20 to 300 per cent of normal sea water for 24 hours.

Seaweeds of the mid tide and lower levels can tolerate a salinity range

of 20 to 200 per cent of normal sea water and 40 to 150 per cent

respectively. These figures indicate that seaweeds living in the

intertidal zone are able to withstand a wider range of salt concentration

than those growing in deeper water (27).

With regard to the hydrogen-ion concentration, present data

indicate that again the algae growing closest to the shore can tolerate

the wider pH range. Some seaweeds can withstand pH as high as 10 while

others thrive in pH as low as 5. During low tide, photosynthesis of

algae may change the pH of the water to a value as high as 9.9. Generally,

many algae can exist in pH ranging from 6.8 to 9.6. In contrast to this

range, the pH in the open sea ranges from 8.1 to 8.3 and is strongly

buffered (49).















CHAPTER III


SCOPE OF PROBLEM


Ever since the introduction of the atomic bomb approximately

sixteen years ago, the peaceful uses of atomic energy have been slowly

but surely growing. From its original use as a means to wreak

destruction, atomic energy has steadfastly grown into a boon to man and

is now not only operable to supply electrical power for domestic use

but is also employed as the source of propelling power for both merchant

and naval vessels (37, 60). As present applications of atomic power

prove to be just as effective, if not more effective than conventional

power based on fossil fuels, and as the economical cost of atomic

energy production becomes comparable to that of conventional power

production, then its use as the chief fuel in power plants will increase.

As of this writing, no less than one hundred land-based power reactors

have been built and are in operation or are being planned for

construction (59). Within the next ten years an estimated three hundred

nuclear marine vessels will be in operation (60).

In addition to the use of atomic energy for power, other uses

are being made of it in such places as industry, research institutions,

hospitals, and agriculture. These are only a few of the broad categories

in which the usefulness of radioactivity is applied in one way or another.

The uses are greatly varied and multiplied in the many-fold subclassi-

fications contained within these broad categories. From all the


-12-






-13-


application of atomic energy as outlined above, there result many varied

waste products which must be cared for properly. The amount of waste

produced in the environment is staggering and extreme care and caution

must be exercised in the disposal of these radioactive products because

of their potential health hazard to both man and his environment.

It has been estimated (15) that by the year 2000, if 700,000

megawatts of installed heat capacity are in operation, there will result

about 280 metric tons of fission products waste alone! This waste will

be largely contained in some 50,000,000 gallons of high-level wastes

per year. The volume of low level waste will no doubt be somewhat

larger. The question arises then, can the environment safely consume

this amount of waste or should the production of such wastes be stopped

and hence sacrifice the untold potential benefits of atomic energy?

Man has chosen to continue his peaceful uses of atomic energy and has

planned to reduce the hazards afforded by the wastes with adequate

treatment and disposal.

The methods of waste treatment and disposal are not many and

are generally similar to the methods used in the treatment of ordinary

chemical wastes (74). Of course, the most ideal form of waste treatment

here would be to actually and positively stop the radioactivity. To

date no such treatment is possible nor does it seem probable in the

near future. Present treatments consist generally of putting the

radioactive waste in a suitable form for either dilution in water or

air, or long time storage whereby the natural decay factor of the waste

will eventually bring the radioactivity to a harmless level. The

available treatment methods now employed are:






-14-


1. Disposal by dilution including both liquid and chemical means are

frequently used. Liquid dilution is merely the dilution of the

radioactive waste by its mixing with a large volume of water (i.e.

the ocean) or some other suitable liquid. Chemical dilution,

however, involves the mixture of the radioactive isotope, whether

it be a compound or element, with the stable isotope.

2. Some wastes lend themselves quite well to the process of evaporation.

Here a large volume of waste can be concentrated into a small

volume of slurry or sludge which can be stored, or buried either on

land or in the sea.

3. Coagulation such as used in water treatment can be used to remove

certain radioactive isotopes from the water. Here also, the

resulting precipitate can be stored or buried.

4. Ion exchange resins are used in column type units through which the

radioactive wastes are passed. The resins have the capacity to

exchange a non-radioactive cation or anion for the corresponding

radioactive ion in the waste. Sometimes the wastes are discharged

to underground cribs from which they leach out gradually into the

soils. Where the soil conditions are favorable, the radioactive

contaminants will attach themselves to the soil and thereby become

fixed.

5. Biological processes have been found to be quite effective in the

removal of certain radioactive isotopes from a given waste. It has

been ascertained that microscopic organisms have a very high capacity

to concentrate certain isotopes whether radioactive or not. After

concentration has occurred, the organisms can be collected and

disposed.






-15-


6. Sand filtration has been used for some radioactive wastes. In most

cases only limited success has resulted from this method. The chief

mechanism of this process is the adsorption of the radioactive

isotope on the sand grains. By appropriate chemical washes the

adsorbed waste is then easily eluted from the sand grains. This

small volume of wash solution is then further treated for ultimate

disposal.

It can be seen from the above that treatment is essentially

containment coupled with isolation from man's environment or dispersion

so that the probability of return to man is extremely small.

Of the above treatments, ocean disposal of low level radioactive

wastes is the major concern in this study. It has been reported that

ocean disposal is not employed to any great extent, but there is no

indication that it will not be. Wolman and Gorman (84) have stated

that low level wastes can be rendered safe by the high dilution of the

ocean. However, they further indicate that ocean disposal of high level

wastes is not recommended because of the uncertainty of the effect in

the ocean and its further use to man. Land disposal of radioactive

waste is practiced to a greater extent than ocean disposal. Land

disposal at the present time is adequate, but as atomic wastes increase,

other disposal areas must be used. When that time comes, there is no

doubt but that the oceans will be used for such disposal purposes.

The ocean affords advantages uncommon to land disposal. For example,

generally any land area used for such purposes cannot be used for

anything else by man until the radioactive waste is rendered harmless.

As a result, further use of such areas is no longer available to man.






-16-


On the other hand, the oceans are not used as much by man. Therefore,

should portions of the ocean become contaminated by radioactive wastes,

the inability to use these areas is of no great inconvenience to man.

Since it seems problematical that the ocean will come into greater use

for the disposal of radioactive wastes, it behooves man to find out the

limitations of this vast water area. One small aspect pertinent to this

sort of investigation is the relationship of macroscopic marine algae to

radionuclides.

The ocean is a natural place for disposal of most any kind of

waste. It has been frequently considered that the ocean affords limit-

less opportunities for waste disposal. The sea is one of the most

attractive areas available to man for use as a receiving medium for his

wastes. Its vast volume affords a very large dilution factor for most

liquid wastes. Saddington and Templeton (64) have indicated that the

oceanic dilution factor will range from 10,000:1 to 1,000,000:1 after

2 and 9-12 hours respectively. However, the high dilution was effected

only when there existed the mixing action of the sea water as caused by

wind movement. Usually, most liquid wastes will mix rapidly along

horizontal planes of comparable viscosity and density and mix rather

slowly in the vertical direction because of the stratifying variations

of both viscosity and density (57).

Portions of the ocean seem aptly suited for waste disposal,

especially in the deep oceanic trenches where the currents and the water

turbulence might be negligible. Here then would be the most likely

location for radioactive wastes to be stored and allowed to decay with

no adverse affect upon man's environment.







-17-


In contrast to this optimistic view, oceanographers have dis-

covered that all the ocean depths are not quiescent but are in some

places alive with motion (76). In deep canyons, for example, mud flows

occur which move at velocities from 15 to 50 miles per hour. The fate

of wastes stored in such canyons cannot be too easily determined nor

can the desired containment of the waste be assured (56).

Currently some low level wastes are fixed in dense concrete

cylinders and blocks which are dropped into the ocean. It was commonly

believed that these concrete containers penetrated the soft ocean floor

and sunk deeply into the bottom ooze where the radioactive materials

became isolated forever from man's environment. This conception for

the most part is in error for the ocean floor is generally hard and

covered with only a thin layer of ooze. Areas of thick ooze do exist

but exist on the continental shelf near land and hence much too close

to man's environment to be used as burial sites (57).

Along with the theory of quiescent trenches and thick bottom

ooze, it has been suggested that the mixing of the colder waters at

the ocean floor with the warmer waters near the surface would take

about 1,500 years. Present evidence in some ocean areas indicate that

a much shorter time, approximately 100 years, is required (56).

Accordingly, waste placed on the ocean floor may rise to the surface

and hence return to man long before it has decayed to a safe value.

Right now this problem is small and of not too much concern. However,

with the reliable knowledge that radioactive wastes to the marine

environment will sharply increase, waste disposal to the sea will become

a real problem. It becomes necessary then in ocean disposal of






-18-


radioactive wastes that the fate of these radioactive materials be known

so that man can determine more accurately whether the wastes will be

returned to his immediate environment by ocean currents and wave action

washing ashore, whether the waste material will be carried to fishing

banks and cause contamination among edible fish, or whether the waste

will be sequestered on the ocean floor harmlessly out of contact with

man (65). Therefore, precaution and controls must be employed in

dispersing wastes even to the vast volumes of the ocean.

In addition to the ocean being purposely and specifically used

as a waste disposal area for radioactive materials from land operations,

it is also the area which receives the radioactive wastes directly from

nuclear powered ocean vessels. Further, some rivers entering the ocean

probably carry the discharged wastes from land based radioactive sources.

Unlike the radioactive waste purposely buried at sea, these two sources

of waste pose a greater threat to man because they are closer to his

environment. Rivers entering the ocean will deposit most of their

wastes in and around the estuarial area as well as in the areas along

the shores and beaches. Nuclear powered vessels while on the high seas

are of no immediate concern; however, when they are inshore and docked

in harbors, the possibility of contaminating coastal waters becomes

very high (58).

Due to the uncertainties of various dynamic physical factors

of the ocean depths and to the increased production of radioactive

wastes from nuclear powered vessels and land based installations, man's

coastal environment in certain locations will be subject to the hazards

of radioactivity.











It is entirely feasible that any dangerously high levels of

radioactivity in these coastal areas could be detected and decontaminated

by certain marine organisms which inhabit the coastal waters. In the

past few years, some studies have been conducted using seaweeds and

other algal forms in radioactive uptake experiments from which it was

learned that algae are capable of concentrating fairly high quantities

of both radioactive and non-radioactive elements (4).

The only apparent disadvantage to the use of seaweeds is that

they are living organisms and subject to whatever toxic constituents

might exist in the waste. Conceivably, under such toxic conditions,

seaweeds would die, thereby releasing whatever activity they had con-

centrated back to the water.

If further studies prove seaweeds to be highly effective

concentrators of non-toxic radioactive nuclides, then there is every

possibility that macroscopic marine algae will not only effectively

protect the coastal environment, but will also render the ocean a more

attractive area for waste disposal. Currently seaweeds are used to

advantage in this respect in the Irish Sea. It is known that most

fission products discharged to the sea usually suffer an isotopic

dilution by the non-radioactive species present, thus reducing somewhat

the hazardous nature of the radioactive species. However, some fission

products such as plutonium do not have a stable isotope in the sea and,

therefore, do not undergo an isotopic dilution (14). The discharge of

active plutonium must, therefore, be made with extreme care. The seaweed

Porphyra umbilicalis is known to concentrate plutonium by a factor of






-20-


several hundred and thereby aids in reducing the potential danger

afforded by this long-lived nuclide (20).

Theoretically, the ocean can accept safely large quantities of

radioactive wastes provided they are discharged in accordance with tested

procedures (57, 52). However, some radioactive waste disposal to the

ocean is not performed in accordance with the rules and regulations

commonly accepted. An example of this is the burial of wastes off the

New England Coast in only fifty feet of water when the accepted code

calls for burial in no less than six thousand feet (6, 7). Wastes from

the Windscale plant in England are discharged, at the estimated daily

rate of 100 curies of mixed fission products, directly into the Irish

Sea from which the Welsh were accustomed to gathering edible seaweed (20).

Man cannot be so sure that the sea is an absolutely reliable and safe

disposal area without exercising judicious care in the discharge of

these wastes. But with proper planning now, and with the ascertainment

of the limitations of the ocean as a disposal area, man can effectively

and safely employ this tremendous area and volume of water for his

radioactive wastes (78).

As a probable means of increasing the potential of the sea as a

disposal area for low-level radioactive wastes, seaweeds no doubt can be

used advantageously for not only removing the radioactive nuclides from

the water but also as a safety factor against careless or accidental

discharge of these hazardous wastes. Since little has been done along

these lines with reference to marine algae, it is thought that this

present study will be both worthwhile and justifiable.















CHAPTER IV


LITERATURE REVIEW


The need for more basic information concerning marine seaweeds

as well as other marine biologic entities has been urgently increased

since the introduction of atomic energy into man's environment. This

desire for more information was brought about through necessity as

aptly stated by Roger Revelle (61):

"Our knowledge of just what share of these fission products
can be safely introduced into the oceans is woefully incom-
plete because we simply do not know enough about the
physical, chemical, and biological processes. If the sea
is to be seriously considered as a dumping ground for any
large fraction of the fission products that will be
produced even within the next ten years, it is urgently
necessary to learn enough about these processes to provide
a basis for engineering estimates. .

"In the next decade we should attempt to learn far more
about the ocean and its contents than has been learned
since modern oceanography began 80 years ago."

Prior to the atomic era, fundamental information such as chemical

composition, the biology and ecology, the rates of growth and the rates

of harvest, the means of reproduction, whether vegetative or sporogenous,

the seasonal development of the algal species, and whether they were

annual or perennial, and the oceanic distribution of seaweed was greatly

lacking (63, 80). The situation at present, however, is not much

improved although it is now receiving greater recognition than ever

before. There were several possible reasons for this apathetic attitude

towards these macroscopic algae. One of the main reasons undoubtedly


-21-






-22-


was that man has had very little use for the seaweed and another likely

reason was that man's methods of analysis were crude and not as well

instrumented as they are today. Due to the advances in the field of

electronics, instrumentations are possible which are able to solve many

of the complex biological problems considered hitherto unsolvable.

At the present time, seaweeds have found extensive application

in such diversified fields as the production of agar, cosmetics, drugs,

prepared foods, cattle feed, fertilizer, and paper manufacturing. The

great potential of ocean farming regarding seaweeds has only been

partially realized. It is not inconceivable that as the world's

population increases more of man's food supply will come from these

marine forms (1, 11, 77). Destroying the usefulness of these fertile

productive areas by the improper discharge of radioactive materials to

the ocean is undesirable and of paramount concern to man (12). It is

for this reason that more studies of not only marine algae but other

aspects of the aquatic environment have been and are being conducted

and especially under radioactive experimental conditions. The following

discussion will be concerned primarily with some of these investigative

studies.


Laboratory Research

The uptake of certain elements by some algae is quite dependent

upon the plant's respiration. Kelly (39), from her experiments with the

brown alga, Ascophyllum nodosum, showed that iodine uptake by this

organism was dependent upon its respiration. She placed several 10 milli-

meter (mm) segments of this alga in a solution of van't Hoff sea water






-23-


131
containing radioactive 131. When the alga's respiration was stimulated

by the addition of substances known to serve as respiratory substrates

such as glucose, sucrose, malic and succinic acids, I uptake increased.

When compared to the control solution, sugars at pH 6.8 and acids at

pH about 4.5 increased the alga's oxygen uptake up to 20 and 25 per cent

respectively. As caused by the sugar effect, the corresponding increases
131
in 131 uptake ranged from about 180 to 400 per cent of control values.

To lend more reliability to her findings, Kelly injected known oxidation

inhibitors such as sodium azide and potassium cyanide and found that not

only was oxygen uptake inhibited up to 87 per cent but that 1131 uptake

was correspondingly inhibited up to 96 per cent of the control value.

When the algal segments were exposed to a non-oxygen atmosphere such

as nitrogen, 1131 uptake decrease ranged from 50 to 75 per cent of the

control. These tests indicate quite well that iodine uptake is an

aerobic process and that maximum 1131 uptake is achieved in the pH range

4.5 to 5.5. This latter finding indicates that only in a limited area

of the ocean will maximim 1131 uptake by A. nodosum be achieved since

the common pH range of the ocean is 6.5 to 10.

In all her samples, Kelly determined the algal uptake by

immersing a number of alga segments for a period of about 20 minutes

in a flask of the radioactive solution. The segments were then removed,

washed with a similar solution but not radioactive, dried with paper

towels and counted. This analytical approach was employed by other

investigators as will be seen later in this discussion. The method

although not precise was evidently accurate enough to produce qualitative

data.






-24-


Shaw (66) also worked with 131 but used the brown alga Laminaria

digitata. He employed sampling and experimental techniques similar to

those of Kelly. In his studies, Shaw proposed to ascertain the mechanism

whereby L. digitata was capable of concentrating iodine by a factor of

about 30,000 (activity per gram of alga/activity per gram of water). From

his studies he concluded that iodine was taken up by the alga as HIO.

Examination of the plant tissues showed that the accumulation was iodide

and not iodine. Shaw believed that the iodine compound HIO had been

converted to iodide within the plant's cells. This mechanism was deemed

valid because HIO, being an undissociated molecule and not in ionic form,

could easily penetrate the alga's cells. The supporting evidence for

this conclusion was found in Osterhout's work completed in 1925 (50).

The uptake of iodine by the alga was found to vary markedly with

the concentration of 12 present in the water. When Shaw exposed his

experiment to an atmosphere of pure nitrogen, the formation of 12 in

the water was greatly reduced and the uptake of 1131 was reduced.

However, when 12 was introduced into the experiment even under a nitrogen

atmosphere, the uptake of 1131 increased. It was evident then that 1131

uptake was an aerobic process -- a conclusion reached by Kelly in her

experiments.

It is known that marine algae discriminate strongly between the

various elements of the alkali metals group. A good example of this is

the larger concentration of potassium over sodium by the algae in spite

of the fact that sodium exists in sea water to a greater extent than

potassium. Since this discrimination exists, and since the alkali metal

cesium is formed in appreciable quantities in nuclear fission products,






-25-


it becomes important to determine the amounts and mode of accumulation

of this radioactive specimen by the marine algae. Scott (65) undertook

such a study using various species of brown and red algae and conducted

his experiments by immersing ten pieces of each algal species in separate

glass aquaria containing 15 liters of filtered sea water enriched with

Cs .34 Temperature of the water was maintained at 10 C and illumination

was provided for eight hours per day by two fluorescent tubes of the

daylight type suspended directly above the aquaria. At intervals, pieces

of the algae were removed from the aquarium, fresh-air dried, weighed

and then wet-ashed with a mixture of nitric and sulphuric acids. The

activity per gram of the residue was then compared to the activity per

gram of the radioactive sea water sampled at the same time from the same

aquarium. This ratio yielded the concentration factor for the various

algae studied. The species studied were Ascophyllum nodosum, Laminaria

digitata, Laminaria saccharine, Polysiphonia fastigiata, Rhodymenia

palmata and Ulva lactuca.

Again, like both Kelly and Shaw, Scott noticed that the uptake

of Cs134 was increased by the addition of certain chemicals. In this

case, the addition of the potassium salts of phosphate and nitrate

stimulated the algal uptake to a slight degree. It was noted, however,
134
that a marked uptake of Cs was achieved when the potassium content of

the sea water was decreased. In this situation, the alga in all

probability absorbed more Cs134 to compensate for the lack of potassium.

The concentration factors thus found ranged from 200 to 50,000 for the

various species examined.






-26-


Rhodymenia palmata, the alga with the high concentration factor

of 50,000 was the subject of further tests. It was found that with no

illumination R. palmata virtually excluded Cs3 but immediately commenced

to concentrate it on being illuminated. The accumulation of Cs134 was,

therefore, found to be dependent upon the illumination. Scott also noted

that no region of the visible light spectrum was more effective in

increasing uptake than any other portion of the total light spectrum. He

further discovered that the concentration factors were independent of and

did not vary with the concentration of the Cs34 initially present in the

water.

R. palmata retained its radioactive cesium for weeks even while

it was kept in natural sea water. The addition of stable cesium to the

same sea water failed to release the radioactive species from the algae.

Cs134 was retained by the plant in the presence of another plant which

contained no detectable radioactivity. However, when the plant containing

the Cs34 was placed in boiling water or was otherwise killed, it

released its concentrated activity almost immediately.

The concentration factors have been found to vary from one algal

species to another and also from one radioactive nuclide to another. As

already mentioned, the red alga, R. palmata, has a concentration factor

of 50,000 for Cs134. Spooner (68), in his studies found that R_ palmata

concentrated strontium-90 and yttrium-90 by a factor of only 2. It is

therefore evident that the results as found for one alga and a given

nuclide cannot be faithfully applied to other algae and other nuclides.

Each must be ascertained by direct experimentation. This is an awesome

task when one considers that there are approximately 40,000 (79) algal






-27-


species to choose from and almost 2,000 radioactive nuclides with which

to contend.

With R. palmata in a solution of sea water containing Sr90

Spooner (68) found that as the Sr90 decayed to its daughter, Y90, the

alga concentrated the daughter nuclide until equilibrium with the Y90

in the sea water was attained and then the Y90 in the plant followed a

decay scheme according to the natural aspects of that nuclide. The alga

absorbed very little of the Sr90 and seemed almost to selectively exclude

990
Sr90 at the expense of selectively concentrating its daughter. Approxi-

mately 99 per cent of the activity found in the alga was due to the Y90

Using other algae such as the brown Fucus serratus and Fucus

vesiculosus, it was found that Sr90 was concentrated at the expense of
90
Y and that the concentration factors for these algae were 40 and 30

respectively. Spooner's studies point out the fact that red and brown

algae react quite differently in their uptake of both strontium and

yttrium.

An interesting aspect of Spooner's method of analysis was that

he placed only one piece of a given alga in his radioactive solution

and instead of assaying the alga he assayed the water. As the alga

concentrated the active nuclide from the water, what amounted to a

dilution of the radioactive species was taking place. From his assay

data, Spooner determined the equivalent weight of water which would

cause the same dilution effect as the alga. Then the concentration

factor was equivalent to the number of milliliters of water required to

cause the same dilution as one gram of alga. This approach in deter-

mining the factor is correct, unique, and no doubt quite rapid as






-28-


compared to assaying the alga for radioisotopes. Any errors brought in

by extraneous uptake caused by the aquarium walls or foreign organisms

were canceled by a carefully monitored control.

In a non-radioactive study, Wassermann (83) has attempted to

explain an aspect of the uptake mechanism of various cations by the

brown algae Ascophyllum nodosum, Laminaria digitata, and Laminaria

saccharine. From his experimental studies he contended that because

alginic acid and other water-insoluble acids occurred in the cell tissue

of brown algae in the form of various metal salts and not in a free

state, then ion exchange between the algal acid salts and ions in solution

was possible. To prove this, he treated the algae with 1 N HC1. The

metals were dissolved, while the H+ were taken up from the HC1 solution.

When this HC1 extracted algae was brought into contact with a metal salt

solution, the reverse happened; namely, the adsorption of the metallic

cation from the salt solution and the release of the H+ back to the salt

solution. Wassermann conducted his studies under both continuous flow

conditions where only the cations appeared to be absorbed by the algae

and under static conditions where anion absorption also could be detected.

So long as the alga was alive to produce alginic acid then the above

chemical reactions occurred to effect the uptake.

Rediske and others (54, 55) have found that with terrestrial

plants such as red kidney bean, tomato, wheat, and Russian thistle,

radioactive uptake was proportional to the concentration of radioactive

substances available to the plant and also to the pH of the radioactive

solution. A similar situation has been noted with uptake by seaweeds,

i.e., the uptake in general is proportional to the radioactive concen-

tration.






-29-


Field Studies

Perhaps to date the greatest opportunity to reap experimental

data concerning several different types of organisms and their relation

to radioactivity has been in the Pacific area around the Marshall

Islands. Ever since the first nuclear bomb was set off in March of 1954

in this area, many studies have been conducted concerning the uptake of

radioisotopes by algae as well as by other marine organisms (17). Studies

and surveys of this nature were generally under the auspices of the

Atomic Energy Commission and were written up in several reports.

Of these reports, only a few dealt with the uptake of radio-

nuclides by seaweeds. Two reports in particular indicated that some

algae do concentrate radioisotopes (16, 53). In some of the island

areas, it was also discovered that algae found in isolated pools onshore

contained more radioactive isotopes than those algae growing on the ocean

floor at depths of 22 to 50 feet. The exact reverse, however, was noted

on other islands in the same atoll (53).

In all these reports concerned with monitoring the Marshall

Islands, not too much work was done with respect to macroscopic seaweeds

as compared to the other environmental entities. Most of the studies

dealt with uptake by fish, invertebrates, birds, plankton, land plants,

soil, and water.

A vast monitoring program for radioactivity in the Pacific Ocean

began in February, 1955, and expired in May, 1955 (31). The area

monitored extended from the west coast of the United States to the

waters around Japan. Some of the significant conclusions reached from

this operation were as follows:






-30-


1. Radioactive assay of sea water and plankton samples indicated wide-

spread activity in the Pacific Ocean as a result of the atomic bomb

tests conducted in the spring of 1954. Activity of sea water ranged

from 0 to 570 disintergrations per minute per liter (d/min/liter)

and of plankton from 3 to 140 d/min/gram wet weight.

2. Concentrations ofradioactive elements were found in the main current

streams which transported the radioactive contaminations over a wide

area.

3. Uptake by plankton offered a sensitive indication of activity in the

ocean. It was further noted that algae and other marine organisms

concentrated more radioelements in the contaminated areas than in

the other areas.

Algae sampled from the Eniwetok area, showed concentration values

ranging from 260 to 223,000 d/min/gram wet weight. Elsewhere in the

Pacific area, the algal uptake varied from 12 d/min/gram of wet weight

to 1,760,000 d/min.

Approximately 2 1/2 years subsequent to the bomb tests of 1954,

Lowman and others (46) performed monitoring surveys in the Marshall

Islands. They examined plants and animals from both land and sea for

radioactive contamination by Mn54, Fe59 55, Zn65, Co60, 58, 57 It

was found in general that these elements occurred in the sea and not on

land, and in marine animals not in marine plants. Only in marine plankton,

invertebrate filter feeders and fish were the radioisotopes found in

measurable amounts. Of course, not all the algae were examined as

only 6 species were investigated in this survey. Of these examined,

some were found to have detectable amounts of Zn65 and Co60 58, 57






-31-


none contained Mn54 or Fe55' 59. Hiyama (35) in his investigation of

radioactive fallout from the hydrogen bomb also found that the concen-

tration of radioactivity was higher in the microplankton than macro-

plankton. This common finding of more activity in microorganisms than

in macroorganisms is in part explained by the fact that since micro-

organisms have more surface area per unit body weight, more radioactive

materials can be adsorbed on these forms than on the larger organisms.

The Marshall Islands area serves as an excellent large scale

radioactive experiment from which most useful data can be collected (19).

However, there are other places, although not as large in extent, which

serve the same purpose. One such area is the Irish Sea which receives

the raw atomic wastes from the atomic reactors of the Windscale Power

Plant in Cumberland, England.

In most coastal areas of the United Kingdom, the problem of

radioactive uptake by seaweeds would ordinarily be of very little

significance. The problem, however, is significant in the Irish Sea

off the Cumberland coast because a seaweed growing there is harvested

for human consumption. The seaweed in question and upon which radio-

active assays were made was Porphyra umbilicalis. Seligman's examination

(20) showed that the concentration factors for this alga based upon wet

weight for various isotopes to be as follows:

Isotope Concentration Factor

Strontium 0.5
Yttrium 30.0
Cesium 5.0
Cerium 25.0
Ruthenium 10.0 (assumed)
Plutonium 500.0 (assumed)






-32-


The concentration factors of yttrium, cerium, and ruthenium from

Seligman's data were lower by a factor of 10 than the findings made by

Spooner (68). Seligman believed that the factor should have been

closer to 50 but because of a paucity of data, the factor of 10 was

assumed to be correct.

To afford protection to those who ate this and other edible

seaweeds, maximum permissible concentrations were established and taken

to be 1/10 the allowable concentration for any radioisotope in drinking

water (22, 47). The allowable limits in units of curies and microcuries

(pc) were as follows:


Mean Mean Assumed
Nuclide Activity Sample Activity Criterion
Discharge of Sample of Safety
(curies) (pc/gm) (jc/gm)

Pu239 0.05 Fish 4 x 10-7 3 x 10-5

Seaweed 3 x 10-8 3 x 10-5

Sr90 7.0 Fish 2 x 107 8 x 10-6

Seaweed 2 x 10-7 8 x 10-6

Ru106 16.0 Fish 1 x 10-5 1 x 10-3

Seaweed 3 x 10-5 1 x 10-3



The mean activity was determined from many months of sampling

from the Irish Sea. The criterion set forth in the table was established

by considering that both fish and seaweed were edible and therefore more

controllable than the external hazard. If it were not for this risk of

ingestion, the amount of radioactive wastes released to the Irish Sea

could be greatly increased to thousands of curies. Uptake of radioisotopes






-33-


by seaweeds was tantamount to a radioactive dilution of the contaminated

water. Dunster has attributed a dilution factor of about 2 for this

uptake (21). This would not only indicate the potentiality of macro-

scopic algae as decontaminating agents but would also emphasize the

precaution to be exercised over the harvesting of edible seaweeds. It

is unlikely that the discharge of small amounts of radioactivity could

cause any significant disturbance of the marine environment. However,

since the effects of internal and external irradiation as would develop

in man by exposure to contaminated fish and seaweeds are not too well

understood, elaborate precautions must be exercised to protect the

environment.


Fresh Water Studies

It has been stated that in the ocean the phytoplankton concen-

trated more radioelements than most other organisms. Krumholz (43, 44),

in his survey of White Oak Creek, Roane County, Tennessee, found that

filamentous algae concentrated radioactive isotopes by a greater factor

than phytoplankton. For example, the phytoplankton Euglena and Pandorina

showed concentration factors of 100,000 and 285,000 respectively. The

filamentous alga Spirogyra, however, was found to have a concentration

factor of 850,000. Krumholz estimated that a mat of Spirogyra weighing

approximately one ton contained well over 100 millicuries (me) of

radioactive material. This study further indicated that in most of the

algae, planktons, and other organisms, radiophosphorous was selectively

concentrated in greater amounts than any other radionuclide. Other

radioelements selectively concentrated were strontium, some rare earths

and cesium.






-34-


In another study, Coffin, et al. (13), dosed a fresh-water lake

with 100 me of P32 and noted the uptake by various aquatic organisms.

It was found that zooplankton, fish, algae and water lilies concentrated

P32 by factors of 40,000, 13,000, 1,000, and 100 respectively. It is

seen by these data that concentration was greater for the animals than

the plants, but more important is that the concentration was greater for

the lower plants than the higher plants.

Foster and Davis (24), during a period when the aquatic animals

of the Columbia River were most abundant, found that the P32 content of

some small fish was about 150,000 times that of the water in which they

were thriving; in caddis fly larve the concentration factor was about

350,000. They attributed these different concentrations in part to the

dissimilarities in chemical composition and to the physiological demands

for specific elements. The isotopes were concentrated by particular

tissues according to need. Bones and scales, for example, were found

to contain more P32 than muscle and fat. Krumholz, as was earlier

stated, also discovered such selective concentration in filamentous

algae.

Adsorption to biological surfaces plays some part in this

concentration ability of organisms. Some radioactive materials readily

diffuse through living membranes and are thereby readily taken in by

the organisms. Plants, for instance, build up protoplasm and thereby

can concentrate many inorganic ions and some organic compounds. Since

animal membranes are more selective, only a few radioisotopes are

absorbed directly in significant amounts.






-35-


Lackey (45), in his examinations of the microscopic algal

population of the Clinch River in Roane County, Tennessee, found that

many algal species concentrate radionuclides. He too, like others

already mentioned, found that filamentous algae have high concentration

factors. He cites, for example, the concentration factor of the

filamentous alga Oedogonium as ranging from 10,000 to 21,000.

Steel and Gloyna (72) examined the possibility of employing

microscopic algae in oxidation ponds to effect decontamination of sewage

containing radioisotopes. Several different species of green and blue-

green algae ranging from unicellular to filamentous were used. In these

studies, no practical effects were noticed by using the many different

organisms. It was observed, however, that the uptake varied greatly

with the particular nuclide in question. It was found, for example,

that about 23 per cent of the original radioactive content could be

removed by the metabolic action of the algae upon wastes containing

mixed fission products only. With Sr89, a 67 per cent removal of the

original radioactive material could be realized in a twenty day period

whereas with Ce41, 45 per cent removal was noted in the same time period.


Physical Characteristics

Ketchum and Bowen (40, 41) have found that the circulation of

isotopes in the sea is different from the natural circulation of the

ocean waters. When marine organisms take up isotopes, the isotopic

circulation is altered not only by the migrational habits of these living

organisms but also by the definitely established patterns of movements

for dead organisms. After the death of organisms, elements will be






-36-


released from their bodies and the elements which will most readily become

well distributed or transported vertically will be those which have a high

exchange rate with the sea water. Those which will settle out to the

ocean floor will be those which are strongly bound to sediments such as

skeletal or tissue material. This biological influence upon the distri-

bution of isotopes in the marine environment depends in large part upon

the species of organisms and upon the elements considered. Such biologic

activity could easily result in the concentration of radioisotopes in

inshore and estuarine waters. As a result, the radioactive concentration

of these waters could conceivably be greater than the radioactive

concentration of the source water.

The water of enclosed basins such as harbors and estuaries does

not as a rule mix rapidly with the water in the open sea. Therefore,

the rate of dilution of a contaminant in these enclosed basins is a

great deal less than the dilution rate in the open sea. Also, if the

entrance to the basin is shallow, there will develop in the basin, below

this entrance depth, a body of water which stagnates seasonally or

permanently. Here, then, contaminants can easily accumulate and create

a potentially hazardous situation to man (60).

On the other hand, radioactive materials discharged to the ocean

will experience almost complete mixing in about 28 hours in the upper

layer of water (surface to depths of 10 to 200 meters). This surface

(mixed layer) or upper layer is separated from the lower layer by a

belt of high density water called the thermocline.

Although there is good mixing in this upper zone, it is not

homogeneous throughout due to concentration by the biologic factors.






-37-


It is also known that when such radioactive wastes are discharged to the

near-surface layer, they are transported away by surface currents. These

currents extend in general through the entire depth of the surface layer.

Some currents move at the rate of 40 to 140 miles per day. Hence, vast

quantities of surface water are capable of making contact with other

surface waters of the ocean.

Since the surface layer is separated from the lower layer by

the thermocline, then wastes discharged to the surface waters will not

generally be diluted by the deeper waters nor will the waste discharged

to the deeper waters be diluted by the surface waters too readily. It

is possible, however, for the areas to become contaminated by organisms

crossing from one layer to the other and also by particulate matter

settling down to the bottom (8, 9).

In coastal areas where precipitation exceeds evaporation, there

is a seaward drift of diluted surface water with a landward subsurface

drift of water from the open sea. Therefore, if radioactive wastes were

discharged in the coastal waters, the surface flow would carry it seaward

but that portion of the material which mixes with the deeper waters will

return towards shore. This coastal and estuarine circulation is rapid

as exemplified by the fact that the water along the coast is a mixture

of which 90 per cent is sea water even off large rivers such as the

Hudson and Delaware (51, 85).

Generally man is not concerned with the contamination of the

ocean as a whole, but only with the water along the coast, within an

arbitrary distance of 12 miles from shore. Within these waters, contam-

ination by land-based operations and also from marine vessels, could occur.






-38-


Of course, contamination could also happen out in the open sea where

the dumping of packaged wastes will take place, but if this is done

discretely in the proper area, the high potential dilution factor

afforded by the ocean can be realized. Since the entire continental

shelf which is the submerged rim of the land mass sloping from the

beach seawards to a depth of about 200 meters and then extending

precipitously into the deep sea, is essentially the ocean farmland, the

disposal of wastes here must be done with extreme caution. It is from

these ocean farming areas where almost the entire harvest of oysters,

scallops, clams, and seaweed is realized. These organisms, as studies

have indicated, concentrate certain elements by large factors (60). It

is important to reduce the contamination and it is perhaps possible to

accomplish this by using seaweeds. In such instances, the plant could

act as the protector of the more edible organisms and thereby reduce

serious contamination in the inshore waters.

Seaweeds grow quite luxuriously on these marine farms. Walker

(81), in a seaweed survey conducted along eleven miles of the Scottish

coast bordering the North Sea found that 15 tons (wet weight) of weeds

per acre, primarily Laminaria cloustoni, were thriving in water ranging

between 0 and 7 fathoms (13 meters). Between the 0 and 5 fathoms depth

(9 meters) 19 tons per acre were found. No measureable quantity was

found in depths greater than 13 meters.

In another survey (82), Walker found 93 per cent of the area

covered predominantly by L. cloustoni with an average seaweed density

of 26.5 tons per acre. The area surveyed was in the 1 to 7 meters depth

and included 4,800 acres supporting 127,200 tons of weed.






-39-


Marine organisms receive their nutrients from the surrounding

ocean water which throughout all its expanse has approximately the same

inorganic concentration. Even in the littoral zone, where there is some

dilution by fresh water from the land, the salt concentration, although

much diluted, remains relatively unchanged. The inorganic material

present in the ocean is chiefly in ionic form with some in colloidal

form as well. The form of some elements in sea water is not known and

since the ocean waters are in a constant state of dynamic equilibrium,

a complete picture of the ocean's chemical composition is as yet not

possible (79). Harvey (32), however, has made an analysis of the salt

content of sea water. Although this analysis may not be completely

and precisely correct, it nevertheless serves adequately for most

marine studies. A portion of Harvey's analysis may be found in

Appendix I.















CHAPTER V


EXPERIMENTAL PROCEDURE


Introduction

Perhaps the most important single aspect in conducting this

present study was the acquisition of algal species to be used.

Before any research study was begun, several field trips were

made to the coastal waters off Cedar Key, Florida, to locate healthy

growing algal specimens. Growths of Sphacelaria sp., Gracilaria as.,

and Enteromorpha intestinalis were found in scattered unattached patches

during the summer months of 1959, and it was thought these algae

representing three different groups would be ideal with which to work.

However, during subsequent field trips during the winter of 1959 and

into 1960, very few healthy algal growths were noted. This indicated

that a reliable yearly growth of the desired algae was not available at

Cedar Key. Attempts were made to culture, under laboratory conditions,

the few good growths which were obtained from that locale. It was no

simple task to raise, successfully, algae under laboratory conditions.

Such culturing attempts failed. Dr. Lackey, Professor of Sanitary

Science at the University of Florida, procurred cultures of Sphacelaria sp.

from the University of Indiana culture collection and under his

guidance, a large, fast growing, healthy growth was realized (71). As

for the other two organisms, the green and the red, luxurious healthy

growths were found in the coastal waters off Marineland, Florida.


-40-






-41-


Beds of Enteromorpha prolifera and Gracilaria foliifera were found in

excellent growing condition all year round. These organisms were found

attached to formations of coquina rock located approximately one mile

south of Marineland (73).

Special trips were made to the Marineland area for fresh supplies

of both sea water and the green and red algae. Sea water, having a salt

concentration of about 36 grams per kilogram of sea water (36 0/oo), was

transported in a 350-gallon stainless steel tank and stored in an outside

translucent fiber glass-covered concrete tank -- a facility of the Earle

B. Phelps Sanitary Engineering Research Laboratory where all the

experimental work for this study was performed. Upon arrival at the

laboratory, the algae were examined and only the most healthy segments

were placed in several glass aquaria holding fresh filtered sea water.

These specimens were in a continuously lighted 200C constant temperature

room until such time as they were needed. The algae were usually held

under these conditions for no more than three days before they were used.


Experimental Procedure

The experimental work concerning these algae consisted of several

different items of interest and was conducted as follows:

1. The determination of how the algal uptake of Zn65 was affected by

changes in both light and temperature.

2. The development of the relationship between growth and uptake of algae.

3. The examination of the retention capacity of the algae for Zn65

4. The ascertainment of whether the algal uptake was achieved by

adsorption, absorption, or both.

5. The measurement of the translocation ability of the algae.






-42-


In determining the effects of light and temperature upon the

uptake by these algae, three different experimental rooms were employed

concurrently. One room was kept at ordinary room temperature of

approximately 250C, another at 180C and the third room was a specially

constructed refrigerator to house the experiments at 5 C. In each room,

the lighting conditions were easily altered so as to produce the desired

light variation. The lighting variations were arbitrarily designated as

bright, moderate, and dim. These variations were controlled by the use

of a Weston light meter, model 703, type-6A, which measured the intensity

of light incident upon the aquaria containing the algae. A meter

reading of approximately 500 foot-candles was selected as the bright

condition, 10 and 1-2 foot-candles were selected as the moderate and

dim conditions respectively. The readings were achieved by placing

Sylvania F, cool-white fluorescent lamps close to or away from the

aquaria containing the photosynthetic algae (33). It was further

considered that the differences in these light intensities were large

enough to create the desired significant changes in the uptake.

In any one given room under a given light condition, the uptake

phase of the experiment was accomplished in the following manner:

Sixteen glass aquaria of at least eight liters capacity each were

arranged in separate groups of fours, as shown in Figure 1. Each group

of aquaria was filled with filtered sea water until the group's total

capacity was about twenty-three liters. With the exception of the

control groups, the first aquarium in each group or the elevated aquarium

on the far right in both Figures 1 and 2 was to contain the algae and

hence receive the light, while the other three aquaria served merely






-43-


Air Air


A .rAir < Light Source -

Direction of -- 1 after Flow



Control
(no organism)









Fig. 1.- Schematic for the arrangement of aquaria, light source,
and algae for a given uptake experiment.



















-"Bubbler" Hoses


Compressed
Air










Light Source Placed in
Air Pump Adapter Front of this Aquarium



Fig. 2.- Arrangement of glass aquaria and appurtenances in the water recirculation
system for the uptake experiments.






-45-


as reservoirs for the sea water. So that all the algae in any one of

the lighted aquaria would receive approximately the same amount of light,

these particular aquaria were positioned so that the incident light

would penetrate the narrow width of each aquarium. All four aquaria in

each group were connected in a series fashion by polyethylene siphons.

Water was pumped from the aquarium on the far left by means of the air

pump through the glass manifold and into the first aquarium containing

the alga. The pumping was facilitated by elevating the first aquarium.

As the pumping began to raise the water level in the first aquarium and

reduce the level in the last, the siphon effect was brought into play,

continuously keeping the water levels in all four aquaria at the same

height. In this manner, the alga located in the first aquarium was not

involved in just five liters of water -- the total volume contained in

the first aquarium -- but rather subjected to the entire twenty-three

liters contained by the chain of four vessels. Under the experimental

conditions, this recirculation system was perhaps the simplest way of

maintaining a sufficient quantity of water as might be required by the

algal portions so as to adequately sustain them for the experimental

period of about eighteen days. This system provided the added advantage

of using the same radioactive-dosed water throughout the life of any one

separate experiment.

After the recirculation systems were operating properly, the

desired radioactive dose of Zn65 in the form of Zn C12 was placed in

the first aquarium of each group and the pumping was allowed to continue

operating to make certain that the radioisotope was equally distrib-

uted throughout the systems. To aid in this distribution, bubblerr"






-46-


hoses were designed so that they would cause an occasional bubble to

agitate the otherwise quiescent water. The algae to be used in a

certain experiment were taken out of the 200C storage room and placed

in freshly filtered undosed sea water. The algae were then subjected to

the desired experimental condition for approximately one week in order

to acclimatize them to the specific experimental situation. At the end

of this period, the algae were then placed in the appropriate aquaria

containing radioactive-dosed sea water, thus marking the beginning of

the uptake experiments.

For the first few days of the experimental period samples of

both algae and water were taken and examined for Zn65. The results were

immediately plotted in order to select sampling times more judiciously.

When the plotted graph indicated that the desired results had been

obtained, the sampling was discontinued and the recirculating system

was dismantled, thoroughly cleaned, and reassembled for the next

experiment. When all the algae had been successfully studied under the

three different light and temperature conditions, the uptake phase was

deemed completed. Both pH and salinity measurements were made before,

ring and after the experiments merely to make certain that no great

differences existed during these tests. As a check for a portion of the

uptake experiments, some replication experiments were made.

So that the interpretation and analysis of the various uptake

curves could be made more clearly, algal growth rates were determined.

Since only two different basic uptake curves were found for the three

algae, it was decided that the growth rate of only one alga responsible

for each different curve would suffice. It was for this reason that






-47-


Sphacelaria and Enteromorpha were selected for the growth rate

determinations.

The growth rate of these organisms was ascertained by plotting

the percentage increase in weight of the organisms against the time at

which the weight was determined. An analytical balance was used in

order to accomplish this measurement. Each specimen used for weighing

was first surface dried by being placed between the folds of thick

absorbent paper. A constant pressure of about 2 grams per cm2 was

applied to the sandwich of absorbent paper and alga for an arbitrary

time of 1 minute. This was done in order to procure reliable reproduc-

ible results. After the dried alga was removed from the paper folds,

placed in a tared weighing bottle, stoppered and weighed to the nearest

0.1 milligram (mg), it was returned to the aquarium and this entire

weighing process was repeated until three successive weighing were

completed. The average of these three weighing was taken as the weight

of the alga for that particular weighing period.

In another phase of experimentation, the retention capabilities

of the algae were measured. This was accomplished by first allowing

the algae to concentrate the radioactive Zn65 and then placing the

organisms in not only plain sea water, but also in plain sea water of

which the non-active zinc content had been increased by the addition

of 5 mg of zinc metal per liter of sea water. Zinc metal was added

after it had been dissolved in 0.2 milliliters (ml) of concentrated HC1.

This acid solution made no detectable pH change in the strongly buffered

sea water. Before the contaminated algae were placed in the non-dosed

sea water, they were first washed off with the plain sea water so as to






-48-


remove any radioactive material which might have been contained in the

water covering the outer algal surfaces. These retention experiments

were carried out under 500 foot-candles of light at 18 C and in the same

equipment used to determine algal uptake. Both algae and sea water were

sampled and a graph plotted to observe whether or not, with time, the

algae's activity decreased as the water's increased.

The next phase of this study was to determine where in the algae

the radioactive nuclide became concentrated. This determination was

made by employing the autoradiograph technique (5, 18). The only alga

that was suitable from a physical standpoint in this particular phase

of the experiment was Gracilaria foliifera. Gracilaria was used because

it was of sufficient size so as to be easily handled in this experimental

phase. The other two algae were morphologically smaller in size and

were composed of several filamentous threads, only fractions of a

millimeter in diameter, ranging from 2 to 4 cm in length and all emanating

from one common holdfast.

Pieces of Gracilaria grown in Zn -dosed sea water were washed

lightly with ordinary sea water, patted dry with absorbent tissues,

gently sandwiched between two sheets of saran wrap and then sealed (30).

Several such sandwiches were made and were carefully placed in contact

with a no screen backing, A A industrial X-ray film. These contact

exposures were allowed to stand for varying periods of time in light-

proof photographic envelopes under conditions of controlled temperature

and humidity. It was only by trial and error that the proper exposure

could be ascertained. After a successful autoradiograph had been made,

the algal specimens used for that purpose were dissected and the






-49-


dissections were examined for radioactive content so that the autoradio-

graph could be read quantitatively.

Since algae in their natural habitat are generally completely

submerged in water, it seemed valid to assume that they perhaps take

up nutrients through all their surfaces, and hence preclude the action

of translocation. If, however, the organisms could translocate

nutrients, then their effective uptake capacity would still continue

while the algae were almost completely out of water. The determination

of the algae's capacity to translocate nutrients can be proven by

placing one end of the organism in a sea water solution of Zn65 and

noting if the other end which is not in the Zn65 solution contains this

nuclide. Such an experiment should be set up so that the only possible

way for the Zn65 to get to the opposite end would be by way of the plants'

ability to translocate the isotope. Many methods, all in non-active sea

water, were tried to test the feasibility of this experiment. One was

to place one end of the alga in a vessel containing water and allow

the other end to remain in air out of contact with the water. The

portion kept out of water desiccated in a matter of a few minutes.

Then two opposite ends were bridged between two vessels containing

plain sea water and because the small bridged portion of necessity had

to be out of water, it soon desiccated. Bridging under an atmosphere

containing 100 per cent relative humidity caused the exposed portion

of the alga to become coated with water droplets and thus prevented

desiccation but created the following new problem. Under the high

humidity condition, the transfer of Zn65 from one portion of the plant

to the other portion could occur by way of surface action along the






-50-


moisture laden bridged portion, thereby defeating the purpose of the

experiment. The ideal solution to solving this problem would be to

place the alga completely in water and have the lower portion of the

plant in sea water dosed with Zn65 and the upper portion in plain sea

water and have the two waters completely separated. This ideal situation

was in effect accomplished as shown in Figure 3. It is seen that two

beakers were used, one an ordinary tall form 400 ml vessel and the other

a 400 ml beaker modified by making a funnel opening in its base. Since,

as it was explained previously, the Gracilaria species was of fairly

workable size, it was used as the test organism.

From this experimental set-up only the water in the top beaker

was assayed for a period of one week, as this was the maximum time

which the alga could survive under the imposed restriction. After that

time, the upper and lower portions of the alga were severed and assayed

for activity. The complete translocation experiment included six

separate examinations. Two were concerned with the alga's holdfast

being submerged into the Zn65 solution, and four with other portions of

the alga submerged in the Zn65-dosed water. This was done to see if

the holdfast was necessary for uptake.


Sample Preparation

The algae were cut up to a minimum size so that forty pieces of

each alga were available for sampling purposes in any one uptake experi-

ment. It was felt that this number of pieces would not create a crowded

condition in the experimental aquarium and would be enough to make three

replicate samples for each of twelve days with four extra portions

included as a precautionary measure against loss.






-51-


Beaker Containing
Undosed Sea Water


Gracilaria foliifera




Petroleum Jelly






-Beaker Containing
65
___ Zn -Dosed Sea Water























Fig. 3.- Arrangement of beakers and alga in translocation
experiment.






-52-


When samples were taken for analysis, they were first cleansed

lightly with distilled water to wash off any loosely attached radioactive

matter, then each single algal sample was placed in a separate mortar
o
and oven dried at 103 C for forty minutes. When sufficiently cool to

handle, these dried organisms were ground to a fine powder and then

made into a very loose paste by the addition of about 1.5 ml of

distilled water. The paste was transferred to prelabled and weighed

planchets and spread out evenly over the entire planchets' area which
2
measured approximately 8.0 cm The average dry weight sample density

was about 5 mg/cm After having slowly and thoroughly dried these

plancheted samples under heat lamps, they were placed in a desiccator

and then weighed to the nearest 0.1 mg on a balance. After weighing,

the algal material on the planchets was fixed with a lucite binder

(containing 0.5 mg lucite per ml of acetone) and then counted. The

counting of sea water samples was accomplished after an 0.8 ml-water

sample was pipetted onto each of several marked planchets, dried

under heat lamps, and fixed with the lucite binder.


Counting Techniques

Since all samples in this study were placed on aluminum planchets

and all were counted in the same Nuclear-Chicago Model D-47 Proportional

Flow Counter, no correction for backscattering or for geometry was

necessary. "PR" counting gas under slight pressure was used in the

counter and counts were recorded on a Nuclear-Chicago Model 186-P

Scaler. Since only relative activity figures were desired, no absolute

counting was done and no such counting correction factors were applied.






-53-


One factor applied to the counting data was the correction factor to

normalize the data from variations as caused by the counter's changing

daily characteristics. This normalizing correction factor was determined

by comparing the value of the standard as counted at the time when the

first sample was counted to the running average of the same standard

counted throughout the experiment. All counting data were corrected for

background and the only other factor applied was a self-absorption

correction for sea water. This factor will be more fully discussed in

Chapter VI.

An examination of the specific activities in Appendices V through

VII will indicate that despite a wide range of algal weights used (5 mg

to 50 mg per planchet), self-absorption corrections for these organisms

were of no concern. Since the counter had a resolving time of 8.0

microseconds and the highest gross activity recorded in any experiment

was approximately 1 x 10 counts per minute, the dead time amounted to

only 8 x 10-2 seconds per minute and was quite negligible.

Counting statistics were maintained so that no greater than

5 per cent error in counts would be realized. As a general rule,

no sample was counted for less than one minute nor counted for a gross

count of less than 500. Mathematical examples concerning the application

of these statistics can be found in Appendix III.















CHAPTER VI


RESULTS AND DISCUSSION


In this study, the counting data (activity) were not corrected

to their true disintegration values. Since all radioactive samples were

counted under similar counting conditions and by the same proportional

flow counter, the relation between the counted activity and the true

disintegrations was virtually a constant. Application of such a constant

correction factor to all the data would certainly change the absolute

values of these data but in no way would this change the relative

magnitude of one value as compared to another. Because it was

essentially this comparative type of data that was sought in this

research, no conversion of actual counts to true disintegration was

made. The constancy between the actual counts and the disintegration

was maintained not only by maintaining the same counting conditions as

mentioned above and as described in Chapter V, but also by making

certain that self absorptions of all samples were corrected either by

proper plancheting or by the application of self-absorption correction

factors. For example, the weight of each plancheted algal sample was

generally maintained at less than 50 mg in order to avoid the self-

absorption effect. Table 1 shows this specific relationship between

weight and activity for the alga Sphacelaria, while a similar relation-

ship existed for Enteromorpha and Gracilaria.


-54-






-55-


TABLE 1

SPECIFIC ACTIVITY OF ZN65
CONCENTRATED BY SPHACELARIA



Weight Activity
(mg) (cpm/mg)

3.1 44

5.6 46

11.9 47

30.6 45

36.9 46

47.6 47

50.7 46

>50 <40



Self-absorption correction factors, however, were applied to

each counted sea water sample. These corrections are listed in Table 2

and were calculated by the use of the graph shown in Figure 4. From

this graph, it can be seen that the activity varies linearly with volume

for each of the three different activity ranges and exhibits no self-

absorption effect below the 0.6 ml volume reading. The extrapolated

straight line plot continued beyond the 0.6 ml mark represents the

activities as if they were ideally not affected by self-absorption. The

actual activities, however, were affected by this absorption and are

clearly noticed as the sample volume increases from the 0.6 ml up to the

1 ml mark. This effect was evidently caused by too much sample material

on the planchet. The material thereby formed such a thick sample that






-56-


TABLE 2

SELF-ABSORPTION CORRECTION FACTORS FOR


ZN65 IN SEA WATER


Water Correction Factors Average
Volume High Activity Medium Activity Low Activity Correction
(ml) Range Range Range Factor


0.1 10.0 10.0 10.0 10.0

0.2 5.0 5.0 5.0 5.0

0.3 3.3 3.3 3.3 3.3

0.4 2.5 2.5 2.5 2.5

0.5 2.0 2.0 2.0 2.0

0.6 1.7 1.7 1.7 1.7

0.7 1.6 1.6 1.6 1.6

0.8 1.5 1.5 1.5 1.5

0.9 1.4 1.4 1.4 1.4

1.0 1.2 1.2 1.2 1.2






-57-


50






40
-4




'-4
30 U

>E
*--i
u

20





10
10 i


I I I i I I I I 0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Volume of Water Sample (ml)


Fig.
sea water
of Zn65


4.- The self-absorption effect and correction curves for
of approximately 35 0/oo salinity for three concentrations


500






400


300






200


100






0






-58-


almost all the weak Zn65 positrons (0.03 Million electron volts Mev)

from within the sample were not counted whereas the strong (1.12 Mev)

gammas not being impeded by the material were detected (3). Therefore,

the deviation of each activity curve from the extrapolated line represents

the loss of the positron and the further almost linear rise represents

the affect of the unimpeded accumulating gammas. Obviously, to avoid

significant self-absorption, a sample volume of 0.7 ml or less would

have had to been taken. Although this would have reduced the self-

absorption, such a small sample might have easily led to significant

sampling errors; for to convert the activity of such a small volume

to its equivalent 1 ml volume activity would have required a correction

factor equal to the reciprocal of the volume fraction sampled. For

example, if a water sample of 0.8, 0.5, 0.2, and 0.1 ml were taken and

assayed, then to determine the activity for 1 ml, a volume conversion

factor of 1.25, 2.00, 5.00, and 10.0 respectively would have to be

applied. It is seen then, that the smaller volumes require the larger

factors, hence, any error in sampling or in counting would be magnified

in the conversion process. On the other hand, a sample volume greater

than 0.8 ml would almost always have resulted in a poorly plancheted

sample because the sea water salts usually crawled over the edges of

the planchet in spite of the application of grease to these edges. It

was experimentally found that a sample of 0.8 ml was small enough so

as not to cause poorly plancheted samples and it was large enough so

that its conversion factor would have less tendency to magnify any

errors. It was for this reason that 0.8 ml sea water samples were used.

A glance at Figure 4 will readily show that the 0.8 ml was greatly






-59-


affected by self-absorption, hence, a factor for self-absorption and

one for volume conversion had to be applied to obtain the equivalent

1 ml activity as represented by the straight line extension.

Further study of Figure 4 indicates that the self-absorption

occurred at about the same volume for the three different ranges of

activity shown and that it is therefore probably independent of radio-

active concentration insofar as it concerns Zn.6 Employing Figure 4,

the following numerical example will show how these correction factors

were obtained for the 0.8 ml sample in the high activity range.

1. Correction factor from actual curve to extrapolated straight line

390
-- = 1.20.
325

2. Correction factor from 0.8 ml mark on extrapolated line to the 1,0

ml mark on this same line 10 = 1.25.
0.8

3. Total correction from 0.8 ml mark on actual curve to 1.0 ml mark on

extrapolated straight line (1.25)(1.20) = 1.5.

One of the first considerations in this investigation was the

determination of the concentration ability of macroscopic algae for the

radioactive Zn65 isotope. This ability was measured by the concentration

factor (C.F.). The factor is a measure of the organism's ability to

take up a specific isotope and for any one given organism, the factor

usually varies with the nuclide in question and with the environmental

conditions to which the organisms are subjected. It was because of such

variations that the three algae used in this study were subjected to

changes of both temperature and light. The C.F. used here is the ratio

of the activity detected in one gram (dry weight) of alga as compared






-60-


to the activity of one gram (approximately 1 ml) of the ambient sea

water. As previously mentioned in Chapter III, the C.F. also represents

the number of milliliters of water necessary to cause by dilution the

same reduction in radioactivity as caused by the uptake of one gram of

alga. For example, suppose that one gram of an alga was placed in

20,000 ml of dosed water having an activity of 40 counts per minute per

milliliter (c/m/ml). Assume that after a definite period of time the

activity of the water was measured and found reduced to 10 c/m/ml. This

reduction in activity represents the uptake of 600,000 c/m for the one

gram of alga (40 c/m/ml 10 c/m/ml)(20,000 ml) = 600,000 c/mj.

Now, at this time when the water has an activity of 10 c/m/ml,

the alga has a C.F. of 600,000 c/m/g = 60,000. The one gram of alga is
10 c/m/ml
therefore equivalent to 60,000 ml of dosed water having an activity of

10 c/m/ml. If this 60,000 ml of equivalent water were added to the

20,000 ml of water containing the 10 c/m/ml, there would then result a

total volume of 80,000 ml having 10 c/m/ml. Under this condition then,

no radioactive matter would have been removed from the original 20,000

ml of 40 c/m/ml. In other words, if 60,000 ml of plain water were

added to the original 20,000 ml of radioactive water, the latter volume

would increase to 80,000 ml with the simultaneous dilution of the water

activity from 40 c/m/ml to 10 c/m/ml -- the same change in activity

that was effected by the one gram of alga.


Uptake Studies

All the uptake studies were investigated in terms of the C.F.

and not in terms of actual uptake values of the organisms. This approach






-61-


was used because the actual uptake varied primarily with the concentra-

tion of radioactive isotopes present in the water medium. Under such

circumstances then, an organism indicating a high uptake value could

be in fact a poorer concentrator than one showing a lower uptake value.

The C.F., however, negates the effect of varied concentration and yields

a truer comparison of the concentration abilities of the organisms.

As previously mentioned, the C.F. is the ratio of the activity

of one gram of alga to the activity of 1 ml (approximately one gram) of

the ambient water. However, this definition is not wholly true when the

C.F. is determined from the dosed water contained in glass aquaria as

employed in this study. From actual experimental work in this research

it was found that Zn65 was absorbed significantly on glass surface.

Hence, a substantial decrease in Zn65 concentration occurred by the

action of the glass surfaces alone. No specific studies were made in

this regard but it was noted that when, for a desired range of activity,

a calculated amount of Zn65 had been added to the undosed water, the

subsequent assayed Zn65 activity in the water varied as much as 30 per

cent from the calculated value. Some of the Zn65 reduction was probably

caused by microbial uptake, but it was felt that most of this reduction

was due to the glass since the radioactive drop-off began within a matter

of ten minutes after dosing. It was for this reason that a larger than

calculated dose was added to the water to insure that the desired activity

of about 25 c/m/ml was maintained. The concentration of Zn65 so added to

the sea water was about 1 x 10"11 times that of the natural stable zinc

ordinarily present in sea water. It is now apparent that by using such

aquaria the algal C.F. for Zn65 could increase substantially with only a






-62-


slight uptake by the algae. The increase in the C.F. ratio could be

caused merely by the decrease in water activity attributed to the glass

effect. In order to negate this effect, a period of approximately five

days was allowed for the glass and Zn65 to equilibrate as much as

possible before the algae were placed in the dosed water. Hence, time

"0" for the experiment was about five days after the water had been

dosed. Complete equilibrium did not occur for there was almost always

a slight drop-off in water activity, about 0.3 c/m/ml/day, as perhaps

still caused by the glass effect. Only an insignificant amount of this

reduction was caused by the natural decay of the Zn65 since its half

life was about 250 days and the life of the experiment was never longer

than 20 days. During this period, the decay would have reduced the

activity by about 0.05 per cent (42).

The C.F.'s were determined by taking the ratio of the graphed-

uptake activity per gram of alga to the corresponding graphed-medium

water activity per ml. The C.F. quantities were not derived directly

by the ratio of the assayed raw data. The plotting of a few C.F. curves

by using the raw assayed data were attempted, but it was found that such

points were highly erratic and the trend of any potential curve was lost.

However, the algae uptake values as recorded in Appendices V through VII

produced fairly smooth plots; the same was generally true with the sea

water activity values. The data taken from these graphs produced C.F.'s

which plotted quite smoothly and delineated curves with distinct forms.

In the display of experimental uptake results, the C.F. curves

for the algae have been arranged according to the three different light

conditions in one figure representing one temperature condition.







-63-


This was done for each alga for each temperature condition. Accordingly

then, the C.F, curves for Gracilaria foliifera shown in Figures 5 through

8 indicate that for any of the three temperature conditions used, the

C.F. was greatest under the condition of bright light with moderate and

dim light following in that order. The highest C.F. was achieved by

Gracilaria under bright lights at 180C. This, however, is a special

case and will be explained later. For the non special case, the highest
o
C.F. was achieved under bright light at 25 C. Under this latter temper-

ature, the C.F. dropped from a high value of approximately 5,300 under

bright light to about 520 and 220 for moderate and dim light respectively.

These curves in themselves point out the fact that the more light there

is available for photosynthesis, the more will be the resulting uptake.

This finding is not in conflict with the accepted understanding that

red algae grow best in deeper waters where primarily the blue end of

the light spectrum reaches them. Under bright light conditions, there

is obviously more blue light reaching the red alga than under moderate

or dim light conditions. Consequently, intense light containing more

blue component should cause more metabolic uptake than light of lesser

intensity. The C.F. curves for Gracilaria show also that the lower the

temperature, the lower the algal uptake and moreover since the C.F,'s

for these low-valued curves were fairly constant throughout almost

their entire length, the indication is that only the adsorption

mechanism had been operating. Figure 8 indicates that under bright

light condition a reduction in temperature from 250 to 5C effected a

marked decrease in the C.F.'s. For the same temperature drop there was

almost no change in the C.F.'s for the other two lighting conditions.







-64-


500 foot-candles

10 foot-candles

1-2 foot-candles


-~- Q--U--- --


0 2 4 6 8 10 12 14 16 18

Time (Days)


Fig. 5.-

in Zn -dosed

250C.


Concentration factors for Gracilaria foliifera

sea water under various lighting conditions at


6,000







5,000


o W
W 41j
-4I C




u --
U
o
,10
0

4-W
u
c







0
C
u


4,000







3,000







2,000


1,000


---1~3---

---~---






-65-


-- -- 500 foot-candles
--3-- 10 foot-candles
---.-- 1-2 foot-candles
5,000
----- 500 foot-candles
algaa with
fruiting bodies)









^ 3,000 -
o










1,000 -
C 0





..___._4 _t --- - --- -I21 ----D t2 --- 2t--

4 I I \ I

0 2 4 6 8 10 12 14 16 18

Time (Days)

Fig. 6.- Concentration factors for Gracilaria foliifera
in Zn65-dosed sea water under various lighting conditions at
180Co
2,000






















180C.






-66-


-- -- 500 foot-candles

--- --- 10 foot-candles
500
------ 1-2 foot-candles




$.4
.i 4 400


ti


o 300

4 ------ -- --



u 200





100
0






0 2 4 6 8 10 12 14 16
Time (Days)


Fig. 7.- Concentration factors for Gracilaria foliifera
in Zn65-dosed sea water under various lighting conditions at
5C.







-67-


----6---


500 foot-candles
10 foot-candles


---.--- 1-2 foot-candles


~-- -
0----- --0


0 5 10 15 20 25 30 35 40

Temperature (oC)

Fig. 8.- The approximate maximum concentration factors
of Gracilaria foliifera for Zn65 in dosed sea water under
different lighting and temperature conditions.


6,0001-


5,000 -


4,000 -


3,000 -


2,000 -


1,000 -


I I I






-68-


This would indicate that the C.F.'s for both moderate and dim light

were not based upon the alga's uptake as caused by an active metabolic

process influenced by light, but rather by adsorption to the external

surfaces of Gracilaria. An examination of all the Gracilaria curves

under the lesser light conditions will show that all these C.F.'s were

approximately the same. The effect of too low a temperature was to

reduce metabolic uptake with the result that concentration was achieved

primarily by adsorption. It seems that the red alga was quite sensitive

to a temperature change as shown by both Figure 8 and Table 3 and was

also quite responsive to reduction in light intensity.


TABLE 3

THE APPROXIMATE MAXIMUM CONCENTRATION FACTORS FOR ZN65 TAKEN UP BY
THREE MARINE ALGAE UNDER VARIOUS CONDITIONS OF
LIGHT AND TEMPERATURE



Concentration Factor (C.F.)
Conditions of Uptake Enteromorpha Sphacelaria Gracilaria


500 foot-candles of light
@ 250C 1,100 9,000 5,300
18C 1,200 13,000 1,200
50C 760 4,000 430

10 foot-candles of light
@ 25C 1,100 8,700 520
18 C 700 4,700 390
5 C 1,400 3,500 260

1-2 foot-candles of light
@ 25C 720 3,000 220
18oC 630 2,800 150
5C 560 2,800 150






-69-


The C.F. curves for Enteromorpha prolifera show uptake variations

quite similar to those of Gracilaria. Referring to Figures 9 through 12

and Table 3, it is noted that the dim light produced little change in

C.F. and, therefore, the uptake was achieved mainly by way of non-metabolic

processes, which may be assumed as adsorption only. The variations of

the C.F. under moderate light condition as shown in Figure 12, fluctuated

oddly as the temperature decreased. It is believed, as will be brought

out later, that this moderate-light curve should have decreased along

the path represented by the dotted line and not increased as experi-

mentally found. With reference to Table 3, it is noteworthy to see

that under the bright light condition the green alga showed far less

uptake than Gracilaria. One reason for the higher uptake by the red

alga could be that it had in addition to chlorophyll at least one more

photosynthetic pigment -- a phycobilin, probably r-phycoerythrin (23).

It is now known that such an accessory pigment can aid in the process

of photosynthesis by absorbing light quanta and then transferring this

absorbed energy perhaps to chlorophyll a, the principal photosynthetic

pigment which then functions as if it had absorbed the original light

quanta. It is then possible for light of such wave lengths that cannot

be absorbed by chlorophyll to become active photosynthetically by this

means (70). Hence, it is very possible that the phycobilin in Gracilaria

was a more effective absorber of light energy than the chlorophyll in

Enteromorpha and this could account for the higher Gracilaria uptake

and therefore higher C.F. However, the higher C.F. for Enteromorpha

under the moderate and dim light conditions as compared to Gracilaria

for the same conditions could be attributed primarily to the fact that






-70-


---- 500 foot-candles

------ 10 foot-candles

----- 1-2 foot-candles


1,200






1,000






800






600






400






200


0 2 4 6 8 10 12 14 16 18

Time (Days)

Fig. 9.- Concentration factors for Enteromorpha prolifera
in Zn65-dosed sea water under various lighting conditions at

250C.


S-----


CU



0




0
2-I






4.


0
CI-)






-71-


1,400 -- 500 foot-candles
----- 10 foot-candles
------- 1-2 foot-candles


1,200 -





1,000 -





0 800 -
800
U4



4/




(0 /
9 600




















0 2 4 6 8 10 12 14 16 18

Time (Days)

Fig. 10.- Concentration factors for Enteromorpha prolifera
in Zn65-dosed sea water under various lighting conditions at
180C.






-72-


-- --- 500 foot-candles

---- 10 foot-candles

---e--- 1-2 foot-candles


1,600




^-----EE-

1,200


0
U


0u
4J
o /







8 400







0 2 4 6 8 10 12 14 16

Time (Days)

Fig. 11.- Concentration factors for Enteromorpha prolifera
in Zn65-dosed sea water under various lighting conditions at
50C.






-73-


--0--

---E}---


500 foot-candles

10 foot-candles


------- 1-2 foot-candles

-E----- 10 foot-candles
(Equivalent C.F.)


,8J


500 -


250 -


I I I I I I I I


5 10 15 20 25 30 35 40


Temperature


(C)


Fig. 12.- The approximate maximum concentration factors
of Enteromorpha prolifera for Zn65 in dosed sea water under
different lighting and temperature conditions.


1,500P-


1,250 -


1,000 -


750 -


-






-74-


Enteromorpha had more surface area per unit weight of organism and was

therefore capable of more uptake by adsorption. So under the poor

lighting situations where photosynthetic pigments of both algae were

somewhat ineffective, the alga with the more area per unit weight

yielded the higher C.F.

The Sphacelaria curves as shown by Figures 13 through 16 depict

C.F,'s which are very high as compared to those of either Enteromorpha

or Gracilaria. It is also seen that the Sphacelaria curves are quite

irregular in shape as compared to the other two algae. Both the high

values of the factors and the irregularities of the curves can be

attributed to the growth rate and surface area of the brown alga.

As already seen, any one of the C.F. curves for Enteromorpha

and all but one for Gracilaria are smooth curves. This, for the most

part, was caused by the fact that these latter algae had small surface

areas per unit weight of the organisms. From measurements made by

using a microscope fitted with a calibrated ocular micrometer, the

average diameter of the individual (nearly cylindrical) branches of

Enteromorpha were found to be approximately 300 microns (j). Those of

Sphacelaria were measured as nearly 25 j. Accordingly then, as

calculations in Appendix VIII show, the surface area of a given weight

of Sphacelaria was in the order of 12 times as large as the surface

area of an equal weight of Enteromorpha under similar growing conditions.

Although the growth rate of the green alga was about 1.5 times as great

as that of the brown (see Figure 17) its surface area increase was

calculated to be slightly greater than one-tenth that of the brown alga.

For all practical purposes then, the brown alga increased its surface






-75-


-0-- 500 foot-candles
----- 10 foot-candles

---.--- 1-2 foot-candles


10,000






S 8,000 -

to ;-I



S 6,000
0/
u


0 /
4,000


U
(a




0 I 1




0 2 4 6 8 10 12 14 16 18

Time (Days)

Fig. 13.- Concentration factors for Sphacelaria sp. in
Zn65-dosed sea water under various lighting conditions at 25 C.






-76-


14,000






12,000






10,000






8,000






6,000






4,000


0 2 4 6 8 10
Time (Days)


12 14


Fig. 14.-
Zn65-dosed sea


Concentration factors for Sphacelaria sp. in
water under various lighting conditions at 180C.


----- 500 foot-candles
----El--- 10 foot-candles

---.--- 1-2 foot-candles














/4
4~ \


CB U


0
4 -

0
u







0

0
1-
4-


2,000







-77-


---- 500 foot-candles

----E-- 10 foot-candles

---*--- 1-2 foot-candles


0 2 4 6 8 10 12 14 16

Time (Days)


Fig. 15.-

Zn65-dosed sea


Concentration factors for Sphacelaria sp. in
water under various lighting conditions at 50C.


5,000


4,000







3,000







2,000


COl





U

$.4
0
4-i


0




U

0
C)
U

0

C.


1,000










----- 500 foot-candles

-- --- 10 foot-candles
---.--- 1-2 foot-candles


-4
0---------- -- --- -


0 t I I I I Ii
0 5 10 15 20 25 30 35 40

Temperature (oC)

Fig. 16.- The approximate maximum concentration factors
of Sphacelaria sp. for Zn65 in dosed sea water under different
lighting and temperature conditions.


-78-


14,000






12,000






10,000


8,000 -


I


6,000 -


4,000 -


2,000 -






-79-


--0-- Enteromorpha prolifera
-- Sphacelaria sp.


0



60 -O


/ o/ C
0 0 -C

4 0 0 00s
40o





20
20 0 36 ae




30 foot-candles at 5C

0 2 4 6 8 10 12 14 16 18
Time (Days)

Fig. 17.- Growth rate curves for Sphacelaria sp. and
Enteromorpha prolifera under various conditions of lighting,
temperature, and medium enrichment.






-80-


area ten times faster than the green alga. It is felt that the peculiar

dips in the C.F. curves for Sphacelaria were caused by this rapid surface

area growth. A glance at Figure 17 will indicate readily that the uptake

by Sphacelaria was not directly related to its growth rate. That is,

the dips in the C.F. curves were not caused by dips in the growth rate

curves. If nothing else, the growth curves do indicate that for a variety

of conditions, the alga continually gains weight by growing and does not

lose weight. In analyzing the dips, there are perhaps two possible

mechanisms whereby an organism which is increasingly accumulating a

radioisotope could possibly lose such an accumulation. An organism can

lose its concentrated radionuclide by simply releasing it back to the

water, or it can appear to have lost it by growing so rapidly that the

new growth had not accumulated its share of the radionuclide and hence

a dilution effect is experienced. In other words, the activity per

gram of the growing organism gets smaller without any radioactive

substances actually being released by the organism. This explanation

for the apparent radioactive reduction seems more feasible than the

one concerning an actual loss of the radioisotope. For Sphacelaria,

this explanation is supported by the fact that the alga was continually

growing in a positive direction. The following is an attempt to explain

the formation of the dips in the C.F. curves. When the brown alga was

first placed in the medium containing Zn65, a large portion, perhaps

70 per cent, of its uptake was caused primarily by adsorption to the

surface with the rest being absorbed. As the surface area increased,

radioactive substances were adsorbed more rapidly than they were being

absorbed. This was feasible since the adsorption process was concerned






-81-


with the surface and not with the plant's interior. The growing

continued and adsorption continually increased until per unit portion

of alga there was a fairly large segment which had adsorbed activity

but comparatively little absorbed activity. Then, per unit weight of

the alga the activity began to level off then decrease. That part of

the alga which up to this time had not fully utilized its absorption

capacity for Zn65 probably began to increase its absorption process.

The increase could have been brought about by the fact that the alga

had experienced a sufficient contact time in the Zn65-dosed medium to

effect the absorption. From this point on, the activity per unit

weight of alga began to climb, level off, and then resume dropping.

This second peak and subsequent depression was caused undoubtedly by

the continued growth of the plant with increasing adsorption and lagging

absorption. It was felt that the uptake would continue in this fashion

so long as the plant remained in a good growing condition. This did

not happen with the green or red alga probably because the rate of

surface increase with weight was small, hence both absorption and

adsorption were either likely in phase or their differences were

negligible.

The dipping fluctuations of the C.F. curves appeared not to be

a direct result of comparable fluctuations in the alga's metabolism

because the dips usually occurred about the same time. If the dips

had occurred as a result of dips in the variations of metabolic processes,

then the metabolic dips must have cyclically occurred for the alga in

the undosed water during the conditioning period just prior to the alga's

being placed into the Zn65-dosed medium. Now the question arises, why,






-82-


if the algal uptake varied directly with metabolic fluctuations, did

not the C.F. dips occur haphazardly at different times instead of almost

uniformly at the same time? Why could not the plants, for example, have

been removed from the non-active water while they were just metabolically

cycling into a dip, placed in the dosed water and so indicate the start

of the uptake curve with a dip? It is for these arguments that the

growth rate idea is preferred. To further substantiate this idea, it was

assumed that if the organism could be made to grow faster, then the dips

should occur much sooner and perhaps be more severe. This very idea was

accomplished by adding approximately 3 grams of KCl to a Zn65 uptake

experiment and plotting the C.F. curve. Figure 18 depicts such a curve

with sharp dips and peaks which do occur earlier as postulated. To

make certain that this was truly caused by the growth rate, three pieces

of Sphacelaria were grown under similar conditions of the above-mentioned

uptake experiment and their growth rates plotted. Figure 17 shows that

the brown alga grown under these conditions had a growth rate which was

about 8 times greater than the growth rate found when no KC1 was used.

Since the growth rate was 8 times as fast, then the first dip in the

C.F. curve should have generally occurred at about 1/8 of the usual time

or in about 1 to 2 days. Figure 18 shows that the dip occurred between

the second and third days. This experiment, it was felt, substantiated

the growth-rate theory. Further, the C.F. curves shown in Figure 15

indicate by their dips that the algal metabolic uptake must have been

somewhat arrested by the low 50C temperature and, hence, the immediate

C.F. increase from time "0" to time "2 days" in all probability was

caused by adsorption. The absorption of Zn65 was, perhaps, retarded







-83-


- -- 500 foot-candles


10,000 -






S 8,000-





u
6,000

0


0
o

I i4,000 -

r.J


0

2,000 -






0 1 1 1 1 1 1 11 1
0 1 2 3 4 5 6 7 8 9

Time (Days)

Fig. 18.- The concentration factors for Sphacelaria sp. at
250C in Zn65-dosed sea water enriched with KC1.






-84-


by the cold and, as the plant grew slowly it was adding on weight

without adding on its proportionate amount of radioactive uptake. It

was assumed, that because of this, the C.F.'s leveled off only to rise

when probably both adsorption and absorption again became operable.

The growth rate curve for Sphacelaria at 5C shown in Figure 17 indicates

a very shallow weight increase which was compatible to the low concentra-

tion factors found at the low temperature. The growth rate for

Sphacelaria under bright light and 18 C temperature was quite high and

this also was compatible to the C.F. curve in Figure 14.

After studying the C.F. curves developed from the uptake experi-

ments, it was desired to ascertain what made one alga a better concen-

trator than another. To do this at least two algae, one having a higher

C.F. value, had to be mutually compared. The organism having the higher

value was then, by the application of certain assumptions, made

equivalent to the one having the lower C.F. value. Having thus made

this transition, the C.F.'s for the equivalent alga were calculated

and compared to those of the actual alga. The above transition was

accomplished by converting Sphacelaria to an equivalent Enteromorpha by

the following approach. Of the probable factors which caused Sphacelaria

to concentrate so much more Zn65 than Enteromorpha the two prime factors

were, perhaps, surface area and the different photosynthetic pigments.

Not only had the brown alga a larger surface area than the green, as

previously calculated, but it also had, in addition to the chlorophylls

at least one more photosynthetic pigment which is common to almost all

Phaeophyceae, namely, fucoxanthin (23). Assume that the concentration

of Zn65 was accomplished by both adsorption to the algal surface and






-85-


absorption within the alga. (These assumptions will subsequently be

proven feasible.) To equalize the surface area effect of Sphacelaria

with that of Enteromorpha, the uptake of the brown alga was first

separated into its adsorbing and absorbing fractions. The adsorbing

fraction, which was assumed to be at least 10 times greater than that

of the green, was then reduced by this amount. Actually, the surface

area had been calculated as being 12 times as great, but not knowing

exactly how this difference operated, the lesser factor of 10 was

considered a valid assumption. Further, the absorption was no doubt

influenced by the surface area and again owing to uncertainty, about

1/4 of the calculated difference was used. This factor for absorption

was, therefore, assumed to be 3. Now, how much of the absorption was

caused by the fucoxanthin? From Fogg's work (23), it was assumed for

the approximate light spectrum range as utilized by plants (4,000 x

109 cm to 7,000 x 10-9 cm) that both the chlorophyll and fucoxanthin

share the light absorption equally. Hence, for this property a factor

of 2 was considered. To summarize briefly: the adsorbing fraction of

the brown alga's uptake was divided by 10 and the absorbing fraction

was divided by 6 (3 x 2). The corrected uptake was then used in the

calculation of the C.F. These new C.F.'s determined from the Sphacelaria

data should then resemble the actual C.F,'s for Enteromorpha. Table 4

shows these comparative data which indicate the proximity of the C.F.'s

figured for both the equivalent and actual organisms. Based on these

data, it would seem that in Figure 12 the C.F.'s for Enteromorpha in

the low temperature range should perhaps have decreased along the

dotted line instead of increasing as found experimentally.






-86-


TABLE 4

COMPARISONS OF THE APPROXIMATE MAXIMUM CONCENTRATION
FACTORS OF THE EQUIVALENT ALGA TO THOSE OF
THE ACTUAL ALGA

Concentration Factor (C.F.)
Conditions of Uptake Actual Equivalent Actual
Sphacelaria Enteromorpha Enteromorpha

500 foot-candles of light
@ 25C 8,900 1,100 1,100
180C 13,000 1,600 1,200
5C 4,000 460 760

10 foot-candles of light
@ 250C 8,700 1,000 1,100
180C 4,700 600 700
50C 3,500 410 1,400

1-2 foot-candles of light
@ 250C 3,000 380 720
18C 2,800 330 630
5C 2,800 320 560



At least one uptake replication for each alga was made under

various conditions of light and temperature. The replications were

made only to provide an approximate check on the initial experiments

and not meant to increase the accuracy of these experiments. The

comparisons of the replicates to the originals are listed in Table 5

and indicate a reasonable variation.

The relationship between an alga's C.F. and the amount of

decontamination afforded by the alga was approximately ascertained.

There seemed to be an interrelationship between the C.F, and the weight

of alga used. It was pointed out that the C.F. was numerically equivalent

to a volume of dilution water which would reduce the activity level of

the dosed water by the same amount as would be realized by the uptake






-87-


TABLE 5

COMPARISONS OF THE APPROXIMATE MAXIMUM CONCENTRATION
FACTORS AS DERIVED FROM ORIGINAL AND REPLICATED
UPTAKE EXPERIMENTS


Organism and Concentration Factor (C.F.)
Conditions of Uptake Original Replication


Gracilaria

500 foot-candles of light
@ 50C 430 390

Sphacelaria

500 foot-candles of light
@ 250C 9,000 7,600

Enteromorpha

500 foot-candles of light
@ 50C 760 720






-88-


of one gram of alga. It is then apparent that more than one gram of

alga would have a greater reduction effect. Also, it seems very

probable that 100 grams of an alga having a low C.F. could cause more

reduction of an active water than perhaps one gram of an alga having

a slightly higher C.F. Therefore, a large C.F. value per se is not

indicative of the amount of reduction possible. The ultimate radio-

active reduction is apparently a function of both C.F. and quantity of

alga used. In support of this theory the following general comparison

was made.

From each uptake experiment, an approximate dried weight of the

total alga used was averaged from the many weighed samples assayed.

Using this very approximated average weight, and the average C.F. value

from the appropriate C.F. curve, an approximate total dilution effect

as caused by the entire amount of alga was determined. This dilution

value was then compared to the dilution value as determined by the

ratio of initial water activity to final water activity. Of course

these latter values must represent the activities of the water as it

was affected by the algae only and not by the glass. In order to

correct for any glass effect, the actual uptake of the glass had to

be determined. As an example as to how this determination was made,

the water data for Sphacelaria under the condition of 500 foot-candles

at 180C will be used. These data as compiled in Appendix VII are

graphed in Figure 19. With reference to the upper half of this figure,

there are plotted three distinct curves. The theoretical activity

level curve represents the level of activity that would probably be

present in the water if no uptake of any kind were realized.






-89-


Theoretical Activity Level
S--- --- ...

0-te se
SUptake Caused
0 by Alga 0


30




Corrected-Medium Activity

20


10 -


SI I I I


0 2 4


6 8 10 12 14 16


Time (Days)


Fig. 19.- Typical working graphs depicting the
of the corrected-medium curve.


determination






-90-


The control curve represents the radioactivity change in the water as

caused by all possible effects other than the alga under consideration.

The medium curve represents the radioactivity change in the water as

caused by all possible effects plus the uptake capacity of the alga.

Therefore, the difference between the medium curve and the control

curve represents in all probability the uptake achieved by the alga.

Now, if this uptake amount is subtracted from the theoretical curve,

there will result the so-called corrected-medium activity curve. This

latter curve insofar as is practical should represent the true activity

in the water as it is affected only by the uptake of the alga and

nothing else. It is the activities as represented by this curve that

were used in the calculations of the dilution factor. If all involved

factors in the uptake studies had been accurately and precisely deter-

mined, then the dilution effects as developed by the two methods

previously mentioned should be similar. The total dry weight of alga

approximated from the C.F. data of each uptake experiment for any one

of the three algae was found to be: 1.1, 1.4 and 3.6 grams for

Enteromorpha, Gracilaria, and Sphacelaria respectively. Table 6

records these dilution factors and indicates fairly closely the

relationship between the C.F. and algal weight in the dilution factor

determination.


Retention Studies

In a prior section, it was mentioned that approximately 69 per

cent of the uptake was achieved by adsorption while the remaining 31 per

cent was by absorption. These data were based upon the retention curves














TABLE 6


COMPARISONS OF THE APPROXIMATE DILUTION FACTORS OF THE MARINE ALGAE AS
DETERMINED BY THE CONCENTRATION FACTOR EQUIVALENT VOLUME AND BY
THE RATIO OF INITIAL AND FINAL WATER ACTIVITY


Dilution Factor Determined by Dilution Factor Determined by
Conditions of Method of Average Concentration the ratio: initial water activity/mi
Uptake Factor Equivalent Volume final water activity/ml
Enteromorpha Sphacelaria Gracilaria Enteromorpha Sphacelaria Gracilaria


500 ft-c
@ 250C 1.0 1.4 1.6 1.1 1.2 2.2
18 C 1.1 1.6 1.2 1.1 1.4 1.2
5C 1.0 1.2 1.2 1.1 1.1 1.2

10 ft-c
@ 250C 1.0. 1.3 1.0 1.1 1.3 1.1
180C 1.0 1.2 1.0 1.1 1.1 1.1
50C 1.1 1.2 1.0 1.1 1.2 1.1

1-2 ft-c
@ 250C 1.0 1.2 1.1 1.1 1.3 1.1
180C 1.0 1.1 1.0 1.1 1.2 1.1
50C 1.0 1.1 1.0 1.0 1.1 1.1






-92-


shown in Figures 20 and 21. The two curves in Figure 20 indicate a

definite, almost immediate release of the Zn65 contained by the brown

alga. Since a surface reaction has a more favorable chance of occurring

before an internal reaction, it is likely that this immediate reduction

was caused by the loss of the alga's adsorbed fraction of Zn65. The

further release of Zn65 progressed along at a much slower rate until it

appeared to have attained an equilibrium. This eventual leveling off was

probably caused by a gradual release of the unsequestered absorbed

Zn65. It is interesting to note that the alga placed in the sea water

containing an excess of zinc released more Zn65 initially than the alga

placed in the sea water not containing excess zinc. This was expected as

it was felt that the exchange between the adsorbed Zn65 on the alga and

the zinc in the water would proceed faster where an excess of zinc was

present. The retention curves show also that after the initial drop,

the release of more Zn65 proceeded at a faster rate in plain sea water

than in stable zinc-enriched water. After a period of approximately

13 days, both curves began to show equal rates of loss for Zn65, and

the algal retention from that time on seems to decrease at a constant

rate of 1 c/m/mg/day. The rates in the first two days were about

45 c/m/mg/day which decreased to 3 c/m/mg/day then decreased to the

constant rate. A likely explanation for the greater algal retention

in the zinc sea water is that the Zn65 absorbed within the alga could

not be released to the water rapidly because the direction of the zinc

concentration gradient was from the water towards the alga. Since there

was an excess of zinc outside the algal cells, the tendency for the

passage of zinc ions would not be from alga to water but rather the






-93-


Total Retention in
Plain Sea Water

Total Retention in
Stable-Zinc Sea Water
Stable-Zinc Sea Water


0 2 4 6 8 10 12 14 16 18

Time (Days)


20.- Retention of Zn65 by Sphacelaria sp.
foot-candles of light, in plain sea water
enriched with stable zinc.


at 180C,
and in


200









100
90

80

70

60


50


40



30




20


Fig.
under 500
sea water




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