- A TREATISE -

Protection Fore Branch No. 93

Copy No.:

Date: November 1970

Forest Research Institute,

Protection Fore Branch,

RANGIORA. 1 ..

"I have yet to see any problem, however

complicated, which, when. you looked at

it the right way, did not become more

complicated.,"

Paul Alderson 2 ..

CONTENTS

PAGE PREFACE 3-5

INTRODUCTION 6

CHAPTER ONE 7-20 SODiill1 MONOFLUOROACETATE - COMPOUND 1080

CHAPTER TWO 21-37 DERIVATIVES OF SODIUM FLUOROACETATE

CHAPTER THREE 38-45 SYSTEMIC TOXICITY AND TRANSLOCATION

CHAPTER FOUR 46-48 REPRODUCTION INHIBITORS AND CHEMOSTERILANTS

CHAPTER FIVE 49-53 ANALYTICAL PRCOEDURES

REFERENCES 54-57

POSTSCRIPT PREFACE

Toxicology, the study of toxins and poisons, is today a multi­ disciplinary system of knowledge which acquires and inter-relates information from many branches of science.

Within the sphere of Protection Forestry, toxicology is concerned not only with biochemical and physiological derangements, but also with their ecological responses in animal and plant communities. Thus, it represents an important aspect of biochemical ecology.

To those acquainted with the problems of wildlife control under the present policy of Protection Forestry it has long been obvious that, in any one of the diverse control programmes, much additional information and benefit could be obtained from a more precise definition and a clearer recognition of the toxicological aspects that form the basis of

the majority of these programmes.

Thus, the responsibilities of a toxicologist confronted with these

problems are many-fold. Not only is he required to select, from the

background of his in the various disciplines, a specific

approach to a specific problem 9 but also, he must be able to inculcate

in other investigators an understanding, or at least an appreciation, of

the reasons for this specificity. In others words, what is required is

that a symbiosis be developed between the several disciplines from which

mutual benefits will acc~e not only the participants, but also at

national and scientific levels. This attitude is not entirely utopian

since an analogous attitude has long been adopted in forestry policy.

The dictum of HMultiple Use" applies also in this instance, with exactly

similar parameters of hopes, ambitions and disappointments. The days

of the one-man research teams are over, and the boundaries between the

several scientific and management disciplines are now almost of historical

The scientific effectiveness of a research team therefore stands or falls with the ability of its individual members to pursue their specific interests and to collate, consultate, collaborate, communicate and compromise. Much can come out of such an effort if it stands, there is little we can do about it if it falls; but does the taxpayer have to subsidise it?

As for my own responsibilities in this research effort it seems important to attempt a cohesive account of the major toxicological applications of toxic fluorine compounds and their consequences in a field that, as yet, lacks structure and form as it also lacks

This situation, so it seems to me, has persisted not so much from choices made as from choices neglected 9 not from malign intention but from failure to take into account the full scope of available alternatives.

After several vaccilations about the purpose and organisation of

this treatise I have rejected the presumption that an account of the

fundamental metabolic events brought about by sodium fluoroacetate in

current animal control programmes would be of little interest to those investigators primarily concerned with population s or pre- and

, or to those who have a share in the process of

in Protection Forestry policy.

I have the treatise as my means of communication since its

form absolves me of several strictures which are required in review or

monograph. It can dispense with exhaustive literature citations since

a good deal of its tone and colour is often derived from casual observa-

tions or personal information. Also a treatise can inc polemical

statements, not intended to confuse, but to portray a state of confusion

in which contending views compete for as if they concerned a

finality rather than a trend. Taken together in the context of biologi-

cal control measures these views suggest a ture of toxicological

marked less success than is normally assumed. This picture

may be somewhat distorted both by hindsight, with its focus on past error, and by inclination 9 for there are few accounts of toxicological management 9 and fewer still that meet standards of scholarship.

My of for this treatise is then the recognition of the need to acquaint puritans, pedestrians and pundits with an outline of the metabolic events which they bring about in their animal control programmes. Our viewpoint will however soon shift closer to the down to more pragmatic considerations of the scope of whilst

the concerns population dynamics and the quality of life within an animal The emphasis becomes more practical, less theoretical. Thus, this treatise to convey the concept of plurality in Protection Forestry. Of course, this term is no more

than another, but we are here merely stating facts, not accounting for them. Nonetheless, the tone of this treatise is one of conciliation, rather than arbitration, between theoretical and practical

, between basic research and field applications. In this manner it may be hoped to abolish the blank wall, or rather the revolving door, of through which both pass without catching up with one other.

tion Forestry Branch

Forest & Range Experiment Station

Rangiora

November 1970 INTRODUCTION

In One, some basic chemical and cal pre s are con- sidered to convey the re of the carbon-fluoride bond of fluoroacetate to the bond in the three halogenated acetates. The toxin-host interactions are then evaluated with to the pharmacodynamic phenomena of fluoroacetate in several animal

The biochemical reactions from which these arise will be examined in some detail because of their role in the of basic and biochemical in the animale Other of tate are also examined in view of their lar ficance to field programmes and medical

Chapter Two discusses the selective toxici of derivatives of fluoroacetate and evaluates the between chemical structure and toxicity. The closes with the enumeration of some toxic fluorine compounds that have al

parti in noxious animal control programmes.

In Three, relationships are evaluated with the aid of some facets of and the effects of translo- cated toxins.

The considerations of on inhibitors ( offer

scope for c are, , of sufficient merit to fy attention. At the least, are a valuable adjunct to control measures in situations where eradication or change of habitat are ticable.

Five is confined to a brief de of

This also conveys an undi sed for facilities to initiate and continue the research proposals described in this treatise. CHAPTER ONE

SODiill'1 MONOFLUOROACETATE - COMPOUND

The introduction in 1945 the U.S. Fish and Wildlife of sodium fluoroacetate (also as 1080, or "ten-

(1)) initiated an extensive of its al biochemical and cal properties fore this however fluoroacetate was recognised as an disease in several

South African states where some ts, si fluoro- acetate in their leaves and fruits, were to stock and natives

Sodium fluoroacetate is to man most warm- blooded animals (2, from the inherent hazard of baits and water, the carcases of deceased animals very to predators,

particularly These facts warrant the need f utmost caution and

fore in the use of this Nevertheless, de its extreme

hazard, the features mark sodium fluoroacetate as being an

in ontrol: high toxici , excelle ace e,

relatively quick action, absence o jectionable taste and

chemical stabili non-volatili , no dermal toxicity or

on under normal safe conditions, tolerance after stion

of sub-lethal amounts. Its one defect (under and

on di its extreme solubili in wate • Hence, its

from baits is a continuous set-back almost every bai or

Fluoroacetic acid, , was first in 1896 ts (

Some of it cal es were rec ded, but no mention was made f 8 .. its In 1 , fluroacetic acid was as a

( Some years after Swarts' Polish workers in p echnic were, the 1935-1939, involved in researches into the preparation and s of fluoro- acetates (6). Their of the toxic effects of fluoroacetate

from their work on iodoacetates and their (

es .. When a bomb me iodoacetate s, a

e cloud results (due to liberation of free iodine) which s easy

to In a search for related c without this un- desirable fluoroacetate was and assessed for

ac ...... 1 be d to the eye of a rabbit. No lacrimation was observed, but the rabbit died This led to more matic studies of fluoroacetates.

A method of involves the reaction of

with fluoride at

\250, 0 C ) under pressuree

The resultant me tate can readily be to sodium fluoroacetate aqueous sodium other different methods of s have been , but the reaction in fluoroacetates is of course the introduction of the fluorine. Once this has been achieved, the stability of the carbon-fluoride link allows a wide varie of common c reactions without loss of fluorine.

fluoroacetates. In 1 , the commercial manufacture of sodium fluoro- acetate was initiated by the Monsanto Chemical , Ste Louis, U.S.A.

(7), but now many other manufacturers the in similar form and similar processes.

11 108011 is a and somewhat both flour and sugar. It has no smell or taste, is soluble in water, but relati insoluble in c solvents .. Most manufacturers supply the material (containing a special metal containers with double fric closurers. The c serves as a means of even distribution in the prepara- tion of bait, as a repellent to , as a bait , and as a bait marker in field

The s in Table 1 present the s of the fluorine atom in fluoroacetic acid with the data for acetic acid and the acetic (8,10). of these

will reveal several intere observations:

(i) The carbon-fluoride bond is both shorter and than the

other bonds.

(ii) The toxicities of the three non-fluorine halogenated acetic acids

are of the order ICH COOH > BrCH COOH > ClCH COOH. 2 2 2 Their toxicities are thus onal to the electron

of the atom.

(iii) The atomic radii of fluorine and are sufficiently

alike to result in a ty of the

radicals ..

e, fluoroacetic acid, FCH COOH, can mimic acetic acid, 2 CH COOH in its biochemical with fatal consequences, 3 1 as will be seen later.

Because of the nature of the cal of the carbon- fluoride bond, as outlined above, the fluoroacetates behave very much like the unsubstituted acetates. This is in direct contrast to the

0 0

..i. ..i.

.. ..

orall orall

7 7

(11,12) (11,12)

63 63

100 100

1 1

salt salt

LD50 LD50

Na Na

of of

( (

90 90

74 74

14 14

.. ..

(1 (1

2.12 2.12

1. 1.

1 1

1.45 1.45

1. 1.

x-c x-c

X X

-

(13) (13)

2.8 2.8

2.5 2.5

4.o 4.o

of of

3.0 3.0

OF OF

1.8 1.8

138 138

155 155

217 217

Constant Constant

of of

TOXICITIES TOXICITIES

.5 .5

.5 .5

.3 .3

13) 13)

AND AND

x-c x-c

( (

1 1

Bond Bond

of of

PROPERTIES PROPERTIES

64 64

Radius Radius

(1 (1

1.,33 1.,33

1.14 1.14

0.99 0.99

0 0

0 0

x x

PHYSICAL PHYSICAL

Atomic Atomic

I I

acid acid

acid acid

acid acid

acid acid

acid acid

fluoroacetic fluoroacetic

bromoacetic bromoacetic

iodoacetic iodoacetic

chloroacetic chloroacetic

acetic acetic

COOH COOH

2 CH 11.

acetates. In fact, from the bond es of the various acetates (Table 1) it is obvious that the C - F bond is considerably

than even the unsubstituted bondo This makes the C F - bond an stable one to resist chemical rupture.

As a consequence, most chemical reactions that can be on the unsubstituted acids behave well on the fluoro-substituted acids. Thus, all the common esters, s, , ketones, salts, amides, and acid halides can be Rather than discuss their we will later examine in more detail the bio- chemical breakdown of a number of these derivatives.

An excellent review (10) deals with the pharmacodynamic actions of the monofluorinated c of which fluoroacetic acid is the

of an extensive series of s and To earlier inve these exhibited a range of reactions in species of animals. Because of their unpredictable (at that time) toxicities and peculiar al responses much work has centred on the elucidation of the actions of fluoroacetate and its derivatives in different species of animals 1943 the main

features structure to function had been re sed.

etates are readi absorbed all common routes of admini-

stration such as oral, ous, ocular, injection (subcutaneous,

oneal, intramuscular, and intravenous) and by inhalation

Thus, it is not well appreciated persons involved in son

programmes that fluoroacetates can be absorbed the naked skin,

and that even solid fluoroacetates can be effective by inhalation as

dusts because of the efficient on the oral and nasal 12.

With regard to reactions caused by the c action of fluoroacetates all can be observed in one or more of the following animals.

~ hour after a lethal dose of fluoroacetate (0.5 mg/kg) the first effect noted is a weakness of neck and front and a decrease in activi This state may progress to a marked but

remains moderate until the occurrence of a sudden violent clonic convulsion. sive relaxation takes e, a few short breaths and death.

the heart immediately after death, the auricles will be found and the ventricles ventricular fibrillation.,

Within hours after a lethal dose of fluoroacetate (about 0.1

the first effect noted is a few minutes of

"Absence" (non-rec tion of human presence) actions of presence

of hallucinations, and a tonic spasm followed qui

movements. Tonic spasms and may alternate

or c cease, and the may appear normal at times.

Ul however, the anoxi assaults the re

centre convulsion will result in The

heart is ften markedly slowed convulsive seizures, but

ceases activi until some time after has ceased. Death

is the result f the effe ts f repeated and on-

vulsions on the centre, and never cardiac in

one two hours after a lethal dose of fluoroacetate

(approx. 2 the animal may vomit and becomes and seclusive few minutes later, actions of audi halluci- nations are followed immediately by Twi of the facial muscles, unilateral, heralds the onset of the convulsive seizure, which is form a It to involve the

and the masseter muscles. of the convulsive ac over the rest of the is then very in a jerking convulsion in which the , violent jerks may occur at a rate of three per second. Tonic c s are seen, but do not

the as do in the dog. The animal is unconscious this but, as the seizure passes off, it will

to its feet and ultimately does so about i hour after the onset of the attack. The animal appears ssed for some

time but often recovers entire from the convulsion* c

second seizure is seldom seen. , the animal becomes weaker

the next few hours, but is often or otherwise itself when stricken by ventricular fibrillation and death. s ous recovery from ventricular fibrillation in the is

uncommon.

Within one or two hours after a lethal dose of fluoroacetate

(5 mg/kg) , associated with to

external stimuli, preceeds convulsions of a tonic nature. Death is the

result of re ssion which occurs after

convulsive ac has decreased ceased. rats

oc develop ventricular fibrillation.

Attention has also been directed (10) to the for

herbivorous animals to manifest cardiac effects, and for carnivores to

central nervous convulsions or ssion. In omnivorous

es both the heart and CNS are affected

vertebrates are insensitive to fluoroacetates. It has been 14 ..

(10A) that fish are unaffected fluoroacetate dissolved in the water in which swim, yet fleas are killed on soned rats, and most insects are very sensitive to fluoroacetates

(see systemic toxicity, 3) ..

These comparisons are instructive since the reactions exhibited by the animal can be in the various noxious animals in New Zealand. Thus, rabbits, hares, possums, deer and all exhibit (with minor modifications) the cardiac, ventricular fibrillation, response. Man's response is similar to that of with mixed cardiac-CNS response.

Table 2 indicates the e of toxi of fluoroacetate in several species of animals (10), and ses the variabi of response.

TOXICITY OF FLUOROACETATE

LD , mg/kg* s 50

Dog 0 .. 06

Cat 0.2

Guinea

t

Horse 1 .. 0

Man 2-10 (should be with considerable reservation) Rat 5 Mouse 7

150

* The term denotes the dose, in of vOmpound per of animal, to kill of a group of animals. A low value therefore a toxicity .. It is clear that the pharmacological phenomena must have their in the reaction of susceptible tissues, at cellular levels, to the toxic insult by fluoroacetate.

1948-1957, many exhaustive ons have unravelled the pathways fluoroacetate involves the into a reactive response from which it is me difficult to recover.

Much of this research has been reviewed by the Sir Rudolf A. Peters (9).

these biochemical elucidations is the extreme fluoro- acetate one of the metabolic cycles concerned with energy and in the normal of the celL In

order to convey the ficance of this on it is to obtain some of this basic metabolic c

which is called 11 the , or "Citric Acid II (which refers to the acid in the cycle), or ttKrebs

Cycle" (in honour of its discoverer, Sir Hans Krebs)o

(39)

The TCA represents the terminal for the oxidati of major nutrients and energy stores in many sms. In addition, the c an role in the sis of many cell constituents from precursors. cal the most cursor is acetate which, derived from acids and other nutrient sources and catabolic reactions, is metabolised the ( Fig. 1)

(i) tivation0

etate is "activated" reaction of the essary co-factors

adenosine and into e

Coenzyme A, adenosine and c Mg++ Acetate + CoA + ATP < Ace + AMP + PP. > i 16.

CH COOH (as CH COS-CoA) 3 3 acetic

CHCOOH oxalo- II CHCOOH CCOOH acetic II cis-aconitic (enol form) C(OH)COOH CHI 2C\R

CH(OH)COOH I CHCOOH iso­ r CH COOH ci tric 2 oxalo­ yH COOH acetic 2 COCOOH -2H j COCOOH -2H f CHCOOH oxalo- 1 CH COOH succinic 2

-co2 yH2COOH I malic CH(OH)COOH COCOOH I ?H 2 o<'. -ketoglutaric CH COOH 2

CHCOOH fumaric II CHCOOH rH2COOH CH COOH 2 succinic

Fig. 1. The normal tricarboxylic acid cycle. The specific enzymes responsible for the formation of the specific acids are not included in this simplified schematic representation. 17 ..

(ii) into the TCA " The ace eacts with the enol form of oxalo-acetic acid to

e citric acid. The reaction is the

enzyme 11 e

(iii) Oxidation in the TCA

The citric acid is se under the action of the various

fie enzymes (not indicated in 1) to dioxide, water

and oxalo-acetic acid., I addition energy is released. Since

oxalo-acetic acid is available for the oxidation of acetate

to carbon dioxide and the essential process

in each individual cell Interference with thi process must

result in gross cal ti to

death.

( 9)

The pronounced simi between the and radicles were out in Table 1. From this it can be inferred that acetic acid fluoroacetic acid bear a close resemblance to each , , other in both their ea_,_ ana. As a

consequence, fluoroacetate can also activation and inc

into the TCA be followed the formation of fluorocitric acid

(as fluoroacetic acid

)H COOH CHCOOH 2 C ( COOH C ( COOH " I acid oxalo-acetic FCHCOOH acid (enol 18 ..

So far so good, but now instead of being metabolised further into cis-aconitic acid (cf. Fig. 1) by the action of the specific enzyme aconitase, the fluorocitric acid itself acts as a potent competitive inhibitor of aconitase. Thus, the inhibition of aconitase results in the blockage of the TCA cycle. This has far-reaching consequences:

(i) The energy supplied by the cycle is gradually reduced to the point

where permeability of cell walls is destroyed, cellular function

becomes impaired, and death ensues.

(ii) The large build-up of citric acid is thus a symptom of the poisoning,

rather than the cause of death.

(iii) The TCA cycle also influences the glycolytic breakdown of diverse

sugars such as glucose, glycogen, lactose, etc •• Because of their

involvement the enzymic blockage of the citric acid also increases

the tissue levels of glycolytic products. Since this situation

resembles insulin diabetes it is often referred to as "fluoroacetate

diabetes".

(iv) The course of fluoroacetate poisoning can be followed by recording

the changes in the tissue/blood levels of citric acid, glucose,

lactose, and the complexing cations Ca++ and Mg++.

(v) Fluoroacetate itself is obviously not toxic, but it is converted to

the metabolite fluorocitric acid which is toxic. This phenomenon

has been called a "lethal synthesis" (9).

(vi) The wide variation in response to fluoroacetate in different species

may be associated with differences in the activating mechanisms

necessary for the formation of fluorocitrate.

(vii) Since the enzyme aconitase is inhibited, and the non-formation of

cis-aconitic acid interrupts the TCA cycle, it seems conceivable that

administration of these compounds to victims of accidental fluoro-

acetate poisoning may be of therapeutic benefit. (See also Chapter 3) Degradation of fluoroacetates in the field

In field applications of 11 1080" it has always been accepted that leaching of the toxin from bait is the only manner in which baits are rendered non-toxic. This attitude has also prevailed in ignoring the

subsequent fate of the toxin in the soil surrounding the bait. It is

apparent that little or no consideration has been given to degradative

changes which augment the physical disappearance of 11 108011 • Two

types of degradation will be considered here:

Chemical degradation

The extraordinary stability of the carbon-fluorine bond has led to

the impression that derivatives of fluoroacetic acid are also very stable.

This is not quite true. Sodium fluoroacetate is a highly hygroscopic

salt and aqueous solutions of the salt or its esters decrease in toxicity

in time. Chenoweth (10) reported that methyl fluoroacetate solution

decreased in toxicity even though refrigerated, while at room temperature

the process was accelerated. Thus, the hydrolysis of the methyl ester

and the salt is rapid, with a half-life of less than one hour at pH 7 (17).

No fluoride ion is released under these conditions, but toxicity studies

(18) have indicated the instability of sodium fluoroacetate in water and

saline solution with a significant progressive loss of fluorine. It

has been suggested (10) that deterioration takes place according to the

reaction

with the formation of sodium bicarbonate and the liberation of the highly

volatile, relatively non-toxic methyl fluoride.

In animal control programmes involving 11 108011 it is common practice

to prepare stock solutions of this compound in advance of actual utilisa-

tion. In many instances these solutions are stored in containers over

considerable periods of time. In view of the degradative changes the

actual concentration of toxin in baits may be considerably less than its

calculated concentration. In addition, storage of these stock solutions 20 .. must be considered hazardous because prevention of escape of the volatile methyl fluoride may build up pressure in the container.

Bacterial degradation

Certain strains of soil micro-organisms have the ability to utilise

fluoroacetate as their sole source of carbon. In particular Pseudomonas

luteochromogens (19) and other pseudomonads (20) are capable of defluorin­

ating fluoroacetates by means of an intra-cellular enzyme which catalyses

the conversion of fluoroacetate to glycolate and fluoride ions according

to the reaction > This is an important reaction since pseudomonads, and possibly other micro­

organisms, can be regarded as active scavengers of fluoroacetate residues

in soil and deteriorating baits.

The fate of fluoroacetate residues in soil and surface water has not

been investigated in an authoritative manner. On the contrary, interest

in its fate has lapsed following its exit (leaching) from baits. This

negligence demands rectification, not only for obvious practical reasons,

but also for its wider medical implications, namely, a possible antidote

for fluoroacetate poisoning.

It is clear that certain pseudomonad micro-organisms possess a

natural or inducible enzyme system (fluoroacetate fluorohydrolase) specific

to fluoroacetates, which can be isolated and purified by established micro-

biological and biochemical techniques. It is conceivable that this enzyme

preparation could have a beneficial effect on the progress of the intoxi­

cation process in victims of accidental fluoroacetate poisoning.

For purely medical reasons therefore this aspect of bacterial fluoro­

acetate degradation is worthy of aggressive investigations since no

effective antidote for fluoroacetate poisoning has yet been devised. 21 ..

CHAPTER TWO

DERIVATIVES OF SODIUM FLUOROACETATE

A large number of analogues and homologues have been synthesised from the simple parent compound, and their chemical, physical and pharmaco­ logical properties determined and recorded (8).

In general, most fluoro-derivatives have the chemical and physical characteristics of their non-fluorinated counterparts. The majority are colourless liquids, their viscocities increase (and their solubilities in water decrease) with increasing molecular complexity so that their physical appearance changes from liquid--....;>• semi-solid---;>"' solid. Deviations from this general trend occur. When the necessary specialised facilities are available it is envisaged that many of these compounds will be evalu­ ated as substitutes of sodium fluoroacetate in dose-effect experiments under controlled laboratory and field conditions. Thus, the situation will arise where a specific toxin can be applied in a specific manner against a specific animal species within a specific plant community under specific climatic conditions. This prediction is entirely feasible and by no means unrealistic. Even in its most restricted sense, it affords a pronounced improvement on current practice ..

Selective toxicity phenomena

The toxicities of fluoro-derivatives to susceptible species depend on several physiological and biochemical factors:

(i) Transport across the cell membrane.

(ii) The presence and ability of hydrolytic and oxidising (detoxicating)

enzymes to degrade the compound into fluoroacetic acid which then

can enter the TCA cycle.

(iii) The efficiency of the hepatic and renal excretory mechanisms. 22.

In their ecological context other factors should be taken into account concerning the palatability of these compounds to their host.

Each one or a combination of these, and other, factors determines the interaction of the toxin and its host. For instance, a water- insoluble compound may be more easily transported across the lipoprotein membrane of the cellular wall into the intra-cellular compartment

(mitochondria) by virtue of its lipid (fat) solubility. This property augments its ready access to intra-cellular enzyme systems responsible for its breakdown to fluoroacetate. On the other hand, the combined processes of cellular transport and enzymic catabolism may be less efficient than the excretory abilities of the liver and kidney by which substances are eliminated via bile and urine. Thus, a compound could be highly toxic if only its cellular transport and enzymic conversion were more rapid than its excretion. This combination of biophysical, biochemical and physiological alternatives may account for differences of toxic action of a compound between different species. In fact, even within a species the range of toxicities encountered may reflect the physiological state

(age, sex, nutrition) of the individual host. Thus, one of the most outstanding features of the toxicology of the fluoroacetates (and deriva­ tives) is the extraordinary wide variation in response between different species of hosts in regard to both sensitivity and also to symptoms.

The essential features of these responses have been described previously; it is important to point out again that these responses follow a long and essentially irreducible latent period after the acceptance of the poison.

As early as 1943 the main features correlating structure and toxicity had been recognised. These may be summarised in the form of a general rule:

Any compound which can form fluoroacetic acid (more correctly

fluoroacetyl-coenzyme A) by some biochemical process is toxic. 23.

The corollary is equally significant in that compounds which cannot form

fluoroacetic acid are usually non-toxic. The rule and corollary may be

used to advantage in predicting the toxicity of new or unknown compounds.

It is interesting to examine this type of structure-toxicity relationship

with respect to the carboxylic acid- and alcohol derivatives of fluoro­

acetate.

In Table 3 are listed some successive members of the homologous

series of fluoroacetic acid (~- fluorocarboxylic acids) and their counter­

parts of the analogues series of fluoroacetate ((»- fluoroalcohols)*.

The toxicities of these series of carboxylic acids and alcohols are

illustrated in Fig. 2, which shows that a high Ln represents a low 50 toxicity and vice versa. It is also apparent that there is a consistent

alternation of toxicity which depends on the total number of carbon atoms

in the chain. This phenomenon can be summarised in the form of a second

general rule:

If the total number of carbon atoms in the chain is ~'

the compound is toxic, if the total number is odd, the compound

is non-toxic.

The corollary from the combination of the first and second rule is

obvious. Compounds with an even number of carbon atoms in the chain are

metabolised to fluoroacetate in the host, unlike those with an odd number

of carbon atoms.

We will now examine the reasons why this is so.

P-oxidation

The metabolic events that lead from the fluoro-derivative to the

formation of fluoroacetate occur via the enzymic process of -oxidation.

For instance, carboxylic acids (also named fatty acids) are degraded by

In chemical nomenclature,(;.> denotes that the fluorine position is on

the terminal carbon atom in the chain. 24 ..

TABLE 3

Total Carboxylic acid Alcohol number of Formula Name Formula Name carbon F(CH )n0H atoms F(CH2)nCOOH 2 in chain

2 FCH COOH fluoroacetic F(CH ) 0H 2-fluoroethanol 2 2 2 F(CH ) COOH 3-fluoropropionic F(CH ) 0H 3-fluoropropanol 3 2 2 2 3 4 F(CH ) COOH 4-fluorobutyric F(CH ) 0H 4-fluorobutanol 2 3 2 4 F(CH ) COOH 4-fluorovaleric F(CH) 0H 5-fluoropentanol 5 2 4 5 6 F(CH ) COOH 6-fluorohexanoic F(CH ) 0H 6-fluorohexanol 2 5 2 6 F(CH ) COOH 7-fluoroheptanoic F(CH ) 0H 7-fluoroheptanol 7 2 6 2 7 8 F(CH ) COOH 8-fluoro-octanoic F(CH ) 0H 8-fluoro-octanol 2 7 2 8 F(CH ) COOH 9-fluorononanoic F(CH ) 0H 9-fluorononanol 9 2 8 2 9 10 F(CH ) COOH 10-fluorodecanoic F(CH ) oH ~0-fluorodecanol 2 9 2 10 11 F(CH ) COOH 11-fluoroundecanoic F(CH ) oH 11-fluoroundecanol 2 10 2 11 12 F(CH ) COOH 12-fluorodoceanoic F(CH ) oH 12-fluorododecanol 2 11 2 12 18 F(CH ) coOH 18-fluoro-octadecanoic F(CH ) oH ~8-fluoro-octadecanol 2 17 2 18 (fluorostearic)

the successive removal of two-carbon fragments from the chain. Essentially,

this involves oxidation of the;.?-carbon atom followed by hydrolytic chain

cleavage. Thus, for example, hexanoic acid would be degraded into the

following fragments: 1i E>tr /Jo<. CH cH CH CH CH COOH > CH CH CH CO.CH COOH 3 2 2 2 2 3 2 2 2 ':> CH cH CH COOH + CH COOH 3 2 2 3 A further oxidation and hydrolysis follows > CH co.cH COOH 3 2 :> CH COOH + CH COOH 3 3 25.

J 100

60

LD Mice 50 mg./kg. 40

20

1 Total number of Carbon atoms in the chain

I w -Fluorocarbpx ylic acids F (CH2 )n COOH El w -Fluoroalcohols F(CH2) 1 ~0H

Fig. 2 Toxicities of f luorocarboxyl i c acids and f luoroalcohols 26 ..

Thus, one molecule of hexanoic acid is broken down into three molecules of acetic acid.

If this process of /f/-oxidation be applied to the M-fluorocarboxylic acids, a clear-cut explanation is evident for the pronounced alternation of toxicity described above. For example, 6-fluorohexanoic acid is degraded in a similar manner as hexanoic acid.

FCH cH cH cH CH COOH ~~~~- FCH CH CH CO.CH COOH 3 2 2 2 2 2 2 2 2 :> FCH CH CH COOH + CH COOH 2 2 2 3 FCH CO.CH COOH > 2 2 FCH COOH + CH COOH > 2 3 So, 6-fluorohexanoic acid (even-numbered carbon atoms) is degraded into

two molecules of acetic acid and one molecule of fluoroacetic acid which now exerts its toxicity on the host.

Alternatively, 5-fluorovaleric acid (odd-numbered carbon atoms) is

degraded in a similar manner

~ FCH CH CO.CH COOH 2 2 2 ">- FCH CH COOH + CH COOH 2 2 3 but now the resulting fragment, 3-fluoropropionic acid cannot be further

degraded, hence its parent compound is non-toxic. With regard to the

fluoroalcohols in Table 3 and Fig. 2 similar criteria apply after the

initial oxidation of the alcohol into the corresponding acid.

Detoxication mechanisms

The degradation of foreign compounds in the body is brought about

by numerous enzymes, at least one for each process, and they accomplish

oxidations, reductions, hydrolyses, etc •• These enzymes are not very

discriminate about the detailed structure of their substrate, hence they

are capable of attacking many toxic compounds not previously encountered.

The site of these "detoxicating" enzymes is in the microsomes of the livercell, their action, broadly speaking, results in the chemical conversion of the foreign molecule into one that is more hydrophilic and thus more easily excreted. In their attack on foreign substances these enzymes sometimes actually increase toxicity (28). With respect to toxic fluorine compounds it is important to point out that degradation to fluoroacetate is an essential prerequisite step before lethal synthesis, fluoroacetate into fluorocitrate, can take place. The metabolic degradative steps of fluorine compounds are similar to those of their non-fluorinated counterparts because of the physical similarity between the fluorine and hydrogen atom in the molecule.

That the fluorine is masquerading for hydrogen is not noticed by

the gullible undiscriminating detoxicating enzymes. But, when fluorine

enters the tricarboxylic acid cycle as fluoroacetate its presence is noticed however by the highly specific enzyme aconitase.

Tables of derivatives

In the tables (pages 29-37) a number of toxic fluorine compounds

have been listed which can be considered as possible candidates for

toxicological surveys in wildlife control. Their physical properties

are summarised as follows: S: solid; L: liquid; LL: low-boiling

liquid (100-120°C); HL: high-boiling liquid (100-120°C); W: soluble

in water; 0: soluble in organic solvents; MP: melting point ( 0 c).

The LD figures are those for mice in mg/kg; 3.0: very toxic; 50 3-25: toxic. The relative toxicities of the compounds to other species

can be deduced, with caution, by reference to Table 2.

Despite the diversity of their chemical structures and physical

properties these compounds all share one fundamental characteristic;

their ultimate fate is their enzymic degradation to fluoroacetate by the

cellular detoxicating mechanisms. For each group of fluorine compounds

these metabolic pathways leading to fluoroacetate have been outlined (8,29). 28.

Applications

The wide range of toxicities and physical properties of these toxic aliphatic fluorine compounds provides ample scope for selective approaches to specific problems in the control or management of noxious animals.

As such, investigations on their practical application will more than fulfill the recommendations for agricultural pest control, as outlined by the Technical Advisory Committee (Animal) in its 1970 Report to the

Minister of Agriculture.

In the wider contexts of Protection and Production Forestry we are concerned with a number of different but related problems, the complete solution of any one of which should provide at least a partial solution to the others. For instance, inclusion of fluorothiocyanates or sulphoryl chlorides may be questioned on account of their repulsive odours. But it is just this repellent property which, apart from their inherent animal toxicity, suggests these compounds as eminently suitable candidates

for erosion plantings or nursery stands where the emphasis is primarily

on plant protection. Selection depends on emphasis. Thus, fluoro­

carboxylate ethyl esters, because of their attractive fruity odours,

contribute to palatability of baits. Their high toxicity and resist-

ance to leaching can be accepted as bonus features. Also, because of

their fungicidal activities, many fluoro-derivatives can prevent deterior­

ation of artificial baits by inhibition of mold growth.

The synthesis of these fluorine compounds has been extensively

documented. Because of their relatively simple chemical structures

it is envisaged that the commercial manufacture of a suitable selection

of these compounds can be readily accomplished by appropriate chemical

manufacturing companies. 29.

FLUOROACETATES

Class Compound Structure LD50 Physical Characteristic

~Cid Fluoroacetic acid FCH COOH 6-10 SW 2 iAldehyde Fluoroacetaldehyde FCH COH 6 LL 2 Sodium fluoroacetate FCH C00Na 6-10 SW Salt 2 Triethyl-lead fluoroacetate FCH COOPbEt 15 swo 2 3 !Ester Methyl fluoroacetate FCH COOCH 6-10 LLWO 2 3 Ethyl fluoroacetate FCH cooc H 6-10 LLO 2 2 5 n-Prophyl fluoro- acetate FCH COOCH cH cH 6-10 HLO 2 2 2 3 Isopropyl fluoro- acetate FCH COOCH(CH ) 6-10 HLO 2 3 2 Allyl fluoroacetate FCH COOCH CH=CH 6 HLO 2 2 2 2-Fluoroethyl fluoro- acetate FCH COOCH CH F HLO 2 2 2 3-5 Phenyl fluoroacetate FCH cooc H 6-10 so 2 6 5 Fluoroacetyl salicylic acid FCH cooc H COOH SW 2 6 4 15 Methyl fluorothiol acetate FCH COSCH 6-10 2 3 HLO foul Ethyl fluorothiol } smellin1 acetate FCH COSC H 6-10 HLO oils 2 2 5 Methylene bis-fluoro- acetate (FCH C00) cH 10 so 2 2 2 Ethylene glycol (FCH COOCH ) bis-fluoroacetate 2 2 2 2 HLO Fluoroacetamide FCH CONH 6-10 SW !Amide 2 2 n-Methylfluoro acetamide FCH CONHCH 6-10 SW 2 3 n-Nitroso-n-methyl fluoroacetamide FCH CON(NO)CH 6-10 SW 2 3 n-2-Chloroethyl fluoroacetamide FCH CONHCH CH Cl so 2 2 2 15 30.

G.>-FLUOROCARBOXYLIC ACIDS AND DERIVATIVES

Compound Structure LD50 Physical characteristic

GV-Fluorocarboxylic acids

Fluoroacetic acid FCH COOH 6.6 SW 2 4-Fluorobutyric acid F(CH ) COOH 0.65 HLO 2 3 6-Fluorohexanoic acid F(CH ) COOH 1.35 HLO 2 5 8-Fluoro-octanoic acid F(CH ) COOH o.64 so 2 7 acid F(CH ) COOH so 10-Fluorodecanoic 2 9 1.5 12-Fluorododecanoic acid F(CH ) COOH 1.25 so 2 11 18-Fluorostearic acid F(CH ) cooH so 2 17 5.7

UJ -Fluorocarboxylate alkyl esters

Methyl 4-fluorobutyrate F(CH ) coocH 0.7 HLO 2 3 3 Methyl 6-fluorohexanoate F(CH ) coocH 1.6 HLO 2 5 3 Ethyl 6-fluorohexanoate F(CH ) cooc H 4 HLO 2 5 2 5 Ethyl 8-fluoro-octanoate F(CH ) cooc H 1. 75 HLO 2 7 2 5 Ethyl 10-fluorodecanoate F(CH ) cooc H 1.65 HLO 2 9 2 5 Ethyl 16-fluoro- hexadecanoate F(CH ) cooc H so 2 15 2 5 7 Methyl 18-fluorostearate F(CH ) coOCH 18 so 2 17 3

UJ -Fluorocarboxylate 2-fluoroethyl esters

2-Fluoroethyl 6-fluoro- hexanoate F(CH ) COOCH cH F 2.5 HLO 2 5 2 2 2-Fluoroethyl 8-fluoro- pleasant octanoate F(CH ) coocH CH F HLO 2 2 7 ' fruity 2 7 smell 2-Fluoroethyl 10-fluoro- decanoate F(CH ) coocH CH F 10 HLO' 2 9 2 2

Acid derivatives

6-Fluorohexanoamide F(CH ) CONH 0.7 so 2 5 2 31 ..

Compound Structure LD50 Physical Characteristic

Unsaturated acids and esters

4-fluorocrotonate FCH CH=CHCOOC H 1.25 HLO Ethyl 2 2 5 12-Fluorododec-2-enoic acid F(CH ) CH=CHCOOH 1.55 so 2 9

Ketoacids and esters

!Ethyl W-fluoroaceto- FCH COCH cooc H 2.5 HLO acetate 2 2 2 5 Ethyl 8-fluoro-3-oxo octanoate F(CH ) cocH COOC H 1.3 HLO 2 5 2 2 5 !Ethyl 12-fluoro-3-oxo dodecanoate F(CH ) cocH cooc H 1.9 so 2 9 2 2 5 32.

'4> -FLUOROALCOHOLS

Oxidation of the alcohols into carboxylic acids is followed by their p-oxidation

3lii F(CH )n_ COOH ~ FCH COOH F(CH2)nOH 2 1 2

Compound Structure LD50 Physical Characteristic

FCH CH 0H 10 LL 2-Fluoroethanol 2 2 HLWO 4-Fluorobutanol F(CH) 4oH 0.9 F(CH ) 0H 1 .. 2 HLO 6-Fluorohexanol 2 6 F(CH ) 0H o .. 6 HLO 8-Fluoro-octanol 2 8 F(CH ) 0H 1.0 HLO 10-Fluorodecanol 2 10 F(CH ) oH 1.5 HLO 12-Fluorododecanol 2 12 18-Fluoro-octanol F(CH ) oH 4.o so 2 18 0 MP 6o c

ESTERS OF W -FLUOROALCOHOLS

Hydrolysis of the esters into alcohols precedes oxidation into carboxylic acids and ,&-oxidation.

Compound Structure LD50 Physical Characteristic

2-Fluoroethyl acetate F(CH ) ococH 18 .. 6 HLO 2 2 3 4-Fluorobutyl acetate F(CH ) ococH 0.9 HLO 2 4 3 4-Fluorobutyl benzoate F(CH ) ococ H 1 .. 7 HLO 2 4 6 5 4-Fluorobutyl nitrate F(CH ) 0N0 0.95 HLO 2 4 3 8-Fluoro-octyl acetate F(CH ) ococH 1.3 HLO 2 8 3 W -FLUOROALDEHYDES

Oxidation into carboxylic acids is similar to that of alcohols. Fluoroaldehydes are unstable, undergoing ready polymerisation. Preparation of their 2,4-dinitrophenyl­ hydrazone derivatives (29) prevents decomposition. These derivatives are solid and stable compounds. Their melting points are listed. 0 Compound Structure LD50 MP c

FCH CHO 6.o ~luoroacetaldehyde 2 F(CH ) CHO 2.0 127 4-Fluorobutanal 2 3 5-Fluorohexanal F(CH ) CHO 0.58 105 2 5 F(CH ) CHO 2.0 97 8-Fluoro-octanal 2 7 10-Fluorodecanal F(CH ) CHO 1.9 94 2 9

{W -FLUOROKETONES

Fluoroketones are also unstable, similar to fluoroaldehydes. The melting points of their stable 2,4-dinitrophenylhydrazone derivatives are listed. Oxidation of the ketones into carboxylic acids is followed by their P-oxidation.

F(CH )n_ COOH [+ RCOOI!J FCH COOH F(CH 2)nCOR > 2 1 > 2

0 Compound Structure LD50 MP c

1-Fluoro-2-octanone FCH CO(CH ) cH 8.o 2 2 5 3 FCH co(CH ) cH 7.5 1-Fluoro-2-decanone 2 2 7 3 1,7-Difluoro-2- heptanone FCH CO(CH ) F 0.7 2 2 5 8-Fluoro-2-octanone F(CH ) cocH 3.0 82 2 6 3 F(CH ) cocH 16 65 9-Fluoro-2-nonanone 2 7 3 F(CH ) cocH 1. 2 83 10-Fluoro-2-decanone 2 8 3 11-Fluoro-2-undecanone F(CH ) cocH 11.8 70 2 9 3 ) COCH 1.5 87 12-Fluoro-2-dodecanone F(CH2 10 F(CH ) co(CH ) cH 4.5 47 12-Fluoro-6-dodecanone 2 6 2 4 3 W -FLUOROALKYL ETHERS

Toxicity is imparted following rupture of the ether link and conversion into alcohols, then carboxylic acids and /3-oxidation.

Compound Structure Physical characteristic

2-Fluoroethyl methyl ether FCH CH 0cH 15 LLO 2 2 3 2-Fluoro-2'-hydroxy diethyl ether FCH CH 0CH CH 0H 15-20 HLO 2 2 2 2 2-Fluoro-2'-cyanodiethyl ether FCH CH 0CH CH CN 10-20 HLO 2 2 2 2 4-Fluoro-4 1 -chlorodibutyl ether F(CH ) o(CH ) Cl 1.32 HLO 2 4 2 4

~-Fluoro-4 1 -cyanodibutyl ether F(CH ) 0(CH ) CN HLO 2 4 2 4 4,4 1 -Difluorodibutyl ether F(CH ) o(CH ) F 0.82 HLO 2 4 2 4 ~-Fluorohexyl methyl ether F(CH ) ocH 4.o HLO 2 6 3

1-FLUOROALKANES

Initial oxidation into carboxylic acids containing same number of carbon atoms, followed by~-oxidation.

Compound Structure LD50 Physical Characteristic n-Fluorohexane CH (cH ) F 1 .. 7 LLO 3 2 5 h-Fluoro-octane CH (cH ) F 2.7 HLO 3 2 7 CH (cH ) F 21.7 HLO n-Fluorononane 3 2 8 h-Fluorodecane CH (cH ) F 1.7 HLO 3 2 9 CH (cH ) F 15.5 HLO 1-Fluoroundecane 3 2 10 1-Fluorododecane CH (cH ) F 2.5 HLO 3 2 11 W , W' -DIFLUOROALKANES

Detoxication mechanisms similar to fluoroalkanes following oxidative cleavage of the chain.

Compound Structure LD50 Physical Characteristic

F(CH ) F 3.4 LLO 1,4-Difluorobutane 2 4 F(CH ) F 18 LLO 1,5-Difluoropentane 2 5 F(CH ) F 21 o3 HLO 1,7-Difluoroheptane 2 7 F(CH ) F 1.6 HLO 1 9 8-Difluoro-octane 2 8 2.1 HLO 1,10-Difluorodecane F(CH) 10F F(CH ) F 2.5 HLO 1,12-Difluorododecane 2 12 F(CH ) F 2.3 HLO 1,14-Difluorotetradecane 2 14 F(CH ) F 10.9 1,16-Difluorohexadecane 2 16 so} ) F 10 so oils 1,18-Difluoro-octadecane F(CH2 18 F(CH ) F 10.2 so 1,20-Difluoroeicosane 2 20

"-'-FLUOROALKENES F(CH 2)nCH=CH 2

Biochemical fate prior to formation of fluoroacetate is not known.

Compound Structure LD50 Physical characteristic

F(CH ) CH=CH 5 .. 4 LLO ~-Fluoro-1-pentene 2 3 2 F(CH ) CH=CH 2 .. 8 LLO 6-Fluoro-1-hexene 2 4 2 F(CH ) CH=CH 9.3 HLO 11-Fluoro-1-undecene 2 9 2 CH=CHCH F 6.1 so 1,4 -Di fluoro-2-butene FCH 2 2 '4> -FLUOROALKYL HALIDES

Fluoroacetate is formed by a sequence of hydrolytic dehydrogenation into alcohols, oxidation into carboxylic acids and their /3-oxidation.

3ID F(CH )nOH ~ F(CH )n-lCOOH ____.,... FCH COOH F(CH2)nX 2 2 2

Compound Structure LD50 Physical characteristic

F(CH ) c1 1.2 HL 4-Fluorobutyl chloride 2 4 F(CH ) Br 8.2 HL 4-Fluorobutyl bromide 2 4 F(CH ) I 5.2 HL 4-Fluorobutyl iodide 2 4 5-Fluoroamyl bromide F(CH ) Br 10.5 HL 2 5 5-Fluoroamyl iodide F(CH ) r 8.5 HL 2 5 F(CH ) c1 5.8 HL 6-Fluorohexyl chloride 2 6 F(CH ) Br 12.8 HL 6-Fluorohexyl bromide 2 6 F(CH ) I 4.5 HL 6-Fluorohexyl iodide 2 6 F(CH ) c1 2.3 HL 8-Fluoro-octyl chloride 2 8 F(CH ) Br 20 HL 8-Fluoro-octyl bromide 2 8 F(CH ) cl HL 10-Fluorodecyl chloride 2 10 5 F(CH ) Br 20 HL 10-Fluorodecyl bromide 2 10 F(CH ) Br 16 HL 12-Fluorododecyl bromide 2 12

(,,) -FLUORONITRILES

Degradation commences with the rupture of the carbon- cyanide bond, and production of hydrogen cyanide, followed by /3-oxidation. '

HCN] ~ FCH COOH F(CH2)nCN-----=> F(CH 2)n-lCOOH [+ 2

Compound Structure LD50 Physical characteristic

F(CH ) CN 10 HL 3-Fluoropropionitrile 2 2 4-Fluorobutyronitrile F(CH 2)3CN 16 HL 5-Fluorovaleronitrile F(CH2)4CN 1 .. 0 HL 7-Fluoroheptanonitrile F(CH2)6CN 2.7 HL 37.

G..> -FLUOROALKYL MERCAPTANS

Transthiolation results in the formation of the corresponding alcohols, followed by oxidation to carboxylic acids and /1-oxida tion. All mercaptans, whether toxic or non-toxic, have the characteristic obnoxious mercaptan odour.

~ F(CH )nOH ~ F(CH )n_ COOH ~ FCH COOH F(CH2)nSH 2 2 1 2 Compound Structure LD50 Physical characteristic

thiolacetate F(CH ) sAc 1.8 HL 4-Fluorobutyl 2 4 mercaptan F(CH ) SH 1.25 HL 6-Fluorohexyl 2 6

W -FLUOROALKYL THIOCYANATES

Metabolic pathway is similar to that of mercaptans following prior reduction and formation of hydrogen cyanide.

Compound Structure LD50 Physical characteristic

2-Fluoroethyl thiocyanate F(CH ) scN 15 HL· 2 2 I 3-Fluoropropyl thiocyanate F(CH ) scN 18 HL vile 2 3 , smelline 4-Fluorobutyl thiocyanate F(CH ) scN 2.6 HL oils 2 4

thiocyanate F(CH ) scN HL 1 6-Fluorohexyl 2 6 5

U>-FLUOROALKANESULPHONYL CHLORIDES AND FLUORIDES F(CH2)nso2x

Rupture of the carbon-sulphur bond is followed by P-oxidation.

Compound Structure LD50 Physical characteristic

2-Fluoroethanesulphonyl F( CH ) so cl chloride 2 2 2 19.5 HL'I 2-Fluoroethanesulphonyl fluoride F(CH ) so F 8.8 HL 2 2 2 pungent ~-Fluorobutanesulphonyl • smelling chloride F(CH ) so cl 18 HL liquids 2 4 2 4-Fluorobutanesulphonyl F( CH ) so F 10 HL fluoride 2 4 2 CHAPTER THREE

SYSTEMIC TOXICITY AND TRANSLOCATION

Several connotations and some confusion apparently exists with respect to the term "Systemic Poison" .. By definition, any compound that is toxic to a physiological system is a systemic poison; it remains to be decided then to what physiological system the poison applies.

A compound may be innocuous to a plant, but toxic to an animal.

If absorbed into the plant it becomes systemic to that plant, on con­ sumption by the animal the compound then becomes systemic to the animal and exerts its toxic action. Thus, the compound is a systemic poison to the animal, but, although systemic, is not toxic to the plant. A compound may be toxic to both plant and animal, or it may be toxic to the plant and non-toxic to the animal. In all cases, its nomenclature requires further definition, such as systemic insecticide, or systemic fungicide or weedicide. Furthermore, a compound may be toxic to parti- cular plant or animal species, or it may be toxic if it gains access via a particular route. In plants, absorption via foliage, fruit, root, seed or stem may determine the degree or extend of toxicity of a compound. In an animal, inhalation, ingestion, or cutaneous absorption are important modes of access where translocation by the systemic circulation to specific tissues or organs may determine the toxic action of a compound.

In order to exert its toxic action a compound must be capable of interfering with at least one biochemical metabolic pathway. It can do this directly, or indirectly by means of its own breakdown products

(metabolites). Many insecticides act in this indirect fashion where the non-toxic parent compound is converted in the insect body into a toxic metabolite which then exerts its lethal action by interference with 39. a specific important metabolic pathway. Alternatively, the metabolite may also be non-toxic but may gain access to a specific metabolic path­ way and be re-synthesised to a compound which is toxic (lethal synthesis).

The majority of fluoro compounds, described in the previous chapter, act in this fashion. They are non-toxic in themselves, nor are their immediate metabolites toxic. Upon reaching the fluoroacetate stage

this metabolite then enters the tricarboxylic acid cycle and is re­

synthesised to fluorocitric acid which then inhibits the enzyme aconitase.

This inhibition then interrupts one of the major energy-yielding metabolic

pathways, with fatal physiological consequences.

From the above preamble a most significant deduction becomes

evident, namely:

A compound is a systemic poison to a physiological system if this

system contains enzymes which render the compound capable of

interfering with normal physiological functions.

In the context of toxicological research and policies relating to

wildlife control systemic poisons are defined as those compounds which,

when applied to plants, become systemic but are not phytotoxic. Instead,

the plant is rendered toxic upon ingestion by the animal. In the

ecological plant-animal relationship this connotation places the toxic

burden on the animal, whilst implying a chemotherapeutic effect on the

plant.

Within the confines of this connotation a systemic poison can be

evaluated according to its mode of translocation in the plant. Its

movements and persistence are factors that determine its effectiveness

for a particular target animal. For instance, a compound may be absorbed

from the surrounding soil and translocated into the root system, but no

further. Its residence there makes it an effective candidate for root-

browsing animals such as pigs. It may be translocated instead to the

stem or foliage system where its residues are effective against bark- or 4o. leaf-browsing animals.

The persistence of a systemic poison must also be taken into account.

Consider for example a small broadleaf bush has perhaps 1,000 leaves of 2 approximately 50 cm total surface each. Consider also a systemic poison with a lethal toxicity to deer of 0.5 mg/kg (32 mg/150 lbs). If 100 leaves are removed by browsing and if this number should contain a lethal dose then this plant requires 320 mg of toxin, provided translocation to the leaves is 100%. This treated bush exposes to the sun and air 2 320 mg of toxin more or less uniformly distributed over 50.000 cm (about 2 54 ft ) of total leaf surface co.3 mg/leaf). This situation is ideally suited to photo-decomposition and volatilisation, including steam- distillation via the transpiring leaves. If a deer were to browse half the bush (500 leaves) then only 64 mg would have to be applied to the plant in order to attain a lethal dosage, assuming that translocation is still 100% (10% would be more realistic). However, exposure would then be even more pronounced.

The concept of systemic toxicity for control of mammalian populations incorporates a wide diversity of compounds which, although primarily introduced as agricultural pesticides, also have effective pharmacodynamic activities in mammals (34). For ethical reasons, however, their mode of action as anti-cholinesterases, convulsants and mucous membrane irritants make these compounds less suitable for control programmes. With some reservations would I therefore recommend the use of tetra-ethyl-pyrophosphate

(TEPP), tetra-ethylene-disulfo-tetramine (TETRAMINE), GOPHACIDE (DRC-714), or similarly-acting compounds in noxious animal control programmes.

Despite their suitability, it is my considered opinion that equal effect- iveness can be obtained by fluorine compounds in a decidedly more humane manner. Moreover, apart from emotional reasons, there are other practical reasons for some measure of restraint in the application of toxic compounds other than those containing fluorine. Sophisticated equipment is required to evaluate their metabolic fate in animals, plants, soil and surface water (40,41). Also, the question of retention of residues demands continuous consideration since little is known about their long-term effects in biological systems. The majority of fluorine compounds, whilst achieving the immediate affects desirable in animal control, are not cumulative. The evaluation of their residues in biological systems is a relatively less complex analytical procedure compared with those for non-fluorinated compounds.

While many different types of aliphatic fluorine compounds have been applied successfully as contact- or systemic insecticides relatively few of these compounds have been applied for plant protection against browsing animals. In overseas countries the principal aims have been to protect selected plants against selected mammals, whilst avoiding intoxication of non-target species. No such considerations are required under our local situations. For this reason aliphatic fluorine compounds, because of their non-selective mammalian toxicity, can be considered eminently suitable as systemic poisons in the control of noxious animals.

Such consideration is the more justified because of the wide diversity of physical properties of these compounds together with their wide range of toxicities.

In our definition of a systemic poison it was implied that its phytotoxic activity should be nil. This aspect must now be examined since a knowledge of the plant's physiological and biochemical reactions will determine the suitability of a fluorine compound as much as its acceptability and toxicity to the browsing animal. An insight is therefore required not only of the metabolic fate of a toxin in the animal, as outlined in previous chapters, but also an appreciation of its metabolic fate in the plant.

There is a wealth of evidence that the cyclic enzymic sequence of the tricarboxylic acid reactions operates in the cells of the higher plants (30) as it does in animals. By inference, it may then be suspected that a compound such as sodium fluoroacetate could be toxic to

the plant as it is to the animal. Practical experience has shown this

to be the case which would seem to eliminate 11 1080° as a candidate for

systemic toxicity, Possible complications imposed by mode of appli-

cation, transport across cellular membranes, sites of enzyme action,

rates of reaction between substrates and enzymes in different plant

species will be ignored in this general outline. Nonetheless, if we

consider the fluoro-carboxylic acids, and other derivatives of fluoro-

acetate, many interesting possibilities arise. The complex detoxicating

enzyme systems in animal cells, leading to the formation of fluoroacetate,

are not necessarily present in the plant cells. Thus, the necessary

enzymes, co-enzymes, co-factors (inorganic anions, cations and metals)

and catalytic amounts of intermediate metabolites that are required

for the formation of fluoroacetate may be quantitatively or qualitatively

different in a specific plant species.

An insight into these biochemical phenomena provides the toxicologist

with a highly effective tool whereby plant-toxin-animal associations

can be evaluated and manipulated (natural baits, bait trees, etc.).

For instance, fluoroacetamide is not as toxic as 1 ~080 11 to plants because

the specific detoxicating enzyme complex (amidase) is quantitatively

less adequate for the conversion into fluoroacetate. As systemic- or

as contact poisons fluoroacetamides have therapeutic value for the

living plant for its protection against sucking and biting insects and

browsing mammals (31, 32). Similarly, the fluoroacetanilides have

considerable mammalian toxicity because of an efficient hydrolytic

enzyme system (amidohydrolase) which is absent in plants. As systemic

and contact poisons the fluoroacetanilides have been intensively investi­

gated in Japan for control of rodents, insects and micro-organisms (33).

From field observations attention has been directed to the attraction of certain animals to certain plants. Thus, some species of willow and poplar are preferred by possums, broadleaf by deer, topdressed grass swards by hares, etc.. Suggestions have been made that phenomena such as these could be manipulated for the development of natural baits or of "bait trees".

Whatever the ecological implications the toxicologist looks at such phenomena in terms of composition and accumulation of constituents synthesised on occasions in the plant cells. Succulance of leaves is often conferred by di- and tricarboxylic acids, and it is the peculiarity of the tissue of higher plants that they often contain one, or a combination, of these acids at what is, biologically speaking, a high concentration.

Prominent among these acids are those of the tricarboxylic acid cycle; citric, aconitic, iso-citric, succinic, fumaric and malic acid, or those outside the cycle; oxalic, malonic and tartaric acid. From conclusions on their preponderance practical benefits may accrue such as anticipated affinity to fluoroacetate toxicity or enhancement of palatability of artificial baits. Also, this type of investigation offers unexplored medical implications. For instance, the juice of sugar cane contains cis-aconitic acid as the main acidic constituent (30). It may be re­ called that this is the acid in the tricarboxylic acid cycle which cannot be synthesised because of the inhibitory action of fluorocitrate on the enzyme aconitase. This knowledge could be exploited in the search for an antidote for fluoroacetate poisoning. (Both cis-aconitic acid and aconitase are now commerciallyavailable from pharmaceutical firms.)

In any discussion on systemic toxicity (and no matter whether the emphasis lies on plant physiology or conservation, animal ecology or control, natural bait or 11 bait-tree 11 phenomena) pride of place must be accorded to those plant species that synthesise and store, in their systemic circulation, toxic fluorine compounds as part of their normal 44. physiological make-up. The leaves of Dichapetalum cymosum contain

fluoroacetate. This South African plant ("gifblaar") has long been known to be a potent cattle poison. The seeds of Dichapetalum toxicarium

contain the fluoro-carboxylic acid, fluoro-octadecanoic acid (35)

(cf. Table 3 and Fig. 2). The fatty nature of this constituent has made

the seeds of this South African plant ( 11 ratsbane 11 ) highly attractive to

animals. Baits of the ground seeds, or the oil obtained by extraction,

mixed with pollard have been considered a better rabbit poison than

fluoroacetate baits. In fact, probably because of physiological

differences in gastric absorption, this fluoro-fatty acid appears less

toxic to ruminants (sheep, cattle) and more toxic to monogastric animals

(rats,rabbits) than fluoroacetate (36).

To the pea-flowered, pod-bearing family Papilionaceae belong the

toxic species of the genera Gastrolobium and Ostrolobium which are wide-

spread in parts of Australia. Ever since the early days of settlement

these leguminous succulents have received notoriety for their attraction

and toxicity to farm animals. In the large number of species described

(37) fluoroacetate, or its derivatives, accumulate in leaves, seeds,

flowers, or young shoots. Their toxicity is considerably increased

under moist conditions or during rapid growth at the flowering and pod

stage.

Galega officinalis ("goats' rue") is also a potentially toxic

member of the Papilionaceae. It is established in New Zealand (38)

on the banks of the Manawhatu River (E.P. White, pers. comm.). Its

toxicity is reputedly due to fluoroacetate. Undoubtedly, other species

exist that have not yet been investigated for their toxic principle.

Whether the introduction of these plant species into indigenous

forest communities or exotic forest plantations could be a practical

venture is, at this stage, not the only consideration. They provide

the soil scientist with unique marker speciment the taxonomist with a chemical classification, the microbiologist with possible rhizobium associations, the enzymologist with unexplored enzyme complexes, the biochemist with a possible avenue for development of fluoroacetate antidotes, the organic chemist with simple systems for the synthesis of fluoro-compounds, the ecologist with plant-animal relationships, the agriculturist with reasonable assurance of more effective rabbit control.

Finally, the toxicologist will borrow the information for his own specific interests. 46.

CHAPTER FOUR

REPRODUCTION INHIBITORS AND CHEMOSTERILANTS

The aim of noxious animal control in relation to agricultural and forestry policies has hitherto been based on destruction of the target­ animal species by increasing mortality rates through the use of mechanical

(trapping, shooting) and lethal chemical (poisons) control measures.

No attention has been given to measures aimed at decreasing fertility rates.

If noxious animal populations could be effectively suppressed with an economically efficient application of non-toxic reproduction inhibitors there would be less need for lethal chemicals. Also, there would be less public concern for contamination of the environment and accidental intoxication hazards.

Reproduction is the only force that can overcome all mortality factors operating against a species. Therefore, the suppression of reproduction will cause the population to decrease as surely as increas- ing one or more mortality factors. Moreover, it seems more practical to prevent animals from being born than to reduce their numbers after they have become established in an environment.

The introduction of compounds with, for example, progestational

(anti-ovulatory) activity cannot permanently change the general equilibrium of a population. Repeated applications are required during several successive breeding seasons to restrain the population density. However, in the context of the ecology of population dynamics, it appears that a given number of sterile individuals in a population exerts a much greater pressure of biological control on that population than removal of that same number of fertile individuals (42). Elimination of fertile individuals may result in a compensating increase in reproduction or survival of the remaining population. The individuals that remain not only fail to contribute to the next generation, but also compete mean­ while for space, food, social order and occupation of territories. The ecologist may ponder whether the social behaviour of the anoestrus or sterile individual remains similar to that of the fertile individual, or how sexual docility and aggressiveness influences the social structure of the community. Nonetheless, when reproduction can be suppressed economically it will be a valuable adjunct to existing control measures since the synergistic effects of this combination may achieve a degree of control greatly exceeding the sum of the independent effects of each measure.

Many compounds with anti-fertility activities, at one or more of several vulnerable stages of reproduction, have been developed overseas, but the full scope of their activities has been exploited in relatively few cases and toward few vertebrate species. Where the aim of the control programme is selectivity of reproduction inhibition, either temporary or permanent, in either male or female of a specific species

then the problems of proper timing, dosage and dispersal of bait far out- weigh the selection of a suitable non-toxic drug. These problems of selectivity and application are considerably simplified under New

Zealand conditions because:

(i) The majority of noxious animals are mono- or di-oestrus. Baiting

operations can therefore be confined to one or two relatively

protracted periods each year.

(ii) Breeding cycles do not normally coincide with those of protected

(e.g. bird) species.

(iii) Selectivity of sex and species is not warranted.

(iv) The hormone balance in the animal is readily upset by oral admini-

stration of synthetic steroid hormones. Since the sterility effect

is temporary, suppression of reproduction can be applied or with- 48.

drawn without permanently affecting either the target-species or

other species that may be exposed.

(v) These compounds are stable and appear to retain their potency in

baits. They are tasteless, odourless, relatively inexpensive,

and capable of being masked in attractive baits.

(vi) Accidental exposure is considerably less hazardous.

A description of the merits of each of these compounds is outside the scope of this chapter since their individual specificities are related to several physiological and hormonal peculiarities of the sus- ceptible animal. Their action can govern the secretion of gonadotrophins, follicle development and maturation, and transport of ova in the oviduct.

They can prevent fertilisation, implantation or gestation, or inhibit spermatogenesis or transport and storage of sperm.

The evaluation of this complex of multiple interactions in relation to control of noxious monogastric, marsupial and ruminant species has led me to believe that investigations on inhibition of the progestational or ovulatory activity could be the best approach. The use of orally active progestagens for the control of ovarian cycles in sheep is a well-established agricultural practice in the management of selected breeding seasons. To this purpose, many long-acting, highly­ potent progesterone analogues have been developed, and their effects investigated in large-scale animal experiments (43). In our pre- occupation with fluorine compounds in wildlife control it is of con­ siderable interest to note that among these steroids the fluoro­ derivatives are prominent for their extreme potency with activities many times that of the native parent steroid, progesteroneo CHAPTER FIVE

ANALYTICAL PROCEDURES

General considerations

The value of any toxicological contribution in any one of the

diverse research or control programmes depends entirely on a precise

and quantitative evaluation of the factors to be measured. Moreover,

the scope of this contribution depends not only on the resourcefulness

of the investigators to select the appropriate analytical parameters,

but also on the availability of the analytical tools by which meaningful

data can be obtained.

In order to evaluate the investigations outlined in previous

chapters with respect to fluoroacetates, it is necessary to devise, in

the first instance, suitable analytical procedures for the determination

of fluorine contents of biological materials. Specific ion electrodes

and instrumentation are required for these types of analyses, but the

equipment is also adaptable far other plant and soil investigations.

Ion electrodes are available for the specific determination of bromide,

cadmium, calcium, chloride, cupric, cyanide, fluoroborate, iodide, lead,

nitrate, perchlorate, sodium, thiocyanide, and water hardness.

Concomittantly with fluoroacetates, the changing levels of citrate,

glucose and lactose should be investigated in states of "fluoroacetate

diabetes" during fluoroacetate poisoning. These procedures require

spectrophotometric facilities and instrumentation.

The translocation phenomena of contact- and systemic poisons, such

as fluorine derivatives, tetraethyl pyrophosphates, tetramines, etc., in

plants and soils cannot be investigated in a meaningful manner without

the acquisition of radioactive tracer- and scintillation equipment.

Investigations concerned with aspects of the reproductive cycle

require suitable gas-chromatography equipment. 50.

The entire spectrum of toxicological investigations is of course dependent on animal-house facilities for sustained evaluations of toxicity phenomena under controlled experimental conditions.

Considering the diversity of aspects that require investigations, and the present paucity of laboratory space, equipment and technical supporting staff, a judicious - if not ruthless - choice of priorities is demanded for the development of analytical methodology. This choice of alternative and conflicting postures in respect to the need for what should be done and what can be done is reflected in the circumscribed nature of this chapter. Nonetheless, I do not wish to overstate my case. I prefer the pragmatic approach whereby one single aspect of a problem is dealt with exhaustively before turning to other aspects. This systematic attention, although unimpressive in its single facets, adds more to the total sum of understanding than a collection of superficial observations and fragmentary data on a variety of aspects

From choice then this chapter will be limited to a description of the analytical procedures concerned with fluorine determination since this aspect forms the toxicological basis of the majority of present and future animal control programmes. Past methods will be briefly evaluated and reasons given for their failure to provide suitable toxicological support. The adoption of more suitable methods will be advocated on the basis of their sophistication, reliability, accuracy and speed.

Analysis of organic fluorides

Analysis of organic fluorides is a vast and rather difficult problem on which extensive reviews and textbooks have been published

(21,22,23). Because of the emphasis on fluoroacetate and its derivatives the analysis of monofluorinated organic compounds containing the carbon­ fluoride bond will only be considered here. 51 ..

The current laboratory glass equipment is usually unsatisfactory for these types of analysis because fluoride ions attack glass. To determine the fluorine content in organic fluoride compounds the fluorine must be converted to the inorganic fluoride ion. In orthodox methodology this involved either fusion with sodium under vacuum at high temperature, or heating under reflux with sodium hydroxide (24). Alternatively, ashing of samples with lime in a platinum crucible in a muffle furnace at high temperature has been recommended (25). Several distillations of the combustion product follow after which the fluoride ion is titrated

against thorium nitrate (25). The entire determination is exceedingly intricate, subject to considerable error, and demands extraordinary skill.

In fact, no entirely satisfactory method has been developed with this

type of methodology, as judged by the number of analytical modifications

offered every year. The number of determinations that can be performed

amounts to about six per week. Clearly, there is room for improvement.

Recent years have witnessed the development of new materials and

more sophisticated techniques which have placed the analyses of fluorine

compounds within range of extensive manipulation by the toxicologist.

Of the materials, high-silica ( 11 VYCOR 11 ) glass and polytetrafluoroethylene

polymers ( 11 TEFLON 11 ) are outstanding improvements to resistance of attack

by fluorine. VYCOR glass is comparable with fused quartz except that

VYCOR glass is transparent. TEFLON is a white opaque polymer with an

amazing chemical resistance and absolute stability towards solvents at

any temperature up to boiling point. It is attacked by fluorine only at

temperatures of 150°c, but is resistant to concentrated acids. Another

interesting property of TEFLON is an absolute hydrophobia and oleophobia;

it does not become wet in contact with water or greasy in contact with

oils or fats, hence its application in domestic kitchen ware. In the

laboratory it has multiple uses.

Of the techniques, three will be outlined since they form part of 52. fluorine analytical procedures presently under development at F. & R.E.S .•

The value of enzymic procedures for the cleavage of the carbon-fluoride bond in fluoroacetates has been alluded to previously.

Freeze-drying technique is a technique of fundamental importance to the biological sciences. Heat-labile materials, including proteins, animal and plant tissues, soils, antibiotics, toxins, vitamin preparations, blood plasma, and bacterial cultures can be preserved for years without significant loss of activity. Many methods and degrees of equipment sophistication are employed for particular applications. The orthodox procedures of fluorine analyses relied for sample preparation on oven drying at elevated temperatures. Unless carefully controlled, this time-consuming technique results in irreplaceable losses of material.

Oxygen-flask combustion techni~ue. Organic elemental analyses have been virtually revolutionised in the past 15 years by the application of the oxygen-flask combustion technique of decomposing organic materials.

For rapidity and simplicity the method could scarcely be bettered, and one of its principal attractions for routine work is that untrained technicians can obtain excellent results after little practice. Apart from macro- and micro determinations of fluorine, the method is also applicable to the determination of chlorine, bromine, iodine, sulphur, phosphorus, arsenic, boron, metals, and carbon (26). Organic fluorides are oxidised into inorganic fluorides by this technique. By contrast, orthodox procedures rely on lime ashing at high muffle temperatures where material losses are unavoidable.

Fluoride-ion electrode technique. Inorganic fluoride ion concentration is measured directly in aqueous or non-aqueous solution by a specific ion electrode (27). The electrode measures the ion activity of fluorine in solution, rather than the concentration of the ion. In many cases, the activity is proportional to the concentration, allowing the electrode to be calibrated in terms of concentration. All specific ion electrodes are subject to interferences from other ions. With the fluoride ion the only significant interference is from hydroxide ion; the electrode is ten times more sensitive to fluoride than to hydroxide ion. It can measure concentrations from 19.000 to 0.02 ppm (i.e. 19 gr/1 to

0.02 mg/1) in a volume of 10 microliters. Compared with the orthodox

technique, the fluoride ion electrode contributes incomparable accuracy,

speed and sensitivity to analyses.

A typical fluorine analysis. A sample (animal or plant tissue or soil)

is weighed, freeze-dried, and stored until required for analysis. An

aliquot is taken and the combustion carried out in a VYCOR oxygen-flask

fitted with platinum electrode. The gaseous products are absorbed in

0.1 N sodium hydroxide and the flask contents transferred to a TEFLON

beaker. The solution is adjusted to pH 4.4, brought to the boil to

expel carbon dioxide, and cooled to room temperature. If the sample

contains sulphur, an amount of barium nitrate sufficient to precipitate

all the sulphur is added. No filtration of this precipitate is necessary.

The pH is adjusted to between 5 and 7 with sodium hydroxide. If the

fluoride concentration is high, the solution is titrated potentiometric-

ally with 0.02M lanthanum nitrate. The emf. is monitored on the fluoride-

ion electrode. If the fluoride concentration is low, the activity is

read directly on the fluoride-ion electrode. The concentration is

determined either from a titration or from a calibration curve. In

contrast to orthodox procedures the number of determinations that can

be performed is in excess of 100-150 per week. REFERENCES

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the Sheep. Sydney University Press, Sydney, 1967. POSTSCRIPT

11 Would you tell me, please, which way I ought to

walk from here?"

"That depends a good deal on where you want to

get to11 , said the Cat.

11 11 I don't much care where 9 said Alice ..

"Then it doesn't matter which way you walk11 , said the Cat.

11 11 - so long as I get somewhere , Alice added ..

11 0h, you're sure to do that 11 , said the Cat, 11 if only

you walk long enoughlt1 9

- Alice in Wonderland -