Mechanism of action of the fungicide copper oxinate

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Title:
Mechanism of action of the fungicide copper oxinate
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108 leaves : ; 28 cm.
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Barnes, David Edward, 1926-
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Subjects / Keywords:
Fungicides   ( lcsh )
Quinoline   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
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bibliography   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Dissertation (Ph.D.) - University of Florida, 1955.
Bibliography:
Bibliography: leaves 105-107.
General Note:
Manuscript copy.
General Note:
Biography.

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University of Florida
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Full Text












MECHANISM OF ACTION OF THE

FUNGICIDE COPPER OXINATE







By

DAVID E. BARNES


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










UNIVERSITY OF FLORIDA
AUGUST, 1955















TABLE OF CONTENTS


LIST OF

LIST OF

Section


TABLES. . .

ILLUSTRATIONS . .


I, INTRODUCTION ,*

A. Literature Review ... .
B. Statement of the Problem .* *

II. PREPARATION OF CARBON-14 LABELED
8-HYDROXYQUINOLINE .

A. General Discussion .....
B. Experimental *

III. RADIOACTIVE MEASUREMENTS. ,

A. General Discussion .
B. Experimental .

IV. STUDIES ON 8-HYDROXYQUINOLINE .

A. ?Jycelial Uptake .. .
B. Mycelial Fractionation ...
C. Radioactive Spores and
Mycelium . .
D. Spore Uptake ... ...

V. STUDIES ON COPPER-8-QUINOLINOLATE.

A. Mycelial Uptake .
B. Mycelial Fractionation ...
C. Spore Uptake ..... *
D. Apparent Breakdown of the
Chelate . .


Page

iv

v












Section Page

VI. SOLUBILITY BEHAVIOR . 86

A. Water-Olive Oil Distribution 86
B. Water-Carbon Tetrachloride
Distribution . 89
C. Water-Octadecanol Distribution. 94
D. Solubility in Aqueous Solution 98

VII. DISCUSSION OF RESULTS .. 100

VIII. SIAARY . 103

BIBLIOGRAPHY l o105

ACKNOWLEDGENTS . 108

BIOGRAPHICAL ITEMS ............. 109

COMMITTEE REPORT .... 110


iii














LIST OF TABLES


Table Page

I Analysis of 8-hydroxyquinoline-9-C14
In 95% HPO4 . 4

II Analysis of 8-hydroxyquinoline-9-C14
in 25% H3PO4 . .42
III Analysis of 8-hydroxyquinoline-9-CI4
in 4i H3PO4 . 43

IV Amounts of Extracted Material and
Their Carbon-14 Activities 76

V A. Niger Spore Uptake . 78
VI Residual Activity in Spores 78

VII A. Nicer Spore Uptake . 79

VIII Residual Activity in Spores 80
IX Radioactive Carbonate Produced j

X Vibrating Reed Data .. 83
XI Vibrating Reed Data ... 84
XII Vibrating Reed Data . 84

XIII Carbon Tetrachloride Distribution
Data . .. 89
XIV Distribution Counting Data 90

XVI Octadecanol Distribution Data 94
XVII Octadecanol Distribution Data 95
XVIII Distribution Data .... ..... 96

XIX Distribution Data . 97














LIST OF ILLUSTRATIONS


Figure Page

1 Apparatus Used In Wet Combustion and
Carbon Dioxide Determination 29
2 BaCO3 Filtration Apparatus 35

3 Calibration Curves of Constant Amounts
of Radioactivity with Increasing Amounts
of Mineral Salt Medium 38
4 Amount of 8-hydroxyquinoline Taken up by
A. nlier, at Different Values of pH 48

5 Amount of 8-hydroxyqulnolino Taken up by
A. niger Mycelium at Different Values of
pH. e # .# 50

6 Amount of 8-hydroxyquinoline washed out
of mycelium ........ .. 53
7 Phosphoric Acid Extracts and Residue.. 54
8 NaOH Extracts and Residue of Mycellum
Grown at Various pH ,... 55
9 Uptake of 8-hydroxyquinoline by A.
nicer Spores . 60
10 Radioactive Material Taken up by Mycelium
In Solutions of Various Copper-8-
hydroxyquinoline ratios . 64

11 Radioactivity of Petroleum Ether (30-40)
Extracts of Mycelium Grown at Various
Copper-8-hydroxyquinoline Ratios o 66
12 Alcohol Extracts of Mycelium Grown at
Various copper-8-hydroxyquinoline ratios 67


_ I ~~~~ ~ ~~~_~~_~











Figure


Page


13 Radioactivity Change in CC14 Phase
for 0.001 Molar Solution of 8-hydroxy-
quinoline with Various CuCI2 Con-
centrations *. . 91

14 Radioactivity Change in Water Phase
for a 0.001 Molar Solution of
8-hydroxyquinoline with Various CuCL2
Concentrations ,* 92















I. INTRODUCTION


A. Literature Review
'When chemical compounds show biological effects,

such as toxicity to bacteria or fungi, they are subject

to speculation and investigation as to their basis of

action. In some cases the mode of action has been

initially explained by an uncomplicated and apparently

straightforward process, but found by further studies

to be very complex. This is the case for 8-hydroxy-

quinoline.

Although 8-hydroxyquinoline has been used as a

bactericide since the early nineteen hundreds, only

recently has its mechanism of action been studied.

The suggestion of Hata (1) that 8-hydroxyquinoline owes

its toxicity to the presence of both a phenol and a

quinoline portion combined in one molecule can not be

seriously considered. On the basis of present evidence

and because the toxicity of 8-hydroxyquinoline is more

than fifteen times that of phenol and quinoline combined

(2), this theory can not be considered valid.

Inasmuch as 8-hydroxyqulnoline acts in very low

concentractlons (100 per cent inhibition of bacteria and









2
fungi at molar concentrations of 0.0001), it appeared
likely that its action was involved in some metabolic
process. From the consideration that 8-hydroxyquinoline
could combine to form bery stable complex compounds, at
biological hydrogen ion concentrations, with important
Ions such as cupric, ferrous, ferric, cobaltous, zinc,
and manganic, Albert (3,4) and Zentmeyer (5,6) suggested
independently that the action was occurring in the form
of chelation between 8-hydroxyquinoline and the essential
trace metals needed for healthy growth conditions. The
complex formed between 8-hydroxyquinoline and copper may

be represented as follows:






OH +





+- O-

^OOH

Albert used bacteria for his experimental work, while
Zentmeyer employed fungi. The basic findings of their
initial work were similar.









3
The toxicity of 8-hydroxyquinoline to bacteria,

was investigated further by Albert and his co-workers (7).
Of the seven possible mcnohydroxyquinollnes, it was found

that on blocking either of the groups essential for chelat-

ion, the oxygen and nitrogen in this case, the toxicity of

the compound was lost along with its ability to chelate.

When compounds were prepared which contained the character-

istic groups of 8-hydroxyquinoline (a hydroxyl group peri

to a ring nitrogen) in other nuclei such as l-hydroxy-
acridlne, 8-hydroxy-5t6-benzquinoline or 6-hydroxy-m-

phenanthrolin, these compounds were found to possess consider-
able toxicity. Among the fifty analogues and derivatives
of 8-hydroxyquinoline tested, only those such as 8-hydroxy-

quinoline-5-sulfonic, -5-carboxylic acids did not show

the relationship between strong chelation and high toxicity.

The two soluble acid derivatives chelate strongly but poss-
ess anionic groups that can prevent a molecule from penet-

rating the cell membrane. This fact supports the theory

that the site of action is in the cell and not in the
medium.

Zentmeyer offered evidence that 8-hydroxyquinoline

inhibition was due to specific trace element immobiliza-

tion for the fungi he studied. He showed first that
8-hydroxyquinoline did not inhibit the fungi below a

pH 3, for this compound does not chelate well in strong









4
acidity. Second, that the toxic effect could be over-

come by adding an excess of metal and third, increasing
amounts of 8-hydroxyquinollne were required for inhibition
in the presence of increasing concentrations of metal ions.
Zentmeyer's work was carried out using only zinc in his
studies. Zinc has been shown to be of lesser importance
than copper or iron (7).
From these experiments It was obvious that 8-hydroxy-

quinoline was acting by chelation, but the site and the
mechanism of action was not established. Albert and his
coworkers have proposed a mode of action based on such
experiments previously described and also on later work

on the 8-hydroxyquinoline-copper equilibrium and the
distribution of 8-hydroxyquinoline in a lipold-wter system

(8,9)t More recently, Vicklund, Manowitz and Bagdon have
proposed a theory that suggests, on the basis of biologic-
al evidence, that the copper complex formed between 8-hydro-
xyquinoline and copper dissociates and the toxicity is due
to the free 8-hydroiyquinoline complexing essential trace
metals in the cells* They state that the copper ions
liberated function synergistically with the 8-hydroxyquino-
line (10). This theory can not be accepted on the basis
of the work of Albert and the experiments to be described
later.









5

From the unusual condition of low toxicity at
high concentration (0.00125 molar) and high toxicity at

low concentrations (0.00001 molar) of 8-hydroxyquinoline,
Albert concluded that the toxic nature of this compound

was related to the metals present in the medium. He
proved this by depleting the medium through chelation of

the metals and extraction of the chloroform soluble

chelates. In this metal free-medium, 8-hydroxyquinoline

was nontoxic toward the gram-positive bacteria used. The

toxicity was rapidly restored by the addition of small

amounts of ferric or cupric ions. From this it was con-

cluded that the true toxic agents were the iron or copper

che 1 ate s.

This conclusion was, however, only partially
correct, for with excess amounts of 8-hydroxyquinoline

and with proper trace metals present the expected tox-
icity was not found. If the 8-hydroxyquinoline concen-
tration was decreased or the metal increased, the solution
showed extreme toxicity. This indicated that the true

toxic agent was a specific metal complex, namely, the 1l1
iron or copper 8-hydroxyquinoline complex. If a large

excess of the trace metal was added the toxicity decreased.

This was considered to be due to the formation of a 111

complex possessing either a single or double positive









6

charge, depending on the ion used, cupric or ferric.

This charged complex would then be unable to enter the

cell,

From this indirect type of information the mode
of action of 8-hydroxyquinolline was thought to be one that

probably occurred within the cell although no direct proof

of the fact was available, For that matter, all evidence

up to the present had been based mainly on indirect

experimentation.












B. Statement of the Problem
The problem undertaken in this research was that
of the establishment of a mechanism of action for the

compound 8-hydroxyquinoline. The research reported here

is concerned with the establishment of certain basic

points, by direct methods such as using carbon-14l labeled

compounds, necessary to either enhance or to change the

hypothesis proposed by Albert and other workers referred

to previously. It was necessary to prepare carbon-14

labeled 8-hydroxyquinoline for the first time. The
carbon-14. was put in two positions. In one case it was
in the angular position (I), using aniline-l-C14, and in

the other in the heterocyclic ring (II) using glycerol-l,-
3-C14 as the carbon-14 source.











(I) (l)
The most essential question to be answered was
whether the 8-hydroxyquinollne was actually entering the
cell or was it adsorbed on the cell wall. Once this









8

question was answered and thus the general site of action

established inside the cell, the effects of various

factors such as hydrogen ion, copper, and 8-hydroxy-

quinoline concentration on the fungus material could then

be studied.

The problem involved further a study of the nature

and properties of the copper complexes of 8-hydroxy-

quinoline. In summary, using carbon-14. labeled compounds

as a direct method of investigation, the mechanism by

which 8-hydroxyqulnoline acts as a toxicant to fungi and

bacteria was studied.


__ __ _~ILI















II. PREPARATION OF CARBON-14 LABELED 8-HYDROXYQUINOLINE


A. General Discussion

It was necessary to synthesize the carbon-14

labeled 8-hydroxyquinoline using the intermediates avail-

able. As stated, the carbon-14 was placed in two positions.

In both cases a modified Skraup's synthesis (11) was used

to yield the desired product. In the synthesis of

8-hydroxyquinoline-9-C14, aniline-l-C14 (a product of

Tracerlabs, Boston, Massachusetts) was reacted with

glycerol and sulfuric acid in the presence of litrobenzene

and ferrous sulfate.

The Skraup reaction frequently takes place with

great violence, and thus the ferrous sulfate is added as

a moderator. In the case of the synthesis of quinoline,

the reaction probably takes place through the following

stages. The glycerol and sulphuric acid react together

to form acrolein, CH2=CH-CHO, which condenses with one

molecule of aniline and combines additively with a second,

thus yielding the anil of B-anilinopropionic aldehyde.

Loss of a molecule of aniline and ring closure then take

place, followed by oxidation of the resulting dihydro-

quinollne (12).


I









10
This scheme may be represented as follows






HHO N

N -7 + HZO











It should be noted that in the preparation of the
quinoline from aniline-l-C14 the observed specific activity
of the resulting 8-hydroxyquinoline-9-C14 was quite low.
This can be explained by assuming some of the reduced nitro-
benzene reacted with the glycerol (or acrolein) to dilute
the product. This point will be discussed later.
The sulfonation of quinoline was carried out accord-
ing to the general method of E. Fischer (13). Using the
procedure described, the major product was the 8-substituted
sulfonic acid with a small contamination of 5-substituted
product.












The 8-hydroxyquinollne-2,L-C04 prepared via
labeled glycerol was of higher specific activity due

possibly to the fact that any dilution effect of the
reduced nitrobenzene would have no effect on the spe-

cific activity of the quinoline.

In these syntheses, using carbon-14, it was
necessary to work in such a manner that none of the

labeled compounds come in contact with the personnel or

equipment in the laboratory. During this research the
apparatus used was completely closed in glass systems
or systems with adequate traps and good ventilation.

It is unnecessary to use shielding with carbon-l4, for the
beta emission is very weak. It is unable to penetrate the

walls of ordinary glass apparatus, and has a range of
approximately 6 cm in air. The greatest hazard is not to

the personnel but to the equipment and results of other

workers. A small contamination could lead one to serious

errors in mechanism studies.











B. Experimental
Individual details for the preparation of the

carbon-14 labeled 8-hydroxyquinoline are given in this
section. All temperatures are in degrees centigrade,
and its symbol is omitted, conforming with present
usage in scientific reports.
Synthesis of Quinoline-9-C14








A one liter, round-bottomed flask was charged successively

with powdered ferrous sulfate (6 grams), anhydrous glycerol*

(50 ml, 0.60 moles), aniline-l-C14 (0.173 moles having a

total activity of one mlllicurie or 5.76 microcuries/
millimole, a Product of Tracerlab, Inc., Boston,
Massachusetts), nitrobenzene (10 ml) and concentrated
sulfuric acid (30 ml of 95%). The flask was swirled to
mix the contents thoroughly and then attached to a water-
cooled reflux condenser and heated over a small flame.


*Commercial glycerol is heated in a porcelain dish in the

hood until the glycerol registers 1800










13
Once homogeneity was obtained the flame was removed and

the reaction allowed to proceed under self-induced reflux.

After the initial reaction subsided, boiling was continued

for four hours over a small flame.

The original flask was then connected for steam
distillation and the unreacted nitrobenzene was removed.

This portion was checked for radioactivity, but it showed

only a very slight trace. After chilling the reaction

mixture was made alkaline by the addition of sodium

hydroxide (50 grams of sodium hydroxide dissolved in

150 ml of water). The mixture was steam distilled again

and the pot residue was checked for radioactivity* It was

found to have activity, thus some quinollne or other

carbon-14 product remained. This portion was saved for

the carbon-14 balance. The unreacted aniline was con-

verted, through diazonlum salt formation and decom-

position, to phenol, The quinoline was extracted from the

distillate with four 25 ml portions of benzene. The benzene

was removed by distillation and the quinoline collected from

233-2400 as a yellow liquid. The crude quinollne further

purified by distillation yielded a pale yellow liquid

(14.3 g) boiling at 236-239 at 763 mm. Inasmuch as the
quinoline was an intermediate, its specific activity was

not determined. The quinoline was dissolved in a mixture


~~









14
of 50 ml of concentrated hydrochloric acid and 200 ml of
water. After warming the clear solution, 30 grams of
zinc chloride in 50 ml of 2 N hydrochloric acid were
added. A quinoline-zinc chloride double salt crystallized
as the solution cooled. The crystals were filtered, washed
with 2 N hydrochloric acid, and decomposed with sodium
hydroxide. Once more the quinoline was steam distilled,
extracted with ether, dried in either over potassium
hydroxide and distilled after removal of ether* A water
white preparation (10.5 grams) in 49 per cent yield and
boiling at 238.5 to 239.50 at 758 rmn was obtained
( n2gl1.6238).
Synthesis of 8-Quinollnesulfonic-9-cl4 Acid




CIL

SO3P


Oleum (40 ml of 20 per cent) was charged into a 50 ml
round bottom glass flask fitted with glass stirrer,
mercury seal, thermometer and dropping funnel. Purified
quinoline-9-C14 (10.5 grams, 77r.5 millimoles obtained
from the previous experiment) was dropped during twenty











minutes into the moderately stirred acid causing a rise
in temperature to 1600. This temperature was maintained

for four hours by application of external heat. After
cooling to about 800, the solution was poured Into 50 ml
of water and the sulfonic acid was allowed to crystallize
for twenty-four hours at room temperature, Ice (25 grams)

was added and after the ice melted, the sulfonic acid was

filtered off and thoroughly sucked dry, Adhering sulfuric
acid was removed by washing with ice water (three portions
of 30 ml). The 8-qulnolinesulfonic-9-C14 acid was dried

on the steam bath. There was obtained 8 grams of light

tan crystals. These were not assayed for radioactivity,

Alkaline Fusion of 8-Quinol nesulfonic-9-C14 Acid
Sodium hydroxide (7 grams) and potassium hydroxide (7
grams) were charged with one ml of water in a thick-walled,

open porcelain dish of 250 ml volume, fitted with a sturdy
stirrer and a thermometer inserted in the dish. The dish
was heated with a Bunsen burner. After the alkali had
become soft, stirring was started and the temperature was
raised to 250, Finely powdered 8-qulnollnesulfonic-9-C14
acid (8.0 grams) was added during ten minutes to the melt
to maintain a temperature of 240-250. The temperature

was not allowed to rise above 250, for low yields resulted
at higher temperatures. The fusion process is exothermic

and the temperature can be controlled somewhat by the









16
addition of the 8-quinolinesulfonic-9-C14 acid. After
the addition of the sulfonic acid)the melt was stirred
ten minutes longer at 240 The melt was then poured on
a metal sheet, allowed to cool, broken up into small
pieces and transferred to a 150 ml round bottom glass
flask fitted with stirrer, mercury seal, dropping funnel
and reflux condenser. Water (40 ml), part of which was
used to clean the porcelain dishwas added; the stirrer

put Into operation and sulfuric acid (95 per cent) dropped

in until a pH of 7.0 was reached* Although the tempera-
ture rose during this process to the boiling point, it

was advisable to reflux thoroughly before testing to make
certain that all alkaline particles were dissolved. The

final pH of 7.5 was determined accurately by means of a

pH meter. After cooling to about 500, the stirrer was
stopped and the contents of the flask were allowed to
remain at room temperature for about ten hours. The solid
consisting of 8-hydroxyqulnollne-9-C14, alkali sulfate, and

a black tar was filtered, washed twice with ten ml of water,
aad dried superficially by air. The 8-hydroxyquinoline-9-
C14 was steam distilled in 250 ml of water and purified
by several reCrystallizations from aqueous ethanol. There

was obtained 4.46 grams (55 per cent of theory) of the
compound with a specific activity of 4.37 microcuries/
millimole and melting at 75-76. The mixed melting point


~__I











with an authentic sample was 75.5-76O. The overall yield
based on aniline-l-C14 was 17.8 per cent.

The radioactive balance is as follows:

Specific Total
activity activil
In.Ac/ml* in mic,


curiLs
1. 000c/4

136/,c


Initial reactant (Anillne-l-C14)
Products (18-hydroxyquinoline-9-C14)
Unreacted Aniline-l-C14 converted
to phenol
Pot residue from first steam
distillation
Quinoline distillation waste
Quinoline-hydrochloric acid
Final steam distillation residue


5.76 AjC/mM
S73/A.Lc/mM


ty
Mo-


122ac


Total


202A4 c

31 l^c
97/4c
57/Ae

925/,c


Per cent accounted for (92.5%)


Synthesis of 8-Hydroxyquinol inem-2,1-C14


* c/,M expresses microcuries per millimole









18
A pear-shaped 100 ml flask was fitted with a condenser,
thermometer, and stirrer, o-nitrophenol (0.5 grams),

o-aminophenol (1.0 gram, 9.18 millimoles) and glycerol-I,
3-CI41, (432 microcuries, 10.8 millimoles) were added and

mixed with a little heating. When the mixture became
homogenous, the condenser was placed on a flask and con-

centrated sulfuric acid (3 ml) was added through the
condenser, After the initial reaction slowed down, the
mixture was refluxed gently for five hours. At this
point the flask was attached to a steam distillation
apparatus and the excess o-nitrophenol was removed from

the acid solution. Sodium hydroxide was added followed
by enough sodium carbonate to adjust the pH to a value

of 7.5. The mixture was again steam distilled to remove

the 8-hydroxyquinoline-2,4-C14. The 8-hydroxyquinoline-
2,4-C14 distilled slowly, and 150 ml of water were required

to remove all the product. The resulting material was re-
crystallized several times from aqueous ethanol. There
was obtained 0.55 grams (41 per cent of theory) of product
with a melting point of 75-76 and a specific activity of
46.6 microcuri eas/mil limo le.




A product of Isotopes Specialties, Pasadena, Calif.










The radioactive balance is as follow:

Total Activity
Initial reactants (glycerol-l,3-C14) h32/U.c
Product (8-hydroxyqulnol ine-2,4-C14) 177/ c
Residue from steam distillation 241 -Fc

Total 418,A.c
Per cent accounted for (97%)















III. RADIOACTIVE MEASUREMENTS


A. General Discussion
Ruben and Kamen (13) were able to isolate and
identify carbon-14 from a graphite probe which had been
bombarded with deuterons of low energy (3-4 Mev). This
offered very small amounts of material for study. The
current production by the reaction Nl4(n,p)Cl4 as
suggested by Reid and Urey (14) is by irradiation of

saturated ammonium nitrate solutions in the Clinton pile
(15). The half-life of carbon-14 based on weighted
averages is 5,568 years (16). Carbon-14 emits beta

particles with a maximum energy of 0.155 t 0.002 Mev. (17)
The approximate range of the beta particle in air is 6 cm.

The beta particles are unable to penetrate the walls of

ordinary glass apparatus. The dangers associated with

the use of carbon_14 are small with respect to personal
health hazards if care is taken to prevent outside con-
tamination of the glass apparatus used. There are some
indications that carbon-l14j can accumulate in certain hard
tissues of the body but this has not been established as
fact (18).









21

The apparatus in which the experiments using

cerbon-14 are carried out can be easily cleaned by vahs-

Ing with water and soaking In hot chromic acid solution.

It is suggested that detergents are not suitable unless

the acid bath treatment is also used. The detergent

particles tend to cling to the glass surface.

For most reactions involving carbon-lt, ordinary

apparatus or slightly modified apparatus was used since

the level of beta activity was not extremely high. Many

compounds used for the preparation of intermediates in tra-

cer work are prepared using vacuum systems (19).

Three types of measurements were used in this

work, Either solids, liquids or gases were used for

counting depending on the applicability and efficiency

demanded by the particular experiment. The solid

samples were of two kinds, pure chemical material and

barium carbonate derived from combustion of the compound

to carbon dioxide, and its subsequent precipitation as

barium carbonate. The combustion was carried out by

Van Slyke-Folch method (20) with several modifications

described in the experimental section in full detail.

The carbon dioxide was then precipitated as BaCO3 and









22
counted as such. The pure chemical materials were

counted by plating on aluminum dishes in very thin layers.

8-Hydroxyquinoline-9-C14 was counted by placing a known

amount in a dish and adding Just enough copper to form

the 2 to 1 copper chelate. It is essential that the

samples used be nonvolatile and the correct self-adsorption

adjustments be made.

The liquid samples were counted according to the

general method of Schwebel, Isbell and Karabinos (21).
The major modification was the use of solvent. They used
pure formamide with less than 10 per cent water while

various solutions of formamide and phosphoric acid and

water were used in this work. The liquid method of count-

ing is superior to the direct solid measurement where

variations in self-adsorption, back-scattering and fore-

scattering, and (in counting on plastic backings) by the

presence of static charges that distort the field. These

complications are not noted in liquid counting because

the environment of the solute is strictly reproducible

and solutions have little tendency to hold static charges.

The drawback to liquid counting lies in the limited number

of solvents that can be used and the low efficiency (1%)
as compared to thin solid samples (50%).

The gaseous measurements were made by first con-
verting the carbon-14 source to carbon dioxide which was











trapped and stored as sodium carbonate. The carbon

dioxide was liberated and allowed to enter an ionization

chamber. The activity of the carbon dioxide thus pro-

duced was measured on a Cary Model 30 Vibrating Reed

Electrometer (a product of Applied Physics Laboratory,

Pasadena, Calif.). The radioactivity determination by

this method is simpler, faster, more accurate and less

expensive than other methods (22, 23, 24, 25, 26). It is

possible to measure as little as 10"12 curies of radio-

activity using the vibrating reed (26). The difficulties

associated with the vibrating reed electrometer are

almost entirely constructional. However, inasmuch as

they are almost always purchased, these are reflected

only In the cost. In the vibrating reed electrometer, the

D.C. Input signal from the ionization chamber filled with

radioactive carbon dioxide is converted to A.C. by using

mechanical energy to move the impressed charge through an

electrostatic field. The A.C. signal thus produced, which

is a measure of the impressed charge, is amplified in a

stable A.C. amplifier and then rectified and used to drive

the recording meter. A portion of the rectified A.C. Is

applied as negative feedback to give very high stability

(27),.


9 -












B* Experimental
Solid Countina Methods
Individual details not included in the general
discussion are given.in this section. Several pro-
cedures which have been found useful in assay of radio-
active carbon Includes 1) a wet oxidation method for
the determination of total carbon and of the specific
activity of carbon in organic materials, and 2) a method
for collecting barium carbonate samples on filter paper

disks for counting.

The combustion fluid of Van Slyke and Folch (20)
and the various reagents were prepared as follows:

Combustion Fluid.

A 500 ml Pyrex Erlenmeyer flask with a glass
stopper is charged with chromic anhydride (12.5 g),
followed by syrupy phosphoric acid (84 ml of specific

gravity 1.7), and fuming sulfuric acid (167 ml of acid

with 20 per cent excess S03). Heat was applied with
occasional gentle agitation and with the stopper off
until the temperature reaches 1500. The flask is cooled
while covered with an inverted beaker. When cool, the
stopper is inserted and the inverted beaker is retained

as added protection from dust. This reagent loses









25

strength on standing. It was tested for Cr03 con-

tent at two-week Intervals as follows: The oxidation

reagent (2,5 ml) was diluted to 25 ml. Three milliliters

of the dilution were placed in a 50 ml Erlenmeyer flask

and water (5 ml) and KI solution (10 ml of a 10 per cent

solution) were added, and the flask was allowed to stand

for five minutes. The free iodine was then titrated with

0.1 N Na2S203. The number of ml of Na2S203 required

divided by 9 gives molar concentration of CrO3 in the

combustion fluid. The Cr03 concentration should be at

least 0.475 N. MJlore Cr03 may be added to make up any

deficiency observed. Upon addition of the extra Cr03, the

solution may be reheated to 1500.

Potassium Iodate.

Reagent grade KIO3 was powdered In a mortar and

stored in a glass stoppered bottle. It was observed on

one occasion that after a month or more in the bottle, the

KI03 failed to dissolve readily in the combustion fluid.

This was corrected by substituting freshly powdered KI03.

Sodium Hydroxide Hydrazine Solution.

A liter of approximately 0.8 N NaOH is made from

carbonate-free saturated NaOH (a saturated solution of

NaOH in a paraffin-lined bottle was allowed to stand

several weeks until the insoluble carbonate settles out)









26

and freshly boiled distilled water. The 0.8N NaOH is

prepared and stored in an aspirator bottle protected

from atmospheric CO2 by a soda-lime absorber* Two grams
of hydrazine sulfate were placed in a second aspirator

bottle, calibrated at 100 ml and protected by a soda-lime
tube. The air above the hydrazine sulfate is replaced

with C02-free gas, and the 0.8N NaOH is drawn in from the

stock bottle to the 100 ml mark. The hydrazine sulfate

is dissolved by agitation, and the resulting reagent is

drawn into a reservoir burette which has been flushed

out with CO2-free gas. The openings of the burette are

protected with soda-lime tubes. This solution loses

hydrazine strength on standing, and should be made up

fresh once a month.

Barium Hydroxide Barium Chloride Solution.

A small portion of a saturated solution of
Ba(OH)2 is standardized against standard HCI. One liter
of 0O3N Ba(OH)2 was then made by appropriate dilution of
a portion of the saturated solution. To 800 ml of the

0.3N Ba(OH)2 was added a BaCl2 solution (160 ml contain-
ing 12 g of BaC12.2H20 per 100 ml). These volumes were

measured in a graduated cylinder. The final solution was

drawn by suction into a bottle previously flushed with
nitrogen gas. The solution was drawn into the burette









27

through a sintered glass filter funnel of "M" porosity

to remove any BaCO3 particles. All openings were pro-

tected with soda-lime tubes.

Methyl Orange Indicator.

A 1 per cent solution of methyl orange in water,

Phenolphthalein Indicator.

A 0,5 per cent solution of phenolphthalein in

95 per cent ethanol.

Standard 0.IN Hydrochloric Acid,

An approximately 0.1N HCI solution was accurately

standardized.

Procedure:

A modification of the method of Van Slyke and

Folch (20), as described by them under the heading "micro

and submicro combustions" was used for the wet oxidations.

In the original procedure, the CO2 formed is pumped into

the Van Slyke chamber and trapped in alkali by repeated

alterations of pressure. In the method described here,

the last portions of 002 are frozen out in the receiver

flask while the generator is being strongly heated.

The sample to be oxidized, which should contain

from 2 to 2.5 mg carbon, and which should be fairly dry

is placed at the bottom of the combustion tube of the

apparatus pictured in Figure 1. The Joint of a receiver

flask is lubricated with stopcock grease and 2 ml of the











NaOH-hydrazine solution are measured into the flask,

using a stream of nitrogen or C02-free air to flush the

flask before and during the addition of the alkali. The

prepared receiver flask is capped and set aside,

Powdered KIO3 (200 mg) was added in a small glass

dish* The dish (30 x 5 x 5 rm) serves as a boiling chip

during the later heating period, The stopcock of the acid

dropper and the glass joint of the combustion are lubri-

cated with syrupy H3P04# The apparatus is assembled

except for the receiver flask.

With the water pump in operation and connected

to the open central stopcock, the receiver flask, previ-

ously filled with alkali, was put in place, Both sides

of the apparatus are evacuated to the limit of a water

pump. The center stopcock was then turned so the two arms

are open to each other but closed to the atmosphere. The

water pump hose is disconnected and the apparatus

supported in such fashion that the receiver flask can be

immersed later in liquid air. Now 2 ml of the combustion

fluid is allowed to run into the tube containing the

sample. The acid cup should be marked at the 2 ml level

to aid in this addition.

A minute or two of heating with a small flame
brings the combustion liquid to boiling, and this










FIGURE 1,























A B

APPARATUS USED IN WET COMBUSTION AND CARBON DIOXIDE

DETERMINATION. Scale: Imm represents 2.5mm in original.

A-Bottom, assembled combustion apparatus; top,

combustion tube with cap. All Joints are 1 24/40.


B-Bottom, assembled apparatus for CO2 collection in.

Ba(OH)2-BaC12; top, flask with cap. All Joints are

- 24/40.









30
boiling Is continued for one to two minutes with some

agitation of the receiver flask. The receiver flask is

then dipped nearly to the glass Joint in liquid air or

a dry ice-acetone mixture and gentle boiling of the
oxidation fluid is continued for three minutes. Finally,

the oxidation mixture is momentarily boiled vigorously,

so that it occupies most of the volume of the oxidation

tube, and the T-bore stopcock is quickly turned so that

the oxidation tube is opened to the water pump while

the receiver flask remains closed. The receiver flask

is allowed to warm to room temperature and finally
heated with warm water and agitated slightly to insure

complete absorption of CO2. Nitrogen gas was allowed to
run into the receiver flask to bring the pressure to

atmospheric, it was capped and set aside for either

precipitation as BaCO3 or liberation of the CO2 for

measurement with the vibrating reed electroeter, The

combustion tube is rinsed with distilled water and set
aside to dry* The rest of the apparatus, unless badly

contaminated with iodine, can be used over again without

cleaning.

It is possible by a modification of the method
of Van Slyke, Mac Fayden, and Hamilton (28) to determine

quantitatively the carbon content of the sample. Samples

of pure benzoic acid gave figures for carbon, by this











acldimetric determination of the total carbon dioxide
produce of 99.4 to 100.3 per cent of the theoretical
values. The original procedure involved the transfer

in vacuo of C02, split by ninhydrin from alpha-amino
acids, to Ba(OH)2-BaCI2 solution. The present method
described provides for introducing acid into a carbonate
solution In the evacuated system. The transfer of CO2
in vacuo to Ba(OH)2-BaCI2 solution is done in the same

way as in the original procedure. This transfer of

CO2 from the NaOH-hydrazine solution serves two purposes
1) acldimetric determination of the total carbon dioxide
produced in the wet oxidation step is possible if desired,
and 2) a pure BaCO3 precipitate Is obtained for counting
purposes. Impure BaCO3 often results from precipitation
of carbonate-hydroxide solutions with BaCl2 (29,30).
This may be guarded against by addition of NHCIl prior
to the BaCI2 (31). Very slight co-precipitation of acid
or alkaline salts occurs in the absorption of gaseous
CO2 by the Van Slyke Ba(OH)2-BaCI2 mixture, as evidenced
by the quantitative results in the back-titration with
acid.
Apparatus (b) in Figure 1 was used for the forma-
tion of the BaCO3 samples. First 2.5 ml of the Ba(O)2-
BaC12 solution was measured into a receiver flask under











a flow of nitrogen gas. A small amount of grease is

used, for particles of grease in the BaCO3 interfere

with filtration onto the counting papers. Second, the
stopcock of the acid dropper is lubricated with syrupy

phosphoric acid and the NaOH-hydrazine solution and the

Ba(OH)2-BaC12 solution was set in place on apparatus (b).
The water pump was put in operation and both arms were

evacuated while the NaOH-hydrazine solution was immersed
in liquid air or dry ice-acetone mixture.

Approximately IN H2SO4. with one drop of methyl
orange, was allowed to drop into the NaOH-hydrazine solu-
tion until the methyl orange Just maintained its acid

color. The acid side was then immersed in boiling water

with agitation. After two minutes the Ba(OH)2-BaCl2
side was immersed in liquid air or dry ice-acetone mixture

for three minutes. The apparatus is then allowed to stand

at room temperature for five minutes or until the ice
melts in the Ba(OH)2-BaCI2 side. This sample may then be

filtered for counting or titrated with standard HC1.

A sample of the Ba(OH)2-BaCl2 solution, measured
out as previously described to exclude atmospheric C02,
was titrated directly with the standard HCI. The differ-

ence between this titration and the titration of the
unknown measures the total BaCO3 through which the









33

radioactive carbon is distributed. A blank run must be
carried through all steps If an exact carbon determina-
tion is desired. It may be necessary to dilute the
NaOH-hydraline solution with "cold" Na2CO3 if the origi-
nal material is of high specific activity.

The collection of BaCO3 and counting of the
sample is of great importance and thus the procedure
will be fully covered, A standard solution of carbon-14
as Na2ClIo3 can be prepared from the BaCl403 as it is
shipped from the Isotopes Division, Atomic Energy
Commission. The specific activity and the isotope ratio
are specified for each shipment* One such shipment is
selected to serve as a standard for comparison and is
diluted with enough C12 so a suitable counting rate is
obtained with a 20-30 mg sample of BaCO3. The most con-
venient method is to obtain a standard Na2C1403 sample
from the National Bureau of Standards and a BaCO3 sample
is prepared as in the method described below. The method
of collection and counting the BaCO3 samples is a slight
modification of a method demonstrated by A, Schwebel at
the National Bureau of Standards. The differences are
in the fact that the quantitative transfer of BaCO3 to the
paper is not attempted, since the total amount of BaCO3
containing the activity to be measured is known when









34

precipitates are prepared by the titration method
described above. Figure 1 and 2 shows the apparatus
used. The stainless steel filter apparatus (a product
of Tracerlab, Inc., Boston, Mass.) was used in combina-
tion with brass mountings. This filter allows the
paper used for collection of the precipitate to be per-
formed and weighed before the BaCO3 is deposited. The
surface area of the paper disk thus formed is 2,895 cm2.

A quantity of BaCO3 suspension containing 20 to 30 mg
BaCO3 is poured in the filter with the water pump on.
The precipitate is washed at least three times with 10 ml
portions of distilled water and finally with two 10 ml
portions of ethanol. The paper is then removed to an
oven at 1100 to dry for one hour, The weight of dried
BaCO3 is obtained by comparing the total weight of the
precipitate plus the paper and the original paper weight.
This gives the fraction of the original total BaCO3
which appears on the paper, and also allows a calculation
of the thickness of the BaCO3 layer, expressed in milli-
grams per square centimeter. The filtering surface area
of the various disks are of equal magnitude. Using
measurements of the disk diameters, the thickness of the
BaCO3 samples in mg per sq cm are calculated, and after














FIGURE 2


BaCO3 FILTRATION APPARATUS




STAINLESS STEEL
FUNNEL












BRASS DISK AND RING


The brass disk is used to mount the

BaCO3 after filtration on preformed paper

in the stainless steel funnel.


Kam ..









36

correcting for background they are translated to counting

rates at "infinite thickness" by means of the BaCO3 self-

absorption curve of Reid et al (32)*

In calculating the specific activity of a BaCO3
precipitate counted in the manner described, the number
of counts per minute observed were first corrected for the
background count, next translated to "infinite thickness"
as described above, then divided by the paper disk area
in sq cm, and finally compared with the number of counts

per minute per sq cm of paper surface given by an
"infinite thickness" of standard BaC14 3.
The carbon-14 standard used was NBS #1* with an

activity of 1280 dps/ml of solution. This sodium carbonate
solution Is the same one used to calibrate the vibrating
reed electrometer.

A second method of solid counting involves direct

plating, of the material either pure or in some biological
substrate, on aluminum dishes. This method does not allow

as quantitative results as the BaC03 method, but by proper
calibration curves to correct for the self-absorption of
the substrate, this method has proved to be satisfactory
for quantitative determinations of biological materials.


*NBS /I is a calibrated Na2CO3 standard from the National
Bureau of Standards.








37
In preparing calibration curves, increasing amounts of

non-radioactive material were added to a series of tubes,
each containing a known constant amount of an aqueous

solution of 8-hydroxyquinollne-9-C14 The contents of

tube were throughly mixed and transferred to aluminum

dishes, dried with an infrared lamp and a hot air blower

on a horizontal turntable. The specific activity of the

8-hydroxyquinollne-9-Cl4 was known so aqueous solutions

were plated with various amounts of substrate. The data

plotted on semi-log scale in Figure 3, indicate that the

addition of very small amounts of substrate to a constant

amount of radioactivity produces an initial decrease in

measured activity. This drop amounts to about 20 per cent

of the original activity determined by plating and count-

ing an aqueous solution of 8-hydroxyquinoline-9-cl4 to
which no substrate has been added. Additional plates
made by the above method, using increasing amounts of

substrate, show a constant decrease in measured activity

consistent with the anticipated absorption curve for

weak beta particles, By using such calibration curves

and interpolating back to the ordinate, better than
90 per cent of the original activity in a system of

In vitro studies with fungi was accounted for.
















FIGURE 3

CALIBRATION CURVES OF CONSTANT AMOUNTS OF
RADIOACTIVITY WITH INCREASING AMOUNTS OF
MINERAL SALT MEDIA


dps

300



200






100
90
80
70
60
50

40

30


20





10


0 1 2 3 4 5 6 7
MILLIGRAMS DRY WEIGHT/CM2


.-cmi









39
Li cuid Counting Method

The usual method of analysis for carbon-14
Involves oxidation of the organic substance and the

collection and precipitation of the CO2 by barium

hydroxide to form BaCO3, In some cases this method is

the only recourse, but it is rather long and difficulties

are encountered in obtaining a uniform sample, A modi-

fication of the method developed by Schwebel, Isbell,

and Karablnos was used in most of this work (21). The

procedure consists of making a solution of the radio-

active material in one milliliter of a suitable solvent.

A suitable solvent would be non-volatile and should have

a relatively high surface tension. This solution is

pipetted into a circular 1 ml cell with an inside

diameter of 37 mm and a depth of 1.1 mm. The cell was

constructed of stainless steel. The cell is used in a
gas-flow proportional counter, Nuclear Measurements

Corporation Model PC-l, The only modification needed

was a cooling system to keep the cell and the sample

cool. By cooling the cell it has been found that up to

10 per cent water can be tolerated without a detectable
change in reproducibility or accuracy. Since the depth

of the liquid for counting purposes is essentially of











"infinite thickness", the counts are proportional to the
carbon-14 concentration of the liquid, It was possible
to calibrate the PC-I proportional counter and determine
its efficiency using a standard solution of D-mannitol-
I-C-14. supplied by the National Bureau of Standards.
This method is not applicable if the activity is very
low since only about one per cent of the total activity
can be determined. It is also important to determine
the effect on efficiency caused by the solvent used in

the analysis. For example, the activity of a sample of
8-hydroxyquinoline-9-C14 was 790 dps/mg in formamide,
505 dps/mg in 90 per cent phosphoric acid, and 600 dps/mg

in an equal mixture of the two solvents. If a solvent
couple such as formamide-phosphoric acid is to be used,

it is possible to determine the efficiencies at several
concentrations and plot concentration vs, activities.

From this graph the intermediate efficiencies can be
determined at any concentration in a manner similar to

that for direct plating. In the analysis of solutions
of 8-hydroxyqulnoline-9-C14, a number of problems arise.
The material counted must be less volatile than the
solvent. 8-Hydroxyquinoline is volatile enough to
contaminate the counter when counted in formamide












solution. For this reason it was necessary to place

0.25 ml of phosphoric acid in each 10 ml sample of

medium taken for analysis. This allowed evaporation

of the water without loss of 8-hydroxyquinoline, now

chemically combined as a non-volatile phosphate. It

was necessary to prepare a control in a medium similar

to the one used in the experiment. The 10 ml sample

taken for analysis was evaporated by blowing a warm air

stream over the solution. When about 0.30 ml remained,

the test tubes were dried further over P205 in a vacuum.

When the volume was 0.25 ml, 1.75 ml of formamide was

added, a one ml volume was placed in the cell and counted.

Generally 5,000 counts were recorded which represents a

one per cent probable error.

The method of analysis was worked out using several

combinations of formamide-phosphoric acid. In Table I the

count of solutions of 8-hydroxyquinoline-9-C in growing

cultures of Asperaillus niger v. Tiegh* at various pH was

made in 95 per cent phosphoric acid.

*
The Asperaillus niger v. Tiegh culture used in this

work was supplied by the Pioneering Research Laboratories

U.S. Army Quartermaster Corps., Philadelphia, Penn.








42
TABLE I
Analysis of
8-hydroxyquinoline-9-C14 in 95. H3P04
(Total Volume 1,0 ml H3PO4)

Control .2i L2 P2
(10-3A 8-hydroxyquinoline-9-Cl4)
71,6 dps/ml 68.5 dps/ml 69 dps/ml 67.5 dps/ml

This method involved pipetting 95 per cent
phosphoric acid, a procedure which was extremely difficult.
In order to facilitate pipetting, 0.75 ml of formamide was
added instead of phosphoric acid and the results in Table
II were obtained.

TABLE II
Analysis of
8-hydroxyquinoline-9-C14 in 251o H3P04
(Total Volume 0.25 ml 95/a H3P04 and 0,75 ml Formamide)
Control .J
(10-3M 8-hydroxyquinoline-9-C14)
95.8 dps/ml 89.8 dps/ml ')0.8 dps/ml 88.8 dps/ml








43
The most critical part of this analysis is proper,
dilution and pipetting so the solutions are of the correct
concentration. The mixture of 0.75 ml of formamide and
0.25 ml of phosphoric acid was still difficult to pipette.
Special test tubes were constructed with a 1 ml bulb at
the bottom. This was done so that after evaporation of the
water from the sample, the 0,25 ml of phosphoric acid
would be in this bulb. Enough formamide was added to
give I ml volume. One more ml of formamide was added
and the contents were well mixed. One ml was withdrawn
and placed in the cell. This method produced a fluid
easy to pipette* The actual efficiency of the counting
was Improved by this method. The following results
obtained are shown in Table III.

TABLE III.
Analysis of
S-hydroxyquinollne-9-C14 in iP H3P04
(Total Volume 0.25 ml H3P04 and 1.75 ml of Formamide)
Control 2H 3 H P H -5 2H7
110 dps/ml 107 dps/ml 106.8 dps/ml 108 dps/ml 101.2 dps/ml

Gaseous Counting Method
The procedure in this method is identical in the
first stages to the BaCO3 method. The Na2C14O3 solution











as prepared in the solid method is placed in a vacuum
line as shown by Calvin (19) and acidified with H2SO4.
The C140o2 is then placed in the evacuated ionization
chamber* Cold CO2 is then flushed into the system by
alternating freezing and thawing of the incoming C02-
Enough CO2 is added to bring the ionization chamber to
a standard pressure, 760 mm for this work. The chamber
is closed, removed from the vacuum line ready for count-

ing. The chamber is connected to the vibrating reed
electrometer and the rate of voltage drift in millivolts
per second is determined, A blank determination of the
chambers with "cold" CO2 only is run to evaluate the back-
ground drift rate. The ionization chambers were calibrated
using NBS # I standard. Using various dilutions of the
Na2C'4o3 the drift rate per second was calibrated in
terms of disintegrations per second. A drift rate of
0,1543 millivolts per second is equal to one disinte-
gration per second. It is possible to convert the ion
current to disintegrations directly if the capacitance of
the electrometer is accurately known. However, this is
not necessary when good Na2C14O3 standards are available
as was the case in this work.










Instruments Used.
An alpha-beta-gamma Porportional Counter, Model

PC-l (Nuclear Measurements Corp., Indianapolis, Indiana,)
was used for the solid and liquid counting. This is a
"P-gas" (90 per cent Argon, 10 per cent methane) windowless
type flow counter. For point sources the geometry is 50
per cent of a solid angle* The other Instrument used was
a Cary Model 30 Vibrating reed electrometer (Applied
Physics Corp., Pasadena, Calif.) with a Brown Electronic
Recorder. The Model 30 was equipped with a No. 3097
turret switch with resistors of 1012, 1010 and 108 ohms.
For the work reported here, the resistors were not used.













IV, STUDIES ON 8-HYDROXYQUINOLINE


A. Mycellal* Uptake
The first condition in determining the manner

in which a fungicide is operating involves a determina-
tion of the actual amounts of the toxicant taken into
the mycelium or spores of a fungus. In an effort to
obtain this information, several series of experiments
were conducted at various hydrogen ion concentration and
at two concentrations of 8-hydroxyquinoline.

The toxicity of 8-hydroxyquinollne toward A. naLer
at various hydrogen ion concentrations increases as the

pH approaches 7. With this in mind the determination of

the amounts of 8-hydroxyquinollne actually taken up by

the fungus at various pH values was determined first.

A salt medium of the following composition was used

0.* g MgSO4 10O g KH2P04 0.5 g KC1 3.0 g KNO3
20.0 g Dextrose 1000 ml water

This medium was selected for its suitability in
allowing macimum growth of A. niger under controlled con-
ditions. Several liquid cultures were inoculated with a
suspension of pre-germinated spores and the volume was

*Mycelia as used throughout sPlid be considered as the
mycellum of the fungus Aserglllua niger.

46












carefully measured In order to maintain uniformity In

cultures. The cultures were shaken at regular intervals

to aerate the medium and allow the fungus to grow below

the surface. After 48 hours growth, the cultures were

adjusted to various pH values, using buffer solutions to

yield the correct hydrogen ion concentration. One ml of

0.1 M 8-hydroxyquinoline-9-CI4 was added to each flask

and to the non-inoculated control. This gave a solution
of 10-3 M or slightly more due to evaporation. The

evaporation, however, was not critical inasmuch as the

control was treated in a similar manner. A 10-3M solution
of 8-hydroxyquinoline counted in phosphoric acid showed

a count of 71.2 dps/ml while the control gave 70.6 dps/ml.

This is good agreement. The cultures were again allowed
to stand with periodic shaking for 24 hours. At this time

30 ml of the flask contents were centrifuged and 10 ml of

the clear mycelia-free liquid were pipetted into a test

tube for analysis as described under the liquid counting
method. Figure 4 shows that the uptake of 10-3 M 8-hydroxy-

quinoline, using two different weights of mucelia, increases

with increased pH. The same series run with 20 g of mycelia

in 100 ml of solution was repeated using mycelia that had











FIGURE 4


AMOUNT OF 8-HYDROXYQUINOLINE TAKEN UP BY A, niqer AT
DIFFERENT VALUES OF pH


( dps dislntegratlons per second)


10 grams of mycelia
20 grams of mycelia


O


O


3 4 5 6
pH AT WHICH MYCELIA WAS GROWN


dPs
ml



110




105




100




95








49
of solution was repeated using mycelia that had been
ground in a Waring blendor. Similar results were obtained.
This indicates that the pH effect does not necessarily
control the ability of the neutral Ion to enter the
fungus mycella but that it could be due to a difference
in reactivity of either the fungal material or the
8-hydroxyquinoline. The specific activity (1,118 dps/mg)
of 8-hydroxyquinoline-9-cl4 was not high enough to go to
lower concentrations so the 8-hydroxyquinoline-2,i4-cc1
prepared from glycerol-l,3-C1l having a specific activity
of 5,110 dps/mg was used in a series of studies run at
concentrations of 10wM.,
Five 20 g portions of A. niger nycelia, grown in
an aereated, shake culture were added respectively to
100 ml of 10"4U 8-hydroxyquinoline-2,4.-C4 at pH of 3, 4,
5, 6 and 7.5. After 24 hours, In which the cultures were
shaken occasionally, the mycelia was filtered and the
resulting solutions analyzed for loss of radioactivity due
to uptake by the mycelia. Figure 5 shows the loss of
8-hydroxyquinoline (10"-4) yj. pH as determined on the
above cultures. This curve is similar to that for
1O3d 8-hydroxyquinoline-9-C14 and the slope is about the
same.












FIGURE 5


AMOUNT OF 8-HYDROXYQUINOLINE TAKEN UP BY A. niger MYCELIA
AT DIFFERENT VALUES OF pH


pH AT WHICH MYCELIA WAS GROWN


dps
ml


3 5 6 7 8











B. Mycelia Fractionation

Once the fact Is established that the

8-hydroxyuinoline actually enters the mycelia, as is

suggested by these data, it becomes necessary to find

the fraction of mycelia into which it Is concentrated,

as for example, fat, protein, or "carbohydrate"

fractions, in order to determine the site of action of

the 8-hydroxyquinoline, a general pattern of fractiona-

tion was used as follows


20 T Mvcelia

S(10"4 8-hydroxyquinolins)


Filtrate
(HO2)

Water wash


(NaOH)-I
(H3P04) NaOH
HP 4(H2SO 4) extract

extract

re1e Red extract
Residue H,SOI.


extract


~








52
The curves in Figures 6, 7 and 8 show the results
of a fractionation of this type on the mycella treated
with 10~M 8-hydroxyquinoline at five different hydrogen
ion concentrations. The mycelia is that used in deter-
mining Figure 5. It should be noted from Figure 6 that
25 per cent of the 8-hydroxyquinoline was removed by water
washing of the mycelia grown at pH 7, while 21 per cent
was washed out from the pH 2 mycelia. This suggests the
greater uptake of 8-hydroxyquinoline at pH 7 may be due
to rather weak bonds. Figure 7 shows a similar trend -

pH 7# smycella giving up the greatest amount of 8-hydroxy-
quinoline to the cold acid extraction. Both 8-hydroxy-
quinoline and its chelates are soluble in this acid solu-
tion. It is interesting to note that upon boiling in
70 per cent H2SO4, Figure 7, no more 8-hydroxyquinoline
could be extracted while the residue, consisting of in-
soluble decomposition products of chitin and proteins,
contained a relatively large amount of radioactivity
with pH 7-.5 mycelia having the lowest specific activity.

The situation as shown in Figure 8 is reversed
from the acid extraction insofar as the relative ratio of
acid extract vs. pH is concerned. Here the greatest amount
of 8-hydroxyqulnoline is extracted at pH 5 by NaOH while
the residue has the greatest activity at pH 7,5. This










FIGURE 6
AMOUNT OF 8-HYDROXYQUINOLINE WASHED OUT OF MYCELIA

























0








2 3 4 5 6 7


pH AT WHICH MYCELIA WAS GROWN











FIGURE 7
PHOSPHORIC ACID EXTRACTS AND RESIDUE
Phosphoric acid extracts

O Residue


dps
mg



0.6









04




0,3




0.2




0o.1 1- I1 I I I L
2 3 4l 5 6 7 8


pH AT WHICH MYCELIA WAS GROWN









FIGURE 8


NaQH EXTRACTS AND RESIDUE OF MYCELIA GROWN AT VARIOUS PH


O Residue from NaOH extract

NaOH extract


)0 L.


pH AT WHICH MYCELIA WAS GROWN


I -~ -r









56
indicates that the 8-hydroxyquinoline is more active
toward the basic extracts of A. niaer mycelia, than
toward the acid extracts. It is also noted from Figure 7
and 8 that around pH 5 the slope of the curves change in
each case.

An important advantage of the radioactive tracer
method is the ability of the experimenter to make a

material balance. A typical radioactive balance of this
series, choosing pH 7 mycelia was as follows:

Radioactive Balance

I. 100 mil 10' 8-hydroxyquinoline-
2,4-C&4 contains 5,260 dps total activity
2. 8-hydroxyquinoline taken up
by mycelia 1,430 dps
3. 8-hydroxyquinoline remaining
in solution 3.830 dps
4. 8-hydroxyqulnoline washed out


H3PO4 extract

H2SO) extract
Residue

NaOH extract
Residue

Total Accounted for

Unaccounted for


0

144
330

-M1
1,228

202
1,430


dps
dps








or 96.1%g


5.

6.

7.
8.

9.
10.

II.











C. Radioactive Spores and Mycelia

In an effort to determine if 8-hydroxyquinoline

or its metabolic products, if any are formed, enter the
growing mycelia and spores, a series of agar plates of

mineral salt medium was adjusted to 10-O' with
8-hydroxyquinoline. This allowed very slow growth after

a prolonged period (4 weeks) and atypical colonies were
formed. Spores were produced which displayed their

characteristic color*. The spores were harvested with-

out touching the medium. They were extracted with hot

H3PO4, and after several hours the black liquid was
diluted with formamide and counted. Only qualitative

results were desired so the efficiency was considered

1,03 per cent since it was counted in 85 per cent form-
amide. The spores were found to contain 117 dps equi-

valent to 0.15 mg of 8-hydroxyquinoline. This evidence
indicates the 8-hydroxyquinoline is able to move through

the mycelia to the spores since at no time were the spores
in direct contact with the medium.



* A. nicer spore color is very sensitive to metals
present. If no copper is present, the spores are white,

turning black in presence of sufficient copper,










Mineral salt medium (100 ml) at a concentration
of 10"3M 8-hydroxyquinoline.-9-CL4 was inoculated with
a few spores. After a long (2 weeks) incubation period,
a single colony grew In atypical raised fashion. About
95 per cent of the mycella and spores were above the
medium. The growth was removed and the mycelia that
had touched the medium was cut off. The mycelia was
washed with water, dried and extracted with H3PO%.
The mixture was diluted with formamide and counted. The
mycelia was found to contain 75.5 dps corresponding to
,097 mg of 8-hydroxyquinoline-9-C14,

D. Spore Uptake

An important consideration in preservation of
material from fungi is the spore. If a fungicide is
active against the spore, the most resistant part, it
will successfully protect the material. A series of
experiments was run in a manner similar to that
described by Miller (33) to determine the amount of
8-hydroxyquinoline taken up by the spores of A. Anier.
Ten ml of 10'3M 8-hydroxyqulnoline-9-C'4were added to
25 and 50 mg of spores. At time Intervals of 5 and 30
minutes and 4 and 30 hours, the mixture was centrifuged











and the 1 ml sample withdrawn for analysis. Figure 9

shows that a large amount of 8-hydroxyquinoline-9-C1~

is rapidly taken up. This uptake is IO5 times more

per unit weight than with the mycelia. A 10.3M solu-

tion of 8-hydroxyquinoline inhibits growth but it would

appear from these data that the spores actually require

larger doses of 8-hydroxyquinoline to prevent their

germination.


I











FIGURE 9


UPTAKE OF 8-HYDROXYQUINOLINE BY A.niger SPORES


0


25 mg of spores

50 mg of spores


TIME IN MINUTES














V. STUDIES ON COPPER-8-QUINOLINOLATE


A, Mycella Uptake
Since copper-8-qulnolinolate is more toxic
than 8-hydroxyqulnoline*, it seems clear that the

true toxicity is due to a copper complex of
8-hydroxyquinoline. According to Albertt work on

bacteria, he suggests that neither the copper-8-

quinolinolate nor the 8-hydroxyquinoline are toxic.

According to his hypothesis the copper-8-qulnolinolate

after entering the cell, breaks down into a charged

complex composed of one copper and one 8-hydroxy-

quinoline molecule. This charged 1ll complex acts

then as the toxic agent. However, if an excess of

copper is added to a solution of 8-hydroxyquinoline,

favoring the formation of such a complex, the solution

shows no great toxicity change. This has been explained

on the basis of the difficulty of charged ions in passing
through a membrane. With this in mind, experiments were
conducted in an effort to determine any difference in
uptake of the two complexes. The previous studies with


* The 8-hydroxyquinoline appears to be only as toxic
as the metals present in the substrate and cells*










8-hydroxyquinoline show that a relatively small amount
of 8-hydroxyquinoline was taken up on a unit weight basis.
It was, however, much larger for the spores than for
mycelia. A series of experiments were conducted with
A. nicer to determine the uptake of various 8-hydroxy-
quinoline-copper ratios by the mycella, If one considers
the complex equilibria involved between copper and
8-hydroxyquinoline, it is possible to write an equilibrium
expression as follows:
Cu4+ + HQ CuQ+ + H* (log k = 12,0) (1)
CuQ+ + HQ Q2Cu + H+ (log k = 11.0) (2)
or
Cu+ + 2HQ Q2Cu + 2H+ (K = 1023) (3)

These formation constants are based on studies
in aqueous solutions (34). Irving and Williams (35),
Meller and Maley (36) and recently Johnston and Freiser (37)
have reported various values from log 12 to 15 for aqueous-
dioxane solutions. From this equilibrium it is obvious
that copper is present in extremely minute amounts and
the formation of copper-8-quinolinolate is greatly
favored. -It Is also clear that this equilibria will be
very sensitive to the pH. This has recently been shown
to be the case (38). At low pH the equilibrium can be
written as follows:
Cu*+ + 2H2Q+ Q2Cu + 4H+










It should be possible to shift the equilibrium
in the desired direction by using an excess of either
copper or 8-hydroxyquinoline. In the uptake experiments
described, a constant amount of 8-hydroxyquinoline was
used and copper varied from a deficiency, favoring

Q2Cu, to an excess, favoring CuQ+.
Equal amounts of mycelia were added to a series
of 10 ml samples of 103M 8-hydroxyquinoline-9-C14.
To each of these flasks different amounts of copper
were added. The copper aliquots were 2, 3, ., 10, 12,
15, and 30 ml of 103M copper acetate. The pH of the
solution was 5. The resulting range of concentrations
would give solutions from 0 to 99 per cent free
8-hydroxyquinoline. The CuQ+/CuQ2 ratio varies, depend-
ing on the equilibria. At the end of twenty-four hours,
the mycelia was filtered and placed in 10 ml of
petroleum ether. Figure 10 shows the uptake of radio-
active material as observed from this experiment. The
maximum shown here corresponds to the highest con-
centration of the 2:1 complex. It should be noted that
the uptake of pure 8-hydroxyquinoline corresponds to
the previous experiments for 8-hydroxyquinoline at this
particular pH.












FIGURE 10


RADIOACTIVE MATERIAL TAKEN UP BY MYCELIA IN SOLUTIONS OF


VARIOUS COPPER- 8-HYDROXYQUINOLINE RATIOS
(Amount Remaining After Removal of Mycella)


MILLILITERS OF 0.001 MOLAR COPPER ACETATE









65

The treated mycelia was extrated with petroleum
ether in which only 8-hydroxyquinoline is soluble.

Figure 11 shows the amount of 8-hydroxyquinoline removed

and this is what one would expect from the uptake curve.

As the copper becomes excessive the equilibrium for the

formation of 8-hydroxyquinoline is unfavorable. This

is borne out by Figure 11. The mycelia was then

extracted with a 50 per cent ethanol solution and the

extracts analyzed to yield the information shown in

Figure 12, The amounts extracted represent about 30 per

cent of the total radioactive material.

These results indicate that CuQ2 is taken up

in larger amounts than CuQ+ or 8-hydroxyqulnollne.

There is about 90 per cent difference in the uptake of

CuQ2 and pure 8-hydroxyquinoline. They also show, as

did previous studies with 8-hydroxyquinollne, that the

greatest amount can also be extracted at the maximum

point of uptake. It appears that uptake could be a

function of lipid solubility, inasmuch as CuQ2 is more

lipid soluble than 8-hydroxyquinoline at the pH used

in this experiment. This further implies that the

material is entering the cell or at least the lipid

layers of the mycella.


__~











FIGURE 11
RADIOACTIVITY OF PETROLEUM ETHER (30-400) EXTRACTS OF MYCELIA

GROWN AT VARIOUS COPPER-8-HYDROXYQUINOLINE RATIOS


10 15 20 25

MILLILITERS OFO.001 MOLAR COPPER ACETATE













FIGURE 12

ALCOHOL EXTRACTS OF MYCELIUM GROWN AT VARIOUS

COPPER-8-HYDROXYQUINOLINE RATIOS


5 10 15 20

MILLILITERS OF 0.001 MOLAR COPPER ACETATE


. .A











The uptake of the species presumed to be
8-hydroxyquinoline, CuQ+, and CuQ2 by A. niaer mycella
as has been described was repeated, using a very large
excess of copper Inasmuch as an Increase in copper
should insure a more favorable equilibrium for the
formation of CuQ*+ It has been suggested by Albert,
that due to its lipid solubility, only the CuQ2 is
able to enter the cell. In an attempt to establish
this here, a large excess of copper was used so that
some CuQ+ would be present and the uptake should be
smaller. In the experiments carried out the uptake
results have not agreed with predictions. In four
separate experiments the average uptake of 8-hydroxy-
quinoline, CuQ+ favored, and CuQ2 favored solutions
was found to be 25 per cent, 85 per cent and 9$ per
cent respectively. In the light of the small amount
of CuQ that would be formed, the difference between
the CuQ4 favored and CuQ2 favored solutions would be

a qualitative measure of the CuQ+ formed, It is not
valid to consider the uptake of CuQ+ as 85 per cent,
actually only the difference (10 per cent) between
CuQ2 and CuQ+ is significant. This difference may be
due to the formation of CuQ+.











The method used for uptake determination was

to place equal amounts of 48 hour A. nicer mycelia

grown In shake cultures In synthetic medium (Czapekis

sucrose, nitrate solution) in a series of flasks con-

taining various cupric chloride-8-hydroxyquinoline

concentration ratios buffered at pH 5. The mycelia

was noted to take up the color of the chelate at once.

The color of the nycelia varied as the concentration

of the respective complexes, becoming greener as pure

CuQ2 was approached, and then changing in color with

increasing copper concentration. These flasks were

then placed on the shaker for 24 hours, after this time

the mycelia was filtered and the filtrate evaporated

and counted in formamide solution. The difference

between the initial count and that for the filtrate

was taken as mycelial uptake. The percentage uptake

given above are based on initial concentration of

8-hydroxyquinoline, and are found for experiments

using 10 g (wet weight) of nycelia in 10"3OM 8-hydroxy-

quinoline solutions.

This high uptake of that copper-8-hydroxy-

quinoline concentration considered to be CuQx is not

in agreement with the idea that charged particles are










deterred from crossing the lipid membrane considered
present in mycelial cells. There are several possible
explanations for the results. First, the membrane may
possess a negative charge, and surface attraction by
electrostatic charges may account for the uptake. Second,
the membrane may simply extract the CuQ2 present, and
shift the equilibrium toward the formation of more CuQ2.
The third and most probable reason for this high uptake
is due to the fact that only a very small amount of
CuQ* is formed, the major constituent being CuQ2, as
suggested in the previous paragraph.
The uptake of CuQ2 has been determined for 8.
niMer on a wet and dry weight basis. Washed mycelia of
A. niger (50 grams wet or 1.4. grams dry) was shaken in
a 10"'AM CuC12 solution after the addition of 8-hydroxy-
quinoline (5 x 10"4M CuQ2 mixture of 136 dps/ml for a
total activity of 0.73/Ac). After the mycelia had been
filtered, the used medium had an activity of 39.2 dps/ml
or a total activity of 0.19/4c. This corresponds to a
75 per cent uptake or 0.38 ,A.c/gram dry weight. This
represents 1.55 mg CuQ2/gram dry weight or a 0.15 per
cent pickup,











B. Mycelia Fractionation
The mycelia grown in the uptake experiments was
fractionated to determine the distribution of the
copper-8-quinolinolate in the material. The fractiona-
tion of mycelia was carried out by growing a relatively
large amount of mycelia and treating it with 5 x l0'W"
solutions of copper-8-qulnollnolate-9-C41 at pH 4*5.

The mycelia was fractionated and attempts were made to
Identify the radioactive materials. The mycelia extracts
from uptake experiments were chromatographed to identify
the species present. Whatman No. I filter paper (one
inch strips) were used with a series of developing
solvents ranging from butanol-saturated water to
chloroform-ethylene chloride mixtures. Butanol-saturated
water was found to give the best Rf value (ca. 0.52) for
8-hydroxyquinoline. Free 8-hydroxyquinoline was found
in the petroleum ether extracts. It has recently been
shown by Hutchison (38) that under certain conditions,
appreciable amounts of the total copper-8-qulnolinolate
in a system of water-chloroform, can be found as free
8-hydroxyquinoline.











After filtration, the mycelia was extracted
(Soxhlet) for 48-hours with 30-400 petroleum ether.
8-hydroxyquinollne Is readily soluble in petroleum

either while copper-8-quinolinolate is quite insoluble
(continuous extraction of 17.5 mg of the pure chelate
for 72 hours removed less than 3 mg). The petroleum
ether extract was a mixture of fatty and crystalline
material. Pure crystalline 8-hydroxyquinoline was
isolated from this fraction. Further fractlonation
with alcohol, water, and acid was carried out, and
each. fraction was counted in formamide solution, or
as solid material. Since activity was found in all

the fractions of this mycelia, only a general picture
could be obtained from this approach. Fractionation

by extraction with different solvents allows only

partial fractionation in a very complex system such

as the mycella of fungi. The fractlonation and
determination of the site of the radioactive 8-hydroxy-

quinollne and Its copper complexes was carried out by
the following scheme of separation:












Mycelia grown in CuQ2


L


Petrol
Ethe.


Extracts, free 8-hydroxyquinoline,
some fats, waxes, and lipides.
Very little CuQ2 extracted. (229)*


Extracts CuQ I some fat$, and
other slightly polar compounds.
(61%)

Many proteins, fats, acids,
lipides, pigment, simple sugars
and CuQ2. (l-4)


Low molecular weight sugars
non-lipides aminoacids, and
coeftzymes. ~l%)


High molecular weight sugars
starches and polyhydric com-
pounds.


Starches, polymers, and gums


eum (30-40)
r


Acetone)



(Alcohol)



(Cold Water)



(Hot Water)


. -


(2%)


Hydrolyzed
Chitin


(Dilute HC1)


(70% H2SO4)


Residue


*The percentages are based on the total radioactivity


-of the mycelia.










Further work was carried out on the fractiona-
tion of mycelia previously treated with copper-8-
quinolinolate. In an effort to determine If this
chelate was undergoing a breakdown, mycelia of A. nlier
was treated with sublethal concentrations and the
resulting mycella fractionated. The various fractions
were assayed for carbon-14. The experiment yielded
interesting information on the distribution of carbon-14
within the mycelia. Actively growing nycelia (net
weight 125 g) was treated with 8-hydroxyquinoline-2,4-CI4
(8 mg) with a specific activity of 0.1375/Ac/mg. The
total activity was lll/ .c, To the mycelia, 5,5 ml of
Q12M CuSCh was added to form the copper-8-quinollnolats.
The mycelia was allowed to remain in this solution
for 24 hours with constant shaking. A small portion of
mycelia was removed and plated on agar. It grew slowly
at first, and finally at a normal rate,
The mycelia was filtered and the filtrate counted
to determine the uptake. The results are as follows












Initial activity 1.10/fc

Filtrate activity 0.82/4c

Activity in mycelia 0.28/,c = 10,400 dps

Weight of dried mycella ll.506l g

Uptake = 2.43 x 10-2 c/g

The mycelia was fractionated by extraction with

various solvents as follows:


Acetone


.. (0.281c M 100%)

Ether

j _15. 820*

Lipides, fats, waxes,
higher alcohols, free
fatty acids, Phopholi-
pides, and sterols
2q. I0W


- a ~


Ethanol


I
Lipides, some fats, and
other polar compounds
13.4o0


Hot H2O



HCl(1-10)


Many protein acids, lipides,
pigments, simple sugars
6.29g%

High and low molecular weight
sugars, non-lipides, amino acids,
polyhydric compounds

12,10%


I
Starches, gums and polymers

Residue 27.105


*Per cent of total activity in mycelia.


|


III











76




in41
O)-4. C o .o o
oo 0 co
41w4< s
440 ,>







t 10 U n rs N C .


04 4 "4 a U
4 1 14
'0 > C co 0 w
n "4 A >0




Q. *- 4)s"4 Wc "4 O M 0 0)
t 1 "" 4M
U>N, 1 m v %0



0 0





cU m X(\ IA fn 0
c% 0 0 0
4 ) "4 CD (7 0 0%




0


Cm fn 04 c) o% ",
to 0 (V "4"4 ( 0 1A 0
< a ft *4 ot 4 a








u "4 014A
406S 0 "4 V $ "4
4"4 4 4 0 Q

I4 4 0 C)
0 s
v? pi- *r












C. Spore Uptake

A parallel series of experiments with the

mycelia uptake were conducted using spores in place

of mycella. The spores were added to solutions of

various copper-8-hydroxyquinoline concentrations and

after a 24. hour period, they were filtered and the

solid spores and the filtrate were analyzed for

carbon-14.. The results were similar to the previous

ones using mycelia, except that more free 8-hydroxy-

quinoline was taken up by the spore than by the

mycelia. This agrees with previous studies on free

8-hydroxyquinoline.

A second series of experiments on uptake

studies of spores were carried out to get a comparison

with the mycella. The spores were suspended In the

solution of toxic materials for 24 hours. At this

time they were centrifuged and the liquid, free from

spores, was evaporated and counted. The following

results were obtained:


~__~ 1_1


_1












TABLE V

A. NIGER SPORE UPTAKE


Ratio of
8-hydroxy-
quinoline
to copper


Uptake (&Lc/gm)


Concentration


55 x 10o2 $c/gm


138 x 10o2 )c/gm

97 x 10-o2c/gm


5 ml 8-hydroxyquinoline-
2,4-C14 (10-3M)

* + 2.5 ml IO'3M CuSO4

" + 5 ml lO^M CuSO4


The spores were washed with ethanol, acetone,
ether, and water, but they retained activity as shown

below.

TABLE VI

RESIDUAL ACTIVITY IN SPORES


Ratio of
8-hydroxy-
quinoline to
copper


Concentration


Specific
activity


5 ml IO-3M 8-hydroxy-
quinoline

+ 2.5 ml 10-3M
C+ 5 ml SO4
+ 5 ml 101M CuSO4


7.1 x 102lO c/gm

11.9 x l102c/gm


34. x 1oi-2c c/gm


It0


2:1

1:10


I:0

2:1


I:I0


_ ____


_~__









79
As It has previously been noted the spores
take up relatively large amounts of copper-8-quino-
linolate and 8-hydroxyquinoline compared to mycella.
A new method for study of spore uptake and removal of
toxicant has been worked out.

A series of experiments have been run to
determine the uptake and ease of removal of 8-hydroxy-
quinollne with various concentrations of copper.
A. niLer spores were placed in 50-ml tubes filled with
the solutions shown in Table VII.


TABLE VII
A. NIER SPORE UPTAKE

8-hydroxy- .
Number Spores (mg) quinoline-9-C4(rmM) CuCl2(mM)

1 41.2 .01 0,
2 32.8 .01 .005

3 37.1 .01 .01

4 42.3 .01 .1

The spores were allowed to stand 24 hours at
300C. The spores were suspended in the solution and
filtered onto paper disks of exactly 2.895 cm2. The
disks were dried and counted. The spores were then
treated, on the paper disks, with ethanol (10 ml of










95 per cent) after which they were dried and recounted,
This was done again using H20(20 ml). Table VIII gives
the tabulated results from this experiment,

TABLE VI II
RESIDUAL ACTIVITY IN SPORES

Specific Activity after Activity al
No. Spores (mg) Activity Ethanol wash water wasl


14.0 dps/mg

32.4 dps/mg
10.5 dps/mg

14.l7 dps/mg


4.4 dps/mg
12.,2 dps/mg
10 .4 dps/mg

14,5 dps/mg


3,9 dps/mg
9.4 dps/mg
8.4 dps/mg
12.8 dps/mg


It is interesting to note the large amount of
carbon-14 activity remaining in 3 and 4 after both treat-
ments. This indicates either the entrance of the
material Into the spores, or a very tight bonding to the
spores. The per cent of carbon-14 activity remaining on

the spores, after treatment with water and ethanol, was
28 per cent, 81 per cent, and 87 per cent, respectively,

for numbers 1, 2, 3 and 4, from the above Table VIIIl
This method of study will allow extensive studies to be

carried out with various types of spores and chemical
agents.


5.8

4.9
5.6
6.4


Peter
h











D. Apparent Breakdown of the Chelate

In an effort to check the possibility of a de-
composition to CO2 of either 8-hydroxyquinoline or its
copper complexes$ an experiment was carried out so all
gaseous products from the growth of A, nDtre nmycella
would be collected.
The apparatus was a flask fitted with a ground
glass Joint, allowing for the entrance of CO2 into a
KOH scrubber bottle.
Actively growing freshly washed A. niaer

mycelia was first placed in three flasks, as described
above. 8-Hydroxyquinoline-9-C14 (10 ml of 10o3M) with
a total activity of 0,028".c was added to all three.
Copper chloride (10 ml of 5 x 10"3 M and 10 ml of 10"3M)
was added to the other two in amounts to give exactly
enough and an excess copper for the formation of CuQ2,
Carbon dioxide free air was prepared by passing it
through 2N KOH. The CO2 produced by the mycella was
collected in 0.IN NaOH. Table IX shows the amount of
CO2 produced, expressed as Na2CO3. The Na2CO3 was
determined by the method of Willard and Furman (40).










TABLE IX
RADIOACTIVE CARBONATE PRODUCED
Ml 10"3M
8-hydroxy- Ml l10M Mg of Na2CO3
Number quinoline CuCl2 produced

1 10 ml 0 3650 mg
2 10 aOl 5s ml 4500 mg

3 10 MI 10 mi 4250 mg

For determination of carbon-14 activity, one-
ml samples were precipitated with a Ba(OH)2-BaCI2
mixture. These, however, gave no Indication of any
activity. For cases of very low activity, the vibrating
reed electrometer is essential. Samples (1 ml) of the
NaOH-Na2CO3 mixture were treated with acid to liberate
the CO 2 which was collected in ionization chambers and
the drift rate was determined. The background drift
rate Is 0.29 milliveolts/second, and 0.1543 millivolts/
second over the background drift is equal to 1 dps.
Table X shows the results of the three samples of OO2.
The amount of activity is small, but it is almost
twice the drift background. Under the experimental con-
ditions used, the activity can be contributed only to
C1402.- Since the activity Is so low, further work was
done in which 8-hydroxyquinoline-2,4-C14 was used.










TABLE X
VIBRATING REED DATA
(1 dp = 0.1543 mv/sec)

Total Activity
Number Drift Rate Background Drift of C0C

1 0.44 mv/sec 0,290 mv/sec 100 dps
2 0.485 mv/sec 0.290 mv/sec 127 dps
3 0.527 mv/sec 0.290 mv/sec 154 dps

A second experiment to determine the decomposition
of 8-hydroxyquinoline and Its copper complexes was
carried out, as described above. Vigorously growing
(48 hours) A. niger mycelia was placed in 250 ml flasks
fitted with a ground glass Joint, allowing for the
entrance of CO2 free air and exit of the 002 containing
air into a NaOH (100 ml of 0.8N) scrubber solution.
Table XI shows the concentrations of 8-hydroxyquinoline
and copper used and the amounts of CO2 produced by the
culture. For determination of the carbon-14 present,
one-ml samples of scrubber solution were treated with
acid to liberate the CO2. The dried 002 was collected
In ionization chambers, and the rate of drift in mv/sec
was determined. The background rate (after cleaning
insulation) was 0.139 mv/sec. One dps is equal to a
rate of 0.1543 mv/sec over the background.











TABLE Xl
VIBRATING REED DATA

1


Number


3


mM 8-hydroxy- 11
quinoline-2,4-C 4 .01 .005 .01

mM CuSO4 0. .0025 .1
Total activity 5,291 dps 2,645 dps 5,921 dj

Ml of scrubber 100 ml 100 ml 100 ma

Milligrams of
Na2CO3 produced 1,741 mg 2,210 mg 2,110 mi

(Specific activity of 8-hydroxyquinoline-2,4-C14 -
5,291 dpa/mg)


Ir


Table XII shows the results from the three samples
as determined by the vibrating reed.

TABLE XII


VIBRATING REED DATA
(1 dps = 0.1543 mv/sec)

Drift Rate Background Drift

1.354 mv/sec 0.139
0.849 mv/sec 0.139
0.889 mv/sec 0.139


Total Activity

786 dps

460 dps

486 dps


No.

1

2

3


~I_~~ ~ ~~~~~~~ ~~ _I~~


3










The amounts of activity were greater than those
found in previous work (4 to 7 times). The high activity
noted for No. I might be explained on the basis of the
growth after the addition of the toxic compounds. The
vigorous growth was continued in No. I while Nos. 2 and
3 were inhibited and finally killed by the mixture used.
Heavy spore formation was observed in the pure 8-hydroxy-
quinoline culture (No. 1). The over-all higher activity
was expected due to the higher 8-hydroxyquinoline-2,4-C14
(5,291 dps/mg) activity as compared to 8-hydroxyquinoline-
2,4-C14 (1014 dps/mg). These results are more signifi-
cant than the ones previously described, for the drift
rate was 8 to 10 times the background. It seems clear
from these experiments that 8-hydroxyquinoline is under-
going some type of decomposition to CO2.















VI. SOLUBILITY BEHAVIOR OF 8-HYDROXYQUINOLINE AND
COPPER-8-QUINOL INOLATE

A. Water-Olive Oil Distribution

To further the uptake and equilibrium studies the
distribution of CuQ2 and 8-hydroxyquinoline between water

and olive oil was studied. The distribution of a dis-

solved substance between two immiscible liquids may be

treated according to the laws of mass action for

reversible physicochemical reactions as follows;

(C1) CuQ2 (dissolved in water)- CuQ2 (dissolved in olive
oil) (C2)


CuQ2 Olive Oil (C2)
Where K a r
CuQ2 Water (CI)


, is true if the active


masses are proportional to concentrations. In an initial

experiment using 8-hydroxyqulnoline-9-C14 in mixture with

copper-8-quinolinolate-9-C14 at different ratios, the

following results were obtained for the water-olive oil

system:


Number 1
(At Equilibrium)
0.013 Mmoles CuQ2
0,004 mMoles
8-hydroxyquinolIne
CuQ2; 8-hydroxy-
quinoline ratio
4 :t


Number 2
(At Equilibrium)

0,003 mMoles CuQ2
0.024 mMoles
8-hydroxyquinol ne
CuQ21 8-hydroxy-
quinoline ratio
1 t 8











Using activity (Carbon-14) as effective concentration,

K = 395.2 dps/17.3 dps K = 399 dps/12.8 dps
K = 23 K = 31

These results agree with the uptake experiments.

They show that free 8-hydroxyquinoline must have a

fairly favorable K inasmuch as the Number 2 value is

eight units greater than Number 1 and the ratio of

CuQ2 is lower. It should be pointed out that the use

of C2/Cl K may be invalid in some cases, for the actual

stoichiometric distribution ratio will depend on the

degrees of dissociation or association of the CuQ2 in

the two liquid phases (39). In these calculations it

is assumed that the CuQ2 will remain essentially as a

single neutral molecule in both water and olive oil*

In order to determine the effect of copper on

this equilibrium by other experimental evidence, solu-

tions of various copper-8-hydroxyquinoline ratios were

mixed, allowed to precipitate and the precipitate was

dried and weighed. From these experiments the follow-

ing data were obtained. The solutions were buffered

at pH 4.


~~_~I











Ratio of copper to
8-hydroxyquinoline Milligrams of Precipitate

Series I
1:1 20.2 mg

1:2 25.2 mg
Series 2
2tl 7.1 mg

Il1 7,4 mg

lt2 8.3 mog

This information indicates that increasing cupric

ion concentration lowers the amount of precipitate formed,

Whether this lowering is due to formation of CuQ+ or to

difference in solubilities of chelates at the various

salt concentrations must be determined by other methods.










B. Water-Carbon Tetrachloride Distribution
An experiment has been carried out to show the
formation of a copper-8-hydroxyquinoline complex that
is soluble in water. A water carbon tetrachloride
system was used due to its similarity to the lipid-
aqueous system of living cells. Carbon tetrachloride
(10 ml), 8-hydroxyquinoline-9-CI4 (10 ml of lO'3m),
and CuCI2 (various concentrations) were mixed in
separatory funfels and allowed to stand for 24 hours
at 200C Table XIII shows the concentration of the
various mixtures. The total carbon-llj. activity of the
10 ml of 8-hydroxyquinoline was 0.028 /Lc. The aqueous
solution was buffered at pH 4-

TABLE XIII
CARBON TETRACHLORIDE DISTRIBUTION DATA
Millimoles UMllimoles
Number 8-hydroxyquinoline CuCl2 Ml of CCli

1 0.01 0 10
2 0.01 0.005 10

3 0.01 0.01 10
4. 0.01 0.1 10
5 0.01 1.0 10
6 0.01 10.0 10

After standing, 2 ml samples of both the CC'4
and water layer were removed, placed in stainless steel










dishes, evaporated and counted. Table XIV shows the
observed activities of carbon-14 in the two phases.
TABLE XIV
DISTRIBUTION COUNTING DATA
CC, layer Water layer Total Per cent
No. dp/mll .dps/ml activity accounted for

1 13.4 42.5 989 97.*
2 17.3 11.3 399 *39.3
(396) solid (98.0)
3 19.7 18.5 567 *56.0
(442) solid (99.5)
4 17.7 41.4 1006 99.3
5 9.2 46.0 1012 99.8
6 4.9 47.6 1003 99.1

*In Nos. 2 and 3 the solubility of the CuQ2 was exceeded
and it precipitated to form a solid phase between the CC!4-
H0O interface. This was collected and the activity
determined, as the bracketed values show. The results are
included, inasmuch as the difference in solubility in the
two phases was the major Interest.
Figures 13 and 14 show the change in solubility
with increased copper concentration. From these graphs
It is clear that great excesses of copper shift the CuQ2+
Cu 2CuQ+ equilibrium to the right. It is clear also
from these data that considerable amounts of 8-hydroxy-
quinoline-copper complex are present in both aqueous










FIGURE 13


L.
0
LO

;-


;7-


cc 0

0 Z
co
. -

0 0
0 U

u
N
O u



0











0
SC
U U


1c-



U>
- H










c co


Disintegrations per second











FIGURE 14




00








o o
- w

0 tn







o 0
0 U
a; 0




uo

o n o4








El l0






0 1
0.. cl
























< 0^










)0 0 0 0 0
IA-t E N -
S ^ ^'-1
3:: *- J
'-4^ -
tiJ ^-























IA 01 ^(j.


r-












and non-aqueous systems. At the peak for the non-aqueous
system, 20 per cent of the complex is in the CC14 layer,
10 per cent in the water layer, and 69 per cent as solid,
The water system, however, at its peak, possesses 94 per
cent of the complex, while the CC14 system holds only 4.8
per cent. This shows that the CuQ+ complex can occur
In higher concentrations in an aqueous phase than CuQ2 can
in a lipid phase. This may be an important force in the
living cell equilibria.









94
C. Water-Octadecanol Distribution
In an effort to conduct an experiment that repre-
sents, as nearly as possible, the probable situation at
a cell membrane, octadecanol and water were used to
furnish the proper type of interface. Octadecanol
(10 ml), 8-hydroxyquinoline-2,4-C14 (1.45 mg), and
CuSO4 (various amounts) were mixed In separatory funnels
and allowed to stand for seven days at 30C. Table XVI
shows the concentration of the various mixtures used.
The specific activity of the 8-hydroxyquinoline was
5,291 dps/mg.

TABLE XVI
OCTADECANOL DISTRIBUTION DATA
mM 8-hydroxy- Ml
Number quinoline mM CuSO Ml H20 Octadecanol

1 10-2 0, 10 10
-2
2 102 .005 10 10

3 10-2 .01 10 10

4 10o2 .5 10 o10
5 o"2 5.0 10 10

The mixtures shown in Table XVI were mixed and
shaken at various time intervals for seven days. After

two days of standing, the water layer was drawn off and
two samples from each mixture were placed in aluminum