Molecular structure and aquatic toxicity — An example with C1 - C13 aliphatic alcohols

Chemosphere, Vo1.13, No.5/6, Printed in Great Britain

MOLECULAR

STRUCTURE

AND

pp 613-622,

AQUATIC

1984

TOXICITY

0045-6535/84 $3.00 + .00 01984 Pergamon Press Ltd.

- AN

EXAMPLE

WITH

C I - C13

ALIPHATICAL(RDHOLS

Bengt-Erik

Bengtsson I , Lars Renberg 2 and Maria Tarkpea I

National 1

Swedish Environment

Protection

Brackish Water Toxicology

Laboratory

Board

Studsvik S-611 82

NYKOPING,

Special Analytical

Sweden Laboratory

Box 1302 S-171

25

SOLNA,

Sweden

ABSTRACT The acute aquatic toxicity of a series of primary aliphatic alcohols (C I - C13) has been tested on the harpacticoid copepod Nitocra spinipes, Boeck, and the bleak, Alburnus alburnus L. The results showed that the toxicity was highly correlated to the number of carbon atoms, ie increasing toxicity with increasing number of carbons. The results are discussed in relation to interspecies differences in sensitivity, correlations between hydrophobicity (log Pow ) and toxicity, and the use of solvents in bioassays.

INTRODUCTION The rapid growth of chemial technology, micals

into commerce yearly,

of laboratory

research and test data concerning

cause damage to living organisms. very expensive

which introduces hundreds

calls for an ever-increaslng

the potential

Many of the necessary

to perform due to long duration,

613

quantity

advanced

of new cheand quality

of chemicals

tests are, however, instrumentation,

to

614

difficult analytical procedures,

etc. This,

t o g e t h e r w i t h the u n s a t i s f y i n g

n u m b e r a n d / o r c a p a c i t y of e x i s t i n g test laboratories, m e n t of r e l a t i v e l y

h a v i o u r of chemicals. its p o s s i b i l i t y effect.

have

led to the develop-

simple s c r e e n i n g tests to p r e d i c t the e c o t o x i c o l o g i c a l beThe p r o g n o s t i c value of such a test m a i n l y depends on

to d e s c r i b e or r e f l e c t the central m e c h a n i s m c a u s i n g the

S e v e r a l a t t e m p t s h a v e b e e n m a d e to e x p l a i n the effects on a more or

less q u a n t i t a t i v e b a s i s and such c o n c e p t s h a v e b e e n put forward u n d e r the name of S t r u c t u r e A c t i v i t y R e l a t i o n s h i p Structure Activity Relationship

A f r e q u e n t l y used p a r a m e t e r eg b i o a c c u m u l a t i o n ,

water

(SAR) or - more a m b i t i o u s - Q u a n t i t a t i v e

(QSAR).

for the p r e d i c t i o n of the e n v i r o n m e n t a l behaviour,

s o l u b i l i t y and s o r p t i o n for o r g a n i c substances,

b e e n the p a r t i t i o n c o e f f i c i e n t

in the n - o c t a n o l / w a t e r

system.

p a r t i t i o n c o e f f i c i e n t as a m e a s u r e m e n t of the h y d r o p h o b i c i t y to as lipophilicity)

has

The use of the (often r e f e r r e d

of an o r g a n i c c h e m i c a l can also be e x t e n d e d into the

field of t o x i c o l o g y as the t o x i c i t y is h e a v i l y d e p e n d e n t on the r e s i d e n c e time of the toxic agent,

which

in turn is o f t e n strongly r e g u l a t e d by the corre-

sponding hydrophobicity.

T h i s report p r e s e n t s alcohols

(C I - C13)

copepod Nitocra

some results on the acute t o x i c i t y of p r i m a r y a l i p h a t i c to the bleak,

spinipes Boeck

tion to the h y d r o p h o b i c i t y

A l b u r n u s a l b u r n u s L, and the h a r p a c t i c o i d

(Crustacea).

The results are d i s c u s s e d in rela-

of the i n d i v i d u a l c o m p o u n d s e x p r e s s e d as the parti-

t i o n coefficient.

EXPERIMENTAL

B l e a k was c a u g h t by seine net in the B a l t i c Sea in the v i c i n i t y of the laboratory. The fish w e r e k e p t tinuous

for at least two w e e k s

in storage tanks w i t h a con-

flow of n a t u r a l b r a c k i s h w a t e r t h e r m o s t a t e d to 10°C. T h e y w e r e fed

o n c e a day w i t h c o m m e r c i a l

fish food (Tetramin HauptfUtter,

u n t i l one day p r i o r to the test.

T e t r a Werke,

FRG)

D i s i n f e c t i o n of test animals was not conside-

red necessary.

T h e b l e a k was t e s t e d u n d e r

static conditions,

w h i c h means that the test solu-

tions w e r e not r e n e w e d d u r i n g the test p e r i o d of 96 hrs. No a e r a t i o n of the t e s t a q u a r i a was p e r f o r m e d .

Glass aquaria

(Rhodorsil 3B, R h o n e Poulenc,

(70 L) sealed w i t h s i l i c o n e rubber

France) w e r e used as test vessels and c o n t a i n e d

60 L of natural b r a c k i s h water.

The w a t e r was p u m p e d from a d e p t h of 40 m in

the n e a r b y T v ~ r e n Bay in the B a l t i c Sea and was then f i l t e r e d t h r o u g h a 300 ~ m

615

filter. ments:

The

following water parameters

salinity

was kept

Adult

7 o/oo,

constant

Nitocra

alkalinity

at 10"C

spinipes

were

were harvested

of N s p i n i p e s

to each c o n c e n t r a t i o n

taining

10 mL of b r a c k i s h w a t e r

surface-water through above

The

a paper

b l e a k was

per

after

Cultivation

laboratory

In this

cul-

of l a b o r a t o r y

test tubes

test,

use,

natural

and pH w e r e

similar

were

con-

brackish

the w a t e r was

were

analysis

filtered

as d e s c r i b e d

Mortality

removed.

The

le a s t

(a s l i g h t l y

were

obtained

toxicity

8 carbon

with 6 carbon

exceeded

up to C11.

series

initial

modified

obtained.

This

was

sources

Due

recorded

and

only

of a s u b s t a n c e

(2) was

determined

Biomedical

by

Computer

or more.

which

and h i g h e r tested.

of 96 % ethyl

e m u l s i o n was

we

Tween

alcohol

added

shaken

80 R

in 10 % again

(by volume)

was

toxicity

on N spinipes

and

that

5 000 ppm.

It was

considered

for the h i g h e r

by o c u l a r

first

without

dissolved inspection,

any ri~k

after

on

of a c e t o n e species

chain

method

FRG) was until

applied

to e x t e n d

dissolved

an e m u l s i o n

at was

to f i l t e r e d b r a c k i s h

of m i n u t e s

tested

until

a new

for its acute

the 96 hrs LCS0 w a s h i g h e r

that 100 p p m of this

alcohols

(C11 - C16) w e r e

however

found

shaken

for a couple

was

with a carbon

(EG Merck,

and w a s

of alco-

for b o t h

t r i e d an a l t e r n a t i v e

This p r e p a r a t i o n it was

concentration

the 96 hrs LC50

qua-

of at

in r e d i s t i l l e d

this p r o c e d u r e

to test a l c o h o l s

series

solubility

first d i s s o l v e d

initial

is far b e l o w

it was p o s s i b l e

For C12

The

and w e r e of pro a n a l y s i

in a l o g a r i t h m i c

to low w a t e r

they w e r e

was obtained.

found,

of the BMD,

tested

control.

and more,

and the m i x t u r e w a s

alcohols

96 hrs

ie 2 x 10

for the b l e a k

was

concentration

version

emulsion

solvent

and the h a r p a c t i -

series,

daily

mortality

during

from c o m m e r c i a l

atoms

of substances

10 % (by weight)

water

recorded

In the tests w i t h Nitocra,

500 ~L/L,

By this m e t h o d

length

ie the

and one

atoms

(pa quality).

alcohols never

was

For N s p i n i p e s

of the a l c o h o l s

6 concentrations with

acetone

in a d o u b l e

025).

alcohols

lity.

in each a q u a r i u m

tube

50 per cent of the test o r g a n i s m s

Programs,

(3).

in each test

96 hrs and the LC(I)50,

probit

hols

laboratory

(i). The h a r p a c t i c o i d s

Before

alkalinity

10 i n d i v i d u a l s

concentration.

individuals

killing

the

tested w i t h

I0 i n d i v i d u a l s

individuals

The

Salinity,

I°C.

used.

old

in the t e s t i n g w i t h bleak.

colds with

dead

filter.

I°C.

earlier

standard

at 21 ±

from a d e p t h of 4 m w a s

temperature

test room.

of 21 ±

in 15 mL

in the experi-

The w a t e r

from 3 to 6 w e e k s

has b e e n d e s c r i b e d

exposed

to c o n s t a n t

pH 7.9.

in a t h e r m o r e g u l a t e d

t u r e s w h i c h w e r e k e p t at a t e m p e r a t u r e cultures

close

1.6 meqv/L,

emulsion

than

c o u l d be used

for the test animals.

as a

The

1 - 24 hrs of stirring.

It w a s

that o n l y C11 - C 1 3 a l c o h o l s

seemed

to

616

r e m a i n in s o l u t i o n d u r i n g the entire test period, obtained

96 hrs. The test results

for C14 - C16 a l c o h o l s w e r e t h e r e f o r e disregarded.

RESULTS AND DISCUSSION

A narcotic

effect,

was d e m o n s t r a t e d was

ie loss of e q u i l i b r i u m by the b l e a k as an early response,

for all a l c o h o l s

in the p r e s e n t investigation.

f o l l o w e d by coma and finally d e a t h of the test animals.

t i o n s h a v e b e e n r e p o r t e d for a l c o h o l s t e s t e d on b a r n a c l e results,

This reaction

Similar observa-

larvae

(4). The

in the p r e s e n t i n v e s t i g a t i o n e x p r e s s e d as 96 hrs LCS0-values,

summarized

are

in F i g u r e 1 and T a b l e I. The t o x i c i t y was h i g h l y c o r r e l a t e d to the

n u m b e r of c a r b o n atoms. For N s p i n i p e s

(Figure la) this r e l a t i o n s h i p is very

w e l l e x p r e s s e d for the w h o l e range of n u m b e r of c a r b o n atoms

(C 1 - C13).

The

c o r r e s p o n d i n g e q u a t i o n s are given in T a b l e 2. The c o r r e l a t i o n c o e f f i c i e n t s (r = 0.986 and 0.978)

show that log LC50 are h i g h l y c o r r e l a t e d to the n u m b e r

of c a r b o n atoms.

A t - t e s t of the two r e g r e s s i o n lines r e v e a l e d that they w e r e not s i g n i f i c a n t l y different

(p > 0.05).

Thus,

the use of acetone

c a u s e any s y s t e m i c change in the b e h a v i o u r

(and T w e e n 80) does not s e e m to

(ie e x p e c t e d i n c r e a s e d toxicity)

by

i n c r e a s i n g number of carbon atoms of the a l c o h o l s to N spinipes.

For bleak,

a s i m i l a r l y good c o r r e l a t i o n exists for all data and the tests per-

f o r m e d w i t h o u t solvent

(r = 0.987 and 0.994,

respectively).

However,

the t-

- t e s t r e v e a l e d that the two r e g r e s s i o n lines w e r e s i g n i f i c a n t l y d i f f e r e n t (p < 0.05). C 9 - C11)

This

is i n t e r p r e t e d as the h i g h e r a l c o h o l s

(C 8 - C11 or rather

t e s t e d in small amounts of a c e t o n e h a v e a lower t o x i c i t y to b l e a k

t h a n c o u l d be e x p e c t e d from the tests p e r f o r m e d w i t h the s u b s t a n c e s d i s s o l v e d in p u r e water.

A comparison

(Student's t-test)

of the r e g r e s s i o n lines for the tests p e r -

f o r m e d in pure w a t e r for bleak and N spinipes s h o w e d that they w e r e significantly different

(p < 0.05).

are i n t e r s p e c i e s differences, t i o n s to this difference.

B e s i d e s the very p l a u s i b l e e x p l a n a t i o n that there there are, however,

also other p o s s i b l e e x p l a n a -

One i m p o r t a n t factor h e r e is the d i f f e r e n c e

of the test animals. N spinipes, w h i c h is less than 1 m m long, v e r y m u c h a "particle" w h e n c o m p a r e d to the b l e a k higher

s u r f a c e / v o l u m e ratio.

(8

-

in size

is in itself

10 cm) and has a m u c h

It is t h e r e f o r e not s u r p r i s i n g that w h e n t e s t i n g

617

Table I. The acute toxicity (96hrs IF.50) of some primary aliphatic alcohols against the bleak (Alburnus alburnus) and Nitocra spinipes after different methods of'dosing the chemicals

Alcohol

Water cnl~ Bleak Nitocra

Methanol

28 000

12 000

-0.82

Ethanol

II 000

7 750

-0.32

l-Propanol

3 800

2 300

0.34

1-Butanol

2 300

2 i00

0.88

1 -Pentanol

470

440

l-Hexanol

120

l-Heptanol

45

l-Octanol

ns*

ns*

16

60

1 -Nonanol

ns

ns

18

25

1 -Decanol

ns

ns

7.2

3.1

1 -Undecanol

ns

ns

4.6

i.i

0.8

ns

ns

ns*

0.91

0.8

ns

ns

ns

ns*

1.0

ns

ns

ns

ns

ns*

1 -Undecanol I -Dodecanol 1 -Tridecanol

'

Water/acetone Bleak Nitocra

Water~h~een 80 Nitocra

io~ P ~

1.40 2.03

317 210

169

*insoluble in the solvent system

Table 2. Some relations between toxicity and physicochemical parameters of 1-alcohols against the bleak (Alburnus alburnus) and Nitocra spinipes.

Nitocra spinipes

CI-C 7 alcohols in water

log iCS0 = -0.31N* + 4.41

r=0.978 r=0.9~

all alcohols tested (CI-C13)

log If_50 = -0.38N + 4.64

CI-C 5 alcohols

log LCS0 = -0.611og P ~

Bleak (Alburnus

CI-C 7 alcohols in water

log LC50 = -0.47N + 5.01

r=0.9~

alburnus

all alcohols tested (CI-C13)

log LC50 = -0.40N + 4.75

r=0.987

CI-C 5 alcohols

log If.50 = -0.611og Pcw + 3.84

r=0.9~

* N = number of carbon atoms in the individual alcohols ** P c w = partition coefficient in the l-octanol/water system

+ 3.64

r=0.9~

618

la Nitocra

4A

.,,J

0 I,D %% 0 %%0

0

-I

%%% %

1-

O %%

% ~.

o

~'Ib Carbon

÷

- -

I+.

atoms (N)

lb 4-

"~

Bleak

,\.

A

~3-

v

""

O u~

~,

2-

O~

%

0 _1

o

1-

%. ~

%.

%~'% o %

,,

,

~,

2 Carbon

:,

~

6

8

atoms

,

, 10

,

~ 1

(N)

Figure i. Relations between the number of carbon atoms of l-alcohols and the toxicity (96hrs LC50) against a) N itocra spinipes and b) the bleak. • = alcohol dissolved in water only o = alcohol dissolved in water/acetone + = alcohol dissoled in water~fween 80 The corresponding mathematic expressions are given in Table 2.

619

a series of s u b s t a n c e s w i t h d e c r e a s i n g w a t e r solubility,

the "affinity" of

s u b s t a n c e s w i t h low s o l u b i l i t y to v e r y small animals w i l l be h i g h e r c o m p a r e d to adult fish.

The use of acetone p r o b a b l y does not i n c r e a s e the s o l u b i l i t y of the substance in a w a t e r - a c e t o n e phase,

but helps to a d m i n i s t r a t e the s u b s t a n c e more evenly

in the test vessels at the m o m e n t of introduction.

S u b s t a n c e s w i t h low w a t e r

s o l u b i l i t y will then be more or less a s s o c i a t e d w i t h of various kinds.

B e s i d e s r e s e m b l i n g a "particle",

"particles" and surfaces

N spinipes

is a l s o a par-

t i c l e - c o n s u m e r and d w e l l s on the s u r f a c e s of the test vessels. s i t u a t i o n for N s p i n i p e s that of p e l a g i c

fish

The exposure

is t h e r e f o r e p r o b a b l y very d i f f e r e n t as c o m p a r e d to

(eg bleak).

The results of the b l e a k study are very s i m i l a r to those p r e s e n t e d by V e i t h et al

(5) w h o found a linear r e l a t i o n s h i p

(Pimephales promelas)

for acute t o x i c i t y to fathead m i n n o w

of alkyl a l c o h o l s b e l o w l-decanol,

t i o n s h i p for h i g h e r alcohols.

Likewise,

t o x i c i t y r e l a t i o n s h i p has b e e n d e m o n s t r a t e d by L u n d a h l and C a b r i d e n c

In this c o n n e c t i o n

structure/aquatic

for anionic s u r f a c e - a c t i v e agents

(6) and for p h e n o l i c c o m p o u n d s by K o p p e r m a n et al

it is w o r t h m e n t i o n i n g that L u n d a h l and C a b r i d e n c

that the t o x i c i t y of a l k y l b e n z e n e s u l p h o n a t e s

deeper penetration

(7).

(6) found

of the same m o l e c u l a r w e i g h t in-

c r e a s e s w i t h l i n e a r i t y of their a l i p h a t i c moieties.

all linear,

but a n o n - l i n e a r rela-

a similar m o l e c u l a r

This can be due to a

into b i o l o g i c a l m e m b r a n e s and as the tested a l c o h o l s are

there s h o u l d be no p r i n c i p a l d i f f e r e n c e

in their p e n e t r a t i o n due

to m o l e c u l a r size.

Ferguson

(8) has p o i n t e d out that toxic a c t i o n is g o v e r n e d by the e q u i l i b r i u m

which distributes where

the s u b s t a n c e s b e t w e e n the e x t e r n a l p h a s e and the phase

it e x e r c i s e s

its effect.

into a lipoid phase,

The r e s u l t of the e q u i l i b r i u m may be p a r t i t i o n

a d e c r e a s e of i n t e r f a c i a l t e n s i o n or an a c c u m u l a t i o n in a

b i o p h a s e w i t h r e s u l t i n g narcosis.

V e i t h et al

(5), as a result of their own experiments,

Ferguson principle" in m a m m a l s

s u g g e s t e d that

"the

of similar c h e m i c a l a c t i v i t i e s p r o d u c i n g similar effects

in e q u a l l y true for fish. T h e i r c o n c l u s i o n s

p r e s e n t investigation,

find support in the

w h i c h gives r e a s o n to e x t e n d t h e m to be v a l i d a l s o for

crustaceans.

In the p r e s e n t case, in a h o m o l o g o u s

the d i f f e r e n c e s

in t o x i c i t y for the i n d i v i d u a l c o m p o u n d s

series can be e x p l a i n e d in terms of d i f f e r e n c e s

in h y d r o p h o -

620

bicity

("lipophilicity").

coefficient

As a m e a s u r e m e n t

has been frequently

as the ratio of the c o n c e n t r a t i o n s p o l a r phase, relative

respectively,

hydrophobicity

coefficients

of h y d r o p h o b i c i t y

used. The p a r t i t i o n of a substance

in a b i p h a s i c

system.

(x) may be d e f i n e d

for two c o n s e c u t i v e

the p a r t i t i o n

coefficient

(P) is d e f i n e d

in the unpolar phase and the A quantitative

measure of the

as the ratio of the p a r t i t i o n

members of a h o m o l o g o u s

series

P x =

n+____~1 P n

(I)

or

log x = log Pn+l

After

the i n t r o d u c t i o n

- log Pn = "

of the . c o n s t a n t by Hansch et al

this term can be regarded as a constant w i t h i n series,

this concept has been u t i l i z e d

bicity

of individual

cients

for at least two individual

Regression

analysis

for m e t h a n o l the number obtained

(II)

compounds

of C H 2 - g r o u p s

(9) who showed that

types of h o m o l o g o u s

for the c a l c u l a t i o n

under the a s s u m p t i o n

of p a r t i t i o n

through h e x a n o l

several

of the h y d r o p h o -

that the p a r t i t i o n

members of the h o m o l o g o u s

coefficients

reported

showed the following

series are known.

in the literature

linear r e l a t i o n s h i p

(N) and the logaritm of the p a r t i t i o n

in the n - o c t a n o l / w a t e r

system

coeffi-

(i0)

between

coefficients

(log Pow):

log POW = 0.570N - 1.410 r = 0.999

This

implies

increases To

(n = 6)

that ~ = 0.570

is a c o n s t a n t and that the h y d r o p h o b i c i t y

linearly w i t h the number of CH2-groups.

investigate

the relation b e t w e e n

lues of log LC50 were p l o t t e d atoms) marised

as seen in Figures

toxicity and the hydrophobicity,

against the number of C H 2 - g r o u p s

la and lb. The c o r r e s p o n d i n g

in Table 2 t o g e t h e r w i t h the relation b e t w e e n

Analogous difference

to e q u a t i o n

(II) w h i c h

in toxicity b e t w e e n

can be e x p r e s s e d

as:

relations

are also sum-

log LC50 and log Pow"

reflects p h y s i c o / c h e m i c a l

two c o n s e c u t i v e

the va-

(or carbon

properties,

members of a h o m o l o g o u s

the series

621

log (LC50) n - log (LC50)n+ 1 = T

T h e t e r m ?,

(III)

r e f l e c t i n g the a d d i t i v e t o x i c i t y of the C H 2 - g r o u p, is thus ana-

logous to ,. A l i n e a r r e g r e s s i o n a n a l y s i s of the t o x i c i t y data for the experiments

in w h i c h the a l c o h o l s w e r e a d d e d as p u r e w a t e r s o l u t i o n s

t h a t Y,

in c o n f o r m i t y w i t h . ,

(Table I) shows

is a c o n s t a n t and can e a s i l y be c a l c u l a t e d from

the slope of the c u r v e s g i v e n in F i g u r e s la and lb. It was thus found that ?=

0.472

for b l e a k s and

?=

0.310 for N spinipes.

F u r t h e r s t a t i s t i c a l a n a l y s i s showed, b e s i d e s that the two r e l a t i o n s h i p s w e r e linear

(ie for b o t h e x p e r i m e n t s w e r e c o n s t a n t w i t h i n each experiment)

that the T - v a l u e

also

for N s p i n i p e s d i f f e r s s i g n i f l c a n t l y from the T - v a l u e ob-

t a i n e d for bleak. As a m e a s u r e of the t o x i c o l o g i c a l fic b i o l o g i c a l system,

the d i f f e r e n t T - v a l u e s

different toxicological

response.

s e n s i t i v i t y of the speci-

for the two species indicate

This m i g h t be due to d i f f e r e n c e s

b u t a l s o to o t h e r i n t e r s p e c i e s differences,

in size,

as m e n t i o n e d above.

T h e use of a c e t o n e as solvent in the t e s t i n g of h e x a n o l and h i g h e r alcohols, can not be a p p r o v e d as judged from this investigation. w i t h N s p i n i p e s are in favour of this procedure, hesitation,

A l t h o u g h the results

there are several reasons for

i n c l u d i n g the results from the p r e s e n t b l e a k study. However,

as

long as we h a v e to p e r f o r m acute t o x i c i t y tests in w a t e r w i t h s u b s t a n c e s of comparatively adjuvants, course,

low w a t e r solubility,

such as acetone,

it m i g h t s o m e t i m e s be n e c e s s a r y to use

to o b t a i n any data at all. S u c h a value should,

of

only be c o n s i d e r e d as a crude e s t i m a t i o n of the acute t o x i c i t y poten-

tial of the s u b s t a n c e

in question.

ACKNOWLEDGEMENT

The i n v e s t i g a t i o n s w e r e

f i n a n c e d by the P r o d u c t s C o n t r o l B o a r d and the Re-

s e a r c h C o m m i t t e e of the N a t i o n a l S w e d i s h E n v i r o n m e n t P r o t e c t i o n Board. We are m o s t g r a t e f u l to Ms M Hansson,

Ms B S a m u e l s s o n and Mrs G A k e r m a n for skilful

technical assistance.

REFERENCES

i.

B e n g t s s o n B-E. M a r P o l l u t B u l l 9

2.

L l o y d R, T o o b y T E. B u l l E n v i r o n Contain T o x i c o l 2 2

(1978) 238. (1979) i.

622

3.

Linden E, Bengtsson B-E, Svanberg O, Sundstr~m G. Ch~-~gsphere 8 843.

(1979)

4.

Crisp D J, Christie A O, Ghobasky A F A. Comp Biol Physlol 22 (1967) 629.

5.

Veith G D, Call D J, Brook L T. Can J Fish Aquat Sci 40 (1983) 743.

6.

Lundahl P, Cabridenc R. Water Ree 12 (1978) 25.

7.

Kopperman H L, Carlsson R M, Caple R. C h e m Biol Interactions 9 (1974) 245

8.

Ferguson J. Proc R Soc London Ser B 127

9.

Hansch C, Quinlon J E, Lawrence G L. J Org C h e m 33 (1968) 347.

(1939) 387.

I0. Leo A, Hansch C, Elkins D. C h e m Rev 71 (1971) 525. (Received in The Netherlands

2 March 1984; accepted 2 April 1984)