THE EFFECTS OF SEJ-ECTXD ALGICIDES AND SOME COO&DIHATION

COMPLEXES tJPOI THE APPARENT PHOTOSYNTHESIS

OF CHIOREILA PYKElfOIBOSA

APPBOTIDs

- ST

Major Professor

nor rroressor

O i m i A l n , Director of the Department of Biolo^v

Dean1of the Graduate School

THE EFFECTS OF SEJECTEB AM1CIBBS M B SOME COOBDIMflON

COMPLEXES OTOI THE APPAREMT FHOTOSTHTHESIS

OF mwmtu FIEiMOXBQSA

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment ©f the Requirements

For the Degree of

MASTER OF SCIENCE

B y

Egbert G» Phelps* B. 3«

Denton, Texas

June, 196$

TABLE OF CONTENTS

Fag#

LIST OF TABLES . . . iv

LIST OF ILL88TMTIGMS . . . . . . . . . . . . . . . . v

Chapter

I. INTRODUCTION . . . . . . . 1 Coordination Complexes Preliminary Screening for Algicidal

Gmpowads History of Manometric Techniques

Concerned in this Study

II. MATERIALS A ® MTHODS . . • . 11

III. RE30LTS . . 23

Coordination Complexes and Miscellaneous Compounds

Algieidss

IV. DISC0SSIQN 36

BI1LIOGMPH1 44

ill

I I I . Effects of

LIST OF TABLES

Tafele Pag#

I , Stock Solution Components fo r BristoIfs Medium * • H

II. Effect of [cHenjJlj on Apparent Photosynthesis Expressed as Per Cent Of Control 24

.CoCenJjJd^ on Apparent Photosynthesis-fxpressed as Per Cent of Control 25

IT. Effects of -[colea^CcDjci on Apparent Photosynthesis Expressed as Per Cent of Control . 26

V. Effects of [Go{ en) p NOg) 2] Br on Apparent Photosynthesis Expressed as Per Cent of Control . . « • • • 27

VI. Effects of Rhodium-Coraplex Compounds , . . . 29

VII. Effecta of Miscellaneous Compounda on Apparent Photosynthesis Expressed as Per Cent of Control . . . . 30

It

LIST or IUUBSTiUTIONS

Figur® Fag©

1. Cult wring Apparatus for GMor#lla pyrenoidoaa • • • • » • • * * « • • « • • • 12

2« Comparison of Pardee*s Method with Pratt m& Warburg Buffers . • • 16

3. Effects of A&iae-d-acet&te m Photosyathetie Rate Expressed as Per Cent of Control # • • 32

4. Effects of Hydrothol 4? (liquid) on Photo-synthotie Hates Expressed in.-Per Cent of Control . . . . . . • « • 34

5* Effects of Hydrothol 191 and Hydrothol 47 (granular) on Photosynthetic f a t ©a Jbqpreewd < la P«r Cent of Contact . . . . . . 35

CHAPTER I

JMT1GB0CTI0N

Many experiments have been performed with the Warburg

apparatus, or variations of this manometric technique, sine#

Warburg's experiments (52, 53) where the effects of cyanides

upon dark reactions and of urethanes upon light reactions of

photosynthesis were demonstrated. The same basic techniques

war® utilised in this research in attempting to determine

the af facts of some coordination complexes upon the apparent

photosynthetie rata of Chlorella pyrenoidosa. A second goal

Of the present papar was to investigate the potential of the

Warburg apparatus as a tool for screening algieidal compounds.

Coordination Complexes

Pasteur was the first to observe the actions of living

organisms upon racemic compounds in which the compound grad-

ually became levorotatory (34). Utilization of this phenom-

enon was aotually one of the method# Pasteur used for resolving

racemic compounds (31). fhe possible importances of the ef-

fects of optically active compounds upon biological phenomena

have been suggested by Bwyer and co-workers (£)• Hunter, et

al* (29) have mentioned the role of cobalt in nucleic acid

construction and possible gene fabrication. These actions

were assumed to take place through the occurrence of cobalt

1

in vitamin The nature of the cobalt link in vitamin

i# unknown but is coordinately bound. Feoktistova (15)

studied the effects of cobalt in relation with vitamin

011 growth and protection from excess cobalt In planktonie

blue-green algae. It was demonstrated that cobalt and

vitamin B^2 were definitely related la the growth requirements

of these organisms. Some coordination complexes * racemates,

diastereoisomers and optical antipodes * were Investigated

to determine if their effects cause a decrease or increase

in the rate of apparent photosynthesis. Since coordinately

bound metals were involved In the complexes used in this

study, it was hoped some outstanding effects si sfc be ex-

hibited by one of the isomers of a coordination complex.

Preliminary Screening for Algioldal Compounds

To date,* the methods of screening potential algicides or

growth accelerators have been by* (a) visual teats involving

color comparisons between control flasks and experimental

flasks containing the compound being tested (3, 6, 16, 17,

It, 19, 26, 2*, 36, 37, 44, 46); (b) cell counts (26, 2*)j

or (c) filtering the experimental cultures through glass fiber

pads and photographing them for comparison with the controls

(16). Actual field tests, after laboratory bioassay experi-

ments, also employed visual observations for determining the

effects of algicides in farm ponds (51) and fish ponds (4$)•

These methods were reliable, especially when aubculturing

techniques (1#, 2$) were employed. However, they were very

time-consuming• Preliminary screening time was usually four

to twenty-one toys (6, 17, Id, 36# 3?) • Subculturing the test

organisms involved, usually, the same amount of time# It was

believed the manometrie methods expressed in this study would

shorten the time considerably for preliminary screening and,

possibly, the subeulturing tine* .

History of Manometrie Techniques Concerned in this Study

Manometrie techniques for determining the effects of or-

ganic and inorganic compounds upon respiratory rates ©f algae

and other* microorganisms have been used by numerous investi-

gators (2, 4, 9, 12, 13, 14, 22, 23, 24, 26, 31, 33). Eespir-

atory rates were variable due to the organisms used, culturing

methods of the organisms, and experimental methods* To exhibit

respiration, the algal cells were nitrogen-starved (1, 4, 7)

and then subjected to the experimental procedure# Perhaps

the most popular method to exhibit respiration was one in

which the cell suspension was placed in a medium to which had

been added glucose or other organic compounds (5, 9* 12, 13,

21, 22, 23, 31, 35, 3*, 49, 50, 54, 55). Upon addition of

the organic compounds, the respiration rate was greatly en-

hanced. The addition of glucose or organic compounds was

used to accelerate aspiration and, in so doing, subjected

the organisms to ^abnormal* conditions. By subjecting the

algae to an organically-effected respiration, the possibility

4

of decreasing the effects of the experimental costpoiand was

introduced. Geo^iagen (24) showed that til® growth and respir-

ation of ChlorelXa vulgaris var. viridis in the presence of

some substituted methylureas was far greater in media with

glucose than in media without glucose* He went ©n to show

that the effects of the substituted raethylureas wart inhibited

to an extent somewhat less than that exhibited with glucose

when the algae, to bo used experiment ally, ware grown on agar*

It follows then that one would not want to use a medium which

would defeat the original purpose in testing a compound for

toxicity#

Manometric procedures al@o were used for determining the

effects of many compounds upon algal photosynthetic rates

(11, IS# 21, 22, 25, 26, 27, 31, 33, 35, 3d, 41, 42, 43,

45, 47, 52, 53, 55) • fhese studies, however, were directed

toward effects produced upon the phot©synthetic mechanisms

for studies in eulturirig techniques, maximum efficiency of

photosynthesis, quantum yields, or specific effects of various

compounds*

Workers, prior to 1949, used various buffering solutions

for maintaining a constant carbon dioxide partial pressure

(10, 20, 32, 33, 41, 43, 45, 52, 53) i& measuring the oxy$m

evolution of photosynthesizing cells* The algal cells were

suspended in these buffer solutions which were completely

foreign to the culture media in which they were growing#

This procedure introduced additional variables which had to

be considered la the final analysis of mmIts* k technique

developed by Pardee (40) in 1949 enabled the worker to wain-

tain the algal cells in the culture medium without til® us# of

buffer. The variables mentioned above were eliminated with

this technique*

Pardee1a method should produce a more accurate determi-

nation of chemical effects# This procedure was investigated

as a possible preliminary screening technique for chemicals

with suspected antialgal properties.

CHAPTER I BIBUOG&APHT

1. Allen. M. B., H. Gest. and !4» D. Karaen, "Differential In-hibition of Kaspiration and Dark QQg-*Fixation In geenedeataus and Chlorellar

M Archives of Biocheg&atry.

. Alien. 1# and F • Skoog, "Phytotoxicity of Imidazoline

OTl'ulIl? "IllSlt!911 C 0 m p 0 U n d 8'" Physiology•

3* Antenides, H* J* and W. S# fanner, "Algieidal and Sanitizing

BRSS# MCROBLOLO<G> 11

4# Bach, K« K. and J* F«lligt "Stimulation of the Respiration of Chlorella vulgaris by Purines and Purine Analogu.es,8

Plant Fhyisioloinr. XXX? (January, I960), 36-44.

5. Bra*k«$t# F. S», R, A# Olson, and R. G. Crickard, "In-spiration and Intensity Bependance of Photosynthesis

n u " , ^ g > j B x n

. Branaon, K. A* and A, F. Bartsch, "Influence of Growth Substances and Cell Division in Green Algae."

£& MftSZ* tMfcy, 1939), 271-279.

7. Casselton, P. J. and P. 4* Syrett, "The Oxidation of C ^ -labeled Glucose by Ghlorella'vulgaris,* Annals of Botany* New Science. lifl (19621.71*4 2*

Dwyer, F. P., E. C* Gyarfas, W. P. Rogers, and J. H. K@®h# "Biological Activity or Complex Ions." Hature. CLXX (August 2, 1952), 190-191. •

9. Smerson, E., *The Effect of Certain Bespiratory Inhibitors on the Respiration of Chlorella." Journal of General PhvalalogyT x (1927), 169-477. -Matm. s_ w»,WfWr

10. • and L. Green, "Effect of H* Cone Kit ration on Photosynthesis," Plant PfayslClogy. XIII

TjSHuary, 1938), 157-168.. «"»-«<*«

11. . . and C. M» Lewis, "Factors Influencing the Ef-IfHmcy of Photosynthesi*,» foartcan of XXfl (December, 1939),

12# tojr, 0* M*$ "Mrngimtim Studiaa.on Okloralla. I« Growth BatpsrlaMBbs with Acid Intemodiatoa, * Plant Phyal-e M y , XX? (1950#, 47i-495. - " * *

13* . *R«a»iration Studies on Chloralla. IX, In-rluenoes of various ©rgaai® A©i4s on 0a§ Mmtoimm** nuafe PhyaielojKy. OTr (April*. 1951),

14# BrlduNm* I»* 8*, I# T« Yodd&ftgj, and ft* I»« BnuutMMtt* "Influanoa of jfl on tod Aaotic Acid Activity | a Cfelorella," Plant PhYaioloffv. XU (Jaeaary, 1955), 69-7C: f w « « w i

15# PaoktiatOYa, 0« X.# "Vliyani# lgobftlMui ta na cJiialannoat* |iariteoiuaykti aintsel«By)& v©4©rol«i 1 na slntasiirmxiit %|#® i W l c i P^S fMfetem i l iaoh, AJted. Hank SShSIIJL X {X/VA# »

X6* Fltagwrftld, G» P», "laboratory Evaluation of f m m s i m Permanganate aa an Algielda for Vfttor JUttttftttinu*

Mmm i t t k a Ismml* ^ Ummtr, ml)

17* -,„. . , ,. f "Loaa of Algieidal Cheaicala in •*!»» MerMcl#gy, f i l l {Slumber,

JJ# _ .^ v.v..u,..., ,.».&«! •» F&ttsfe, "Paotora Affecting tho

If* _ - 0* 0« Oerloff, and P. Skooc, *8tudioa on Chitaicala with Selective Toxicity to Bl t t^ re tn Al«y«» p k laditi igltl Wastes. XXIV {.July, iir5^ I n #©#**0 ^ ii

20. Fleischer, t# 1»» *The Eolation Between Chlorophyll Content an4 Hate of Photosynthesis,* Journal of Oaner*l Physiology. XTO1 {March 20* IfflT, 573-5977'^^^

21# Gaffron, H«# "tffcer anonalien de« arntmssquotieisteii von al§@a m& 4 suckerkalteren, * .IloJtoiAsAo 2entralblatt. XXX (1939)* 2t§~302U .ramiJTimr-1-T-.r,-jniJX, r"TO™3,mjt*

22# Galloway, R. A# and ft. W* Krausa. "The Differential Action of Chemical Agent®, Especially Folynyxin-B, m Certain Alga** Bacteria ama Fungi,* Journal of Botany. XL (January, 1959), 40*49*

23* aeaevoia. 2»», *Uber Atsung wd Oarung in granoa Pflanger,* ^ t ,< i53u f t , CLXXX (1927), 451-473.

$

24. Geoghagen, M. J#, wThe Effect of Sum® Substituted Methyl-ureas on the Respiration of Chlorella vulgaris yar. virldla," New S S 2 1 M B * LVI (MarS, 1957), 71-#).

25. Govindjee, I#, J# B. Thomas, and £• Eabinowitch, ««Second Emerson Effect' is the till Enaction of Chlorella Calls with Qwinone as OxidantScience. CIXXII (Auguat, I960), 421. .

26. Green, L. P., J« F« McCarthy, and C. G. King, "Inhibition of Inspiration and Photosynthesis in Chlorella Pyrenoidoaa by Organic Compounds that Inhibit Copper

C ^ ^ | t f i a y f l 9 f | , ^ ^ ^ J't 27. Greenfield, S# S». "Differential Inhibition of Photo*

chemical and Bark B©actions • in Photosynthesis by In-organic Compounds,* Science. <2C,0 IXjE (January 6# 1941), 550-551.

2ft» Greenfield, S. 3., "Inhibitory Effecta of Inorganic Com-pound® on Photosynthesis in C&orella.» American Journal $£ Botany. XXIX (Pebruax7, 194277 151-131.

29. Hutner, S. J., 3U Proyasoli, A. 3 char z, and 0# P. Haskins, "Some Approaches to the Study of the Role of Metals in the Metabolism of Microorganisms,11 Proceedings of

I|l-l76hil° &:i S ° C — y > XClfllpHir

5 30. Jaeger, F. M., O p t i ^ Activity

feaaure»entaf McGraw-Hill BooleCompany, Inc., New ork,

31. Handler, 0., "fhe Effect of 2#4-Dinitrophenol ©n Respira-tion, Qxydative Assimilation, and Photosynthesis in Chlorella f» Physiologla Plantarua. XI (195#)# 675-6^4.

32. Kennedy, S. B*. "Jhe Influence of' Magnesium Deficiency, Chlorophyll Concentration, and Heat Treatments on the Bate of Photosynthesis of Chlorella." American Journal of Botany, XXViII (February 2, 3-940), 68-73 •

*

33. Kohn, H. I., "Inhibition of Photoayntheaia in Chlorella ayrenoidosa by the lodo-acetyl ladical,* Journal of leneral Physiology. XIX (September, 1935), 23-24.

34* I*andolt,

oiapany,

35» Lindahl, P. I. B« (Royal Agricultural College of Swedes, Uppsala, Sweden), "The Effect of Sodium Dimethyl-dithiocarbamat e and T etramethylthiour ©a Bisulfide onPhotosynthesis and Endogenous Respiration in Bnteremonaha^linza. « Physiological Plant arum. XVI

36# Haloney, T. K., "Control of Alga* with Chlorophenyl Dimethyl ttrea^" ^ f l j | J & e * *"*

37. . , and C. M« Pala*r, "Toxicity of Six Chemical Compounds to Thirty Cultures of Al*ae." Water and Sewage Works, CIII (November, 1956), 501-505.

)#• Myers, J», "Oxidative Assimilation in Relation to Photo-

^2sasEsi iMi2flc' 39. Palmer, C. M. and T. C. Maloney* "Preliminary Screening -

for Potential AIgicides,tt the Ohio Journal j>f Science. LY (January, 1955), 1-3.

40, Pardee, A* B., "Measurement of Oxygen Uptake Under Controlled Pressures of Carbon Dioxide," Journal £f Biological Chemistry. CLXIII (1949), i m - l W l .

41* Pratt, JUt "Studies on Chlorella vulgaris. VI. Retardation of Photosynthesis by a Growth-Inhibiting Substance from Chlorella ydg«rl«." i£BJ2Si 2£ M S S Z . XXX (1943), 32-33.

42. _ _ _ _ _ , "Studies on Chlorella vulgaris. VII. Influence of tie Age of the Culture on the Rates of Photosyn-thesis in Respiration," American Journal of Botany, XXX (1943), 404-403.

„ "Studies ©a Chlorella vulgaris. YIII. Influence ETSotosynthesis of Prolonged Exposure to Sodim Carbonate and Potassium Carbonate.11 American Journal St£ Botany, XXI (1943), 626-629. .

, "Studies on Chlorella vulgaris. IX. Influence of Growth of Chlorella with Continuous Removal of Chlorollia from the Culture Solution," American Journal o| Botany, XXXI (1944), 413-421.

45. and 3. P. Trelease, "Influence of Deuterium Oxide on Photosynthesis in Plashing and Continuous U | h t ^ American of Botany, XXV (1933),

10

46# Safferm&n, R, 3» and M. I* Morris, "Evaluation of Natural Products for Algicidal Properties, * Ainllfd Micro-M ^ S C * X (July, 1962), 2S9-292# -

if

47* Shiau. X* G. and J. Franck, "Chlorophyll Fluorescence and Photosynthesis la Algae, Leaves and Ghloroplastss," Archives of Biochemistry. XIV (194?), 253*395•

4&* Snow, J. R#, "Siaasiiia as an Algicide for las® Ponds." ||9^te0grwive fish Culturigt. XXV {January, 19&3),

49# Syrett, P. J., "The Effect of Cyanide on the Inspiration and the Oxidative Assimilation of Glucose by Chlorella vulgaris.*« Annals of lotany. New Series. XV (Octoier, 195X1, '47I-491*

50# Tang, P. §*, "Studies on the Kinetics of Cell Respiration. VI•Respiration of Ghlorella ovrenoidosa in Presence

Phyaiology8>X jf

|1, Walkar, C# R», "Control of Certain Aquatic Weed® in Mis-souri Farm Ponds,* Weeds. VII (July, 1959), 310-316.

53# Warburg, 0#, "Geischwindifkeit der photoehemlsche Kolen-s&uersetiung* XI,« Biocheaische Zletschrift, GUI (1920), 18S-217#

54* Wjtt&n&fee, A#* "Uber die beeinflussung der atmmg von elnigen

frunen algen durch Kalinmycyenid un methylenblau. eitrage zur stoffweehselphysiologi© der algen* I."

Aotfe Phytochimica. VI (1932), 315-335* 55* Weia, D# and A. H. Brown, "Relation Between Respiration

and Photosynthesis in the Green Algae, Ankistrodearous feramil," Plant Physiology. XXIV (May, 1959)# 224-234#

CHAPTER II

MATERIALS AND METHODS

Tests were mad® with a bacteria-free unicellular alga,

Chlorella pyrenoidosa Chick #26, obtained from the Indiana

University Culture Collection# Cells were cultured in Bristol's

Medium as modified by Bold (1)# The medium was prepared fro®

six stock solutions, each 400 milliliters in volume (Table I)•

Tea ailliliters of each stock solution were added to 940

milliliters of Pyr ex-distilled water. One drop of 1 per cent

FeGl^ solution was then added to the medium# The final pH

was between 6#6 and 6.& after autoclaving and cooling.

TABLE I

STOCK SOLUTION COMPONENTS FOE BRISTOL'S MEDIUM

Salt Weight in drams

Ia» 3 10.0

KHgPO^ 7.0

K2HP04 . . . . . . . . . . 3*0

WgS04 • TUgO 3.0

CaCl2 . . . . . . . . . . 1.0

NaCl . 1.0

The culture was maintained in a one-liter, double side-

armed, filtering and media storage flask (Figure 1). The

11

1 2

E, C, culture/ flask——- j

-Et

E, E-t, Ep, compressed ax

filtering apparatus

A, Media storage flask

compressor clamp

D, auto-matic pipetting outfit

F, overflow flask

Fig. 1—Culturing apparatus for Chlorella pyrenoidosa*

13

entire culturlng apparatus was autoclaved and placed into &

plant growth chamber (15). Compressed air was filtered through

distilled water, copper sulfate solution, acid dichromate

solution and a fourteen-inch dry, sterile, cotton filter be*

for® entering the culture flask. Fresh medium was added to

the culture flask with an li-gauge hypodermic needle attached

t® a tube from tk® lower arm of a media storage flask. The

hypodermic- needle m e inserted into the "fresh media" line with-

out contamination by swabbing the media line with absolute

ethanol several times* Gravity flow of the medium was con-

trolled by a screw compressor clamp on the aedia line*

The culture flask was Inoculated with ceUss of a young

culture transferred fro® a Bristol*s Agar slant. Cells were

washed from the agar surface by adding Bristol^ liquid

medium to the culture tube and agitating with a Vortex Jr.

Mixer# The cell suspension was drawn into a sterile syringe

and injected into the "fresh media" line# The cells were

then washed into the culture flask by fresh media after re-

leasing the cla»p on the media line. After a period of one

week, the culture had reached a growth dense enough to yield

approximately 0.007 milligram ;@f cells per milliliter.

Physical environmental culturing conditions were main-

tained as constant as possible with the plant growth chamber.

A sixt ©en-hour light period was followed by an eight-hour

dark period. The light intensity was approximately 175-foot

candles as produced by three twenty-watt fluorescent bulbs.

The temperature varied from 24 to 2& degrees centigrade.

14

Previous workers cited instances of physical and chemical

environmental factors bearing directly upon the development

@f the respiratory and photosynthetie systems in algal cells*

Dvorakova-Hladka (3) concluded that taking of a culture

affects the respiration considerably# When compared with a

non-shaken culture, the shaken culture displays a higher

oxygen consumption and a different respiratory quotient. Light

intensities were also shown to have definite effect# upon the

photosynthetie mechanism development (14, &). Cells grow

under low light intensities had relatively high chlorophyll

content and low photosynthetie rates# The reverse was true

of cells grown under hi# light intensities. These physical

factors Halted the accuracy of results# However, chemical

influence® might have been present and could have acted in

conjunction with the physical factors. Pratt (9, 10, 11, 13)

and Shiau (16) have shown that respiration and photosynthesis

were inhibited by a water-soluble, membrane-penetrating poison

which Pratt called "chlorellin." For obtaining reproducible

results, one must limit these factors. Myers (17) devised a

method of culturing cells which held these limiting factors

to a minimum and enabled the worker to use cells which yielded

the same degree of photosynthesis.

The method of culturing used in this study was planned

with the aforemeationed factors in mind. Cells were taken %

from the same growth phase and "chlorellin" levels were

negligible.

15

Cells were concentrated for the respiratory flasks by

centrifugation. Distilled water was then added to the packed

calls and agitated with the Vortex mixer. The process was

then repeated and the packed cells were suspended in fresh

medium and diluted to a concentration of approximately 0*3

milligram per milliliter. A comparison of the distilled

water washing with a medium wash yielded m difference in

phot©synthetic rates*

Photosynthetic rates were measured manoiaetrically by the

method of Pardee (4, 5, 6# $$ 17) with a Bronwill Warburg

apparatus* This method utilised a solution of diethanolamine

(2,2*~iminodiethanol) which is used in Industry for absorbing

carbon dioxide* However, if diethanolamine la acidified with

6H HC1 in the i m m . of KHOOj, th* r«eU<m i. reyarS.d and

carbon dioxide is liberated until a constant pressure la at*

tained. the amount of carbon dioxide liberated la dependent

upon the Mount of KHCO^ present* A greater weight of KHCO^

was supplied to the solution because of the low temperature

(25® centigrade) of experimentation. Pardee pointed out

experiments by Irlch Hirschberg that equilibrium pressures

were reduced by approximately one-fourth at 19® and one-half

at 27°. A trial run for comparing Pardee9# method with the

buffering salts - 0*111 KHGQj (2) and a mixture of G.035M MQO^

with 0.065M MalCQj (12) - waa made* It was found that Pardee*f

method yielded approximately twice the volume of oxygen as

the other methods (Figure 2)* Pardee*a method had the added

16

o 70

"> r\

3 OU

111.05 Pardee's

# 66.01 Warburg1s

^^•63.66 Pratt's

Time in Minutes U f " , t . . i i i—r. L- . - 1 1„

0 15 30. 45 60 75 90 105 120

Fig. 2—Comparison of Pardee's Method with Pratt and Warburg Buffers.

17

advantage of maintaining the cells in the culturing

medium.

The coordination rac©mates and their isomers used were

Co(«n)^ Clj, Co(en)2 (Cl)2 CI, Cr(tn)5 I^# Co(en)2

(lOgJtjlr* The group ©f raceaiates used wer«|~Rh(en)2(l~

valine)] Xg, [aii(®»)2(l-leu«siii«)]x2# [RManJgtl-tyroslne)] I2*

[^(ea)g(*alaaine)]l2# RhfaiOgd-glyei&elJXgj aild [lh{ea)2

<l-phenylalanine)]l2* One optical isomer, gJVo{l~aspartic

acid)2 , with an optical rotation of -31.3° was used* Mis-

cellaneous compounds used were CuSO^, CoCl2*6H2Q, thiourea,

and n~phenyl urethane. These compounds were supplied in

crystalline form and one-tenth gram samples were added to

XO~s&lliliter volumetric flask®# Distilled water ma added

to th© mark yielding a concentration of 1 per cent by weight»

Serial dilutions ware made to yield concentrations of 0*01

par cent. n-Phenyl urethane was prepared in concentration of

0*005 per cent.

The algicides used in this study war® Amine-d«acat at e»

Eydrothol 191# Hydrothol 47 (liquid), aad Hydrothol 47

granular)* Thes© compounds war® prepared for experimentation

by adding ©a® milliliter of the commercially prepared com-

pounds to nine milliliters of distilled water* Thia yielded

a concentration of 1 per cent by volume of th® preparation

and not toy the active compounds in the preparation# Serial

dilations war© made to yield the desired concentration. The

U

granular form of Hydrothol 4? was saturated at less than 0*01

per cent by adding 0,01 gram to 100 milliliters of distillad

water*

Preparation of the Warburg vessel® and manometers had

to fee done carefully and correctly If gas-tight connections

ware to fee achieved* Erroneous results may be reported if

the seals are incomplete* Celvaeene light vacuum grease was

used as the sealant for all flask-sanometer arid venting arm-

flask connections* A thin film was evenly distributed 'with

a tightly rolled cotton swab over one surface of the con-

nection. After the vessels were prepared, they ware easily

sealed with slight pressure by the fingers* A thin film of

Celvaeene was applied to the rim of the center well to prevent

creeping of the algal suspension into the well and splashing

of Pardee*a solution into the suspension*

Two milliliters of the algal suspension were added to the

large well of the Warburg vessel with a volmefcrie pipette#

One-half milliliter of the chemical to be tested was added to

the side ana* Immediately before attaching the vessel to its

manometer* one-half milliliter of Pardee's solution was added

to the center well* The vessel was then attached to the

manometer by pressing lightly with the fingers until all air

bubbles were forced out of the seal. The vessel was then

secured with a rubber band*

The prepared vessels were placed in the water bath set at

a temperature of 25° centigrade and maintained to within plus

If

or minus 0.01 degrees# Cooling water was pumped through

refrigeration coils with a diaphragm pump or city tap water

was used when the lights were turned on dm© to m undesired

increase in temperature., A bank of incandescent 45-watt

bmlbs beneath the water hath served as the light source at an

intensity of approximately 1150-foot candles m measured with

a Weston light meter. The lights were turned on when the

vessels were submerged. Fifteen minutes were allowed for the

equilibration of temperature la the flask. fhe vessels were

shaken at a rate of 100*110 tiaea per minute through a distance

of 6.5 centimeters. At the and of the equilibration period,

the manometer columns were set at 15 centimeters and the

stop-cocks closed. One hour was allowed for complete equili-

bration at which time the rate of oxygen evolution became

constant. At the end of one hour, the chemical was added from

the side era into the algal suspension. leadings were taken

every 15 minutes during the first hour of equilibration and

for three hour® of contact tiae after the chemical was added#

Cell weights were taken by filtering two milliliter* of

the suspension with a tared Millipore membrane filter (G.45/<

pore site)* The filter pad waa placed in a vacuus deasicator

with aeid phosphate as the deasieant# After the pad waa

dried completely, the vacuum was released and the pad allowed

to remain for a few houra before the second weighing. The

eelis were weighed to the nearest tenth milligram.

m

Two milliliters of the algal suspension were placed in

sterile test tubes and 0.5 milliliter of the experimental a©*-

pound added# These tubes were placed is til® plant growth

chamber and incubated for at least two weeks* Th© cells were

then transferred to a Bristol*s Agar slant and incubated for

a m a days* Comparison of this suboulturins technique with

the manometric technique was made to determine whether the

QOfflfOUM was algistatic or algicidal.

The rate of photosynthesis was recorded as apparent be**

cause a© respiration could be exhibited by which the rate

could be corrected. Data were reported as per cent of the

control for oxygm evolution.

CHAPTER II BIBLIOGRAPHY

iran 1. Bold, H* C., "The Morphology of Chlajnydomonaa .

??949)T

2. Dam. H., "Vitamin K in 0aie@13.iOar Photoayatheaising , Organisms," American ©£ ISSSBZ# rai {October, 1944), 4 9 1 - ^ 3 . '

3» Dvorakova-Hladka, J.# «Th# Effect of Hiysical Conditions of Cultivation on the leapiratory Metabolism ®f Algae," Biological Flantarum, I? (19©2), 141-146#

4. Brickson, L. C., R« T. Wedding, and B. L. Brannaaan, "laflutaee of pi on 2,4-M ehlorophenoxya c et i o and Acetic Acid Activity in Chlorella,» XXX (JanuaryI 1955), 69~?4*

5. Galloway, 1. A« and R. M, Kraiiss, "The Differential Action of Chemical Agents, Especially Polymyxin-B. on Certain Algae Bacteria and Fungi," American 2l M » Ufl (January, 1959), 40-49.

6. Horamersaxid, M. H.t "Some Effects of Monochromatic Ligfrt on Oxygen Evolution and^Carbon Dioxide FiMtion in QllSr rella Pyrenoidosa "

S S r e r f S S n c l l (Oeteber 1V18, 1963), 3S1-390.

7# Myers, J* and h. B# Clark, "Culture Conditions and the De-velopment of the Fhotosynthetic Mechanism. II# An Apparatua for the Continuous Culture of Chlorella**

& Sg^aral Physiology. XXVXXX (November 20,

Pardee, A# B«

9. Pratt, R* and J. Feng, "Studies on Chlorella vulgaris* II. Further Evidence that Chlorella Cells Jforia a Growth-i inhibiting Substance,* American Journal M . XXVII (1940), 431-430#

21

22

10. Pratt* I., "Studies on Chlorella vulgaris. VI. Ettardation of Photosyntheal a by a Growth-inhibiting Substance

Mfflrii," i a s s l si M m t » XIX i19#3# t 3**'33* *•

11- ... . « '•Studies ©a Chlorella vulgaris. VII. Influence orthe Age of the Culture 011 the Rates of Photosyn*

xxxs(i%3? B « i o a ' " h * s r A w J o a r n* 1 Masse.

12. "Studies On Chlorella vulgaris. YIXI« In-' fluence on Photosynthesis of Prolonged Exposure to-

HOOj and ftoOj," iSHBal a£ SS£2££. XXX (1943), 626-629.

13• . "Studies on Chlorella vulgaris. XX* Influence on Growth of Chlorella of Continuous Bemoval &f Chlorellin from the Culturet Solution.* American Journal ©£ Botany, m i (July, 1944)» 4Xi~421*

15# Schlichting, H. 8*. Jr., "Construction of an Inexpensive Plant Growth Chamber,* Turtox Mews. XLI (August, 1963), 214*21$. -

16. Shiau, I« G« and J. Franck, "Chlorophyll Fluoreioenee and Photosynthesis in Alga®,-leaves and Chloroplasta,w

M *x? (1947), 253-295• *

17. Bmbreit, W* W., R. I. Burris, and F.'stsuffer, Manoaeta

fechniouea. Minneapolis, Minn., Burgess P«Wi«iiag ornpany, "1957*

1#, Winokar, M., Photosynthesis Relationships of Chlorella Speciea.w American Journal of igtany. XXX? (April, 194&) 1 4W7*214 •

CHAPTER III

BITOT®

Coordination Complexes and Miscellaneous

Compounds

The data for results of coordination complexes* effects

©a the apparent photosynthetic rates were recorded la tallies*

Graphical interpretation of the results would not be feasible

b@eau.se of the close approximation of points ami subsequent

difficulty in reading them. Subculturing results art listed

at the bottom of the tables• On# pirns (*) indicates poor

growth, two (•+) fair growth, three (+•+) good growth, four

(+•••) excellent growth and a negative sign (-1 no growth.

Table II shows results Of the effect which [Cr(en)^]l2

had upon the apparent photosynthetic rata. The 1 par cent

solutions of the isomer3 and racemate exhibited depressing

action on the rata and were supported 'by the subculture tests.

The 0.01 per cent solutions also had a depressing effect on

the rate but subeulturing tests exhibited a non-toxic level.

Cells were "dead* i. e.f the color had faded, after twenty-

six days contact time in the 1 .per cent concentration of the

d-isomer.

The effects of [Co(en)3]Cl3 are listed in Table III. A

depressing effect was noted in all three 1 per cant concen-

trations# However, subculture growth rates were positive for

23

24

TABLE II

EFFECTS OF [Cr<en)3]l3 ON APPA&ENT PHOTOSYNTHESIS

EXPRESSED AS PER CENT OF COITEOL

Tine Rae estate 1~isomer d-iaomer la

Kin* 1$ *01$ 1$ .01$ 1* .01$

15 109.7# 106.20 #6.50 33*n 99.43 109.36

30 104.53 93.29 93.58 97.17 96.50 93.43

45 105.05 97.2# 94.93 90*20 92.06 36.95

60 99.50 93.91 93.5* ;66.15 37.04 30.06

75 96.74 92.61 102.97 90.34 93*57 73,02

90 99.52 95*2$ 95.62 89.73 97.34 . 85.95

105 99.93 97.05 94*&3 £9.25 93.43 33.26

120 99.49 97.36 $4.67 #4. tO 93.06 30.32

135 99.37 99.32 90.25 33*96 92.45 33.74

150 94*63 95.01 03.79 : #5*^1 Qj>« /C> ; 31.53

165 97.96 93.30 34*95 36.54 34.39 32.76

ISO 33.76 93.74 $4*7$ $7.57 77*15 35.14

PH

S us-es til"* ture

' 6*35

•k

7*00 +

7.20

<*r

7.15 6.90 6.90

*

the race-mate and d-isomer and negative for the 1-isomer.

Thirteen days of contact time with the 1 per cent d-isomer

showed the cells t© fee "dead#8 Fair growth was exhibited by

the Q.GX per cent concentrations of the raeemate and d-iaomer.

25

TABLE III

EFFECTS 0? fCo(en)j]cij 01 APPABEBT FHOTOSXNTHBSIS EXP8ESSIB AS HR GEM OF S0NTE0L

fim© In

Mia*

Raceraat© l~iaoner

1$ ,01$

d-isonar

w >01>

IS

50

45

60

75

90

105

120

135

150

165

100

pH

Sub.

95.90

91*55

92*40

9*«13

93.70

90.68

#9.41

19.76

$$.3$

06*97

103.81

90*17

103*31

102.31

100.23

99.64

96.49

100.37

97.75

97.29

97.17

96*14

100*24

90*21

S4.ll

#3*43

95.25

03*43

61*56

66*36

65.03

61.27

62.50

62*73

97.2#

97*75

100*02

109.61

120*05

117*07

119*30

112*12

117*09

110.5

110*12

109*02

111* 24

91.51

#0*19

71.56

70*09

64.32

62*50

59*70

50.94

54.39

52*00

52*02

116*40

110*25

115*07

121.33

1360,97

124*40

110.55

122.46

123*43

123.40

121*95

121*50

6*35 6*30

+•*>

6*45 5.95 +

6*15

*

6*00

4+

Table IY lists results &£ [Co(en)2(Cl)2Jci* A depressed

photo synthetic rate was observed only in the 1 per cent con-

centration of the rac estate* Subculture growth rates were all

positive with excellent growth in the 0.01 per cent concen-

tration of the 1-isoaer. Good growth was noted in the 1 per

26

TABLE If

EFFECTS OF •[C©{#»)2(C1)2]C1 OM APPARENT PHOTOSYNTHESIS EXPRESSED AS PER CENT Of CONTROL

Time in

Racemate 1-isomer d-isomer

Kin* 1$ *01$ 1$ 1 .01* 1$ .01$

15 83,69 96.39 104.37 103.76 33.19 93.00

30 34.35 107.65 IO4.5$ 105.21 86.56 91.34

45 79.53 97.02 103.21 103.76 102.57 99 #77

60 63.11 95. #2 101.23 104.55 93.26 94.21

75 72*95 93.29 101.27 IO4.I4 96.69 90.42

90 74.72 103.21 101*59 105.79 92.04 90.33

105 69 #74 107.33 102.52 109.46 99.77 91.25

120 59.42 33.43 100.14 107.57 73.36 69.17

135 ; 71.99 101.46 9$. 04 105.67 91.75 39.79

150 63.65 93.40 97.34 106.17 33.03 30.01

165 73.29 102.00 95.72 104*49 39.91 35.07

130 76.32 ! 142.5# . 94.67 104.33 95.12 : 36.74

pH 4.00 4.50 4.00 4*60 6.35 r ft*

Sub* 4 4" • + ++ +

cent d-isoaer wmmtr&tlm which differs fro» the earn® con-

ctatratio&s of the 1-isomer and racemate* The cells were

"dead" at the end of twenty-two days in the 1 per cent con-

csntration of the l~igomer.

The results for [co{«n)2(K02 ]®r ar® listed in Table ¥*

No extreme depressant effects were not ed for any concentration®•

27

TABLE ?

1FTOTS OF rG©Un)2(N02)2]Br ON APPAEIHT PHOTOSYNTHESIS EXPRESSED AS'PIR CEST OF CONTROL

fine in

Sac mate 1-isomer d- i somer

Mitt# 12 •0156 & .01$ ; xi .om

15 104.54 119.53 105.51 116.69 101.02 101.46

30 110.51 119.05 107-ia 112.14 99. #3 .102.36

45 119.20 U 7 . 4 4 124#7$ ;1X2.49 100.37 109.03

60 105.49 11#.23 103.59 100.7# 9##44 103.96

75 106*39 119.76 99.26 ; 101.56 99.13 103.79

90 110.13 123.74 9#.12 102.33 96.27 103.12

105 1O9*S0 122.57 96.13 100.34 94»65 104.23

120 : 106.23 121 .31 94.62 5S.71 97.93 107*1#

135 106.09 121 .61 90.0# 97 . *5 93*27 9 9.2#

150 109.62 132.57 89.91 9#.#6 : 91.90 104.9#

165 124 .51 148.97;' #7.59 ! : 96 .67 #9*64 104.2#

i#o 106./^ 130.95 #6«5& : 95*95 92 .91 104.#7

pH l 6#90 6.75 6 . 9 0 6.60 6.75 6 . 0 5

Sub* * + +-T - + * *

However, subculture tests showed a negative growth for the 1

per cent concentration of the 1-isomer. It will be seen later

that the #6.5# #er cent oxygen evolution level, which was

reached at the end of three hours, lies within an estimated

%&m of negative or positive growth# Good growth wai noted

m

for the racemlc 0.01 per cent concentration which also

exhibited a high oxygen ©volution rate*

fable ¥1 list® the results of six rh©diua~coiaplexes.

The concentration* are varied due to the very small quantities

available* Inhibited phot©synthetic rates were noted for all

1 per cent concentrations. A subculture was made for the 1

per cent concentration of [Rh(lwtyroaine)]l2 which was nega-

tive. All 0*1 per cent concentrations exhibited growth in

the subcultures and above the control level for oxygen

evolution*

The effects of several "aisceHaaeoua* compounds are re-

corded in Table VII. K|Co(l-asparfcie acid)2]exhibited no

effect on the phot©synthetic rate and all subcultures were

positive. One per cent and 0.01 per cent concentrations of

CuSO^ depressed the photosynthetie rate and yielded negative

subculture results. During the course of the Warburg experi-

ment and after a few hours in the subculturing tubes, the cell

suspension turned fro® a deep green to grayish-green due to

the action of the 1 per cent concentration. Microscopic

examination under oil immersion showed the cells to be greatly

decolorised and clumped together* The same effect was not id

in the 0.01 per cent GuSG^ concentration after six days of

subeulturing. GoGlg«6l20 exhibited a mild inhibiting effect

on the fhoyosynthetie rate and a negative subculture was re-

corded. Color faded from the cell suspension at twenty-six

m

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31

days. Thiourea displayed positive subcultures for 1 per cent

and 0*01 per cent concentration. A strong inhibition of the

phot©synthetic rate was exhibited by the 1 per cent concen-

tration* n-Phenyl urethane also showed a strong inhibiting

eft est on the photosyrithetic rate and good growth in subculture.

A comparison, was made of all the subculture results by

using the last recorded per cent value in each concentration

of every compound. The median was calculated for each growth

rate sign# The median value for negative growth was found to

be ?1«$2 per cent oxygen evaluation, 90.49 per cent for poor

growth, and 103*0$ per cent and above for fair, good, and

excellent growth.

Algieides

Figure 3 represents the results of Amine-d-acetat e on

the photosynthetic rate* The general shape of the curves for

the 1, 0.1 and 0*01 per cent concentrations was the same but

inhibition of the photosynthetic rate was greatest at the

highest concentration and least at the lowest concentration.

The 0.0001 per cent concentration depressed the rate to 37

per cent during the contact time* This concentration line,

although somewhat erratic, shows the inhibiting effect* The

suspension of cells turned light green at five days contact

time and white at seven days with the 0.0001 per cent con-

centration. Microscopic examination showed the cells to be

lysed and many cell walls present in clumps. The other

32

a o •H •P O 3 TJ O u

PL,

CM O

100

90

ao

70

60

50

40

30

20

10

0.0001%

0.01%

Time in Minutes

_i J I l 15 30 45 60 75 90 105 120 135 150 165 ISO

Fig. 3—Effects of Amine-d-acetate on Photosynthetic Rate Expressed as Per Cent of Control.

n

eoncentrations were toxic within thirty-six hours, Subcul-

tures for the concentrations were negative#

Hats for Hydrothol 47 liquid are represented in Figure

4. The curves are very similar in shape to those shown for

Amine-d-acet&te. The 0.01 per cent concentration produced

a yellow-green cell suspension after two days contact time#

At 10 days, the greenish color was completely absent and the

cells were assumed "dead". Cell suspension color faded to

grayish-green after three days contact time with the 0*0001

per cent concentration. All green color was absent after

fifteen days# Microscopic examination of the cells showed the

sm® effects as Aoine-d-acetate. Subcultures were negative

for both concentrations.

Figure 5 represents the data for Hydrothol 191 and Hydro-

thol 47 granular. The typical curve is shown for Hydrothol

191. Cells in the culture tubes exhibited a marked color

reduction after one day. the color was .completely absent

after three days contact time and the cells were assumed

•dead". Cells were found to be disrupted upon microscopic

examination. Subculture results were negative* The granular

Hydrothol 47 was non-inhibiting according to the median

toxicity photosynthetie rate levels. Cells in contact with

the less than 0.01 per cent concentration showed no inhibition

of growth after thirty-two days. Excellent growth wa« ex-

hibited in subculturing.

34

£ O •H •P O d •xS p M

Cm C\J

o X

100

90

so

70

60 L

50

*0

30

20

10 0.0001%

iime m Minutes

105 120 135 150 165 ISO 15 30 45 60 75 9

Fig, 4—Effects of Hydrothol 47 (liquid) on Phot©synthetic Rates Expressed in Per Gent of Control.

35

100

d o •H "p o "d o u Dh

o

30

20

10

Hydrothol 47

Hydrothol 191

Time in Minutes J I I I L

15 30 45 60 75 90 105 120 135 150 165 18,0

Fig. 5—Effects of Hydrothol 191 and Hydrothol 47 (granular) on Photosynthetic Rates Expressed in Per Cent of Control.

CHAPTER If

DISCUSSION

The data presented indicate effectual treads of eaordi-

nation complexes, inorganic compounds, organic compounds, and

commercial algicides upon the apparent photoeynthetic rat#

of Ghlorella pyreaoidosa. These treads obtained from the

Warburg apparatus war# supported generally by tuba cultures

and subculturing techniques. The method of action of the

compounds was not known but a®*# possible actions were

discredited.

Deleterious effects were not due t© osmotic pressure,

temperature, light intensity, depletion #f nutrients from the

culture medium, or pH. One per cent concentratioaa yielded

final concentrations of 2500 parts per million after dilution

in the Warburg Teasels and culture tubes. At this concentra-

tion, mm® flasks showed a greater rate of oxygen evolution

than those with 0.1 and 0#01 per cent concentratioaa. The

temperature had no effects on the cells for it was maintained

fairly constant from the culture chamber to the Warburg

Teasels and was within the optimum range for Chlorella

pyrenoidosa (6). Light intensity effects in the Warburg ap-

paratus were negligible due to the low intensity used. De-

pletion of nutrients during the course of the Warburg analysis

and culturing method was not exhibited. pH effects were also

36

37

found to be «f no consequence. A pi range of 4.00 to 7# 20

was within the growth range for Chlorella ovrenoidosa (6),

Whether ©r not the results were due to portions of the com-

pounds by dissociation, Absorption, adsorption, membrane

transport interference, or metato&Lie phases interruption was

not investigated. Hone of these phenomena can be discredited

and each should be considered in future work with these

compounds.

[cr(en)jJlj {Table 21) was the most effective of the co-

ordination complexes# The subcultures were negative for each

1 per cent concentration. Results -showed also that twenty-

six days were required for the culture to be killed in the

presence of the 1 per cent d-isoiaer. It is interesting to

note that the subcultures for the r&cemate, and isomers in a

1 per cent concentration were negative but only the cells in

the d-isoraer culture tube were "killed.* Warburg analysis

showed this to be true at the end of three hours.

Table III lists the results for [c©(on)J CI3. Warburg

analysis shows the 1 per cent concentration of the 1-iaomer

and d~iso®er to depress photosynthesis# This initial effect

could be due to a cell surface phenomenon or metabolite in-

terference. long range results show, however, that the

l-iso»er was toxic or inhibited reproduction as verified by

the negative subcultures. It was also seen that thirteen

days of contact with the 1 per cent d-isoaer "killed- the cells,

3B

but subculturing was positive. Evidently, the d-isomer can

be toxic if it remains in contact with the cell* for a long

period of tiae.

[00(011)2(01)2 CI (fable IV) shows a depressed photo-

synthetic rate in the 1 per cent racemic mixture which was

very much below the values for the 1 per cent isomers# It

is conceivable that an antagonism may exist between the isomers

although this was not supported by subcultures at the 1 per

cent concentration# Growth in the 0.01 per cent isomeric

subcultures was good and doss support the antagonism possi-

bility when compared with the racemic subculture. The cul-

turlng tube results were again contradictory to Warburg

analysis and subculturing tests. The cells in contact with

the 1 per cent 1-iaoaer were discolored at the end of twenty-

two day® and assumed •dead*"

Warburg analysis of [Co(en)2(^2)2] liable ?) showed

little effect of the raceiaate and d-isomer on the photo syn-

thetic rate. The 1 per cent 1-isomer exhibited a slight

inhibition and was supported by the negative subculture* A

good "synergistic effect" was noted for the 0*01 per cent

racemic concentration. Accelerated growth was seen in the

subculture as well as exhibited by the oxygen evolution. It

is possible that the racemate in the 0.01 per cent concentra-

tion was dissociated at the cell surface yielding the nitrite-

portion of the molecule. It is also possible that the complex

39

was absorbed and yielded the nitrite-portion directly to the

intermediate steps of nitrat© reduction in the formation of

amino acids or related organic nitrogenous compounds.

Table VI represents the data for rhodium-amino acid

complexes# All 0.1 per cent concentrations exhibited an in-

crease in the oxygen evolution rate* It is not known nor can it

be assumed that this acceleration was due to any portion of

the complex. It was noted that inhibition of photosynthesis

occurring at the 1 per cent level was prevalent. One sub-

culture showed a negative growth which indicateda toxic level*

Beckwith {1) showed that all the amino acids used here were

either beneficial* stimulatory, or non-influencing to the

growth rate of Chlorella species. It was assumed that any

toxic effects were due to the rhodium interfering with

metabolic processes.

Copper sulfate has been said to form copper chlorophyll

which causes the cells to retain their color for a period of

tisae but inactivates the photosynthetic process* The same

action was shown to occur as Greenfield (5) reported. For

verification of the Warburg technique in screening procedures#

CuSO^ was used in the study because of its universal use as

an algicide. The results are recorded in fable VII.

CoClg'&^O (Table VII) showed toxic characteristics in

the Warburg analysis, tube cultures and subculture tests.

Again,, the action was not known but the depressed ll£it

40

reaction suggested a retardation of energy transfer#

n-Phenylurethane (fable VII) is in a class of chemicals

called narcotics and has been found to be inhibiting to energy

transference in the photosynthetic mechanism (Z$ $§ 7, $)*

Emerson and X*ewis (2) found that 0*005 per cent concentration

inhibited photosynthesis by 50 per cent* The same concen-

tration was used in this study for comparison of results with

the reported effects# Apparent photosynthesis was inhibited

68 per cent. The difference in results was probably due to

the differences in methods! i# e., Pardee's method as compared

to Warburg* s method* The culture tube tests and subculture

tests showed good growth which possibly mean# that the inhibit-

ing effect was dissipated after a period of time and respira-

tion was not affected at all.

Green, McQarthy, and Xing (I*) reported thiourea to be

inhibiting to respiration and photosynthesis# fable Til

shows their findings to be false# Photosynthesis was decreased

in the 1 per cent concentration but culturing was found to be

positive. Photosynthesis was not depressed to a lasting degree

in the 0.01 per cent concentration* Thiourea effects were

reported to be reversible which may account for the results

in Table VII. The 0.01 per cent column shows a fluctuation

in percentage levels* This fluctuation suggested metabolic

shunts which enabled the cells to continue photosynthesis.

More pronounced results were obtained from experiments

with the commercial algicides* These compounds were used to

kl

determine the feasibility of Warburg analysis for screening

algicides. fiesults support the initial hypothesis that

manometry is a more rapid procedure and, when used in con-

junction with subculturing techniques, will yield ®or© con-

clusive evidence for eradication chemicals.

Figures 3, k$ and 5 illustrate the effects of algicides

on the apparent photosynthetic rates* Hydrothol 191 was

somewhat less pronounced in its depressing action but, was

lion-affect ire to photosynthesis. It was very insoluble in

water and would probably be more effective as a contact poison#

This fact introduced a limitation for the use of manometry as

a screening tool. The compound being tested must be soluble

in water which is the usual case in hydrophytic pesticides.

Cells used in all three phases of testing with the

algicides were bleached of all color. Microscopic (anamination

under oil isnersion revealed cellular disruption and chloro-

phyll breakdown. At first notice, the algicides* actions

appeared to be mechanical but noa~ruptured cells showed the

chlorophyll to be degraded* Culture tube cells showed no

pyreaoids present and the chloroplasta to be greatly de-

natured in intact cells. Presumably, entrance into the sells

was made either by active transport or some autoc&talytlc

enayae. At any rate, the action was vary definite and photo-

chemical reaction systems were completely destroyed.

42

la conclusion, the results indicate definite effects

upon apparent phot©synthetic rates by coordination complexes,

the inorganic compounds used, and the commercial algicides.

However, specific action should not be expected to be deter-

mined by Manometry unless a compound is broken down into its

component parts and each of these tested.

gffeetual treads should be emphasized and particular ©are

should be taken in procedures* The work of Geoghagen (3)

which shewed "protective" effects by medium organics and

agar, points to a very simple mistake made by many workers

who have tried to determine deleterious effects of various

chemical compounds* If the Warburg apparatus is t© be used

for preliminary screening techniques, «ell culturing, buffer-

ing solutions, and vessel preparations should all be very

carefully considered before experiments are begun*

It should not be concluded fro* this study that bacterial-

free, unialgal, and unicellular clones must be used. Iy

using modifications of the Warburg apparatus, i. e., light

filters, shaking rates, and temperature controls, it is en-

tirely possible to screen many algicides for particular

problem algae in different ecosystems, fhe method described

enables a worker to go to a pond where a problem exists, pro**

cure a sample of pond water and the plant or plants and

nearly duplicate the environment in the Warburg vessel. In

so doing, haphazard trial and error control measures in the

field may be eliminated.

GHAPTE& If BIBLIOGRAPHY

1. Beekwith, T* S. , "fh® Metabolism of Ohlorellaf" Scientific P r o p h a s e . rroo»«djL?Kf of Socl«tr for gjtj>w

2. Intraon* 1# awl C« M. Lewis, ftCo2 Exchange and th« Measure-ment of til® Quantum Yield of Photo synth eni0. t t Anariean Joanwl of Botany. OTXIX (Her«b.r 9, 19Wi, W W :

3* Geoghagen, M. J . , «Th« Effect of Some Substituted Methyl-areas ©a tha Baspiration of Chlore£l& vulgaris f a r . v i r id is f

w Mm Fhytoloidat. LTI (March, 195f>, 71-$®.

4. Grmnt l$* F . , J . ? . McCarthy, and C« J . King. "Inhibition of Expirat ion sad Photosynthesis in Chiorella Pyreaoidosa by Organic Coaroownds that Inhibit Copper Satalysis.* fha C * " c m i l l {May7l9j

Graenfiald, S. 3 . , "Inhibitory I f f ao t s of Inorganic Cos-" san

TOu

Joarntl of Biological Ctumdntry.

i# s * s • 0 pounds on PhotosymthBsia InChloralla « Ajjj.rl««n Journal s i Botany. I H I (February, 1942), izi-

6 - 3 S 8 8 t S i i S ^ ! ; ' w ! ^ e I i I M « 4 n ^ W # i S f i H - a i l -

7« Warburg

8. . .... f "Gesehwindigkeit dar photochemigche Kohlea« ?x920f^xlif2171 *# M l a t e l H # c m

43

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