RESIDUE REVIEWS

VOLUME 23

RESIDUE REVIEWS Residues of Pesticides and Other

Foreign Chemicals in Foods and Feeds

RUCKST ANDS .. BERICHTE Riickstande von Pesdciden und anderen

Fremdstoffen in Nahrungs- und Futtermitteln

Edited by

FRANCIS A. GUNTHER Riverside, California

ADVISORY BOARD

F. BAR, Berlin, Germany • F. BRD-RAsMUSSEN, Copenhagen, Denmark J. W. COOK, Washington, D.C .• D. G. CROSBY, Davis, California

S. DORMAL-VAN DEN BRUEL, Bruxelles, Belgium C. 1. DUNN, Wilmington, Delaware • H. FREHSE, Leverkusen-Bayerwerk, Germany

J. C. GAGE, Macclesfield, England· H. GEISSBUHLER, Basel, Switzerland S. A. HALL, Beltsville, Maryland • T. H. HARRIS, Bethesda, Maryland

L. W. HAzLETON, Falls Church, Virginia • H. HURTIG, Ottawa, Canada o. R. KLIMMER, Bonn, Germany • G. K. KOHN, Richmond, California

H. F. LINSKENS, Nijmegen, The Netherlands • H. MAIER.BoDE, Bonn, Germany N. N. MELNIKOV, Moscow, U.S.S.R. • R. MESTRES, Montpellier, France

P. DE PIETRI·ToNELLI, Milano, Italy • R. TRUHAUT, Paris, France

VOLUME 23

SPRINGER-VERLAG

BERLIN • HEIDELBERG • NEW YORK

1968

ISBN-13: 978-1-4615-8439-1 e-ISBN-13: 978-1-4615-8437-7 DOl: 10.1007/978-1-4615-8437-7

All rights, especially that of translation into foreign languages, reserved. It is also forbidden to reproduce this book, either whole or in part, by photomechanical means (photostat, microfilm and/or microcard) or by other procedure without written permission from the Publishers.

© 1968 by Springer-Verlag New York Inc.

Softcover reprint of the hardcover 1st edition 1968

Library of Congress Catalog Card Number 62-18595.

The use of general descriptive names, trade names, trade marks, etc. in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by anyone.

Title No. 6625

Preface

That residues of pesticide and other "foreign" chemicals in foodstuffs are of concern to everyone everywhere is amply attested by the reception accorded previous volumes of "Residue Reviews" and by the gratifying en­thusiasm, sincerity, and efforts shown by all the individuals from whom manuscripts have been solicited. Despite much propaganda to the contrary, there can never be any serious question that pest-control chemicals and food­additive chemicals are essential to adequate food production, manufacture, marketing, and storage, yet without continuing surveillance and intelligent control some of those that persist in our foodstuffs could at times conceivably endanger the public health. Ensuring safety-in-use of these many chemicals is a dynamic challenge, for established ones are continually being displaced by newly developed ones more acceptable to food technologists, pharma­cologists, toxicologists, and changing pest-control requirements in progressive food-producing economies.

These matters are also of genuine concern to increasing numbers of governmental agencies and legislative bodies around the world, for some of these chemicals have resulted in a few mishaps from improper use. Adequate safety-in-use evaluations of any of these chemicals persisting into our food­stuffs are not simple matters, and they incorporate the considered judgments of many individuals highly trained in a variety of complex biological, chemi­cal, food technological, medical, pharmacological, and toxicological dis­ciplines.

It is hoped that "Residue Reviews" will continue to serve as an integrat­ing factor both in focusing attention upon those many residue matters requiring further attention and in collating for variously trained readers present knowledge in specific important areas of residue and related en­deavors; no other single publication attempts to serve these broad purposes. The contents of this and previous volumes of "Residue Reviews" illustrate these objectives. Since manuscripts are published in the order in which they are received in final form, it may seem that some important aspects of residue analytical chemistry, biochemistry, human and animal medicine, legislation, pharmacology, physiology, regulation, and toxicology are being neglected; to the contrary, these apparent omissions are recognized, and some pertinent manuscripts are in preparation. However, the field is so large and the in­terests in it are so varied that the editor and the Advisory Board earnestly solicit suggestions of topics and authors to help make this international book-series even more useful and informative.

"Residue Reviews" attempts to provide concise, critical reviews of timely advances, philosophy, and significant areas of accomplished or needed en­deavor in the total field of residues of these chemicals in foods, in feeds, and in transformed food products. These reviews are either general or specific, but properly they may lie in the domains of analytical chemistry and its method­ology, biochemistry, human and animal medicine, legislation, pharmacology, physiology, regulation, and toxicology; certain affairs in the realm of food technology concerned specifically with pesticide and other food-additive problems are also appropriate subject matter. The justification for the prepara­tion of any review for this book-series is that it deals with some aspect of the many real problems arising from the presence of residues of "foreign" chemicals in foodstuffs. Thus, manuscripts may encompass those matters, in any country, which are involved in allowing pesticide and other plant­protecting chemicals to be used safely in producing, storing, and shipping crops. Added plant or animal pest-control chemicals or their metabolites that may persist into meat and other edible animal products (milk and milk products, eggs, etc.) are also residues and are within this scope. The so-called food additives (substances deliberately added to foods for flavor, odo!', ap­pearance, etc., as well as those inadvertently added during manufacture, packaging, distribution, storage, etc.) are also considered suitable review material.

Manuscripts are normally contributed by invitation, and may be in English, French, or German. Preliminary communication with the editor is necessary before volunteered reviews are submitted in manuscript form.

Department of Entomology University of California Riverside, California May 3, 1968

F.A.G

Table of Contents

Use and residues of mercury compounds in agriculture By N. A. SMART . 1

Pesticide residues in Canada By A. B. SWACKHAMER 37

Ueber den Abbau von Dazomet im Boden By N. DRESCHER and S. Orro . 49

Examinations of Danish milk and butter for contaminating organo­chlorine insecticides By F. BRO-RASMUSSEN, Sv. DALGAARD-MIKKELSEN, Th. JAKOB-SEN, Sv. O. KOCH, F. RODIN, E. UHL, and K. VOLDUM-CLAUSEN. 55

The fundamental kinetics of cholinesterase reaction with substrates and inhibitors in an automated, continuous flow system By G. Voss.

The dipyridylium herbicides, paraquat and diquat By A. A. AKHAVEIN and D. 1. LINSCOTT

Subject Index .

Manuscripts in Press

71

97

147

152

Use and residues of mercury compounds in agriculture

By N. A. SMART*

Contents

I. Introduction . II. Mercury compounds used

III. Diseases controlled and rate of use of mercury compounds IV. Formulation and extent of use

(a) Formulation (b) Extent of use

V. Methods of residue analysis (a ) Wet oxidation-thiocarbazone methods (b) Schoniger flask combustion methods (c) Neutron·activation analysis (d) General

VI. Residues in edible crops and tissues (a) Apples . (b) Pears (c) Tomatoes ( d) Potatoes (e) Grain (f) Animal material, including eggs (g) Water . (h) Soil. (i) Other crops and materials

VII. Movement of mercury in plants and trees VIII. Regulatory Summary Resume. Zusammenfassung References .

2 3 4 9 9 9

10 10 14 15 15 15 16 19 19 20 21 22 25 26 26 27 29 30 31 32 32

.. Ministry of Agriculture, Fisheries & Food, Plant Pathology Laboratory, Hatching Green, Harpenden, Hertfordshire, England.

2 N. A. SMART

I. Introduction

Mercury compounds were first used in Germany as seed dressings to con­trol seed-borne diseases of cereals about 1914. 'Uspulun', described as a 'chlorophenolmercury' compound was placed on the market in 1915 by Bayer A.G. as a liquid dressing and was soon widely used. Dusts, such as 'Ceresan', at first having phenylmercury acetate as the active ingredient and, subsequently, a methoxyethylmercury compound, and 'Agrosan', a tolylmercury acetate formulation, became more widely used ten years later. Liquid treat­ments, using alkylmercury active ingredients, were reintroduced about the time of World War II because of the reduced hazards and inconvenience to operators dressing the grain in specially designed machines, although alkyl­mercury compounds are more toxic than arylmercurials. In Sweden it has recently been found that the extensive use of alkylmercury compounds can lead to harmful contamination of the environment and there they have been replaced by other organomercury compounds. In most countries both liquid and dust treatments are commonly used. Formulations containing organo­mercurials have been found to give a better control of fungal diseases of grain than non-mercurial formulations.

Mercury compounds are now also used as foliar sprays, as aerosols in glasshouses, in the wood pulp and paper industries, and in a variety of other ways. The monetary value of mercury compounds used in world agriculture is at least five million pounds sterling per annum.

The possible hazardous nature of mercury residues in foodstuffs was emphasized in the 1950's at Minamata, Kyushu, Japan. An efHuent system from a chemical factory had been opened for passing waste containing, among other products, amounts of mercury into Minamata Bay. The bay was regularly used as a source of seafood for many of the families inhabit­ing the eleven small villages along its shores. In 1953 severe neurological disorders among people living in the area were recorded, most ending fatally or with severe disability. All the persons affected had eaten fish or shell­fish, which contained high levels of mercury, caught on the bay. Cats and fish-eating birds were also affected. A methylmercury compound in the fish was considered to be the main toxic agent.

In January, 1966, the Swedish Royal Commission on Natural Resources held an international symposium in Stockholm on a number of facets of the mercury residue problem; the extent to which grain is dressed, mercury levels in the Swedish aquatic environment, modern methods of determining mercury residues such as neutron-activation analysis, the chemical nature of mercury residues, and other related topics were covered. Attention was drawn to the importance of distinguishing between alkyl, aryl, and inor­ganic mercury in order to evaluate the potential hazards of mercury residues

Residues of mercury compounds 3

and chromatographic procedures for achieving these separations were pre­sented. Although proceedings of this interesting symposium have not yet been published, some of the work presented there is discussed in this pres­ent review.

Information on the diseases controlled, mode of application and residues of mercury compounds in crops and foodstuffs, and methodology of residue analysis are widely dispersed in numerous publications; this review brings together essential data for those concerned with crop protection and its problems.

II. Mercury compounds used

Organomercury compounds are widely used in world agricultute con­trasted with lesser amounts of inorganic mercury compounds. The former may be divided, chemically, into three groups: alkylmercury, alkyloxyalkyl­mercury, and arylmercury compounds. Many of the organomercury com­pounds may be regarded as salts of the moderately strong bases methyl­mercury, ethylmercury, and alkyloxyalkylmercury hydroxides, or the weaker base phenylmercury hydroxide, with acids such as hydrochloric, hydrobromic, hydriodic, nitric, acetic, propionic, lactic, salicylic, benzoic, and silicic. Com­pounds known to be used in world agricultute at the present time are:

Alkylmel'cul'Y compounds Methylmercury sulphate, acetate, nitrile, propionate, 8-hydroxyquinolate,

2,3-dihydroxypropyl mercaptlide, pentachlorophenolate, p-chlorobenzoate, benzoate, dicyandiamide

N -Methylmercury I,2,3,6-tetrahydro-3,6-endomethano-3,4,5,6, 7 ,7 -hexachloro­phthalimide

Ethylmercury silicate, chloride, bromide, phosphate, acetone, urea, oleate, stearate, pentachlorophenolate, hydroxide, thiouronium chloride, p­toluene sulphonamide, 8-hydroxyquinolate

N -Ethylmercury 1,2,3 ,6-tetrahydro-3 ,6-endomethano-3 ,4,5 ,6, 7,7 -hexachloroph­thalimide

Ethyl phenethynylmercury Mercury pentanedione

Alkyloxyalkylmel'cury compounds Methoxyethylmercury ohloriide, silicate, dicyandiamide, benzoate, lactate,

acetate Ethoxyethylmercury ohloride, silicate, hydroxide I-Carboxy-3-ethoxyethylmercury propandicarboxylate Chloromethoxypropy lmercury acetate p- (t-Oetyl) phenoxyethoxyethyl dimethylmercury benzyl ammonium chloride

Al'ylmel'cury compounds Phenylmercury acetate, dimethyl dithiocal1bamate, chloride, dinaphthyl methane

sulphonate, urea, nitrate, iodide, benzoate, pyrocatechinate, triethanol ammonium lactate, 8-hydroxyqUlinolate, hydroxide, lactate, oleate, pro­pionate, salicylate, salicylanilide, formamide, naphthenate

4 N. A. SMART

N -Tolylmercury-p-toluene sulphanilide, tolylmercury chloride Hydroxymercurichlorophenol, ihydroxymerourinitrophenol Cresolmercury naphthenate Diphenylmercury dodecenyl succinate o-(Hydroxymercury) benzoic acid

Inorganic mercury compounds Mercurous chloride Mercutlic chloride Mercuric oxide

III. Diseases controlled and rate of use of mercury compounds

Many fungus diseases of seeds, bulbs, plants, fruits, and vegetation are controlled by mercury compounds, as shown in Table 1. Mercury compounds are used for control of soil-borne fungi as well as of seed-borne diseases in the United States whereas in most European countries they are predomi­nantly used to control the latter.

Phenylmercury acetate is widely used in the wood-pulp and paper-making industry: about 90 percent of ground wood pulp exported from Norway, Sweden, Canada, and the United States contains up to 20 p.p.m. of mer­curial and the compound is also widely used in slime control. A minor use of organomercury compounds in forestry is in helping to preserve cut timber.

Table I also gives the range of rate of use of mercurials in world agri­culture as far as can be ascertained. The ranges are large in some cases re­flecting the different practices of countries.

Tab

le I

. D

isea

ses

cont

rolle

d by

mer

cury

com

poun

ds a

nd r

ates

and

tim

e of

app

lica

tion

for

con

trol

Nam

e of

dis

ease

St

age

of

grow

th

Rat

e of

app

lica

tion

of

mer

curi

al

whe

n m

ercu

rial

C

omm

odity

C

omm

on

Scie

ntif

ic

appl

ied

g. H

g/kg

. g.

Hg/

ha.

:ere

als

Whe

at

Bun

t T

illet

ia c

arie

s T.

foe

tida

Bar

ley

Roo

t ro

t, se

edlin

g bl

ight

Fu

sariu

m s

p.

Cov

ered

sm

ut

Usti

lago

hor

dei

Lea

f st

ripe

py

reno

phor

a gr

amin

ea

Net

blo

tch

Pyre

noph

ora

tere

s O

ats

Loo

se s

mut

U

stila

go a

vena

e Se

ed

0.0

1-2

.5

Lea

f sp

ot

pyre

noph

ora

aven

ae

-C

over

ed s

mut

U

stila

go h

orde

i R

ye

Stri

pe s

mut

U

rocy

stis

occu

lta

Snow

mou

ld

Fusa

rium

ni

vale

B

unt

T il

ktia

car

ies

Mai

ze

HeI

min

thos

pori

um

leaf

spo

t H

elm

inth

ospo

rium

spp

. R

ice

Bla

st

Piric

ular

ia o

ryza

e Se

ed, d

urin

g gr

owth

0

.02

-0.4

1

0-6

0

mal

l gr

ains

} ·{;C~~·~

Sorg

hum

Se

edlin

g bl

ight

, dr

y C

anar

y se

ed

rot,

seed

born

e di

s-Fu

sariu

m s

p. a

nd o

ther

s Se

ed

0.1

-0.4

-

Lin

seed

ea

ses,

pre

-em

erge

nce

Mil

let

rots

s: P: ib

res

Flax

L

eaf

spot

M

ycos

phae

rella

lin

orum

Se

ed

Seed

ling

blig

ht

Col

leto

tric

hum

lin

icol

a 0

.02

-0.3

-

Cot

ton

Ant

hrac

nose

G

lom

erel

lrl g

ossy

pii

Soil,

fu

rrow

, se

ed

0.0

2-0

.3

11

~ f a. J j 8 a. Ul

Tab

le 1

. (C

onti

nued

)

Nam

e of

dis

ease

Com

mod

ity

Com

mon

Sc

ient

ific

Top

fru

it

App

le

Scab

(bl

ack

spot

) V

entu

ria

inae

qual

is

Can

ker

Nee

tria

gal

ligen

a B

lotc

h G

loeo

des

pom

igen

a Fi

re b

ligh

t E

rwin

ia a

myl

ovor

a P

ear

Scab

V

entu

ria

piri

na

Scab

V

. pi

rina

A

pric

ot

} C

herr

y B

row

n ro

t M

onil

inia

fru

etie

ola

Peac

h

Nut

s Alm

ond

--

Wal

nut

Lea

f sp

ot

Sept

oria

nig

ro-m

aeul

ans

Soft

fru

it S

traw

berr

y L

eaf

blig

ht

Den

drop

hom

a ob

seur

ans

Lea

f sp

ot

Mye

osph

aere

lla f

raga

riae

Cue

urbi

ts

Cuc

umbe

r,

rock

mel

on

Gum

my

stem

bli

ght

M.

mel

onis

W

ater

mel

on,

pum

pkin

A

nthr

acno

se

Col

leto

trie

hum

lag

enar

ium

Sq

uash

, re

late

d pl

ants

Se

ed r

ot,

dam

ping

-off

P

ythi

um s

p.

Seed

rot

, da

mpi

ng-o

ff

Rhi

zoct

onia

sp_

Stag

e of

gr

owth

· w

hen

mer

curi

al

appl

ied

} Preb

loss

om,

earl

y fr

uit

grow

th,

post

-har

vest

To

peta

l fa

ll

Dor

man

t

Pet

al f

all

Pop

corn

sta

ge

to

peta

l fa

ll

- - - - - - -

Rat

e of

app

lica

tion

of

mer

curi

al

g.H

g/kg

. g.

Hg/

ha.

-1

5-4

00

-4

5-2

50

-

800

{ 20

-

20

-40

2

0-4

0

-1

20

-60

0

--

--

--

--

1-2

-

--

--

0\ ?: i> ~

Tab

le 1

. (C

onti

nued

)

Nam

e of

dis

ease

Com

mod

ity

Com

mon

Sc

ient

ific

Trop

ical

cro

ps

Pea

nut

Cro

wn

rot

-P

inea

pple

B

ase

rot

-Su

garc

ane

Sett

rots

M

aras

miu

s sp

. Se

tt r

ots

Pyt

hium

sp.

Roo

t cro

ps

Pot

ato

Pot

ato

blig

ht

Phy

toph

thor

a in

/est

ans

Pot

ato

root

ee

lwor

m

Bet

erod

era

rost

ochi

ensi

s B

lack

scu

rf

Cor

ticiu

m s

olan

i G

angr

ene

Pho

ma

sp.

Skin

spo

t O

ospo

ra p

ustu

lans

D

ry r

ot

Fus

ariu

m c

aeru

leum

Sw

eet

pota

to

Bla

ck r

ot

Cer

atoc

ystis

fim

bria

ta

Scur

f M

onilo

chae

tes

in/u

scan

s C

arro

t

} C

eler

y Se

ed·r

ot,

dam

ping

-off

P

ythi

um s

p.

Par

snip

S

ugar

bee

t B

lack

leg

P

leos

pora

bet

ae

Bee

t, si

lver

bee

t Se

ed·r

ot,

dam

ping

-off

P

ythi

um s

p.

Stag

e of

gr

owth

w

hen

mer

curi

al

appl

ied

Seed

- - - F

olia

r sp

ray

Soil

cult

ivat

ion

- - - - - - Seed

Seed

Se

ed

Rat

e of

app

lica

tion

of

mer

curi

al

g.H

g/kg

. g.

Hg/

ha.

0.3

-0.4

-

--

--

--

-20

(w

ith

copp

er)

-6,

000

--

--

--

--

--

--

--

0.0

4-0

.12

-

--

~ f a. I I g. '" ......

Tab

le I

. (C

onti

nued

)

Nam

e of

dis

ease

Com

mod

ity

Com

mon

Sc

ient

ific

Vege

tabl

es

C.b

boge

, a""-}

flo

wer

, B

russ

els

Bla

ck r

ot

cam

pestr

is sp

rout

, tu

rnip

, C

lub

root

'a

bra

ssic

ae

radi

sh,

rela

ted

plan

ts

Pea

} Se

ed d

isea

ses

Bea

n T

omat

o L

eaf

mou

ld

fulv

um

Oth

er p

lant

s G

rape

vine

D

ead

arm

vi

ticol

a H

ops

Bac

teri

al d

ecay

of

setts

Bulb

s N

arci

ssus

B

asal

rot

po

rum

f. n

arci

ssi

Gla

diol

us

Bac

teri

al s

cab

narg

inat

a Tu

rf

Fusa

rium

pat

ch

Bro

wn

patc

h ni

L

eaf

blig

ht

'ium

sat

wum

C

urvu

lari

a :p

p

a 40

mg.

Hg/

1,00

0 cu

. ft.

gl

assh

ouse

.

Stag

e of

gro

wth

w

hen

mer

curi

al

appl

ied

Seed

ling

- Seed

Gro

win

g,

mat

ure

plan

t

Dor

man

t Se

tts

Bul

b - - - - -

Rat

e of

app

licat

ion

of m

ercu

rial

g.H

g/kg

. g.

Hg/

ha.

--

--

0.0

6-2

-

_a

-

-1,

300

0.1

-0.2

-

--

--

--

-5

00

-10

,00

0

--

--

00

~

l> ~

Residues of mercury compounds 9

IV. Formulation and extent of use

a) Formulation

The types and uses of organomercury formulations in agriculture, and the range within which mercury is present in these formulations, are given in Table II. Some inorganic mercury formulations are included for the sake of completeness.

Table II. Formulation of organomercury compounds in agricultural use

General use and type of formulation

Organic Mercury

Seed dressing Dry Wet Slurry

Bulb dips Liquid

Seed potato dips Soluble powder Liquid

Sugar cane dip

Glasshouse aerosol

Orchard canker paint

Orchard spray Dispersible powder Emulsifiable concentrate

Lawn fungicide

Inorganic merCMY

Lawn fungicide

Soil fungicide for potatoes etc.

b) Extent of use

% Hg in formulation

0.2-10 0.4-6 1.3-6

1.5-6

3 -9 0.5-6

6

0.35

2

0.6-40 0.4-40

3.4-6.7

45

10

Table III gives tonnages (in metric tons = 1,000 kg.) of mercury com­pounds used in or sold to agriculture in some countries of the world. Some figures are taken from the F.A.O. Production Yearbook 1965, while that for Great Britain is given by STRICKLAND (1966). The total tonnage of mer­cury compounds used in agriculture throughout the world is thus at least 2,100 metric tons.

10 N.A. SMART

Table III. Quantities of mercury compounds currently used in or sold to agriculture

Country

U.S.A.

Denmark

Germany

Great Britain

Bulgaria

Finland

Italy

Poland

Austria

Norway

Portugal

Turkey

Spain

Sweden

Morocco

Israel

New Zealand

Japan

Metric tons of Hg compounds

400

3.5

41

20

5

1

26

9

4

0.4

0.2

22.5

7.1

2

1

0.2

0.5

1,600 (approx.)

V. Methods of residue analysis

All the methods for mercury residue analysis given in the groupings be­low determine the total mercury and not that of an organomercurial as such. Information on organomercury residues, as distinct from total mercury resi­dues in vegetable tissue, is only just beginning to be available. WIDMARK

(1966) in Sweden is engaged in working out analytical techniques for iden­tification of organomercury compounds in vegetable and animal material using thin-layer and gas chromatography. Some methods are available for determining organomercury compounds in animal tissue (GAGE 1961, KIM­

URA and MILLER 1964).

a) Wet oxidation-thiocarbazone methods

One of the earliest methods for determining traces of mercury in vege­table material was that published by the Association of Official Agricultural

Residues of mercury compounds 11

Chemists in 1945. It consisted of digesting the sample, under reflux, with nitric acid and potassium permanganate and then with nitric acid followed by hydrogen peroxide. The mercuric ions were extracted with a chloroform solution of dithizone and, after employing a thiosulphate reversion tech­nique to remove oxidized dithizone and interfering metals. the mercury dithizonate was measured absorptiometrically. ARTHINGTON and HULME

( 1951) found the recoveries disappointing, however, when the method was applied to apple peel and described a nitric-sulphuric acid wet oxidation of freeze-dried apple peel in a long-necked flask fitted with a cold-finger con­denser. Mercuric ions were extracted with dithizone and, after purification by reversion, the amount of mercury was determined spectrophotometrically; 94 to 111 percent recoveries were reported. KUNZE (1948) had also in­vestigated earlier methods for determination of mercury residues in apple peel and obtained low recoveries. He found that addition of selenium to the digestion, either as the metallic powder or aqueous sodium selenate, led to almost quantitative recovery using both 1: 1 nitric-sulphuric acid and potas­sium permanganate-sulphuric acid wet oxidation.

More complete wet oxidation of the sample was obtained by the tech­nique introduced by KLEIN (1952) and adopted as an official method by the Association of Official Agricultural Chemists (1952). Digestion of the sample was carried out with nitric-sulphuric acids in a flask fitted with a trap on top of which is seated an efficient reflux condenser. As wet oxida­tion proceeded at the reflux temperature the aqueous condensate collected in the trap and the contents of the flask became more concentrated and a more vigorous oxidation took place enabling a more complete destruction of organic matter (no selenium was used). Mercuric ions were isolated by dithizone extraction and the extract cleaned up by a thiosulphate reversion technique. The absorption of the mercury-dithizone complex was measured at 490 mll. The digestion was modified for different materials: dried fruits, seeds, and grain required addition of water before the nitric acid; meat, fish, and other biological material should be left in contact with concentrated nitric acid for half an hour before diluting and heating to minimize froth­ing. Collaborative study of the method by recovering mercuric chloride from tomatoes gave excellent results.

ABBOTI' and JOHNSON (1957) found the KLEIN (1952) method sat­isfactory for tomatoes but not for apples, probably due to mercury being volatilized in the larger amount of carbon dioxide evolved in the latter case. They used the modified nitric-sulphuric acid digestion of KUNZE

( 1948) without a trap in the presence of selenium and extracted and deter­mined the mercury by the method of KLEIN; 90 to 95 percent recovery of added phenylmercury chloride at the 0.1 p.p.m. level was obtained. The method was sensitive to 0.01 p.p.m. of mercury in a 50g. sample. It should be noted that recovery of an organomercury compound is a fairer test of a residue method that recovery of mercuric salts, as the former are, in fact,

12 N. A. SMART

the compounds used in agriculture in most cases and are more volatile (PHILLIPS et al. 1959). ASHLEY (1959) also obtained satisfactory recovery data with apples at the 0.1 p.p.m. level by this method. The method was studied collaboratively by the loint Mercury Residues Panel (1961) of the Scientific Subcommittee on Poisonous Substances used in Agriculture and Food Storage, the Society for Analytical Chemistry, and the Association of British Manufacturers of Agricultural Chemicals, and recommended for use in determining mercury residues in tomatoes and apples. It was also recom­mended by the European Plant Protection Organisation (1963). SMART (1963 a and b, 1964) has successfully used the method, with slight modi­fication of the digestion stage, for determining mercury residues in grain, eggs, pullet muscle and liver, and potatoes.

BEIDAS and HIGGONS (1957 and 1959) in their work on mercury resi­dues in tomatoes and apples used the digestion method of KLEIN (1952). They oxidized the extracted mercuric dithizonate with permanganate, de­stroyed the excess of permanganate with sodium nitrite, and titrated the mercuric ions with dithizone; about 90 percent recovery of added mercury was obtained.

PICKARD and MARTIN (1960 b) employed wet oxidation with nitric­sulphuric acids in the presence of selenium using a trap, and then with hydrogen peroxide, following KLEIN's (1952) method. Ions interfering in the dithizone colorimetric determination were removed by complexing them with ethylenediaminetetracetic acid, and sodium metabisulphite was used to reduce oxidation of dithizone. Extraction with dithizone in carbon tetrachloride rather than chloroform, which is normally used, was pre­ferred. Recovery of mercury added to apples, tomatoes, and coffee beans at residue levels was 85 to 100 percent.

The International Union of Pure and Applied Chemistry (1965) have recommended a method for determination of mercury residues in food­stuffs. The organic material is digested with acids according to KLEIN ( 1952). Urea and hydroxylamine hydrochloride were added to remove oxidizing substances and the mercuric ions were extracted with dithizone in chloroform. A nitrite reversion procedure was then used and the mer­cury finally determined by a titrimetric or photometric method. Recovery data for the method were not given.

The Analytical Methods Committee of the Society for Analytical Chem­istry (1965) has recommended a method for determination of small amounts of mercury in organic matter after extensive investigations, in part con­ducted with radiotracers. After destruction of the organic matter by wet oxidation with nitric-sulphuric acids (no selenium) in an apparatus fitted with a trap, the acidity of the digest was adjusted to approximately normal and hydroxylamine hydrochloride added to destroy oxides of nitrogen. Dithizone in carbon tetrachloride was used to extract the mercuric ions which, after purification using a sodium nitrite reversion technique and

Residues of mercury compounds 13

addition of ethylenediaminetetracetic acid, were determined at 485m(J.. The method gave excellent recoveries and is sensitive to 0.5 (J.g. of mercury.

There seems little to choose between the four recommended or official methods of analysis for mercury residues although the ABBOTI and JOHN­

SON (1957) method has been widely used and no adverse comment has appeared in the literature. All the methods are lengthy and there seems to be scope for devising a more rapid wet-oxidation procedure.

TRUHAUT and BOUDENE (1961 and 1963) have described wet-oxida­tion methods in which purification of the extracted mercuric complex was effected by distillation. In one, the sample was first digested with nitric­sulphuric acids and hydrogen peroxide. After refluxing until free from solids the digest was distilled, until fumes appeared, into a receiver con­taining acidified potassium permanganate. After further oxidation, excess of oxidant was removed with hydroxylamine hydrochloride and urea and the pH was adjusted to 1 to 2. Mercuric ions were extracted with di-~­naphthylthiocarbazone and thence into acidified potassium permanganate. This solution was mixed with aqueous stannous sulphate and steam-dis­tilled. The distillate was titrated with di-,B-naphthylthiocarbazone solution. The authors claim that 0.02 to 0.002 p.p.m. of mercury can be determined with an accuracy of -t- five percent. KIMURA and MILLER (1962) have also used reduction with stannous ions followed by distillation to effect quan­titative separation of traces of mercuric ions from nitric-sulphuric acid digests. Dithizone was used in a photometric measurement. The method has been applied to samples of wheat, barley, and soil.

A more complete wet oxidation of vegetable and plant material than that obtained with nitric-sulphuric acids is obtained when perchloric acid is used with these acids. HORDYNSKA et at. (1961 and 1962) described this approach with a Gorsuch-Gnrust apparatus and used a dithizone pho­tometric measurement of the mercury. Recovery of phenylmercury acetate (20f.!,g.) from grain was 96.5 -t- 3.8 percent and of phenylmercury-8-hy­droxy-quinolate from apples was 97.7 percent. WARD and MCHUGH (1965) have also used this approach as has EPPS, Jr. (1966). The latter worker used it for rice and obtained 95 to 100 percent recovery of phenylmercury acetate.

In all the methods discussed above the mercury thiocarbazone has been measured spectrophotometrically or visually in titration. In the method of JACOBS and GOLDWATER (1961) the mercury dithizonate, obtained by ex­tracting the digest of the sample, was transferred to a furnace where it decomposed and the mercury vapour in the effluent was measured with a mercury vapour meter. This method has been modified by KUDSK (1965). LINDSTROM (1959) had earlier used a mercury vapour meter to determine residues in aqueous extracts of grain and wood pulp without using dithizone extraction.

In the cases of some vegetable materials it is difficult to srart the wet-

14 N. A. SMART

oxidations described above smoothly and losses of mercury can occur through traces being carried away in a vigorous evolution of gases. PIECHOCKA

(1961), in his method, first digested the sample (milk, meat, vegetables, beer, vinegar) with pancreatin at pH 8 or pepsin at pH 1 and then wet­oxidized it with acidified permanganate. Mercuric ions were determined using a titrimetric endpoint with dithizone. The sensitivity was given as 0.5 /Ag. of mercury.

Wet ashing-dithizone methods for determination of mercury in soils have been described by PICKARD and MARTIN (1963) and by VASILEVSKAYA

and SHCHERBAKOV (1963).

b) Schoniger flask combustion methods

Schoniger flask combustion was first used for determination of mercury residues by GUTENMANN and LISK (1960). Apple tissue (10g.) was dried on cellophane overnight under vacuum and then burned in a five-I. oxygen­filled flask having a balloon attachment for pressure control. The mercuric ions were determined spectrophotometrically after extraction with dithizone. Re­covery of added mercury from apples averaged 84 percent. Twelve sam­ples could be analysed in a day.

BERCK (1963) used Schoniger flask combustion for determining mer­cury residues on treated seed. As little as one seed could be analysed but greater accuracy was obtained with larger amounts. Mean recovery of phenyl­mercury acetate from wheat, barley, oats, rye, and flax at 1 to 7 /Ag. was 89 to 104 percent with s.e. 8.4 to 8.5 percent for wheat and flax. JONES

and SCHWARTZMAN (1963) have also used Schoniger flask combustion for determining mercury residues in wheat.

RAJAMA et at. (1964) have used the oxygen-flask method for deter­mining mercury residues in eggs. The sample was first freeze-dried and tablets of 1 to 1.5 g. were prepared from the city product for ignition. The ignition products were extracted with dithizone and the mercury deter­mined spectrophotometrically. The method was sensitive to 0.1 p.p.m. of mercury. It may be mentioned that no report has appeared in the literature, as far as the reviewer is aware, of an investigation into whether mercury is lost during freeze-drying samples containing organomercury residues. Several workers have used this or a similar technique and until the point is elucidated such work must be treated with caution.

PAPPAS and ROSENBERG (1966 a) have also used Schoniger flask com­bustion for determining mercury on grain. One g. samples were burned and the combustion products were absorbed in hydrochloric acid. After neutral­ization the mercuric ions were collected as the sulphide and co-precipitated on cadmium sulphide-impregnated asbestos pads. The pads were pyrolysed at 650°C. and the mercury was determined in a cold vapour atomic absorp­tion cell (SCHACHTER 1966). Mercury recoveries from wheat at levels be­tween 0.01 and 2.00 p.p.m. averaged 86 percent.

Residues of mercury compounds 15

c) Neutron-activation analysis

Several workers in Sweden have described the application of neutron­activation analysis to the determination of mercury residues (LJUNGRBN

and WESTERMARK 1960, SJOSTRAND 1964, CHRISTELL et al. 1~65). A sam­ple of the order of one g. was sealed in a quartz tube and irradiated for twO to four days in a thermal neutron flux of about 1012 neutrons/ cm.2 / sec. The contents of the vials were dissolved by digesting with nitric-sulphuric acids using a Bethge apparatus. Separation of the mercury was achieved by dis­tilling it with nonradioactive carrier material into the Bethge trap with perchloric acid and glycine, followed by electrolysis onto a gold foil kathode. The 77 ke V-radiation of the 197Hg was measured using a multi-channel analyser. Recovery of added mercury was 85 to 90 percent. The great ad­vantage of the neutron-activation method over the classical wet-oxidation­thiocarbazone methods previously described lies in its greater sensitivity (0.5 ng.,! g. sample).

SZKOLNIK (1965) has performed neutron-activation analysis of mer­cury residues in apples by SchOniger flask combustion and compared the results obtained with those from a dithizone method. The agreement be­tween the two methods was not always good, although the number of sam­ples compared was relatively small.

SMITH (1963) has described analysis of mercury in biological material by a neutron-activation method but he did not specify the actual sample type examined. The 203Hg was determined by scintillation counting. TOMIZAWA (1966) has developed a neutron-activation method for deter­mining 0.01 to 1.0 !lg. of mercury in rice grains. The samples were irradiated for 130 hours in a thermal neutron flux of 2 to 3 X 1013 neutrons/cm.2/sec. The mercury was separated as the mercurous iodide-copper ethylenediamine­tetracetic acid complex.

d) General

BRISKI (1966) has surveyed qualitative, semi-quantitative, and quantita­tive analytical methods for the determination of 1 to 10 !lg. of mercury in various seeds treated with mercurial fungicides. Qualitative methods, having a sensitivity of about two p.p.m. of mercury, are based on the formation of amalgams with copper or aluminium. A paper chromatographic semi-quan­t~tative test, having a sensitivity of 0.5 to 2.0 p.p.m. of mercury, can be car­tIed out after mineralization of organic matter. A microbiological method using Sarcina /lava is useful.

VI. Residues in edible crops and tissues

All vegetable and animal materials contain traces of mercury. These traces are very small, often being of the order of nanograms (ng. ) or tens of

16 N. A. SMART

nanograms per gram. The actual amount depends on the locality from which the sample was taken, its species, and other factors. It is, therefore, necessary to evaluate the mercury residue arising from the use of mercury compounds in agriculture against the background of the naturally occurring mercury. In reviewing the residues of mercury found in agriculture, background levels of mercury will therefore be noted, where these are known. Finally, in this section some data on the background levels of mercury in water and in soils are given.

a) Apples

The earliest paper on mercury spray residues in apples is that of HOWARD

(1947) in the United States who found residues of 0.02 to 0.19 p.p.m. at harvest in 1944 and 0.023 to 0.357 p.p.m. in 1945. However, in view of the early stages at which methodology in analysis of mercury residues was at this time the figures should be treated with some reserve. The same is perhaps true of the residues given by COHEN (1951), although these are on firmer ground as a collaborative recovery data for the analytical method used (that of KUNZE 1948) are given. COHEN found an average residue of 0.06 p.p.m. of mercury with a range of 0 to 0.36 p.p.m. FORD (1952) re­ported spray residues on apples determined by dissolving the surface residues in boiling three percent nitric acid. We now know that appreciable amounts would have also been found in the flesh of the samples so that his values were low through incomplete extraction. Further, losses of mercury would have occurred in the several evaporation stages involved in his method so that for this reason also his results were likely to be low.

Early authoritative work in Great Britain was done by MARTIN and PICKARD (1957) who reported residues resulting from the experimental spraying of Bramleys Seedling apples with a 1.6 percent mercury formula­tion at the normal rate of two lb./lOO gal. (0.003 percent mercury) using five to six gal./tree to ensure complete wetting. Seven applications were given from green cluster stage to within eight weeks of harvest. A mean residue of 0.06 p.p.m. of mercury, in the range 0.02 to 0.12 p.p.m., was found in the apples, 75 percent being in the flesh. Trees on a commercial fruit farm also receiving seven applications, mostly at 0.002 percent mercury, gave apples having a residue of 0.02 to 0.09 p.p.m. An unsprayed control contained 0.01 p.p.m. of mercury. Initial tests showed that the mercury was present as phenylmercury nitrate and chloride after a limited period between spraying and harvest at the end of the season. Subsequently, PICKARD and MARTIN

(1959) found 0.04 and 0.03 p.p.m. of mercury in Bramleys Seedling fruits from an orchard which had been given a normal commercial spray treatment of organomercurial. Fruits harvested from an orchard that had received three full-coverage pre-petal-fall applications of phenylmercury nitrate at full strength, followed by seven post-petal-fall treatments at half strength con-

Residues of mercury compounds 17

tained 0.04 p.p.m. of mercury (fow: determinations). In the first of these cases 70 to 80 percent of the mercury was in the peel whereas in the second case, the reverse was found, almost 70 percent being present in the fleshy tissue. PICKARD and MARTIN also described determinations of the levels of the mercury on apple leaves, bark, and wood after spraying. Most of the mercury falling on shoots and entering the tree is retained in the bark. These workers described further work in 1961 showing somewhat higher residues than the 0.05 p.p.m. they had previously reported in apples.

BEIDAS and HIGGONS (1959) have reported residues from the application of phenylmercury nitrate sprays. In 1956, 5 to 10 sprays were normally applied at two-week intervals from April to July giving a twelve-to-sixteen week interval between last application and harvest. Residues of less than 0.01 p.p.m. of mercury were found in each of the fow:teen samples analysed. Two control samples also showed less than 0.01 p.p.m. In 1957 dessert varieties were given five to ten sprays of phenylmercury nitrate. In ten samples, five to six weeks were allowed between last application and harvest when residues of mercury were 0.08 p.p.m. or less. In five samples, seven to

fifteen weeks elapsed between last application and harvest and residues were all less than 0.01 p.p.m. of mercury. An untreated control showed less than 0.01 p.p.m.

ASHLEY (1959) reported residues of 0.04 to 0.12 p.p.m. of mercury in apples from seven commercial orchards treated with 'PP' Liquid Mercury Plus. An untreated sample of Cox's Orange Pippin contained 0.02 p.p.m. of mercury. MILLER (1956) described residues of mercury in seven com­mercial samples of apples having received up to ten applications of organo­mercurial fungicidal sprays ten weeks or more before harvest; residues were 0.07 p.p.m. or less, and a control sample showed 0.01 p.p.m. SMART (1961) found residues well below 0.1 p.p.m. five or more weeks after the last of up to eight applications in eight samples from commercial orchards. Controls contained less than 0.005 p.p.m. of mercury.

TEW and SILLIBOURNE (1965) have examined the residues occurring when phenylmercury compounds are applied to apple trees dw:ing the dormant season to suppress ascospore production by Venturia inaequalis. In the absence of organomercury sprays in the succeeding summer, mercury could not be found in the fruit at harvest. Even when post-harvest or dormancy applications preceded conventional summer schedules of organomercurials, the residues at harvest were not increased: after fow: summer sprays of phenylmercury nitrate at 0.003 percent mercury the residue was 0.05 + 0.01 p.p.m. and after nine summer sprays of phenylmercury oxinate at 0.002 percent mercury the residue was 0.02 p.p.m.

In New Zealand, STONE et al. (1957) sampled apples from experimental orchards receiving various spray schedules of 2 to 2Y2 percent phenylmercury chloride at two Ib./lOO gal. up to closed calyx stage and one Ib./lOO gal. afterwards. The total mercury content of the peel and pulp ranged from

18 N. A. SMART

0.011 to 0.135 p.p.m. When no mercury was applied after the closed calyx stage the level was generally less than 0.05 p.p.m. STONE et al. (1964) later reported residues in Sturmer Pippin apples treated with phenylmercury di­methyl dithiocarbamate (Phelam) at one Ib./lOO gal; the residues were less than 0.05 p.p.m.

NARDIN (1965), in Australia, analysed nineteen commercial samples of apples that had received organomercurial sprays and found 0.001 to 0.052 p.p.m. of mercury. The mean, over two seasons was 0.021 p.p.m.

In the United States GUTENMANN and LISK (1960) determined the mercury residues in eleven plots after several applications of phenylmercury 8-hydroxyquinolate, mainly in the spring at 1.25 pt. of 20 percent active ingredient/lOa gal. and diphenylmercury ammonium S-hydroxyquinolate at 0.5 pt. of 10 percent active ingredient/lOO gal. Up to O.lS p.p.m. of mercury was found in the apples. JACOBS and GOLDWATER (1961) reported decay curves of mercury residues in Stayman and Red Delicious apples after an application of alkymercury fungicide. At harvest, eleven weeks after the ap­plication, residues of 0.07 and 0.06 p.p.m. respectively, were obtained. Un­sprayed control apples contained 0.01 p.p.m. at harvest although earlier in the season values as high as 0.16 p.p.m. had been obtained. The latter values are very high indeed for background mercury. The maximum residue at the end of the growing season for other sprayed samples was 0.09 p.p.m. of mercury while that for controls was O.OS p.p.m. It is difficult to evaluate the real meaning of JACOBS and GOLDWATER'S figures in view of the variable and sometimes abnormally high background of mercury reported in their trials. SZItOLNIK et al. (1965) investigated residues of phenylmercury acetate after one to five applications, usually between the delayed-dormant and petal­fall stages, and found residues of up to 0.05 p.p.m. of mercury; 0.01 p.p.m. was found in apples from trees known not to have received any application of mercurial for ten years.

In Canada, ROSS and STEWART (1960) found residues of up to 0.125 p.p.m. of mercury in the peel of apples receiving phenylmercury acetate and phenylmercury dimethyl dithiocarbamate commercial formulations at 0.5 pt. and one lb. of 10 percent and three percent of active ingredient, respectively, per 100 gal. Less mercury was found in the flesh of the apples. The total residue in Cortland apples after summer spraying was 0.05 p.p.m. of mercury. Control McIntosh apples showed 0.003 p.p.m. Data were also given in this paper of the decline of mercury residues in apple leaves after spraying. These workers subsequently found residues of about 0.05 p.p.m. in apples from experimental plots after spraying once with phenylmercury acetate at 0.5 pt. of 10 per cent active ingredient/lOO gal. in the summer (STEWART and ROSS 1960).

BERNSTEIN et al. (1962) reported that apples from trees sprayed three times with phenylmercury S-hydroxyquinolate before petal-fall showed no

Residues of mercury compounds 19

mercury residue in the apples. One spray on small fruit, which was not effective in controlling scab, gave a residue of 0.008 p.p.m. of mercury.

b) Pears

There are no published data on residues of mercury in pears. However, information from Western Australia (1967) gives 0.04 p.p.m. in an un­sprayed sample and 0.14 to 0.26 in pears sprayed four times in the spring and early summer with 0.002 percent mercury formulation. Two to four months elapsed between the last application and harvest.

c) Tomatoes

BEIDAS and HIGGONS (1957) investigated the mercury residues arising from the use of an aerosol containing 0.2 percent organically-combined mercury, as phenylmercury chloride, at three fluid oz./ 4,000 cu. ft. in glass­houses. Residues were found to be variable, up to 0.50 p.p.m. being obtained; up to 90 percent was found in the pulp of the tomatoes.

LLOYD (1958) described a similar investigation into residues arising from using an aerosol containing 0.3 percent w/w organically combined mercury, as phenylmercury salicylate, at a standard rate of one fluid oz./1,700 cu. ft., and also a more concentrated formulation at a proportionately lower dosage, in glasshouses. He found that the mercury residue is not governed by the date of application, the residues hardly decreasing with time, but it is gov­erned by the rate of application; as an example of the latter a residue of 0.014 p.p.m. was found after application at 0.9 time the standard rate compared with 0.033 p.p.m. at 1.3 times the standard rate. Path samples always con· tained more mercury than eaves samples. The number of weekly applications that could be made to a particular fruit varied from ten on fruit maturing early in the season to six on fruit maturing late in the season. A maximum level of 0.17 p.p.m. of mercury was found in one path sample after seven applications at 1.3 times the standard rate (the corresponding eaves sample contained 0.035 p.p.m.); 0.09 p.p.m. was found after six applications at the standard rate. Only when the method of application of aerosol, which is normally ejected down the central path, was abused by directing the spray onto the fruit were levels in excess of 0.2 p.p.m. recorded.

STONE and CLARK (1958) investigated residues arising from one of the aerosols used by Lloyd, 40 m!. of 0.56 percent phenylmercury salicylate formula­tion being applied to 5,000 cu. ft. glasshouse space. A number of applica­tions of aerosol was made at intervals and the mercury content found in treated tomatoes was 0.010 to 0.075 p.p.m. They concluded that if residues were to be kept to 0.05 p.p.m. or below, the number of applications would have to be limited to seven. The mercury content of untreated tomatoes

20 N. A. SMART

ranged from 0.012 to 0.044 p.p.m. In these experiments skin and pu1p of tomatoes were analysed separately and resu1ts for each part of the tomato are given. In 1954 STONE et al. published resu1ts of similar experiments using a phenylmercury salicylate aerosol but only the skins of the tomatoes were analysed for residues so that the residues given are all too low.

EGAN and LIDZEY (1960) and BLAND and EGAN (1963) published re­su1ts of mercury residue analyses on tomatoes from crops treated with various numbers of applications of phenylmercury salicylate aerosol. The dosage per application did not exceed one fluid oz. of 0.3 percent w/w formulation per 1,700 cu. ft. glasshouse space. The residue on whole fruit on plants receiving up to eight applications was approximately 0.01 (n + 1) and for nine to twelve applications 0.090 to 0.010 (n - 8) where n is the total number of applications. These formu1ae are only a rough ru1e-of-thumb guide to the level found and should not be regarded as a statistically proved relationship. EGAN suggests that this maximum in residue is connected with the decline in leaf growth and the increase in the rate of fruit volume production.

SMART (1960) found a maximum residue of 0.11 p.p.m. of mercury in thirteen samples of commercially treated tomatoes which had received up to eight treatments.

d) Potatoes

PICKARD et at. (1962) have shown that potatoes grown in soil treated with mercuric oxide or mercurous chloride for control of potato root eelworm at 3.3 lb. of mercury/acre a few days before sowing gave rise to a negligible amount of mercury residue in the tubers at harvest. The roots, however, con­tained appreciable amounts. Workers at Long Ashton (PICKARD and MARTIN

1961) had shown appreciable residues in tubers of potatoes after the foliage had been sprayed with 0.005 percent phenylmercury acetate with added wetter.

Residues arising in tubers at harvest after applying a phenylmercury chloride fungicide (0.6 percent w/w mercury) on foliage during the grow­ing season were investigated by SMART (1964). In a preliminary plot experi­ment, Majestic potatoes were sprayed three times during the growing season at ten times the normal rate. If the difference in mercury content between treated and control plots could not be detected in this case then there would be no residue from normal applications. A residue of up to 0.17 p.p.m. was in fact found, even after peeling the potatoes. A randomized block experi­ment was therefore undertaken the following year spraying at the normal rate. The maximum number of applications that could possibly be made in a year would be about eight, and four would be on the generous side of normal. Four treatments of eight applications, four early and four late ap­plications, and controls were randomized in five blocks of King Edward potatoes and tubers harvested half-way through the growing season and at the normal time. Residues at harvest are shown in Table IV.

Residues of merauy compounds 21

Table IV. Residues of mercury in the flesh of potatoes after foliar application of a mercury·copper fungicide

Treatment

Half-way through growing season 4 applications Unsprayed

At the end of the growing season 8 applications 4 applications early 4 . applications late Unsprayed

Mean for blocks

(p.p.m.)

0.021 0.003

0.031 0.032 0.031 0.003

S.E. of treatment mean between blocks

(p.p.m.)

± 0.001 ± 0.001

± 0.002 ± 0.004 ± 0.002 ± 0.001

Residues in the peel were not noticeably higher than those in the flesh. Potatoes grown on ground where phenylmercury chloride had been sprayed the previous year were not contaminated.

STEWART and ROSS (1962) and ROSS and STEWART (1964) also in­vestigated the residues arising from the use of phenylmercury chloride-copper oxychloride foliar spray on potatoes. They found that 0,1, and 4 sprays at the normal rate in August led to <0.01, 0.01 and 0.05 p.p.m. of mercury, respectively, in the whole tubers. When different numbers of sprays were applied at different times during the growing season, the accumulation of mercury in the foliage was approximately proportional to the number of sprays and the interval following application. This was not the case for residues of mercury in the tubers, where it accumulated during the period of rapid increase in tuber size. Varying the number of sprays early in the season did not significantly change the amount of mercury in the tubers but smaller tuber residues were present where only late sprays were applied. About twice the level of mercury was found in the peel of tubers of sprayed plants as in their flesh.

DAS et al. (1966) have reported 0.006 -+- 0.001 p.p.m. of mercury in potato flour.

e) Grain

The residues occurring in the ears of grain grown from dressed seed are very small. Thus, in the case of wheat dressed normally, the harvested grain contained 0.01 p.p.m. of mercury (Victoria Department of Agriculture Australia 1967). WESTERMARK (1967) gives the mercury level in grains of wheat or barley as 0.008 to 0.012 p.p.m. whether or not they have been grown from dressed grain.

FURUTANI and OSAJIMA (1965 a and b) have studied the mercury con-

22 N. A. SMART

tent of dusted rice. They compared the mercury concentrations in rice dusted with mercury fungicides and in undusted rice. There was about a 50 percent increase over a natural mercury level of 0.2 p.p.m. after dusting. A poorly­drained soil gave rise to a higher mercury content. GOTO and SATO (1966) found 0.077 to 0.185 and 0.056 to 0.065 p.p.m. of mercury, respectively, in unpolished and polished rice. TOMIZAWA et al (1966) found that the mercury contents of polished rice from paddy fields, where phenylmercury acetate had been applied, were mostly in the range of 0.1 to 1.0 p.p.m.; rice from unsprayed fields showed 0.227 to 0.238 p.p.m. TOMIZAWA used the sensitive neutron-activation technique in his analyses and, although these background values are high compared with those found in wheat, barley, fruits, and vegetables, they must be accepted, at least until further evidence is forthcoming. SMART and HILL (1968) found that levels of mercury in rice imporred into the United Kingdom are often negligible (0.005 p.p.m. or below) although they may rise to 0.01 p.p.m. and occasionally to 0.015 p.p.m. (Japan does not export rice.)

The levels of mercury on dressed grain are sometimes of interest. SMART

(1963 a) has determined some levels both from dry-dressed and liquid­dressed wheat and barley. They varied from six to 23 p.p.m. in some samples taken in Great Britain. Moderate washing of the dressed seed removed only about 10 percent of the fungicide except in the case of one dry dressing when 50 percent was removed. LEGATOWA (1963) found 34 p.p.m. of mercury in a sample of fungicide-treated grain; washing reduced this level to 20 p.p.m. It is thus clear that even when dust dressings are used appreciable amounts of mercury are absorbed into the seed coat.

f) Animal material, including eggs

Excessive residues of mercury in birds and their eggs can arise from them eating dressed grain. In almost all countries the advice of government de­partments and manufacturers is that mercury-dressed grain should not be fed to birds or other livestock, but cases arise from time to time where this has deliberately or unintentionally been done, sometimes with fatal results. Wild birds, however, may pick up dressed seed from the surfaces of fields, and in time such birds may become prey of others so forming a mercury residue chain. Fish are known to accumulate mercury and residues of mercury above the background level can sometimes be found in sea birds.

Residue data therefore comprise those obtained from deliberate feeding trials, which will be mentioned first, and also those from chance contact with mercury fungicides.

BORG (1958) fed birds of different species with mercury-dressed seed daily. Deaths did not occur until about one month after the experiments be­gan when the mercury content of organs was 3 to 100 p.p.m. In non-fatal cases mercury could be detected up to six to seven months after ceasing to

Residues of mercury compounds 23

feed dressed grain. Feeding pheasants mercury-dressed grain for nine days did not affect egg production but reduced hatchability.

SMART and LLOYD (1963 b) investigated the mercury residues in hens fed on a ration containing wheat treated commercially with methylmercury dicyandiamide. The mercuty content of the eggs rose steadily from zero to 8 p.p.m. over a period of four weeks and limited evidence indicated a continued rise for another four weeks. Over 75 percent of the mercury in the eggs was found in the albumen and was first detected three days after starting to feed treated grain. The residues in the thigh muscle of the hens were about four p.p.m. with slightly more in the breast muscle. Livers contained 8 to 24 p.p.m., and kidneys eight p.p.m., after eight weeks. Tissue from hens fed on untreated grain contained 0.01 p.p.m. or less. The laying and general health of the birds was normal and autopsy did not reveal characteristics attributable to mercury poisoning. It would be interesting to enquire whether cockerels fed on a similar diet would remain unaffected as two-thirds of the mercuty entering the hens was discharged in eggs. SVENSSON and ULFV ARSON (1967) have studied the distribution of several types of mercury compounds in cockerels.

TEJNING and VESTERBERG (1964) have also fed grain containing methyl­mercuty dicyandiamide to hens. One hen was fed for 4.5 months on grain containing 14 p.p.m. of mercury and at the end of this time the eggs being laid contained 0.3 mg. of mercuty each and the carcass 8.2 mg. A second hen was fed on grain containing one p.p.m. of mercury and was then laying eggs containing about 0.03 mg. each. The carcass contained 0.2 mg. Both hens were apparently healthy.

WESTOO et at. (1965) have closely investigated the mercuty content of eggs on the open market in Sweden and in some other European countries. A summary of their results is shown in Table V.

Eggs sampled from farms in the north of Sweden contained 0.019 to 0.052 p.p.m., mean 0.033 p.p.m., and higher concentrations, far in excess of 0.05 p.p.m., were found in eggs from some small private farms.

WESTOO (1966 a) has given figures for the mercury residues in normal Swedish commercial samples of poultry meat. Several series of determina­tions were undertaken and the means of each were 0.021, 0.023, 0.009, and 0.015 for breast meat, 0.021, 0.005, 0.005, and 0.011 for leg muscle and :::;;0.Q28, and 0.D31 p.p.m. for liver.

Some pheasants, partridges, pigeons, corvids, finches, and owls found dead in the Swedish countryside contained up to 200 p.p.m. of mercury. In 196451 percent of a sample of 200 normal seed.eating birds (pheasants, pigeons, corvids) in Sweden had more than two p.p.m. of mercury in their livers.

PAPPAS and ROSENBERG (1966 b) found that eggs of hens in the United States on a feed containing no dressed grain showed 0 to 0.003 p.p.m. of mercury.

24 N. A. SMART

Table V. Residues 0/ meI'CU1'J in eggs sampled on the open market in Sweden and other European Countries

Range of residues found

Country No. of samples (p.p.m.)

Sweden 79 0.015-0.043

mean 0.029

Holland 3 0.005-0.007

Belgium 3 0.006-0.008

W. Germany 2 0.005

Austria 8 0.006-0.013

Italy 4 0.005-0.006

Denmark 2 0.004

Norway 3 0.015-0.020

CONDER (1961) was one of the first to report levels of mercury found in dead birds. In the case of some birds in Great Britain in 1961 levels be­tween 0.6 p.p.m. in a partridge to 7.1 p.p.m. in a woodpigeon were found. KOERNAN and VAN GENDEREN (1965) reported levels of 0.33 to 0.75 p.p.m. in some dead or dying birds of prey in the Netherlands. ULFARSSON (1965) has obtained data of the mercury residues in pheasants after they have eaten dressed seed grain. EADES (1966) has given residues in pheasant, pigeon, and kittiwake found dead or dying in Ireland and also guillemot eggs.

In view of the levels of mercury that have been found to have no de­leterious effect when fed to birds, it is difficult to ascribe to mercury poison­ing the death of many of the birds reported above as having a mercury content above the background mercury level. The situation is, however, complicated by the presence of residues of organochlorine pesticides that exert their own toxicological effect alongside the mercury residues.

WESTOO (1966 b) investigated the mercury content of pork, veal, ox, and reindeer meat. A summary of her results is given in Table VI.

PAPPAS and ROSENBERG (1966 b) in the United States found a back­ground level of 0.017 to 0.023 p.p.m. of mercury in haddock. CHRISTELL

et al. (1965) have found 0.026 to 0.041 p.p.m. in herring from the Baltic Sea. Fish are known to accumulate mercury (BOETHUSS 1960), and this is reflected in the higher background levels reported above compared with those for most fruits and vegetables. Accumulation of mercury in fish has been particularly noticeable in rivers and lakes in Sweden as a result of mercurial effluent, arising from use in agriculture and in paper-making,

Residues of mercury compounds 25

Table VI. Mercury content of various meats

Residue (p.p.m.)

No. of deter-Commodity minations Range Mean

Swedish pork cutlets, raw 35 0.016-0.130 0.030

Danish pork cutlets, raw 6 0.002-0.007 0.003

Swedish bacon 21 0.005-0.048 0.Q18

Danish bacon 5 0.002-0.009 0.004

Pigs liver, Swedish, raw 26 0.014-0.183 0.060

Pigs liver, Swedish, fried 5 0.028-0.140 0.091

Pigs liver, Danish, raw 3 0.005-0.020 0.009

Ox fillet, Swedish, raw 23 0.002-0.074 0.012

Calf and ox liver, Swedish, fried 5 0.014-0.023 ~0.Q18

Ox liver, Danish, raw 6 0.003-0.007 0.005

Reindeer, raw 11 0.005-0.023 0.013

Reindeer liver, raw 4 0.009-0.044 0.026

draining, or being discharged into them. LUNDHOLM (1966) reported mean levels of 0.3 and 0.8 p.p.m. in fish in two Swedish lakes compared with cor­responding values of 0.07 and 0.1 p.p.m. in Norway and Switzerland, re­spectively. ]OHNELS (1966) found up to 20 p.p.m. in pike in Swedish rivers, the levels of mercury increasing with the weight of the pike. WESTOO

(1966 c) has found residues of methylmercury compounds in river fish; she has also (WESTOO 1967) given the values of mercury residues found in determinations on fish eaten in Sweden, as summarized in Table VII.

LIHNELL and STENMARK (1967) have described the occurrence of mer­cury in small rodents caught during or shortly after spring and autumn grain sowings; 130 out of a total of 184 analyses of the rodents gave values of 0.5 p.p.m. or lower and 42 were above this figure and below 2.0 p.p.m.

g) Water

Various figures have been given for the trace-levels of mercury present in river and sea water. According to STOCK (in MONIER-WILLIAMS 1949) sea water contains 0.03 ng./g. More recent information from Japan (HOSAHARA

1961) gives 0.15 ng./g. for surface water, the mercury content increasing slightly with depth. Some Russian figures indicate 0.4 to 2.8 ng./ g. in river water and 0.7 to 2 ng.! g. in sea water.

26 N. A. SMART

Table VII. Mercury levels in fish eaten in Sweden

Residue (p.p.m.)

Species No. of determinations Range Mean

Halibut 1 - 0.089

Haddock 10 0.016-0.060 0.031

Plaice 4 0.037-0.064 0.050

Cod 9 0.024-0.110 0.050

Whiting 1 - 0.048

Perch 25 0.13 -3.95 1.30

Bream 1 - 0.37

Pike 28 0.15 -5.20 1.09

Pike perch 6 0.17 -2.55 1.00

Carp 1 - 0.11

Burbot 9 0.010-0.74 0.42

Salmon trout 6 0.022-0.13 0.07

Roach 1 - 0.20

Whitefish 12 0.035-0.46 0.29

h) Soil

MARTIN ( 1963) gives the natural mercury content of some English soils as between 0.01 and 0.06 p.p.m. Earlier STOCK (in MONIER-WILLIAMS

1949) had reported 0.1 to 0.3 p.p.m. in soils. ANDERSON (1967) has found 0.01 to 0.9 p.p.m. in 200 analyses of some Swedish soils although lower values of 0.05 p.p.m. in French and Sudan soils. The actual values will, of course, vary appreciably with the locality from which the sample was taken.

FURUTANI and OSAJIMA (1966) have given figures for the mercury content of paddy field soil where organomercury compounds are applied as dusts. PICKARD et al. (1962) found 2.6 p.p.m. of mercury in soil treated once with yellow mercuric oxide to control potato root eelworm.

The mode of degradation of organomercury compounds in soil has been discussed by KIMURA and MILLER (1964).

i) Other crops and materials

GOLDWATER (1964 and 1965) has given some general environmental levels. WARD and MCHUGH (1965) have given levels of mercury in a num-

Residues of mercury compounds 27

ber of samples of vegetation and STOCK (in MONIER-WILLIAMS 1949) also analysed a wide range of materials. WESTOO (1965 a) investigated the total mercury content of fourteen diets which included vegetables, milk, cheese, :fish, meat, egg, bread, butter, coffee, tea, beer, sugar, etc., and found between 0.004 and 0.014 p.p.m. of mercury.

VII. Movement of mercury in plants and trees

Several groups of workers have found evidence, which is summarized below, of mercury compounds applied to some parts of plants and trees being translocated to other parts. Movement within the foliage, fruits, tubers, and stems is greater than that from the roots upward. The amount of translocation may well vary with the mercury compound used; for ex­ample, phenylmercury acetate has a higher water-solubility than some other organomercurials commonly used as foliar sprays and the amount of trans­location reported with this compound by PICKARD and MARTIN (1959) and by ROSS and STEWART (1962) with apples (see below) may be greater than that occurring with, say, phenylmercury chloride.

PICKARD and MARTIN (1958) found mercury (0.58 p.p.m.) in newly­emergent coffee foliage of shrubs previously sprayed so that the level of mer­cury was 17 p.p.m.

In 1959 these workers reported the movement of mercury residues as a result of foliar spraying of apple trees. Residues were found in the flesh of apples as well as in the peel. Table VIII shows the distribution of mercury in an apple fruit.

Table VIII. Distribution of mercury in a sprayed apple fruit

Mercury

Wt. Fruit part (g.) p.g. p.p.m.

Peel 9.5 21.3 2.21

Flesh 0.5 em. deep 17.4 0.63 0.04

Flesh 0.5-1.5 em. deep 30.0 0.20 0.01

Flesh 1.5-2.5 em. deep 32.2 0.17 0.01

Central core 23.3 0.23 0.01

Calyx and stem 45.0 0.50 0.01 cavity ends

Within a few days of spraying Worcester apple trees most of the mercury on the fruit and leaves had penetrated beyond the reach of surface washing.

PICKARD and MARTIN (1960 a) also described the residues found in

28 N. A. SMART

apples that had been bagged to prevent them. receiving direct deposits of seven applications of a 0.005 percent mercury as phenylmercury acetate formulation. The mercury residue on whole unbagged fruit was 0.20 to 0.25 p.p.m. and in bagged fruit 0.04 to 0.08 p.p.m., almost all the residue being in the flesh of the apples.

STEWART and ROSS (1960) found that mercury residues in apples from trees receiving phenylmercury acetate cover sprays in late spring and sum­mer declined over a period of about one month to approximately one-third of the initial deposit. At this time, a large part of the residue was concen­trated in a small portion of the apple at the calyx end. This stage was followed by a rapid accumulation of mercury in the fruit throughout the remainder of the growing season until at harvest the residues (in terms of !1g. of mercury) were greater than the initial deposits. Fruit bagged to pre­vent direct contact with the organomercury spray, as in PICKAlID and MARTIN'S work, accumulated mercury. Further studies by ROSS and STEWART

( 1962) showed that mercury moved into the fruit from the foliage mainly by translocation rather than by volatilization. There was little or no uptake through the roots.

CORKE (1962) has reported that application of organomercury formu­lations to the bark of apple trees led to mercury in all parts of the tree, with some concentration in the region of the phloem.

SMART (1964) briefly investigated translocation of mercury residues in potatoes using a phenylmercury chloride formulation and the results sug­gested that traces of mercury compounds can be both translocated from the foliage and taken up from the soil into tubers. PICKAlID and MARTIN (1961) have found downward-translocation of phenylmercury acetate residues in potatoes.

PICKAlID and MARTIN (1959) showed that dwarf bean plants grown in a nutrient solution containing 0.06 to 0.28 !1g./mI. of mercury took ap­preciable amounts (about 10 p.p.m.) of mercury into the roots and 0.09 to 0.90 p.p.m. was found in the tops. Similar results were obtained with let­tuce. When the lower leaves of broad bean plants were painted with a 50 !1g./ mi. of mercury solution, mercury was taken into the higher leaves and seeds. Determination of the mercury content of broccoli treated at the seed­ling stage with mercurous chloride or mercuric chloride showed some in the roots but curds from treated plants were identical with untreated curds when analysed. Carrots grown in soil treated with mercurous chloride or mercuric chloride at sowing showed no accumulation of mercury (PICKARD and MARTIN 1960 a). Similarly, potatoes grown in soil treated with inorganic mercury showed that although the fine root hairs contained traces of mer­cury the peel and flesh of tubers were uncontaminated (PICKARD and MARTIN 1961). Little or no mercury accumulated in turnips grown in soil treated with mercuric oxide or mercurous chloride (PICKARD et al. 1962). The latter workers also found that lettuce and dwarf bean plants grown in

Residues of mercury compounds 29

soil treated with mercurous chloride in vermiculite dust at 5.7 lb of mer­cury / acre contained little or no mercury in the leaves of the lettuces or pods of the beans. Some mercury «0.1 p.p.m.) was found in the roots.

VIII. Regulatory

For official control purposes seed dressing formulations have often a red or orange dye added to them so that mercury-dressed seed can be readily recognised. In some countries there are regulations forbidding the use of dressed seed for human or animal consumption whereas in others, farmers are merely warned or advised not to use dressed seed in this way.

Restrictions governing the use of mercury compounds in various coun­tries are summarized below:

Australia There is a residue tolerance of 0.03 p.p.m. of mercury in fruit and vegetables.

Benelux countries There is a residue tolerance of 0.03 p.p.m. of mercury. Organomercury compounds are used in fruit culture until June or after leaf fall.

Brazil Mercury residues must not exceed 0.05 p.p.m. Denmark The proposed WHO/FAO maximum acceptable resi­

due level of 0.05 p.p.m. of mercury is used as a guide (there is no legal figure). Last application of orchard sprays must not be later than 'dense cluster'. In spring not more than two applications are allowed and the maximum dosage/application is 30 g. of mercury /ha.

Germany The sale of food containing residues of mercury whose origin is in the use of mercury-containing pesticides is forbidden.

Israel All organomercury pesticides have to be registered. They are not directly used on edible crops.

Japan There are no regulations at the moment but it is con­templated banning the use of organomercury pesticides in two years' time.

New Zealand The sale of fruit and vegetables containing greater than 0.05 p.p.m. of mercury is forbidden. No mercury is per­mitted in other food. Stock must be removed from any orchard before trees are sprayed with any organomer­curial and must not be returned to the orchard until six weeks after the date of application. There is a two week waiting period between last application to pota­toes and harvest. A maximum of five weekly applica­tions may be made to tomatoes. No more than five

30

Norway S. Korea

Sweden

United Kingdom

United States

N. A. SMART

applications may be made between greentip and har­vest on apples and pears. The use of alkyl-mercury compounds is banned. The concentration of mercury in the formulations is regulated depending on its use. The sale and use of organomercury pesticides is regu­lated. Treatment of spring cereal seed must not take place unless the need for its treatment is verified by the National Central Seed Testing Institute. The use of alkylmercury compounds is forbidden. The proposed WHO/FAO maximum acceptable residue level of 0.05 p.p.m. of mercury is used as a guide except for drink­ing water, fish, and shell fish. For fish a maximum level of 1.0 p.p.m. is used as a guide. The sale and use of mercury pesticides are regulated by voluntary schemes. The uses and rates of uses on the labels of the marketed formulations are such that proper use should not lead to residues exceeding 0.1 p.p.m. of mercury. Phenylmercury salicylate should not be applied to tomatoes more than five times in a growing season or at intervals of less than seven days. There should be a minimum interval of twelve hours between last appli­cation of aerosol and harvesting an edible crop. An in­terval of at least six weeks should elapse between last application of arylmercury foliar sprays and harvest. Livestock should be kept out of a sprayed area for at least-two weeks. All mercury formulations must be registered before be­ing put on the market. No mercury residue is allowed in foodstuffs.

Summary

The diseases in world agriculture commercially controlled by mercury compounds, the rates of application and the nature of the mercury formula­tions used are listed. The extent of production of mercury compounds for agricultural purposes in various countries is given.

Methods for analysis for residues of mercury compounds in foodstuffs are reviewed with special consideration of 'recommended', 'official' or agreed methods.

Foodstuffs normally contain traces of mercury, the amount depending on the environment in which they are grown or bred and on any external application of mercury-containing compounds. Evidence is presented for the translocation of mercury compounds in growing plant material. In pome

Residues of mercury compounds 31

fruit, the background levels are normally 0.04 p.p.m. of mercury or below; in tomatoes, up to 0.02 p.p.m.; in potatoes, up to 0.01 p.p.m.; in wheat and barley, up to 0.02 p.p.m.; and in eggs and meat up to 0.05 p.p.m. In rice limited evidence suggests background levels up to 0.2 p.p.m. Fish tend to accumulate mercury and have a resulting higher background level.

When a crop or foodstuff is treated in accordance with good agricultural practice, residues of mercury are generally not greater than the following: apples, 0.1 p.p.m.; tomatoes, 0.1 p.p.m.; potatoes, 0.05 p.p.m.; wheat and barley, 0.02 p.p.m.; and eggs and meat, 0.1 p.p.m.

Finally, an outline is given of the regulations governing use and residues of mercury compounds in agriculture in different countries.

Resume * Usage et residus des composes du mercure en agriculture

On donne la liste dez maladies qui, dans Ie domaine mondial de l' agri­culture, sont combattues commercialement par les composes du mercure, les doses d'application et la nature des formulations utilisees. On presente, pour divers pays, 1'importance de la production des composes du mercure destines a des fins agricoles.

On examine les methodes d'analyse des residus de composes du mercure dans les dentees alimentaires en accordant une attention particuliere aux methodes "recommandees", "officielles" ou approuvees.

Les denrees alimentaires contiennent normalement des traces de mercure dont 1'importance depend du milieu dans lequel les denrees sont cultivees ou produites et de toute application externe de composes contenant du mercure. On presente la preuve de la translocation des composes du mercure dans les matieres vegetales en croissance. Dans les fruits a pepins, les niveaux de base du mercure sont normalement de 0,04 ppm ou moins; ils atteignent 0,02 ppm dans les tomates; 0,01 ppm dans les pommes de terre; 0,02 ppm dans Ie froment et l'orge; 0,05 ppm dans la viande et les oeufs. Dans Ie riz, Ie niveau de base atteindrait 0,2 ppm, selon des donnees limitees. Les poissons tendent a accumuler Ie mercure et les niveaux de base qui en resultent sont plus eIeves.

Lorsqu'une recolte ou une denree alimentaire est traitee selon les bonnes pratiques agricoles, les residus de mercure ne depassent generalement pas les valeurs suivantes: 0,1 ppm pour les pommes; 0,1 ppm pour les tomates; 0,05 ppm pour les pommes de terre; 0,02 ppm pour Ie froment et 1'orge; 0,1 ppm pour les oeufs et la viande.

Enfin, on donne un aper<;:u des reglementations regissant 1'usage et les residus des composes du mercure en agriculture, dans differents pays.

"Traduit par S. DORMAL-VAN DEN BRUEL.

32 N. A. SMART

Zusammenfassung*

Anwendung und Riickstande von Quecksilber­Verbindungen in der Landwirtschaft

Die Arbeit gibt eine Obersicht iiber die auf we1tweiter Basis in der Land­wirtschaft mit Quecksilber-Verbindungen bekampften Pflanzenkrankheiten, iiber die angewendeten Mengen und iiber die Art der verwendeten Formu­lierungen. Auch die Produktionsziffern von Quecksilber-Verbindungen fiir landwirtschaftliche Zwecke in verschiedenen Landern werden angefiihrt.

Methoden zur Bestimmung von Quecksilber-Riickstanden in Lebensmit­teln werden referiert, wobei insbesondere "empfohlene", "offizielle" oder bewahrte Methoden beriicksichtigt sind.

Lebensmitte1 enthalten normalerweise Spuren von Quecksilber, deren Menge abhangt einmal von der Umwelt, in denen die Pflanzen wachs en oder angebaut werden, zum anderen von jeglichem Kontakt mit quecksilber­haltigen Verbindungen von aussen her. Quecksilber-Verbindungen konnen in wachsenden Pflanzen transportiert werden; hierfiir werden Belege an­gefiihrt. Die natiirlichen Quecksilber-Gehalte liegen normalerweise in Ker­nobst bei 0,04 ppm und darunter, in Tomaten bis zu 0,02 ppm, in Kartoffe1n bis zu 0,01 ppm, in Weizen und Gerste bis zu 0,02 ppm, in Eiern und Fleisch bis zu 0,05 ppm. Weniger umfangreiche Daten an Reis lassen "Blindwerte" bis zu 0,2 ppm vermuten. Fische neigen zur Anreicherung von Quecksilber und haben daher hohere Blindwerte.

Mit Quecksilber-Verbindungen sachgemass behandelte Erntegiiter oder Lebensmittel enthalten im allgemeinen folgende Hochstmengen an Queck­silber: Apfel 0,1 ppm, Tomaten 0,1 ppm, Kartoffeln 0,05 ppm, Weizen und Gerste 0,02 ppm, Eier und Fleisch 0,1 ppm.

Abschliessend wird ein Oberblick gegeben iiber die in verschiedenen Landern flir die Anwendung und die Riickstande von Quecksilber-Verbind­ungen in der Landwirtschaft herrschenden Regulierungen.

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.. Vbersetzt von H. FREHSE.

Residues of mercury compounds 33

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GOLDWATER, J.: Absorption and excretion of mercury in man. VII. Significance of mercury in blood. Arch. Environ. Health 9, 735 (1964).

- Mercury in our environment. J. Food Cosmetic Toxicol. 3, 120 (1965).

34 N. A. SMART

GOTO, T., and A. SATO: Determination of mercury in various foods by quantitative isotope dilution analysis. Eiyo To Shokuryo 19, 245 (1966).

GUTENMANN, W. H., and D. J. LISK: Rapid determination of mercury in apples by modified Schoniger combustion. J. Agr. Food Chem. 8, 306 (1960).

HORDYNSKA, S., B. LEGATOWA, and 1. BERNSTEIN: Colorimetric determination of microgram quantities of mercury in grains and apples. Roczniki Panstwowego Zakladu Hig. 12, 105 (1961).

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HOSAHARA, K.: Mercury content of deep-sea water. Nippon Kagaku Zasshi 82, 1107 (1961).

HOWARD, F. 1., and F. S. SCHLENKER: Magnitude of residues on apples from orchards sprayed with organic mercurials. Proc. Amer. Hort. Sci. 50, 81 (1947).

International Union of Pure and Applied Chemistry: Determination of the mercury content of foodstuffs. Pure Applied Chem. 10, 77 (1965).

JACOBS, M. B., and 1. J. GOLDWATER: Ultramicrodetermination of mercury in apples. Food Technol. 15, 357 (1961).

JOHNELS, A. G.: The mercury content in fish and some other aquatic organisms in Sweden. Symposium of the Swedish Royal Commission of Natural Resources, Stockholm, (to be published) (1966).

Joint Mercury Residues Panel: Determination of mercury in apples and tomatoes. Analyst 86, 608 (1961).

JONES,1., and G. SCHWARtzMAN: Determination of mercury in pink wheat. J. Assoc. Official Agr. Chemists 46, 879 (1963).

KIMURA, Y., and V. L. MILLER: Mercury determination at the microgram level by a reduction-aeration method of concentration. Anal. Chim. Acta 27, 325 (1962).

- - The degradation of organomercury fungicides in soil. J. Agr. Food Chem. 12, 253 (1964).

KIRBY, A. H. M., and M. BENNETT: Phytotoxic effects of phenylmercuric compounds upon certain pear varieties. J. Hort. Sci. 38, 68 (1963).

KLEIN, A. K.: Report on mercury. J. Assoc. Official Agr. Chemists 35, 537 (1952). KOEMAN, J. H., and H. VAN GENDEREN: Some preliminary notes on residues of

chlorinated hydrocarbon insecticides in birds and mammals in the Netherlands. Proc. 17th lnternat. Symposium Phytopharmacy, Gent. p. 1879 (1965).

KUDSK, F. N.: Determination of mercury in dithizone extracts by ultraviolet photo­metry. Scand. J. Clin. Lab. Invest. 17, 171 (1965).

KUNZE, F. M.: Addition of selenium to wet-ash procedure for the determination of mercury in apple peel. J. Assoc. Official Agr. Chemists 31, 439 (1948).

LEGATOWA, B., S. HORDYNSKA, and 1. BERNSTEIN: Mercury content determination in grains not treated with mercuric fungicides. Roczniki Pzh. 14, 221 (1963).

LIHNELL, D., and A. STENMARK: Bidrag till kannedomen om forekomsten av kvick­sihner i smagmagure. Statens Vaxstskyddsanstalt Meddelanden 13, 11 0 (1967).

LINDSTROM, 0.: Diffusion of mercurial in the fruit coat of treated seed. J. Agr. Food Chem. 7, 562 (1959).

LJUNGGREN, K., and T. WESTERMARK: Method for the detection of mercury by radioactivation analysis. Pure Applied Chem. 1, 127 (1960).

LLOYD, P. W.: Mercury residues in tomatoes. Private Communication (1958). LUNDHOLM, B.: The mercury problem. Symposium of the Swedish Royal Commis­

sion of Natural Resour.ces, Stockholm (to be published) (1966). MARTIN, J. T.: Mercury residues in plants. Analyst 88, 413 (1963). -, and J. A. PICKARD: Spray application problems. XLII. Mercury deposits on apple

fruits and foliage. Ann. Rept. Agr. Hort. Research Sta. Long Ashton, Bristol, p.76 (1957).

Residues of mercury compounds 35

MlLLBR, E. J.: A note on mercury spray residues in apples. Plant Pathol. 5, 119 (1956).

MONIER-WILUAMS, G. W.: Trace elements in food. London: Chapman & Hall (1949).

NARDIN, H. F.: An investigation of spray residues on apples and pears. N.S. Wales Dept. Agr. Chem. Br., Bull. S36 (1965).

PAPPAS, E. G., and L. A. ROSENBERG: Determination of sub-microgram quantities of mercury by cold vapour atomic absorption photometry. J. Assoc. Official Anal. Chemists 49, 782 (1966 a).

- - Determination of sub-microgram quantities of mercury in fish and eggs by cold vapour atomic absorption photometry. J. Assoc. OfIicial Anal. ChemiSts 49, 792 (1966 b).

PI~, J. A., and J. T. MARTIN: Spray application problems. XLVI. Mercury resi­dues on coffee leaves and berries in relation to the control of berry diseases. Ann. Rept. Agr. Hort. Research Sta., Long Ashton, Bristol, p. 88 (1958).

- - Spray application problems. LX. The uptake of mercury by plant tissues. Ann. Rept. Agr. Hort. Research Sta., Long Ashton, Bristol, p. 93 (1959).

- - Spray application problems. LXIV. The absorption and movement of mercury in plants. Ann. Rept. Agr. Hort. Research Sta., Long Ashton, Bristol, p. 85 (1960 a).

- - Determination of mercury in plant material. J. Sci. Food Agr. 11, 374 (1960 b).

- - Spray application problems. XLVII. The distribution of mercury in plant tissues after leaf applications of phenylmercury acetate. Ann. Rept. Agr. Hort. Research Sta., Long Ashton, Bristol, p. 106 (1961).

- - Determination of mercury in soil. J. Sci. Food Agr. 14, 706 (1963). - -, and J. GRAINGER: Spray application problems. LXVIII. Mercury residues in

ground crops. Ann. Rept. Agr. Hort. Research Sta., Long Ashton, Bristol, p. 65 (1962).

PmcHOCKA, J.: Determination of microgram amounts of mercury in food. Roczniki Panstwowego ZakIadu Hig. 12, 101 (1961).

PlllLLIPS, G. F., B. E. DIXON, and R. G. LInZEY: The volatility of organo-mercury compounds. J. Sci. Food Agr. 10, 604 (1959).

RAJAMA, ]., S. HIL'I'UNEN, and A. HILPI: An oxygen.flask combustion method for the determination of mercury in eggs. Valtion Tek. Tutkimuslaitos, Tiedotus, Sarja, IV, No. 65 (1964).

Ross, R. G., and D. K. R. STEWART: Mercury residues on apple fruit and foliage. Can. J. Plant Sci. 40, 117 (1960).

- - Movement and accumulation of mercury in apple trees and soil. Can. J. Plant Sci. 42, 280 (1962).

- - Mercury residues in potatoes in relation to foliar sprays of phenylmercury chloride. Can. J. Plant Sci. 44, 123 (1964).

SCHACHTER, M. N.: Apparatus for cold vapour atomic absorption of mercury. J. Assoc. Official Anal. Chemists 49, 778 (1966).

SJOSl"RAND, B.: Simultaneous determination of mercury and arsenic in biological and organic materials by activation analysis. Anal. Chem. 34, 814 (1964).

SMART, N. A.: Mercury spray residues in tomatoes. Internal report (1960). - Arylmercury spray residues in apples. Plant Pathol. 10, 158 (1961). - Retention of organomercury compounds on dressed grain after washing. Nature

199,1206 (1963 a). -, and M. K. LLOYD: Mercury residues in eggs, flesh and livers of hens fed on wheat

treated with methylmercury dicyandiamide. J. Sci. Food Age. 14, 734 (1963 b).

36 N. A. SMART

- Mercury residues in potatoes following application of a foliar spray containing phenylmercury chloride. ]. Sci. Food Agr. 15, 102 (1964).

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ysis. Anal. Chem. 35, 635 (1963). STEWART, D. K. R., and R. G. Ross: Note on mercury residues in potatoes. Can. J.

Plant Sci. 42, 370 (1962). STONE, H. M.: A note on the mercury content of apples treated with phenylmercury

dimethyl dithiocarbamate. New Zealand]. Agr. Research 7, 439 (1964). -, and P.]. CLARK: Mercury content of tomatoes. New Zealand J. Sci. 1,373 (1958). - -, and H. JACKS: Mercury residues on tomatoes. New Zealand J. Sci. Technol.

B 35, 301 (1954). - - - Mercury content of apples. New Zealand ]. Sci. Technol., B 38, 843 (1957). STRICKLAND, A. H.: Some estimates of insecticide and fungicide usage in agriculture

and horticulture in England and Wales, 1960-64. ]. Applied Ecol. 3, 3 (1966). SWENSSON, A., and U. ULFVARSON: The distribution and the excretion of organic

mercury compounds from the organs of the cockerel. Private communication (1967) .

SZKOLNIK, M., K. D. HICKEY, E. J. BRODERICK, and D. ]. USK: Mercury residues of apple fruit as related to application schedules and to the colorimetric and the neutron activation analyses. Plant Disease Reporter 49, 568 (1965).

TE]NING, S., and R. VESTERBERG: Mercury in tissues and eggs from hens fed with grain containing methylmercury dicyandiamide. Poultry Sci. 43, 6 (1964).

TEW, R. P., and ]. M. SILLIBOURNE: Mercury residues in apples from trees receiv­ing organomercurial fungicide in the previous autumn and winter. Ann. Applied BioI. 56, 293 (1965).

TOMlZAWA, c.: Residue analysis of pesticides in plant materials. I. Behaviour of mercury in rice plants treated with organomercury 20SHg disinfectants. Shokuhin Eiseigaku Zasshi 7, 26 (1966).

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- - Micro-determination of mercury in foods. Ann. fals. et fraudes 56, 225 (1963). ULFARSSON, U.: Mercury, aldrin and dieldrin in pheasants. Svensk Kem. Tidskr. 77,

235 (1965). VASILEVSKAYA, A. E., and V. P. SHCHERBAKOV: Determination of mercury in soil.

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dithizone. A single extraction procedure. U.S. Geol. Survey, Professional Papers No. 501 D, p. 128 (1965).

WESTERMARK, T.: Private communication (1967). West6'1"n Australia Depetrtment of Agriculture: Private communication (1967). WESTOO, G.: Kvicksilver i mat-iir forgiftningsrisken stor? Var foda 17, 4 (1965 a). - Kvicksilver i agg fran svenska hons i kott fran svenska hons, broiler och kycklingar

samt i svensk kycklinglever. Var foda 18, 85 (1966 a). - Kvicksilver i kott och lever fran svin, ka1 och oxe samt reno Var fOda 18, 88

(1966 b). - Methylmercury compounds in fish. Acta chim. scand 20, 2131 (1966c). - Kvicksilver i fisk. Var fOda 19, 1 (1967). -,B. S]OSTRAND, and T. WESTERMARK: Kvicksilver i agg. Var foda 17, 1 (1965). WIDMARK, G.: Analytical problems; mercury in the general environment. Symposium

of the Swedish Royal Commission of Natural Resources, Stockholm (to be pub­lished) (1966).

Pesticide residues in Canada

By

A. B. SWACKHAMER*

I. Introduction II. History

III. Authority IV. Legal requirements

a) Need for pesticides b) Chemistry of the pesticides c) Methods of analysis . d) Residues . e) Acute toxicity f) Short-term chronic toxicity g) Long-term chronic toxicity h) Reproduction studies

V. Residue tolerances VI. Enforcement program

VII. Legal action Summary Resume Zusammenfassung References .

Contents

I. Introduction

37 38 39 41 41 41 41 42 42 43 44 44 45 46 46 47 47 47 48

Under the terms of the Food and Drugs Act of Canada, an Act enforced by the Food and Drug Directorate of the Department of National Health and Welfare, "No person shall sell a food which has in or upon it any poisonous or harmful substance." A pesticide, because of its purpose, is toxic to some form of life, whether it be an insect, a fungus, a rodent, or a plant. Hence, if a pesticide either directly or indirectly becomes part of a food which may be consumed, a complete and thorough assessment must be made to ensure that the amount remaining on the food is not harmful to man.

"Chief, Division of Standards, Additives and Pesticides, Bureau of Scientific Ad­visory Services, Food and Drug Directorate, Ottawa, Canada.

38 A. B. SWACKHAMER

II. History

The authority to restrict the sale of a food containing a harmful sub­stance has been in existance in Canada since 1860 when "an Act for the preventing of Adulteration of Articles of Food and Drug" was passed. In 1884 this became known as the Adulteration Act and in 1920 the name of the legislation became the Food and Drugs Act. This Act stands basically unchanged today, providing authority and the responsibility to ensure that food offered to the consumer in Canada is safe.

It is understandable that the amount and manner of use of a pesticide may determine the amount of a residue which may be present in a food. It is also understandable that certain levels may be without hazard to health and. providing these levels are not exceeded, no objection need be taken to the proper use of the pesticide. There are provisions under the Food and Drugs Act which permit the establishment of tolerances for pesticide residues which will allow their use under good agricultural practice and, at the same time, not constitute a hazard to the health of the consumer.

In 1911 there was established the first provision for a food to be sold containing in its composition a substance which if present in sufficient quantity would be considered to be harmful. This was a provision for the presence of arsenic as an impurity in a prepared food product, namely baking powder.

While arsenic itself had always been of concern as a potentially harmful substance, it wasn't until 1942 that provision was made for the presence of arsenic in fresh fruit and vegetables at a level that would not be considered to be a hazard to health. A level of 1.4 parts per million (p.p.m.) of arsenic calculated as As20s was established for such products (Food and Drugs Act and Regulations 1942). This level should not be exceeded under the normal agricultural use of arsenicals in the control of insects.

Now that consideration had been given for tolerance levels of a sub­stance such as arsenic, regulations were also promulgated to provide for the presence of lead, copper, zinc, and fluorine in foods within prescribed limits. Hence, in 1949 (Food and Drugs Act and Regulations 1949) the limits for these five substances were provided for in a list of foods as in­dicated in part in Table 1.

As the world population began to grow at an excessive rate and the need for food increased, it became the concern and the practice of agri­culture to use newer and more sophisticated chemical control agents in the production of better quality fruits and vegetables. Since the prescribed use of these materials, in some instances, would result in residues in the marketed food, it seemed desirable to make provision for such residues, provided of course, that the amount was not harmful. It was agreed that a tolerance was the best means of control since this had already been introduced for arsenic

Residues in Canada 39

Table I. Canadian tole1'ances as of 1949

Tolerance (p.p.m.) Commodity

Arsenic Lead Copper Zinc Fluorine

Marine and fresh water animal produas 5 10 100 100 25

Liver 1 2 150 100 2

Fresh fruits 2 7 50 50 2

Fresh vegetables 1 2 50 50 2

Gelatin 2 7 30 100 2

Dried herbs and spices 5 10 50 50 20

Apple juice, cider, wine, and beer 0.2 0.5 2 5 2

Fruit juice except apple juice 0.1 0.2 2 5 1

Beverages as consumed and bottled water 0.1 0.2 2 5 2

and the heavy metals. Hence, in 1956, by an Order-in-Council, provision was made for tolerances for pesticides in Part II of the Regulations under the Food and Drugs Act. This listing has expanded but the format has re­mained unchanged. Table II shows a segment of the 87 pesticides as listed in Part II of the Regulations.

III. Authority

Authority under the Food and Drugs Act only extends to the residue limits which may be present on a food as it is sold. No authority is exercised over those materials which may be used during the production of food crop or food and not leave a residue when it is finally offered for human con­sumption. There is also no authority under the Act over those pesticides which may be used on animal feeds. There is concern, however, over the possibility of these pesticides entering the food chain and becoming a prob-

40 A. B. SWACKHAMER

lem in animal or poultry products if the level is in excess of those considered to be safe for man. Authority under the Food and Drugs Act is restricted, however, and may only be applied to the sale of the animal product when it is offered for the use of man.

Table II. Examples of Canadian tolerances as of 1956

Pesticide

Toler-ance Foods

Common or (p.p.m.) trade name Chemical name

Demeton O,O-diethyl-O 0.2 Potatoes (Systox) (and S)-(2-

Beans, muskmelons, ethylthioethyl) 0.3 phosphorothio ates tomatoes

0.5 Grapefruit, lemons, oranges, straw-berries

0.75 Almonds, apples, apricots, broccoli, Brussels sprouts, cabbage, cauli-flower, celery, grapes, lettuce, peaches, pears, peas, pecans, peppers, plums, prunes, walnuts

Maleic 1,2-dihydro-3, 15 Onions hydrazide 6-pyridazinedione (MH)

30 Beets, carrots, rutabagas

50 Potatoes

Sodium a-phenyl phenol, sa Cherries, ortha- sodium salt nectarines phenyl

loa Citrus fruits, phenate (Stop cucumbers, bell Mould B) peppers, pine-

apple, tomatoes

lsa Sweet potatoes

20G Carrots, peaches, plums

25a Apples, pears

l25a Cantaloupes

G Calculated as a-phenyl phenol

Residues in Canada 41

IV. Legal requirements

Legislative control over the pesticides which may be used with respect to animal feeds is maintained by the Canada Department of Agricttlture and the Provincial Departments of Agriculture. A very good working rela­tionship with these agencies, however, provides an opportunity of entering into discussions as to the pesticides which mayor may not be used, the rate at which they may be used, and whether or not they are likely to constitute a health hazard.

a) Need for pesticides

Before provision is made for a residue tolerance on a food in Canada, it is necessary to provide evidence that such a pesticide is needed in the production of that food. The conditions of use must be such as will provide the control required by the agriculturist and must not result in the presence of a harmful residue. A general outline is given of the information required to support a request for a residue tolerance.

First of all it is necessary to insure that the pesticide will be useful for the purpose intended. Will it provide satisfactory control of certain insects or of certain diseases? Decisions on the usefulness of a pesticide are reached in consultation with other agencies, such as the Department of Agriculture, which can provide competent advice and assistance on the subject of agri­cultural practice and needs.

b) Chemistry of the pesticides

The details of the chemistry of the material must be provided in order that there is a clear understanding of its chemical activity. It is necessary to know if the chemical will remain unchanged or if it changes, what chemical form is it likely to take? Is there a possibility that if degradation products occur, they may constitute a further residue problem? The possibility of impurities resulting from the manufacturing process is carefully studied. The stability of the compound, both in its pure form and in the technical from that may be used for pest control formulations, must be provided.

c) Methods of analysis

With this information as a starting point, a method of analysis is re­quired which will determine in a satisfactory manner the amount of residue which may occur in any of the foods concerned. The ability of the method to satisfactorily recover all active components of that pesticide must be demonstrated. Any significant variation from a 100 percent recovery must be explained. In addition to the primary method of analysis, a confirmatory method is required which wIll ensure that there is no chance of misinter-

42 A. B. SWACKHAMER

pretation of results. The methodology provided must be capable of determin­ing without any doubt the extent of a residue, if one is present.

A very important factor in pesticide analysis is the "cleanup" of the substance being examined. Since minute quantities of a pesticide may be involved, it is necessary to ensure that naturally occurring factors in the food or pesticides which may be present will not interfere with the pesticide being analysed for and provide false values.

d) Residues

In order to understand the extent to which the pesticide may be present in food, sufficient residue smdies must be carried out on all types of crops to establish the residue levels which are likely to occur both at the rate of application recommended and at higher rates. Consideration must be given to residues likely to result using the recommended interval be­tween application and harvest, as well as shorter intervals in order to provide information in the event the pesticide is used too closely to harvest. If the material is to be applied to a crop which is likely to be diverted, either in part as crop waste or by the use of the crop itself for animal feeding, information is required on the possible residue which might occur in dairy, animal, or poultry products used for human consumption. If the material is considered likely to accumulate in the soil, information is required on the persistence in soils and the availability of soil residues to be taken up by subsequent crops.

If the material is water soluble, or if its area of use is likely to allow contamination of watersheds, data are required on the effect that residues in the water may have on fish or cattle, or on growing crops which may be irri­gated from such a water supply.

In the event the material is to be applied to a crop in a manner where a residue is unlikely to occur, data from the examination of treated crops must be provided to demonstrate that indeed no residue will be found. This requires, first of all, recovery checks on simulated crops to show that the method will detect a residue if one exists. Secondly, residue studies on crops which have been treated in a normal manner will have to be checked in order to confirm that no residue is present.

e) Acute toxicity

In the establishment of a tolerance, since human safety is involved, it is essential to have complete and comprehensive toxicological data. This data must be developed by competent groups who are able to both conduct and evaluate the toxicological smdies in a manner which will produce meaningful results.

The first smdies normally carried out to evaluate the safety of the

Residues in Canada 43

pesticide are with respect to the acute toxicity. Initially, oral LD50 determina­tions are made on a minimum of three species of animals, using both sexes. At least one of these three species should be a non-rodent. The active in­gredient in either pure or technical form is used for these determinations. In addition, oral LD50 determinations should be made in at least one animal species for each formulation. Dermal LD50 should also be established in at least one species, usually the rabbit, and where the nature of the pesticide, or the mechanics of its application, indicates possible inhalation hazards, an inhalation LD50 should be determined for at least one species. In all LD50 determinations, symptoms should be fully recorded and details of gross pathological changes in both deceased and surviving animals should be submitted to allow proper evaluation.

f) Short-term chronic toxicity

Short-term chronic oral toxicity studies are essential. The tests should be performed on both sexes of at least two species of animals, one of which should be a non-rodent. The tests are required to be of not less than 90-days' duration. Choice of dose levels should be such that the top dose level initiates a toxic response while the lowest dose level should result in no effect. Intermediate dose levels are desirable to permit estimations of the dose-response effect. Observations should provide information regarding the effect on general appearance, mortality, growth rate, food consumption, haematology, blood chemistry, and pathology. The pathology should re­port the organ weight, organ-body weight ratios, and the histology of the major organs.

In addition to the oral studies there should also be provided eye irrita­tion tests involving repeated exposures to small numbers of animals, pre­ferably using a species with similar central corneal thickness to that found in man. Details of any effects should be reported and should indicate the time of appearance, as well as the disappearance, of such effects. If possible, fluoroscein irrigation should be used to detect corneal damage. A re-assess­ment of the condition of the eye should be made after a short interval to

determine the degree of persistance of symptoms. Dermal irritation studies are also of value. They would consist of re­

peated exposures on consecutive days generally employing a relatively small number of animals. Observations of all signs of irritancy during exposure, together with dermal histopathology on a proportion of the test animals at the termination of exposure, should be provided. Again, there should be a re-assessment during the period of recovery.

If pertinent to the material involved, inhalation tests should be per­formed. The experimental protocol usually requires the repeated exposure of a number of animals of one species to several dose levels of the pesticide for a specific period on a number of consecutive days. A conttol group is

44 A. B. SWACKHAMER

also necessary. Again observations on all toxic symptoms and histopathology are required, in this case, on the respiratory tract and lungs.

In addition to the above short-term tests, data derived from personnel in­volved in the production, processing, or handling of the pesticide are of considerable value and regardless of whether they demonstrate positive or negative effects, they should be submitted whenever possible.

g) Long-term chronic toxicity

In the establishment of a tolerance, since we are concerned with the extended ingestion of a toxic substance, long-term feeding studies are required. There are authorities in the field of toxicology who might dispute this need, however, except where there may be concern over carcinogenic properties. Such long-term studies involve a similar program to that used for the 9O-day oral tests. The long-term studies should continue for not less than 18 months. The groups of animals should be as large as practical in order to give a proper and realistic assessment of the effect and in order to ensure over the long span of 18 months that sufficient animals will survive to provide adequate information on their condition at the termina­tion of the studies Again the dose levels should be selected to give an effect at the highest level and no-effect at the lowest level. The observations to be made on this study should be the same as the 90-day test but with the addi­tion of urinalysis and organ function tests. Observations should be made at the commencement and throughout the program, as well as at its termination.

In the general examination of the toxicology of the material, wherever possible the breakdown products and the metabolites should be identified. If there is any suspicion of significant toxicity of these metabolites, short­term tests should be initiated to establish the degree of toxicity. Data should be provided on the absorption or excretion of the compound or its breakdown products. If there is any suspicion of tissue storage or deposition, this should be investigated. If enzyme studies could be carried out, this would also be of assistance in the evaluation of the safety of a material.

h) Reproduction studies

It is also essential that reproduction studies be performed. These should involve oral feeding of at least two dose levels, with a third group as con­trol. Such a study should extend through three generations with at least two litters per generation. The breeding line should be continued through the Flb and F 2b litters. If possible the F3b litter should be terminated after 20 days' gestation and examined following Caesarean section. Data which should be submitted for Fo adults include body weight and gross pathology at sacrifice. For all generations, mating index, fertility index, gestation in­dex, lactation index, foetal birth weights, survival rates, weanling weights,

Residues in Canada 45

litter size, and sex ratios as well as data on any abnormalities should be provided together with any other data believed relevant to reproductive performance.

In those pregnancies terminated prior to completion, examination for possible teratogenic effects should be undertaken, corpora lutea of pregnancy / ovary, implantations/uterine horn, resorptions, dead embryos, embryo weights, and embryo sexes should be reported. In addition, a proportion of the embryos should be examined for soft-tissue abnormalities, the remainder being examined for skeletal abnormalities. Of course all embryos should first be examined for gross abnormalities.

V. Residue tolerances

If a tolerance is not required, but the material is to be used on an edible crop on a no-residue basis, then the long-term chronic studies, the reproduction studies, and the teratogenic examinations may be omitted. The 90-day studies are usually all that are required to demonstrate what the no­effect level might be and should provide satisfactory information when only incidental human ingestion may occur.

When a tolerance is required, and sufficient toxicological data are avail­able, it then becomes necessary to establish an acceptable no-effect level for man. It should be possible to determine the ampunt of pesticide that the most sensitive animal tested can accept without any harmful effect for a period of time approaching its normal life span. By applying a reasonable safety factor, which is normally taken to be one hundred, the amount that man should be able to withstand for a prolonged period of time without any harmful effect can be established. This information in terms of mg./lday is compared to the amount of pesticide which man could be expected to receive if the residue levels requested are present in all the foods upon which a tolerance has been requested. If the no-effect level is large enough to ac­commodate this load, then it may be possible to accept the tolerance levels which have been requested by the industry and supported by the agricultural agencies. The appropriate recommendations may then be made to have this residue tolerance considered for establishment for the appropriate crops by the Govemor-in-Council. Only when this recommendation has been ac­cepted may the pesticide be used in the manner designated and the crops so treated offered for sale. Providing the tolerance levels designated are not exceeded, there should not be a violation of the Food and Drugs Act. The several safety factors used generally provide a tolerance level that will allow the agriculturist to protect his crops even when the needs are extreme. During normal conditions of use residues rarely are found which approach the established tolerances. Using this system, it is considered that the con­sumer is well protected.

46 A. B. SWACKHAMER

VI. Enforcement program.

At the present time in Canada, there are tolerances established for some 87 pesticides which may be applied to 180 different foods or crops. The responsibility for developing a program to check on possible violations of these tolerances rests with the Field Inspection Staff of the Bureau of Opera­tions of the Food and Drug Directorate. This Bureau maintains a program including both surveillance and monitoring activities. The surveillance pro­gram is directed principally to those areas where pesticide problems with respect to residues are considered most likely to occur. These may be areas of heavy use, or perhaps areas where climatic conditions may create problems conducive to excessive pesticide use. The monitoring phase of the program is designed to establish what the normal use of pesticides without the in­fluence of adverse conditions is likely to produce in the way of residues. This program involves the examination of food samples without specific background knowledge regarding spray histories or area of production.

The entire pesticide program for surveillance, enforcement and monitor­ing comprises the examination of some 3,000 samples per year. By examina­tion of this number of samples it is felt that a reasonable assessment of the levels of pesticides in foods both of domestic or of imported origin can be provided. Emphasis has been placed for a period of years on examining for organochlorine pesticides since these materials, because of their wide use and cumulative properties, have been a cause for concern. But as the use of the organophosphates and the other newer pesticides become more pre­valent, laboratory facilities are being turned to checking for these materials.

Considerable interest has always surrounded the presence of residues in dairy products because of the important position that milk plays in the diet of young children and invalids. The surveillance program has produced a small percentage of residues which may constitute a hazard to health. In­vestigation of these incidents has pointed to the improper use of the pesticide, such as an application which was not warranted, or the use of a level of application which was not in accordance with that recommended. A con­tributing factor to many residues being found in milk is from contamina­tion of soil and of water supplies. This may be as a result of applying a persistant pesticide to a prior crop. Another factor is the use of contaminated crop wastes. Or, it may be as a result of spraying practices which have not taken spray drift into account.

VII. Legal action

In the event that a residue is discovered which is in excess of permitted tolerance levels, the first action by enforcenrent officers is to return to the area of origin and to investigate the reason for the violation. In the event the residue found is in excess of that permitted, the enforcement officer may,

Residues in Canada 47

if health of the consumer could be endangered, order the remainder of that food to be withdrawn from sale. If it can be adequately demonstrated that the producer had knowingly produced a food containing objectionable resi­dues, legal proceedings could be instituted and prosecution could result.

The excellent co-operation with the agricultural chemical industry and with the federal and provincial government agencies has made it possible for the legitimate needs of agriculture to be met. At the same time the public is protected from poisonous substances on the food that may be consumed.

Summary

The regulation of pesticide residues in Canada originated with the introduction of legislation dating back to 1860. This has now become the Food and Drugs Act and Regulations with amendments to provide for the safe use of the modern pesticide. By means of the compilation and review of complete chemical and toxicological data, residue tolerances are established which provide for the use of pesticides in accordance with good agriculture practices which will not endanger the health of the consumer. Monitoring and surveillance of food supplies by the enforcement agency of the Food and Drug Directorate ensure that any violation of established residue levels are dealt with in a practical and effective manner.

Resume*

La reglementation sur les residus de pesticides au Canada a son origine dans l'introduction de la legislation qui remonte a 1860. Ceci est devenu maintenant Ie "Food and Drugs Act and Regulations" avec des amendements pour assurer l'usage sans danger des pesticides modernes. Par la compilation et l'examen de donnees chimiques et toxicologiques completes, on etablit des tolerances residuelles qui assurent un usage des pesticides, selon les bonnes pratiques agricoles, qui n' entralnepas de danger pour la sante du consommateur. En procedant par avertissements et en surveillant les ap­provisionnements, les services de controle du Food and Drug Directorate veillent a ce que toute infraction des teneurs residuelles fixees soit traitee de fa~on pratique et efficace.

Zusammenfassung* *

Die Regelung der Pflanzenschutzmittel-Riickstiinde geht in Canada bis auf die Gesetzgebung des Jahres 1860 zuriick. Aus ihr ist das heutige Lebens­mittel- und Arzneimittel-Gesetz mit seinen Verordnungen und Verbesse­rungen im Hinblick auf den wirksamen Gebrauch der modernen Pflanzen­schutzmittel entstanden. Nach SammIung und Oberpriifung aller chemischen

• Traduit par S. DORMAL-VAN DEN BRUEL • •• 'Obersetzt von H. MAIER·BoDE.

48 A. B. SWACKHAMER

und toxikologischen Daten sind Riickstandstoleranzen festgelegt worden, die in Verbindung mit einwandfreier landwirtschaftlicher Anwendung der Mittel eine gesundheitliche Gefiihrdung der Verbraucher dutch diese ver­huten sollen. Standige Dberprufung und Beaufsichtigung der Lebensmit­tellieferungen dutch den 'Oberwachungsdienst des Lebensmittel- und Arznei­mittel-Direktorats gewahrleisten, dass jede Dberschreitung der festgelegten Ruckstandstoleranzen praktisch und wirksam verhindert wird.

Acknowledgment

I wish to acknowledge the assistance and advice of Mr. David Clegg with respect to the section of this paper pertaining to toxicology.

References

Food and Drugs Act and Regulations (1942). Food and Drugs Act and Regulations (1949).

Uber den Abbau von Dazomet im Boden von

N. DRESCHER* und S. Qrro*

Inhalt

I. Einfiihrung 49 II. Abbau von Dazomet im Boden 50

III. Isolierung und Aufkliirung eines weiteren Dazomet-Metaboliten 51 IV. Biologische Eigenschaften des l,3,5-Trimethyl-hexahydro-triazin-thions 52 Zusammenfassung 53 Resume 53 Summary 53 Literatur 54

I. Einfiihrung

Dazomet (3,5-Dimethyl-tetrahydro-l,3,5,2H -thiadiazin-thion; DMTT; 3,5-D) ist der wirksame Bestandteil des Bodenentseuchungsmittels ® BASA­MID-Pulver, das in Aufwandmengen von 50-60 g. / m.2gegen pflanzenparasitare Nematoden, Bodenpilze und keimende Unkrauter eingesetzt wird. Die reine

s· H C/ 'C=S

2 I I CH 3-N..... ........N- CH 3

CH2

Dozomet

Verbindung ist im trockenen Zustand stabil, sie kristallisiert aus Wasser in farblosen Nadeln vom Schmelzpunkt 105 ° bis 106°C. In saurer Losung zerfallt die Verbindung bereits in der Kalte langsam in Schwefelkohlenstoff, Methylamin und Formaldehyd. Diese Reaktion verlauft in der Wiirme quan­titativ und dient zur Bestimmung des Wirkstoffs in formulierter Ware: Man absorbiert den Schwefelkohlenstoff in alkoholischer Lauge und titriert jodome-

.. Landwirtschaftliche Versuchsstation der Badischen Anilin- & Soda-Fabrik AG, Limburgerhof/Pfalz.

50 N. DRESCHER und S. Ono

trisch das gebildete Xanthogenat. Die alkalische Hydrolyse verIauft anders: Es entstehen gleichfalls Formaldehyd und Methylamin, jedoch wird kein Schwefelkohlenstoff gebildet. Man findet den Schwefel hauptsachlich als Sulfid wieder.

Eine wassrige, neutrale Losung von Dazomet, die bei Zimmertemperatur gehalten wird, beginnt nach ein bis zwei Tagen infolge der Abscheidung von elementarem Schwefel triibe zu werden. Gleichzeitig tritt der stechende Geruch von MethylsenfOl auf und in der Losung lassen sich wiederum Methyl­amin und Formaldehyd nachweisen.

II. Abbau von Dazomet im Boden

1m Boden verIauft der Abbau des Wirkstoffs in ahnlicher Weise wie in neutraler Losung (MUNNECKE et al. 1962, MUNNECKE und MARTIN 1964). Die Bildung von Methylsenfol im Boden erfolgt tiberraschend schnell: Mischt man einer feuchten Komposterde (ca. 12 Prozent Humusanteil) bei Zim­mertemperatur reines Dazomet zu, so kann man bereits nach 15 bis 20 Mi­nuten MethylsenfOl am Geruch deutlich erkennen. Entsprechend schnell ver­Iauft der Abbau des Wirkstoffs im Boden: Wir verfolgten den Abbau tiber die Schwefelkohlenstoff-Bestimmung der noch intakten Substanz und fanden in der obengenannten Komposterde-bei Zusatzversuchen mit 125 p.p.m. Wirkstoff-Abbauzeiten von vier Stunden (bei 20 0 C.) bzw. 12 Stunden (bei 30 C.). Dieser Befund scheint uns darauf hinzudeuten, dass der erste Abbauschritt in einer Ringspaltung an der S-CS-Bindung besteht. Schwefel­kohlenstoff wird beim Dazomet-Abbau im Boden anscheinend nicht ge­bildet, hingegen liessen sich geringe Mengen Schwefelwasserstoff nachweisen.

Methylsenfol ist als das wesentliche fungizide und bakterizide Agens anzusehen (MUNNECKE et al. 1962, SCHADE und RIECHE 1966). GOKSOYR

( 1964) weist auf die gute Wirkung des Formaldehyds als Bodenbegasungs­mittel hin und schreibt diesem gleichfalls eine desinfizierende Wirkung im Boden zu.

KOETIER et al. (1961) konnten mit 35S-markiertem SenfOl zeigen, dass dieses im Boden gleichfalls schnell abgebaut wird, wobei ein Teil des Schwefels bis zum Sulfat oxydiert wird.

Methylsenfol setzt sich mit Methylamin nach bekannter Reaktion zurn symmetrischen Dimethylthioharnstoff urn. In wassrigen Ausztigen von BOden, die einige Tage zuvor mit Dazomet behandelt worden waren, liess sich der Harnstoff praktisch immer nachweis en. Daneben fanden wir gelegentlich in geringer Menge auch den Monomethylthioharnstoff, der aus Methylsenfol und Ammoniak im Boden gebildet wird.

Flit unsere weiteren Untersuchungen wahlten wir einen Boden, der den Wirkstoff besonders langsam abbaute. Es handelte sich urn einen hurnusarmen (0,24 Prozent), schlecht versorgten Sandboden (pH 6,8; abschlammbare Teile: 9,85 Prozent; P20 5: sieben mg./IOO g.; K20: zwei mg./l00 g.; Mg: zwei mg./IOO g.), dem wir ca. 2000 p.p.m. BASAMID-Pulver zusetzten.

Dazomet im Boden :51

Das ist etwa das 15-fache der Konzentration, die im Boden bei iiblicher Be­handlung erreicht wild.

Den Abbau verfolgten wir diinnschichtchromatographisch. Hierzu wurde zu bestimmten Zeiten ein Tei! des Bodens mit Wasser extrahiert, die wass­dgen Ausziige wurden mit Chloroform ausgeschiittelt und die Chloro­formextrakte chromatographiert. Am Ende des Versuchs wurden die gesam­melten Chloroform-Extrakte noch einmal gemeinsam chromatographiert (Abbildung 1).

o o

• Abb. 1. Abbau von Dazomet im Boden. Links: 1 = Testgemisch (Mono-, Di- und

Trimethylthioharnstoff), 2 = 3 Tage, 3 = 8 Tage, 4 = 15 Tage, 5 = 23 Tage nach Versuchsbeginn. Rechts: 1 = Testgemisch, 2 = 30 Tage, 3 = 37 Tage, 4 = 44 Tage, 5 = 52 Tage nach Versuchsbeginn. Kieselge1 G (Merck); Laufmitte1: Methanol-

Chloroform (5 :95); Indikator: Reagens nach GROTE fur Thioharnstoffe

Auf den Chromatogrammen ist zu erkennen, dass sich im gleichen Masse wie der Wirkstoff (umrandete Flecken) abgebaut wurde, eine neue Ver­bindung bildete.

III. Isolierung und Aufkliirung eines weiteren Dazomet-Metaboliten

Wir konnten diesen Metaboliten aus dem Boden isoHeren und seine Konstitution aufklaren: Der Boden wurde, nachdem diinnschichtchromato­graphisch kein Dazomet mehr nachweisbar war, mit Wasser extrahiert. Aus der wassrigen Phase wurde die Verbindung mit Chloroform ausgezogen und nach Einengen des organischen Losungsmittels mit Aether gefallt. Aus Cyc­lohexan kristallisierte die Verbindung in farblosen Stabchen vom Schmelz­punkt 115 0 bis 116 0 C. Aus der Elementaranalyse Hess sich die Summen­formel C6HlaNaS berechnen. Die Substanz enthielt somit ein S-Atom weni­ger und CHaN mehr als das Ausgangsprodukt. Dieser Befund Hess sich am besten erklaren, wenn man im Dazomet-Ring den Schwefel durch eine N-CHa-Gruppe ersetzt dachte.Fiir die Struktur eines 1,3,5-Trimethylhexa­hydro-triazin-thions (1) sprachen auch das Ultraviolett- und NMR-Spektrum.

52

S II

N. DRESCHER und S. Orro

CH 3""" /C" /CH 3 N N I I H H -2HzO

H'CHO OHC'H

HNH I CH 3 I

Den Konstitutionsbeweis erbrachten wir durch Synthese der Verbindung: Man erkennt aus der Konstitutionsformel (I), dass man das Triazinderivat als Kondensationsprodukt des symmetrischen Dimethyl-thioharnstoffs mit Formaldehyd und Methylamin auffassen kann. Wir brachten diese Kompo­nenten in wassriger Losung in stochiometrischem Verhaltnis zusammen, stellten den pH-Wert mit Natronlauge auf acht ein und liessen das Gemisch bei Zimmertemperatur stehen. Nach drei Tagen kristallisierte die erwartete Ver­bindung spontan aus (77 prozentige Ausbeute) und erwies sich als identisch (Schmelzpunkt, Misch-Schmelzpunkt, UR-Spektrum, Dlinnschicht-Chroma­togramm) mit dem Dazomet-Metaboliten.

Die Verbindung entsteht im Boden offenbar in gleicher Weise wie wir sie synthetisiert haben, denn alle erforderlichen Komponenten werden beim Zerfall des Dazomets gebildet. Es ist dabei nur wesentlich, dass das Methyl­amin in Freier Form vorliegt. In stark sauren, humusreichen Boden konnten wir den Metaboliten nicht nachweisen. Allerdings ist die Bildung des Triazin­Derivats keine spezifische Bodenleistung. Lasst man namlich eine wassrige Dazomet-Losung bei Zimmertemperatur stehen, so kann nach einigen Tagen sowohl der symmetrische Dimethyl-thioharnstoff als auch das Triazin-Derivat in der Losung nachgewiesen werden.

IV. Biologische Eigenschaften des 1,3,5-Trimethyl-hexahydro­triazin-thions

Es war noch zu prlifen, ob der Dazomet-Metabolit, wenn er im Boden in grosserer Menge entsteht, Kulturpflanzen schadigen kann. Der Verdacht lag insofern nahe, als Triazin-Derivate als herbizid wirksame Verbindungen bekannt sind. Die Verbindung besitzt jedoch keinerlei herbizide, fungizide oder insektizide Wirksamkeit. Selbst Zusatze von 50 p.p.m. zu verschiedenen BOden-das ist eine Konzentration, die bei normaler Anwendung von BASAMID-Pulver bei we item nicht erreicht wird-verursachten bei Salat, Lauch, Kohl und Tomaten lediglich eine geringfligige Wachstumsdepression, sonst aber keinerlei Schaden. Die Verbindung ist im Boden nicht persistent. Untersuchungen liber ihren weiteren Abbau sind jedoch noch nicht ab­geschlossen.

Dazomet im Boden 53

Zusammenfassung

Beim Abbau von Dazomet (3,5-Dimethyl-tetrahydro-l,3,5,2H-thiadiazin-2-thion) im Boden entstehen neben den bereits bekannten Abbauprodukten Methylsenfol, Formaldehyd und Methylamin durch Resynthese N,N'-Di­methyl-thioharnstoff und ein weiterer Metabolit, der als 1,3,5-Trimethyl­hexahydrotriazin-thion identifiziert werden konnte. Die Konstitution der Verbindung wurde durch Synthese bewiesen. Die Bildung des Triazin­Derivats ist allerdings keine spezifische Bodenleistung, es entsteht auch beim Zerfall von Dazomet in neutraler, wassriger Losung. Die Verbindung ist biologisch inaktiv und im Boden nicht persistent. Gewachshauskulturen zeigten keine Schadigungen, wenn sie auf Boden wuchsen, denen bis zu 50 ppm der Substanz zugemischt waren.

Sur Ia decomposition du Dazomet dans Ie sol

En plus des produits de decomposition deja connus, l'isothiocyanate de methyle, Ie formaldehyde, la methylamine, 1a N.N'-dimethyl thiouree et un autre metabolite plus lointain resultent de la decomposition du Dazomet (3,5-dimethyltetrahydro-l,3,5,2H -thiadiazine-2-thione) par resyntheses. La constitution de ce compose a ete demon tree par synthese.

Cependant, la formation de ce derive triazinique n'est pas specifique au sol, car il se forme egalement lors de la degradation du Dazomet en solution aqueuse neutre. Ce compose est biologiquement inactif et peu remanent dans Ie sol. Les cultures en serre ont lieu sans dommage lorsque les sols en ren­ferment jusqu'a 50 ppm.

Summary * * The decomposition of Dazomet in soil

In addition to the already known decomposition products methylmustard oil, formaldehyde, and methylamine, N.N' -dimethylthiourea and a further metabolite result from the decomposition of Dazomet (3,5-dimethyl-tetra­hydro-l,3,5,2H-thiadiazine-2-thione) by resynthesis. The constitution of this compound was proved by synthesis. Indeed, the forming of this triazine­derivate is not specific to soil for it also results from the decay of Dazomet in neutral or aqueous solution. This compound is biologically inactive and not persistent in soil. Greenhouse cultures did not show any damage when they grew on soils mixed to contain up to 50 p.p.m. of this substance.

*Traduit par R. MESTRES. • ·Translated by MARGARETE DUSCH.

54 N. DRESCHER und S. OTro

Literatur

GOICsOYR, J.: Chemical and fungicidal reactions of 3,5-dimethyl-tetrahydro-l,3,5-thia­diazine-2-thione (3,5-D). A comparison with sodium N-methyl dithiocarbamate and methyl-isothiocyanate. Acta Chem. Scand. 18, 1341 (1964).

KoETI'ER, K., J. WILLENBRINK, und K. JUNKMANN: Der Abbau von 35S-markiertem Methylsenfol in verschiedenen Boden. Z. PB.-Krankh. PB.-Path. PB.-Schutz 68, 407 (1961) .

MUNNBCKE, D. E., K. H. DOMSCH, and J. W. EcKERT: Fungicidal activity of air passed through columns of soil treated with fungicides. Phytepatho1. 52, 1298 (1962) .

-, and J. P. MARTIN: Release of methylisothiocyanate from soils treated with Mylone (3,S-dimethyl-tetrahydro-l,3,5,2H-thiadiazine-2-thione). Phytopatho1. 54, 941 (1964).

SCHADE, W., und A. RIECHE: Synthetische Senfolbildner, VII. Arch. Pharmaco1. 299, 589 (1966).

Examinations of Danish milk and butter for contaminating organochlorine insecticides

By

F. BRo-RAsMusSEN*, Sv. DALGAARD-MIKKELSEN**, TH. JAKOBSEN***, Sv. O. KOCH***, F. RODIN***, E. UHL****, and K. VOLDUM-CLAUSEN*

I. Introduction II. Kinds of material examined

III. Methods IV. Results

a) Milk and butter b) Animal feed

V. Discussion Summary Resume Zusammenfassung References .

Contents

I. Introduction

55 56 57 58 58 61 65 67 68 68 69

In 1961 the Municipal Food Control Laboratory at Arhus, Denmark, started analytical examinations of Danish milk and butter to establish the content of organochlorine pesticide residues. The material chosen was milk delivered to six local dairies, and the analytical work was based on paper­chromatographic methods. The results obtained through this initial work showed that certain contaminations were prevalent, and it was decided to extend the work to include both examination of salIliPles from the whole of the country and more thorough local examinations. At the same time, more efficient methods of analysis by means of gas chromatography were intro­duced. Furthermore, as part of the extension of the work, a close co-opera­tion was established with the National Pesticide Laboratory, Copenhagen. This laboratory directed the country-wide examinations and afforded an in-

.. National Pesticide Laboratory, Copenhagen, Denmark. .... Department of Pharmacology and Toxicology, Royal Veterinary and Agri­

cultural College, Copenhagen, Denmark. ." .. Arhus Municipal Food Control Laboratory, Arhus, Denmark.

.. "." National Health Service, Copenhagen, Denmark.

56 F. BRO-RAsMUSSEN et at.

creased analytical capacity for supplementary investigations, especially for the analysis of animal feed.

The purpose of the examinations, which are still going on, has been to establish whether Danish dairy products are contaminated by contents of persistent organochlorine pesticides, and, if so, in which quantities, and also, if possible, to trace some of the sources which might account for a contami­nation demonstrated. The results presented here include all samples drawn until the end of 1966.

II. Kinds of material examined

Throughout the period of examination the sampling prograIIl1lle was as follows:

Milk delivered to six local dairies in the Arhus area was sampled regu­larly every three months during the period 1962-1966. These samples were compositions representing about 100 producers delivering to the dairy on the same day. For practical reasons samples were taken from each producer at the delivery to the dairy and taken to the laboratory where the final sam­ples were composited and mixed in the ratios in which the individual pro­ducers delivered to the dairy on that same day, A total of 179 fluid milk samples representative of the production of the six dairies was taken during the period in question.

Milk samples from individual farms. During a period of 12 to 14 months, one monthly sample of milk was taken from each of 20 selected farms which had daily deliveries of milk to dairies in Arhus. In these well-organized farms with controlled working conditions, records were kept on the animal feeding as well as on the use of pesticide chemicals, which afforded very good co­operation and the best possibilities of obtaining further information. About 260 samples of fluid milk were taken from the farms during this period.

Butter samples from the whole of the country were obtained by the as­sistance of the inspectors of the State Quality Control of Dairy Products and Eggs, etc. through a monthly sampling programme during the period 1964-1966. The country was divided into six regions having almost the same total productions, and butter samples were taken monthly from one or two dairies chosen at random within each region and sent to the labora­tory; 381 butter samples representative of the Danish butter production dur­ing the three-to-four year period of examination were taken.

Animal feed. Supplementary to the milk and butter sampling a consider­able number of animal feed samples for analysis were taken at different points of the programmes. From the 20 farms mentioned above, samples of feeding beets (swedes) of own growing as well as samples of commercial feed mix­tures used at the farms were taken in October 1964 and June 1965. Further­more, samples were drawn from a variety of different plant material used for the feeding of cows in two selected farms, for example, alfalfa, silage,

Danish milk and butter 57

straw, etc. And, finally, a number of imported single feed components such as oilseed cakes and expellers were included in the programmes. These im­ported products are usually main constituents of the commercial feed mix­tures.1

III. Methods

All samples were taken into analysis or stored at a temperature of 20°C. immediately after being received at one of the laboratories.

For the paper chromatographic analyses in the early period the method described by MILLS (1959) was used without modifications, whereas the method of MAUNDER et al. (1964 a and b) was preferred for the gas chrom­atographic work, except for an initial period until the beginning of 1965 during which one of the laboratories used the method of MILLS (1959) for extraction and cleanup.

EGAN's method (Maunder et al. 1964 a and b) for determination of organochlorine insecticides uses gas chromatography with electron-capture detector, the samples first having been prepared through isolation of the butter fat, followed by liquid/liquid partitioning between DMF (dimethyl­formamide) and hexane, and chromatographic cleanup on a column of stand­ardized aluminium oxide. For the analysis of plant material, extraction with methylene chloride followed by chromatography on aluminium oxide was used.

Since the analytical work was divided between two laboratories, it was most essential that the methods were tested and that the results obtained were proved to be directly comparable. After a period of working in of the gas chromatographic method and with the opportunity of making necessary modifications, a collaborative testing of the method was arranged between the laboratories. Samples of butterfat, milk, and extracts of carrots with or without insecticides added in known amounts were divided between the lab­oratories for anlayses and evaluation of the results. As this test proved that the method was satisfactory at both of the laboratories, all later examinations were made by this method without further modifications.

The results of the testing as well as a more detailed description of the method will be found elsewhere (Bro-Rasmussen et al. 1968). It should only be mentioned here that the limit of detectability was found to be 0.007 p.p.m., and the quantitative determination limit was 0.020 p.p.m. in the case of organochlorine insecticides, both values found on a butterfat basis. Accord­ingly all results are corrected to two decimals and results between the two sensitivity limits, meaning identified traces, are recorded as 0.01 p.p.m. Any results below 0.007 p.p.m. are recorded as not detectable (n.d.)

1 Samples of imported animal feed constituents were kindly supplied for this pur­pose by the firms of ]ysk Andelsfoderstofforretning, Arhus, and Korn- og Foderstof­kompagniet, Ltd., Arhus.

58 F. BRO-RAsMUSSEN et at.

IV. Results

a) Milk and butter

Table I. Organochlorine insecticides in milk delivered to six local dairies in Arhus (1962/1966)

Year Insecticide in butterfat (p.p.m.) a

(no. of

\ Method samples) u-BHC Lindane DDT DDE

\ Dieldrin

Paper 1962 chroma- (36) 33 samples (-92%) containing one or more insecticides tography

1963 (22) 16 samples (-73%) containing one or more insecticides

Gas 1964 0.06 0.04 0.11 0.04 _11

chroma- (33) [n.d.-0.18] [n.d.-0.18] [n.d.-0.271 [0.02-0.14] tography

1965 0.05 0.03 0.08 0.04 _11

(44) [0.02-0.09 ] [n.d.-O.071 [0.02-0.16] [0.02-0.071

1966 0.05 0.03 0.05 0.03 0.03 (44) [0.02-0.10] [0.01-0.071 [0.02-0.08] [n.d.-0.06] [n.d.-D. 11 ]

a n.d. = not detectable (i.e., <0.007 p.p.m.). Figures in brackets represent low­est-highest contents in individual samples.

b Not determined.

Table I shows the analyses of milk from the six local dairies in the Arhus area from the period 1962-66. The paper chromatographic examinations of the first years indicate that a-BHC, lindane, DDT, DDE, and dieldrin were present in a majority of the samples whereas the results of the more sensi­tive gas chromatographic analyses from 1964-1966 show that one or more of these insecticides are present in all of the samples. The same conclusion could be drawn from the gas chromatographic analyses of 381 butter sam­ples from dairies all over the country as shown in Table II.

Quantitatively, the mean contents of the individual compounds exceed 0.10 p.p.m. in a few cases only, and there is a relatively good agreement between the results of Table I and Table II for milk and butter, respectively, confirming the general experience that insecticide residues follow the but­terfat of the milk. There is, however, a tendency to find slighdy higher resi­dues in milk delivered to the six local dairies (Table 1) than in the butter from all over the country, which is probably due to a higher degree of level­ling in the country-wide material. Furthermore, there is a tendency to find decreasing contents of all insecticides from 1964-1966 as indicated both by the mean values and by the highest content found in an individual sample. This tendency is hardly of statistical significance, but once noticed it may be interpreted as an increased attention on the part of the pesticide users towards the risks of contamination.

Year

1964

1965

1966·

Danish milk and butter 59

Table II. Organochlorine insecticides in butter from 381 Danish dairies (1964/66)

Insecticide in butterfat (p.p.m.) a

No. of

I samples n-BHC Lindane DDT DDE Dieldrin

118 - 0.01 0.08 0.05

[0.01.0.071 [0.01·0.42] [0.01·0.20]

137 0.05 0.02 0.05 0.02 0.04

[0.02·0.18] [0.01-0.04] [0.01-0.22] [0.01·0.06] [0.01-0.18]

126 0.05 0.01 0.04 0.02 0.03

[0.02-0.14] [0.01·0.03] [0.01-0.171 [0.01·0.05] [0.01-0.17]

a Figures in brackers represent lowest·highest contents in individual samples.

To get closer to the sources of contamination, milk samples from 20 selected farms were obtained through a monthly sampling programme, di­rectly from the producers during a one-year period. The results of the analyses of these samples are compiled in Table III, showing a rather uniform pic­ture as far as the mean content is concerned and showing no substantial dif­ference from the results in Tables I and II. There is, however, as might be expected, a greater variation and a greater difference between the highest and the lowest values.

Among the results in Table III there is one diverging value, the DDT content in milk from farm no. 4 being considerably higher than the others. This finding caused an increased sampling programme of milk (as well as of animal feed-see below) especially from that farm, followed by inter­views and increased control of the use of DDT chemicals. No direct proof of excessive or illegal use of DDT in connection with the animal husbandry was found; however, it was noticed that the farm had a comparatively large stock of a 50 percent DDT emulsion concentrate. During the later part of the sampling programme, the DDT /DDE content in milk from farm no. 4 decreased and reached the same level as that found in all the other farms. Thus, one cannot preclude the possibility that a certain excessive use of the DDT preparation occurred which, however, came to an end on account of the enquiries and the search.

The increased level of DDT /DDE in milk from farm no. 4 as compared to other farms is also illustrated in Figure 1 which shows the variations in DDT /DDE contents throughout the one-year period. The curve reaches a peak value in the course of the autumn months. Such seasonal variations have not been demonstrated in the case of the other insecticides found.

60 F. BRO-RASMUSSEN et aI.

Table III. Organochlorine insecticides in milk from 20 farms in the Arhus area during a 13 months' period (1964-1965)

Farm no. Insecticide in butterfat (p.p.m.)a a-BRe Lindane DDT DDE I DDT/DDE

1 O.OS 0.01 0.04 0.03 0.07 [0.02-0.2S] [n.d.-0.04] [n.d.-0.21] [n.d.-0.09] [n.d.-0.24]

2 0.07 0.02 0.05 0.02 0.07 [ 0.02-0.14] [n.d.-O.ll] [n.d.-0.26] [0.02-0.05] [O.02-0.2S]

3 0.03 0.01 0.03 0.02 0.05 [n.d.-0.05] [n.d.-O.Q3] [n.d.-O.OS] [0.01-0.03 ] [0.01-0.11]

4 0.04 0.01 0.34 0.06 0.40 [0.02-0.071 [n.d.-0.02] [0.12-0.70] [0.02-0.35] [0.14-0.75]

5 0.05 0.02 0.09 0.04 0.13 [0.02-0.10] [n.d.-O.Q3] [n.d.-0.32] [0.02-0.23] [0.02-0.37]

6 0.06 0.03 0.05 0.03 0.09 [0.03·0.11] [n.d.-0.09] [n.d.·0.23] [n.d.-0.06] [n.d.-0.271

7 0.03 0.01 0.02 0.02 0.05 [n.d.-O.13] [n.d.-0.03] [n.d.-0.09] [n.d.-0.04] [0.02-0.12]

8 0.05 0.01 0.05 0.03 O.OS [n.d.-0.09] [n.d.-0.02] [n.d.-0.23] [n.d.-O.OS] [n.d.-0.2S]

9 0.04 0.01 0.03 0.03 0.05 [n.d.-O.OS] [n.d.·0.05] [n.d.-0.I5] [n.d.-0.14] [n.d.-0.15]

10 0.06 0.02 0.03 0.03 0.06 [0.02-0.171 [n.d.-0.05] [n.d.-O.lO] [n.d.-0.09] [ 0.02-0.16]

11 0.06 0.02 0.03 O.OS 0.11 [0.02-0.16] [n.d.-0.05] [n.d.-0.09] [n.d.-0.5S] [O.02-0.5S]

12 0.06 0.02 0.04 0.04 0.10 [n.d.-0.14] [n.d.-0.05] [n.d.-0.09] [n.d.-O.14] [ 0.02-0.19]

13 0.05 0.02 0.06 0.07 0.13 [n.d.-O.lO] [n.d.-0.03 ] [n.d.-0.44] [n.d.·0.19] [n.d.-0.54]

14 0.04 0.02 0.03 0.02 0.05 [n.d.-0.16] [n.d.-0.07] [n.d.-0.12] [n.d.-0.04] [0.02-0.14]

15 0.05 0.01 0.03 0.02 0.05 [0.02-0.11] [n.d.-O.Q3] [n.d.-0.09] [n.d.-0.05] [n.d.-O.13]

16 0.05 0.01 0.06 0.04 0.10 [n.d.-O.lO] [n.d.-0.03] [n.d.-0.26] [n.d.-0.12] [0.02·0.26]

17 0.04 0.01 0.D3 0.D3 0.06 [n.d.-O.lO] [n.d.-0.03] [n.d.-O.071 [n.d.-O.OS] [0.02-0.13 ]

IS 0.03 0.01 0.05 0.03 O.OS [n.d.-O.071 [n.d.-0.03] [n.d.-0.34] [0.02-0.05 ] [ 0.02-0.36]

19 0.06 0.01 0.04 0.D3 0.07 [n.d.·0.17] [n.d.-0.05 ] [n.d.-O.lS] [n.d.-O.071 [n.d.-0.2l]

20 0.04 0.02 0.04 0.D3 0.07 [n.d.·O.ll] [n.d.-0.06] [n.d.-0.16] [n.d.-0.05] [0.02-0.lS]

Av.b 0.05 0.02 0.06 0.04 0.09

a Figures in brackets represent lowest-highest contents in individual samples; n.d. = not detectable (i.e., <0.007 p.p.m.).

II 260 samples. .

0.8

0.7

0.6

~ 0.5 ci.

~ 0.4 o

§·0.3

0.2

0.1

o

Danish milk and butter 61

r-

f- n Farm no.4 (June 1964 10 July 1966)

I-~ Average of 20 farms (June 1964 to June 1965) 1

September value only approximate

l-

t-

l-

f-

l- I I .~ n nnnnn • June Aug. Oct. Dec.· Feb. Apr. June Aug. Oct. Dec. Feb. Apr. June 1964 a 1965 1966

Fig. 1. Seasonal variation of DDT-DDE-content in butterfat

b) Animal feed

The feeding plans for cows in Denmark are highly dependent on the season. During the pasture period of the summer (from May to the end of October) some supplements of commercial feed mixtures and concentrates are given, usually amounting to about 10 to 15 percent of the total feeding. They consist of imported products such as oilseed cakes, expellers, etc., and, to some extent, of Danish grown grain, mostly barley. In the winter feeding plan, these feed mixtures and concentrates are still essential, whereas the pas­ture feeding is replaced by other products, predominantly feeding beets (for example, swedes) covering 25 to 50 percent of the feeding plan, with hay, straw, silage, etc. as the remaining components. Practically all of these com­ponents are of the own growing of the individual farms.

Tables IV to VI contain the results of analyses of a total of about 160 samples of animal feed, all of which are major components in cow feeding in Denmark in accordance with these plans. The samples were obtained from the 20 farms mentioned above. Only the feed concentrates of foreign origin (Table V) and a variety of Danish grown crops (see Table VI) were sampled from the importers or from two individual farms, respectively, of­fering better possibilities for such samplings.

The material in the tables should not be taken as final or fully covering of Danish cow feeding or of the risk of contamination derived from the feeding. It does, however, give an indication of the problem, as shown by the tables, that residues of the insecticides found in milk can be traced back to feed samples of foreign as well as of Danish origin. Most significant is

62 F. BRo-RASMUSSEN el ill.

Table IV. 01'ganochlO1"ine insecticides in animal feed mixtMes containing both impo1'ted and domestic components (1964-66)

Insecticides in feeds (p.p.m.)a

No. of Farm Aldrin/ samples no. Date a-BHe Lindane DDT DDE dieldrin

19 1-19 Oct. '64 0.02 0.01 0.02 0.01 0.04 [n.d.-0.10] [n.d.-0.03] [n.d.-0.06] [n.d.·0.02] [0.01-0.14]

19 1-19 June'6S 0.02 0.01 0.05 0.01 0.02 [n.d.-O.OS] [<0.01-0.03] [0.02-0.10] [n.d.-0.02] [0.01·0.02]

1 4 Oct. '64 0.03 0.02 0.04 0.01 0.02

1 4 June '65 0.02 0.01 0.10 0.01 0.02

1 4 Oct. '65 n.d. n.d. 0.02 n.d. n.d.

2 4 Nov.'6S <0.01 <0.01 0.03 n.d. 0.05

2 4 Dec. '65 <0.01 <0.01 0.03 n.d. 0.04

2 4 Feb. '65 <0.01 <0.01 0.02 n.d. 0.02

1 4 May'66 <0.01 <0.01 0.01 n.d. 0.04

1 4 Oct. '66 <0.01 n.d. 0.04 n.d. 0.01

2 21 Sept. '65 <0.01 <0.01 0.02 0.01 0.01

2 21 Nov.'6S <0.01 <0.01 0.06 0.01 0.05

2 21 Dec. '65 <0.01 <0.01 0.02 n.d. 0.03

2 21 Jan. '66 n.d. <0.01 0.03 n.d. 0.01

2 21 Mar. '66 0.04 0.02 0.03 n.d. 0.03

2 21 Apr. '66 0.04 0.02 0.03 n.d. 0.03

2 21 May'66 0.04 0.02 0.02 n.d. 0.03

2 21 June '66 <0.01 <0.01 <0.01 n.d. 0.02

1 21 July'66 0.01 <0.01 <0.01 n.d. n.d.

2 21 Dec. '66 0.04 0.02 0.08 n.d. 0.01

a n.d. = not detectable (i.e., <0.005 p.p.m.). Figures in brackets represent low­est/highest contents in individual samples.

Danish milk and butter

Table V. Organochlorine insecticides in samples of imported feed concentrates (1964-1966)

63

Insecticides in feed concentrates (p.p.m.) a

Product Exporting Aldrin/

country a-BHC Lindane DDT DDE dieldrin

Coconut expellers - 0.02 0.05 0.37 0.02 0.01

Coconut expellers - 0.01 0.01 0.01 0.01 n.d.

Coconut expellers Philippines 0.02 0.01 0.01 n.d. n.d.

Cottonseed cakes Syria 0.01 0.02 0.D7 0.01 n.d.

Cottonseed cakes Nicaragua 0.08 0.02 0.10 0.01 <0.01

Cottonseed cakes U.S.S.R. <0.01 <0.01 0.01 n.d. <0.01

Cottonseed cakes Turkey <0.01 <0.01 0.02 n.d. n.d.

Cottonseed expellers - <0.01 <0.01 0.04 0.01 <0.01

Cottonseed expellers India 0.05 0.02 0.05 0.01 n.d.

Cottonseed expellers Iran <0.01 <0.01 0.01 n.d. n.d.

Cottonseed expellers Peru <0.01 <0.01 0.01 n.d. <0.01

Cottonseed expellers Tanzania 0.01 0.01 <0.01 n.d. n.d.

Cottonseed expellers Uganda 0.01 0.02 0.04 0.02 0.01

Groundnut cakes Iraq n.d. n.d. n.d. n.d. n.d.

Groundnut cakes India 0.05 0.02 0.04 n.d. <0.01

Groundnut expellers Dakar n.d. 0.01 n.d. n.d. <0.01

Hazelnut expellers Turkey 0.03 0.02 0.13 0.01 n.d.

Linseed cakes W.Germany <0.01 <0.01 <0.01 n.d. n.d.

Rapeseed cakes W.Germany <0.01 0.02 n.d. n.d. n.d.

Rapeseed expellers Pakistan 0.03 0.02 0.12 0.01 0.01

Rapeseed & groundnut Denmark & 0.01 0.10 0.08 0.01 <0.01 expellers (1: 1 ) Dakar

Soyabeans U.S.A. 0.01 n.d. 0.01 n.d. 0.01 (or China?)

Soyabean cakes - n.d. n.d. <0.01 n.d. <0.01

Soyabean cakes U.S.S.R. <0.01 <0.01 n.d. n.d. n.d.

Soyabean expellers Argentine 0.03 0.02 0.D7 0.01 <0.01

Soyabean meal U.S.A. n.d. <0.01 n.d. n.d. n.d.

a n.d. = not detectable (i.e., <0.005 p.p.m.).

Tab

le V

I. O

rgan

ochl

orin

e in

sect

icid

es i

n s

ampl

es o

f do

mes

tic a

nim

al f

eed

com

pone

nts

(196

4-66

)

Inse

ctic

ides

in

feed

com

pone

nts

(p.p

.m.)

a

No.

of

Farm

A

ldri

n/

Prod

uct

sam

ples

no

. a-

BH

C

Lin

dane

D

DT

D

DE

di

eldr

in

Bee

ts

21

1-21

n.

d.

«0

.01

n.

d.

n.d.

n.

d.

[n.d

.-n.

d.]

[n.d

.-0.

02]

[n.d

.-n.

d.]

[n.d

.-n.

d.]

[n.d

.-n.

d']

Alf

alfa

mea

l 1

1-21

<

0.01

n.

d.

0.01

n.

d.

n.d.

A

lfal

fa p

elle

ts

1 1-

21

<0.

01

n.d.

0.

01

n.d.

n.

d.

Gra

ss p

elle

ts

1 1-

21

n.d.

n.

d.

0.02

n.

d.

n.d.

Bar

ley

1 4

n.d.

0.

01

n.d.

n.

d.

0.01

B

eets

7

4 n.

d.

«0

.01

<

0.01

n.

d.

«0

.01

[n

.d.-

n.d'

] [n

.d.-

<O.O

l]

[n.d

.-0.

03]

[n.d

.-n.

d.]

[n.d

.-<O

.Ol]

B

eets

gre

ento

p 2

4 n.

d.

n.d.

n.

d.

n.d.

n.

d.

Bee

ts w

aste

3

4 n.

d.

n.d.

«

0.0

1

n.d.

<

0.01

[n

.d.-

n.d'

] [n

.d.-

n.d'

] [n

.d.-

<O.O

l]

[n.d

.-n.

d.]

[n.d

.-<O

.Ol1

G

rass

6

4 n.

d.

n.d.

n.

d.

n.d.

«

om

[n

.d.-

n.o.

] [n

.d.-

n.d'

] [n

.d.-

n.d'

] [n

.d.-

n.d.

] [n

.d.-

<O.O

l]

Lin

seed

cak

e 1

4 0.

08

0.08

0.

07

n.d.

0.

02

Sila

ge g

rass

1

4 <

0.01

n.

d.

n.d.

n.

d.

n.d.

St

raw

5

4 «

0.0

1

«0

.01

0.

01

<0.

01

0.02

[n

.d.·O

.Ol]

[n

.d.-<

O.O

l1

[n.d

.-0.

04]

[n.d

.-0.

02]

[ <0.

0 1-

0.04

]

Bee

ts

17

21

«0

.01

«

0.0

1

0.01

«

0.0

1

<0.

01

[n.d

.-<O

.Ol1

[n

.d.-

<O.O

l]

[n.d

.-0.

04]

[n.d

.-O.O

l]

[n.d

.-0.

02]

Bee

ts g

reen

top

2 21

n.

d.

n.d.

0.

01

n.d.

n.

d.

Bee

ts s

ilage

1

21

n.d.

n.

d.

0.01

n.

d.

n.d.

G

rass

3

21

n.d.

n.

d.

<0.

01

n.d.

«

0.0

1

[n.d

.-n.

d']

[n.d

.-n.

d']

[n.d

.-0.

02]

[n.d

.-n.

d.]

[n.d

.-<O

.Ol]

H

ay

8 21

<

0.01

<

0.01

0.

03

«0

.01

<

0.01

[n

.d.-

0.04

] [n

.d.-

0.03

] [<

0.01

-0.1

2]

[n.d

.-0.

02]

[n.d

.-0.

02]

Stra

w

4 21

n.

d.

n.d.

0.

12

<0.

01

<0.

01

[n.d

.-n.

d']

[n.d

.-n.

d']

[0.0

1-0.

40]

[n.d

.-O.O

l]

[n.d

.-0.

02]

a Fi

gure

s in

bra

cket

s re

pres

ent

low

est-

high

est

cont

ents

in

indi

vidu

al s

ampl

es.

The

sig

natu

re «

0.0

1 i

s us

ed o

nly

for

arith

met

ical

mea

n va

lues

in

dica

ting

calc

ulat

ed f

igur

es b

elow

0.0

05 p

.p.m

.; n.

d. =

no

t de

tect

able

(i.e

., <

0.00

5 p.

p.m

.).

~

!-.:I I 1.\ ~

Danish milk and butter 65

the residue level of all of the insecticides in feed mixtures (Table IV) and in the feed concentrates (Table V). This level is sufficiently high to account for detectable residues in milk, and as the components are used all the year round, they may give rise to a certain "background contamination" (see the discussion below).

Quantitatively, the major parts of the feeding plans are filled by prod­ucts of the farmers' own growing. The analyses of some of these products are shown in Table VI and-in comparison with Tables N and V-it is interesting to notice that residues are found here, too, but usually they are more occasional and the amounts found are smaller. A most important thing is that a dominating product, the swedes, is not a carrier of insecticide resi­dues except for a few samples containing lindane at the level of 0.02 p.p.m. or less. This is in agreement with the general experience that organochlorine insecticides have gained no wide acceptance for pest control purposes in Danish beet growing.

Some of the grass and silage samples, etc., do contain residues, especially of DDT, but generally in a limited number and in small amounts only. Because of their significance in the feeding plans these types of feeding stuff may, however, contribute to the general contamination level, with residues in excess of the "background contamination" (see below).

V. Discussion

Contamination of milk and milk products by pesticide residues has been a question of increasing interest in recent years. In American investigations from the latter part of the 1950's contamination of milk by such compounds as DDT, BHC, toxaphene, and chlordane (HEINEMAN and MILLER 1961) could be proved. These findings were confirmed and further elaborated through the large projects of Henderson and coworkers (1964), analyzing more than 30,000 samples of milk and milk products.

The results of analyses of milk from other countries have been published, i.a., from Germany (KIERMEIER et al. 1964) and Great Britain (Report of the Government Chemist 1964). The English work is of special interest be­cause it demonstrates a general and relatively uniform level of contamination by the insecticides dieldrin, DDT jDDE, and BHC in butter from several countries, including Australia, Denmark, and New Zealand, as well as 10

butter from the production of the United Kingdom itself. The investigations described here have likewise-within the limits of

the Danish production--aimed at elucidating the extent to which Danish milk and milk products are contaminated by organochlorine insecticides. The analytical work comprises 750 samples of milk and butter, partly sampled from dairies all over the country and partly from dairies and selected farms in a local area near Athus, Jurland. In agreement with the English results, a general contamination caused by the compounds dieldrin, DDT jDDE, a-BHC, and lindane was found. The concentrations vary from one com-

66 F. BRO·RAsMUSSEN et at.

pound to another, averaging between 0.02 and 0.10 p.p.m. on a butterfat basis, the DDT /DDE residues being at the higher end and the lindane residues at the lower end of the range.

From the more detailed material sampled directly from 20 farms through a one-year period it is evident that a seasonal variation, in particular in the case of DDT /DDE, may be seen, reaching peak residues during the m,onths from September to December. Such seasonal variations have been observed also in other places, for example, in the U.S.A. (HEINEMAN and MILLER

1961). Among the 20 farms one was found to produce milk with an increased

level of DDT /DDE, indicating that an excessive use of DDT might have preceded the sampling period. Inspections and interviews did not reveal any misuse, but during the continued programme the content of DDT /DDE in milk from that farm decreased and reached the same low level as was found in rnJlk from the other farms.

The sampling of animal feed under this programme was not fully sufli­cient or representative of the cow feeding plans. The samples are, however, obtained from the same farms as the monthly milk samples, and they cover a series of feed and feed components of Danish as well as of foreign origin which are all of major importance in Danish cow feeding. The results of the analyses are noteworthy because they confirm the suggestions made by HENDERSON (1964) that the contamination of milk is mainly caused by the intake of the cows through the feeding.

At an evaluation in this direction it should be borne in mind that a considerable concentration and accumulation occurs during the transfer of insecticides from the feed to the milk fat. In the feeding experiments made by WILLIAMS et al. (1964) a concentration factor of about five was demon­strated for DDT (recalculated from WILLIAMS et al., on the assumption of a content of four percent of fat in the milk) whereas the corresponding factor for dieldrin may be calculated to be about ten.

About 10 to 15 percent of Danish cow feed is given in the form of mixed feed and feed concentrates, mainly imported products, and if it is assumed that these feeds are carriers of insecticide residues at an average level as shown in Table IV, it may be estimated that 60 to 100 percent of the dieldrin which is found in the milk and the butter originates from that source, whereas, in the case of DDT, only 30 to 50 percent of the content of the milk can be accounted for. This is a considerable contribution to the general contamination and as the feeds are given all the year round, they may be considered a major source of a "background contamination" which is beyond the control of the individual farmers.

Accordingly, the remaining parts of contaminating insecticides in milk and butter should be traced back to the use in domestic agriculture. As far as dieldrin is concerned, this is a minor part only in agreement with the fact that dieldrin is scarcely found in Danish feed samples (Table VI), and

Danish milk: and butter 67

that dieldrin is not recommended or registered for use in Danish agriculture, while the parent cOlilipound, aldrin, gained small acceptance only before its use for soil treatment, seed dressing, etc. was stopped in 1964.

In contrast to this, the estimate for DDT (and probably for BHC) shows that 50 percent or more of the content of this compound in milk may be caused by the use in own farming. There is nothing in the results or in the feeding plans to indicate which types of pesticide use or which crops are most essential in this connection. Table VI shows that grass, alfalfa, and hay as well as straw may all carry DDT residues to account for the con­tent in milk above the "background contamination," in spite of the fact that these crops are not usually the objects of pest control with organochlorine insecticides. There is reason to believe that an aerial contamination caused by wind drift may be of significance. In a country like Denmark, with in­creased use of pesticide chemicals during the summer months, such contam­ination would of course be seasonal, and this gives a reasonable connection to the observation of an increased content of DDT JDDE in the milk during the autumn months immediately following.

Summary

About 750 samples of Danish milk and butter as well as 160 samples of animal feed and feed concentrates were analysed by paper and gas chroma­tographic methods during a five-year period up to the end of 1966. The milk and butter samples are considered representative of the Danish production of milk, and all of the samples were found to be contaminated by one or more of the insecticides: a.-BHC, DDT /DDE, dieldrin, and lindane in the order of 0.02 to 0.10 p.p.m. calculated on a butterfat basis. The content of DDT is the highest, and the content of lindane the lowest with dieldrin as an intermediate contaminator at the level of 0.05 p.p.m. or less.

A final and quantitative evaluation of the sources of contamination could not be given. The analyses of a variety of animal feed products, however, justify the interpretation that feed mixtures and concentrates mostly of for­eign origin cause a considerable part of the contamination throughout the yeat. In the case of dieldrin this "background contamination" is estimated to be of the order of 60 to 100 percent and in the case of DDT (and prob­ably BHC) 30 to 50 percent of the total contents.

At the sampling of Danish grown feed crops, residues were generally found in fewer samples and the amounts found were smaller. Nor1l¥llly, or­ganochlorine pesticides are not used intentionally on such crops in Danish farming. Among the samples there were, however, some containing substan­tial residues, especially of DDT, and it is assumed that the remaining part of the contamination, in addition to the "background contamination," may originate from such sources, wind drift being considered the most probable pathway for the contamination of the feed crops.

68 F. BRO-RAsMUSSEN et al.

Etude sur la contamination du beurre et du lait Danois par les insecticides organochlores

Environ 750 echantillons de lait et de beurre danois ainsi que 160 echantillons de fourrages et d' aliments concentres ont ete analyses par chro­matographie sur papier et par chromatographie gazeuse, au cours d'une periode de 5 ans precedant la :fin de 1966. Les echantillons de lait et de beurre sont consideres comme representatifs de la production laitiere danoise et tous les echantillons ont ete trouves contamines par un ou plusieurs des insecticides: a H.C.H., zeidane/D.D.E., dieldrine et lindane a des taux de l'ordre de 0,02 et 0,10 ppm dans la matiere grasse. Les residus de zeidane sont les plus importants, ceux de lindane les plus faibles tandis que ceux de dieldrine sont intermediaires a des teneurs inferieures ou au plus egales a 0,05 ppm.

L'evaluation definitive et quantitative des causes de contamination n'a pu etre donnee. Les analyses de divers aliments pour Ie betail, cependant, justifient de donner aux melanges d'aliments et aux concentres, pour la plupart d'origine etrangere, une importante responsabilite pour la pollution permanente au cours de l'annee.

La contribution de cette pollution de base par rapport a l'ensemble est evaluee a 60-100 p. cent pour les residus de dieldrine et a 30-50 p. cent dans Ie cas du zeidane (et probablement de l'H.C.H.).

Avec les fourrages originaires du Danemark des residus furent general­ement trouve dans un plus petit nombre d' echantillons et en quantites plus faibles. Normalement, les pesticides organochlores ne sont pas utilises in­tentionnellement dans ces cultures.

Certains echantillons ont cependant renferme des residus substantiels, surtout de zeidane. II est presume que Ie reste de la contamination, en plus de la pollution de base, puisse provenir de telles sources: les entrainements par Ie vent etant consideres comme la voie la plus probable de contamination des fourrages.

Zusammenfassung* *

Untersuchung von daenischer Milch und Butter auf Verschmutzung mit chlorierten Insektiziden

Ungefaehr 750 daenische Milch- und Butterproben, sowie 160 Proben von Tierfutter und Futterkonzentraten wurden mit Hilfe von papier-und gaschromatographischen Methoden waehrend einer Zeitspanne von fuenf Jahren bis zum Ende des Jahres 1966 analysiert. Die Milch- und Butter­proben werden als Vertreter der daenischen Milchproduktion angesehen, und man fand, dass alle Proben durch ein oder mehrere Insektizide verschmutzt waren: a-BHC, DDT /DDE, Dieldrin und Lindan in der Groessenordnung

.. Traduit par R. MESTRES . .. .. 'Obersetzt von MARGARETE DUSCH.

Danish milk and butter 69

von 0.02 bis 0.10 ppm, berechnet auf einer Butterfettbasis. Der Gehalt an DDT ist der hoechste und der Gehalt an Lindan der niedrigste, waehrend die Verschmutzung von Dieldrin, in Hoehe von 0.05 ppm oder weniger, da­zwischen liegt.

Eine endgueltige und quantitative Auswertung der Verschmutzungsquel­len konnte nicht angegeben werden. Die Analysen von verschiedenen Tier­futterprodukten rechtfertigen jedoch die Auslegung, dass meistens Futter­mischungen und -Konzentrate auslaendischer Herkunft einen beachtenswerten Teil der Verschmutzung ueber das gauze Jahr hinweg verursachen. 1m Falle von Dieldrin wird diese "Blindwertverschmutzung" auf 60 bis 100 Prozent und im Falle von DDT (und wahrscheinlich BHC) auf 30 bis 50 Prozent des gesamten Gehaltes geschaetzt.

Bei der Bemusterung von in Daenemark angebauten Futtermitteln worden im allgemeinen in weniger Proben Rueckstaende gefunden, und die gefundenen Mengen waren kleiner. Normalerweise werden chlorierte Pesti­zide in der daenischen Landwirtschaft auf solchen Ernten absichtlich nicht verwendet. Dnter den Proben waren jedoch einige, die wesentliche Rueck­staende, hauptsaechlich von DDT, enthielten; und es wird angenommen, dass der restliche Teil der Verschmutzung zusaetzlich zur "Blindwertverschmutz­ung" durch solche Quellen wie Winddrift entstanden sein kann, der als der wahrscheinlichste Weg der Verschmutzung von Futtermitteln angesehen wird.

References

BRO-RAsMUSSEN, F., F. RODIN, and K. VOLDUM-CLAUSEN: Bine iiberpriifung gas­chromatographischer Bestimmung chlorierter Insektizide in Butter und pflanzlichen Erzeugnissen. Dispatched for publication in Zeitschrift fiir Lebensmittelunter­suchung und -forschung. To be published (1968).

CLIFFORD, P. A., J. 1. LASSEN, and P. A. MILLS: Chlorinated organic pesticide resi­dues in fluid milk. Public Health Reports 74, 1109 (1959).

FAUBERT MAUNDER, M. J. DE, H. EGAN, and J. ROBURN: Some practical aspects of the determination of chlorinated pesticides by electron-capture gas chromatography. Analyst 89,157 (1964a). .

- -, E. W. GODLY, E. W. HAMMOND, J. ROBURN, and J. THOMSON: Clean-up of animal fats and dairy products for the analysis of chlorinated pesticide residues. Analyst 89, 168 (1964 b).

HEINEMANN, H. E. 0., and C. B. MILLER: Pesticide residues and the dairy industry. J. Dairy Sci. 44, 1775 (1961).

HENDERSON, J. L.: Insecticide residues in milk and dairy products. Residue Reviews 8, 74 (1964).

KIERMEIER, F., A. GANZ, and G. WILDBRETT: Zum Problem der Riickstiinde in­sektizider Chlorwasserstoffe in Milch. Z. Leb. Vnter. u. Forschung 124, 252 (1964).

MILLS, P. A.: Detection and semiquantitative estimation of chlorinated organic pesti­cide residues in foods by paper chromatography. J. Assoc. Official Agr. Chemists 42, 734 (1959).

Report of the Government Chemist: pp. 44 ff. (1964). Published by Ministry of Tech­nology. H. M. S. 0., London (1965).

WILLIAMS, S., P. A. MILLS, and R. E. McDOWELL: Residues in milk of cows fed rations containing low concentrations of five chlorinated hydrocarbon pesticides. J. Assoc. Official Agr. Chemists 47, 1124 (1964).

The fundamental kinetics of cholinesterase reaction with substrates and inhibitors in

an automated, continuous flow system

By

GUNTHER VOSS*

I. Introduction

II. Experimental

III. Results .

a) Glutathione standard curve

Contents

b) Enzyme concentration and reaction velocity

c) Substrate concentration and reaction velocity

d) Energy of activation

e) Enzyme activity and hydrogen ion concentration

f) Pre-inhibition experiments

IV. Practical considerations for residue analyses

V. Discussion

Summary

Resume.

Zusammenfassung

References .

,. Agrochemical Division, eIBA Limited, Basel, Switzerland.

72

75

77

78

78

79

81

82

83

85

85

92

92

93

93

72 GUNTHER VOSS

I. Introduction

In the decade between 1950 and 1960 certain cholinesterase inhibiting substances such as many organophosphates and carbamates became im­portant insecticides1 in hygiene and in plant protection. The toxicological properties of these compounds stimulated the research on cholinesterases in many scientific disciplines; the development of reliable methods for cho­linesterase activity determinations has been of particularly great importance. Today one can distinguish between two different categories of practical approaches. The first deals with the experimental determination of the un­known enzyme activity in healthy, unhealthy, and/or poisoned human beings and animals; studies of this kind are important in physiology, toxicology, and clinical chemistry. The second approach, however, is of more interest to the analytical chemist, to whom cholinesterases are noth­ing but tools for quantitative determinations of unknown amounts of inhibitors; such procedures are frequently applied for residue determina­tions of certain organophosphates ( GAGE 1961). A further important point to be mentioned here is the determination of inhibitor potencies in vitro, which are usually expressed in terms of molar inhibitor concen­trations causing 50 percent enzyme inhibition (I50-values). Here again the inhibitor concentration is the factor to be determined and the particular type of cholinesterase chosen for the experiment only serves as a necessary reagent for the experiment.

Many review articles on cholinesterases have been published and the reader is referred to the summary listed in Table 1. Although this summary does not claim completeness, it may serve as a guide to those fields of research where, according to the author's opinion, an automated procedure could successfully be applied.

The development of automated procedures plays an important role in modern analytical chemistry. This tendency has also affected cholinesterase measurements. The first publications on this subject are those of WINTER

(1960) and WINTER and FERRARI (1964). Their method is based on a colorimetric determination of a pH change in the enzyme-substrate solu­tion by means of an appropriate indicator. GUNTHER and OTT (1966) recently modified this procedure for a residue screening method for organo­phosphates.

Another approach for automated cholinesterase activity determinations and inhibition studies has been chosen by LEVINE et at. (1965) and by voss (1966 and 1967) and voss and GIDSSBUHLER (1967), respectively. They automated the colorimetric method of ELLMAN et al. (1961), using acetylthiocholine (ASCh) as substrate and dithiobisnitrobenzoic acid (DTNB) as reagent (the use of these two compounds will be circum-

1 Chemical designations of insecticides mentioned in text are listed in Table V.

Tab

le I

. Li

tera

ture

rev

iew

s on

cho

lines

tera

ses

and

antic

holin

este

rase

s

Scie

ntif

ic s

ubje

cts

whe

re a

utom

ated

pro

cedu

res

coul

d be

app

lied

Met

hods

of

chol

ines

tera

se d

eter

min

atio

na

Fun

dam

enta

l in

vest

igat

ions

Kin

etic

s an

d m

ode

of a

ctio

n of

cho

lines

tera

ses

Cla

ssif

icat

ion

of c

holin

este

rase

s an

d co

mpa

rativ

e en

zym

olog

y

Phys

iolo

gy a

nd t

oxic

olog

y

Nat

ural

var

iati

on o

f hu

man

blo

od c

holin

este

rase

s

Mea

sure

men

t of

hu

man

bl

ood

chol

ines

tera

se

in

conn

ectio

n w

ith

occu

patio

nal

expo

sure

to

inse

ctic

ides

St

ruct

ure-

activ

ity

rela

tion

ship

of

an

ticho

lines

tera

ses

Rea

ctiv

atio

n of

pho

spho

ryla

ted

chol

ines

tera

se

Met

abol

ism

of

orga

noph

osph

ates

Inse

ct c

holin

este

rase

s

Ana

lytic

al b

ioch

emis

try

Cho

lines

tera

ses

and

inse

ctic

ide

resi

due

dete

rmin

atio

nsb

Aut

hori

ty

AU

GU

ST

INS

SO

N

(195

7 an

d 19

63),

WIl

TE

R

(196

3)

O'B

RIE

N

(19

60

), H

EA

TH

(1

96

1),

CO

HE

N a

nd

OO

STE

RB

AA

N

(196

3)

AU

GU

ST

INS

SO

N

(196

3)

AU

GU

STIN

SSO

N

(195

5)

GA

GE

(196

7)

O'B

RIE

N

(19

60

),

HE

AT

H

(19

61

),

HO

LM

ST

ED

T

(19

63

),

LO

NG

(1

963)

HO

BB

IGE

R

(196

3)

O'B

RIE

N

(19

60

), H

EA

TH

(1

96

1),

MO

UN

TE

R

(196

3)

CH

AD

WIC

K

(196

3)

GA

GE

(196

1)

aAut

omat

ed m

etho

ds h

ave

been

des

crib

ed b

y th

e fo

llow

ing

auth

ors:

WIN

TE

R

(19

60

), W

INT

ER

and

FE

RR

AR

I (1

96

4),

LE

VIN

E et

at.

(19

65

), S

ER

RO

NE

et

at. (

19

65

), G

UN

TH

ER

and

OlT

(1

96

6),

vos

s (1

96

6),

vos

s an

d G

EIS

SBU

HL

ER

(1

96

7).

b A

utom

atio

n al

low

s th

e an

alyt

ical

use

to

be e

xten

ded

to r

outi

ne s

tabi

lity

tes

ts o

f ca

rbam

ates

and

org

anop

hosp

hate

s in

sol

utio

ns o

r in

fo

rmul

atio

ns.

[ ~.

li ~ I>

i" ~.

a.

r;l

-..j

<.

»

74 GUNTHER VOSS

scribed by the term "ASCh/DTNB method" in this paper). Thiocholine, formed upon enzymatic hydrolysis of ASCh, reduces DTNB to the yellow anion of thionitrobenzoic acid, whose absorbance is measured at 420 mil. It is the author's opinion that the principle described by ELLMAN and co­workers (1961) represents one of the most straightforward methods ever developed for cholinesterase experiments. Unfortunately, the attention paid to it was not very great until recently. The main advantages are simplicity, high precision, pH-constancy, modifiability, adaptability for microdeter­minations and routine analyses, short incubation periods, continuous in­crease in color density as a function of incubation time, and measurement of a reaction product instead of the remaining intact substrate.

Because of their high speed and reproducibility, automated techniques are particularly suited for serial analyses. Routine studies on insecticide residues, experiments on temperature and hydrolytic stabilities of anti­cholinesterases in solutions and in formulations, measurements of their evaporation rates, I50-screenings, and many other applications may serve as examples. Furthermore, with automation a degree of standardization is obtained which is unknown with manual methods. This fact is of the great­est importance for all enzymatic methods, because it automatically solves the problem of exact timing of preinhibition and incubation periods. The deter­mination of ho-values of anticholinesterases, for instance, requires standard­ized conditions if the inhibition potencies are to be compared with each other. Thus all compounds should experimentally be treated in exactly the same way, but every worker in the field knows that this standardization has not been achieved. Many divergent results on inhibitor potencies described in the literature are caused by this lack of standardization; others are diffi­cult to interpret because experimental conditions, such as preinhibition periods or temperature, are not given. There can be no doubt that auto­mated cholinesterase techniques could reduce certain shortcomings of pre­vious manual methods and contribute to a high degree of standardization in any laboratory.

In his fundamental publication on cholinesterase inhibition analysis, GAGE (1961) described the kinetics of cholinesterase action and inhibition. Indeed, some knowledge of enzyme kinetics is essential for the develop­ment of analytical procedures based on enzyme inhibition and for a correct interpretation of the results. Therefore the reactions between enzyme, sub­strate, and inhibitors were also studied in detail under automated condi­tions by the present author to provide the analyst with some information on the theoretical background. These studies may also be regarded as model experiments, demonstrating that a great variety of problems connected with enzyme kinetics may be solved by using continuous flow systems. Data so obtained are often superior in their precision to results manually obtained, as will be discussed in a later section.

Cholinesterase kinetics 75

II. Experimental

A Technicon®-AutoAnalyzer®2 consisting of Sampler II, proportioning pump, heating bath, colorimeter and recorder was used. For further details see the standard flow diagram (Fig. 1) which is essentially that described

9min. 16 min. r--10.045 Sample I

10.045 Enzyme/buffer II

10.045 Air

10.056 Buffer B.O :m: Discard

t 10.040 ASCh/DTNB

I : 0.073 Water Ill:

Discard I , ,O.OBI

Colorimeter 10 1 L __ J

Discard Proportioning [I~e batJ pump

Fig. 1. Standard flow system for the automated ASCh/D1NB method according to voss and GEISSBUHLER (1967). I, II, III, and IV describe the main tubings used for the different solutions. For the kinetic experiments these tubings were used as out-

lined in Table II and in the subsections under "Results"

by voss and GEISSBUHLER (1967)3. Depending on the problem under in­vestigation, however, the standard flow system had to be modified to a certain extent. To avoid confusion, the four main tubings are numbered I, II, III, and IV in Figure 1, and the same numbers are used in Table II and in the different subsections under "results" for a brief description of these modifi­cations.

In the present paper all concentrations given in the text mean final con­centration in the reaction mixture. These were calculated by using the flow rates of the pump tubings, which were also experimentally rechecked and found to be well within the range given by the manufacturer. Reaction velocity (v) was always expressed as increase in absorbance during the incubation period of approximately nine minutes. To check the linearity between incubation time and absorbance a mixture of AChE solution and 10-3 MASCh and DTNB was directly pumped through the flow cuvette from a beaker (37°C.). The increase in absorbance was found to be propor­tional to time over the incubation period used in the automated system.

2 Available from Technicon Instruments Corporation, Chauncey, N ew York. 3 With the standard flow system described in Fig. 1 the incubation periods of

cholinesterase with inhibitor and substrate are constant parameters which depend on the length of the delay coils in the heating bath and on the diameters of the pump tubings. It is, however, possible to apply this automated procedure also for kinetic experiments, where reaction periods are varied.

Tab

le I

I.

Mod

ifica

tions

of

the

sta

ndar

d /lo

w s

yste

m o

f Fi

g.

1 us

ed

for

stud

ying

diff

eren

t pr

oble

ms

on c

holin

este

rase

ki

netic

s

Solu

tions

a in

tub

ings

I I

I I

Fir

st

incu

b.

Exp

erim

ent

I II

II

I IV

co

il us

ed

Glu

tath

ione

sta

ndar

d cu

rve

H2O

B

(S)

B(S

) G

lut.

/D1

NB

b N

o

Enz

yme

conc

entr

atio

n an

d re

actio

n A

SC

hjD

1NB

velo

city

E

(8)1

1 H

2O

B(8

) B

SC

hjD

1NB

N

o

Subs

trat

e co

ncen

trat

ion

and

reac

tion

AS

Chj

D1N

BlI

velo

city

H

2O

E(S

) B

(S)

BS

Chj

D1N

B

No

Ene

rgy

of a

ctiv

atio

n H

2O

E(S

) B

(S)

AS

Chj

D1N

B

No

Enz

yme

activ

ity a

nd p

H

B(6

.6-S

.0)

B(6

.6-S

.0)

E i

n R

oO

AS

Chj

D1N

B

No

Pre

inhi

biti

on e

xper

imen

ts

III

E(S

) B

(S)

AS

Chj

D1N

B

Yes

B

SC

hjD

1NB

a L

ette

r ab

brev

iati

ons:

B (

buff

er),

E (

enzy

me,

AC

hE o

r C

hE),

I

(inh

ibit

or),

ASC

h (a

cety

lthi

ocho

line

), B

SCh

(but

yryl

thio

chol

ine)

.

num

ber

in p

aren

thes

es a

fter

B o

r E

indi

cate

s pH

val

ue o

f th

e bu

ffer

ed s

olut

ion.

II Fr

om s

ampl

er.

~

0\

G) ~: ~ ~ gJ

Cholinesterase kinetics 77

The following solutions and chemicals were used: Soerensen phosphate buffers, 1/15 M, pH 7.0 and 8.0. Purified bovine erythrocyte acetylcholinesterase (AChE; Serva Entwick­hmgslabor, Heidelberg, Germany). This enzyme preparation was dis­solved in buffer pH 8.0 and a concentration was applied which gave an absorbance reading of approximately 0.5 on the recorder chart. This concentration was determined in a preliminary experiment. Human plasma cholinesterase (ChE). Outdated human blood was cen­trifuged for 15 minutes at 3,000 r.p.m. The supernate was stored in a deep freeze (-25°C.). For the automated experiments one part of plasma was diluted with 250 parts of pH 8.0 buffer. L-Glutathione (Fluka, Switzerland). Acetylthiocholine iodide (ASCh; Fluka Switzerland). Butyrylthiocholine iodide (BSCh; Fluka, Switzerland). 5,5'-Dithiobis-2-nitrobenzoic acid (DTNB; Aldrich Chemical Co., Mil­waukee, U.S.A.): 200 mg. of DTNB were dissolved in 100 mi. of phos­phate pH 7.0 buffer and stored in a refrigerator as a stock solution. For a substrate-reagent working solution 10 mi. of this DTNB solution was added in a lOO-mI. volumetric flask to crystalline ASCh or BSCh, the amount of which depended on the final substrate concentration wanted, and made to volume with distilled water. The substrate-reagent solution was kept in an ice bath during the experiment to minimize self-hydroly­sis of the thiocholine esters. During the enzymatic hydrolysis, ASCh is split into thiocholine and acetic acid; the former compound reduces DTNB to the yellow anion of 5-thio-2-nitrobenzoic acid, whose absorb­ance is determined at 420 mlJ.. Acetylcholine iodide (ACh; Fluka, Switzerland). Eserine salicylate (Fluka, Switzerland). Monocrotophos (proposed common name of the organophosphorus in­secticide AZODRIN@, NUV ACRON@). Dicrotophos (common name of the organophosphorus insecticide BID­RIN@, CARBICRON@).

III. Results

Different experiments with the two known types of mammalian cho­linesterase, abbreviated as AChE (purified bovine erythrocyte acetylcho­linesterase) and ChE (human plasma cholinesterase) will be described in the following section. AChE as a commercially available enzyme preparation was chosen for most of the experiments because it is a relatively pure product. Since for analytical purposes ChE from crude human plasma is often pre­ferred because of its higher sensitivity towards many organophosphates, this enzyme was also utilized. Most of the results are presented in form of figures and the original experimental data are always indicated in the graphs

78 GiiNnmR Voss

to demonstrate the high degree of precision obtainable by suitably automated methods.

a) Glutathione standard curve (see Fig. 2)

Tubing No. I: water No. II: buffer pH 8 No. III: buffer pH 8 No. IV: Glutathione/DTNB (from sampler)

First incubarion coil omitted

Reduced glutathione served as a calibration substance for the prepara­tion of a standard curve (Fig. 2), which allowed the absorbance determined with DTNB at 420 m!-t to be expressed as final concentration of SH groups. The relationship between these two parameters may then be used to describe the enzyme activity in terms of final concentration of thiocholine produced from substrate hydrolysis, as has been done later in Figure 4.

8 c: o

0.8

0.6

-e 0.4 g .c .:t

0.2

2 3 4 5 X lO-5M Glutathione

Fig. 2. Glutathione standatd curve with final concentration of glutathione plotted against absorbance

b) Enzyme concentration and reaction velocity (see Fig. 3)

Tubing No. I: AChE or ChB, pH 8 (from sampler) No. II: water No. III: buffer pH 8 No. IV: ASCh/DTNB or BSCh/DTNB

First incubation coil omitted

The rate of ASCh hydrolysis by AChE (Fig. 3) and that of ASCh and BSCh (butyrylthiocholine) by ChE was proportional to the enzyme con­centration under the automated conditions for both substrate concentrations studied.

Cholinesterase kinetics

0.3

~ "g 0.2 Q; > c o

:;:: <.> o £ 0.1

'/ /

/ II

/ /

'/ V

/ /( .-

'/

'/ /

J> .­'/

20 40 60 80 100 Percent AChE

79

Fig. 3. Linear relationship between AChE concentration expressed as percentage of a working solution in buffer, and enzymatic hydrolysis of ASCh. Final substrate con­

centrations were 10-3 M (upper curve) and 2 X 10-4 M (lower curve)

c) Substrate concentration and reaction velocity (see Fig. 4)

Tubing No. I: water No. II: AChE or GhE, pH 8 No. III: buffer pH 8 No. IV: ASCh/DTNB or BSCh/DTNB (from sampler)

First incubation coil omitted

The inhibition by excess of substrate is one of the special properties of AChE being used for distinguishing this enzyme from ChE. Both types of cholinesterases were therefore studied under this aspect with the automated flow system. Since ChE preferentially hydrolyzes butyrates, BSCh was also investigated with this enzyme.

1. AChE.-The results obtained with a commercial sample of purified bovine erythrocyte cholinesterase on AChE are summarized in Figure 4. The substrate concentration optimum for ASCh was found to be in the magnitude of 10-3 M. As can be seen from the figure, a pronounced peak was not ob­tained. Under the concentrations shldied, 8 X 10-4 and 1.6 X 10-3 M ASCh gave the highest enzyme activities, so that this range may be re­garded as optimal. Because of this finding the substrate concentration for most of the experiments on AChE was 10-3 M. Substrate concentrations higher than 3.3 X 10-3 M were not tested, because self-hydrolysis of ASCh caused blank values which were too high for precise evaluations of enzymatic hydrolysis rates.

80

0.3

>-

g 0.2 Q; . > c: ~ g 0.1 a:

GiiNTHER VOSS

4 3 pS

1:5 ~

u <J)

1.0 ::E It) I

2 x

0.5

Fig. 4. Relationship between ASCh concentration (pS = negative logarithm of molar substrate concentration) and reaction velocity. The latter is expressed as absorbance recorded after the standardized incubation period on the left ordinate

and in terms of final thiocholine concentration on the right ordinate

In Figure 4 the reaction velocity has been expressed not only as absorb­ance over the incubation period but also as concentration of thiocholine. This was possible by using the glutathione standard curve presented in Figure 1. Under optimal conditions, i.e., with 10-3 MASCh, the final con­centration of the hydrolysis product thiocholine was less than 2 X 10-5 M. This indicates that under the conditions chosen for most of the experiments less than two percent of the substrate was actually hydrolyzed. The substrate concentration may therefore be regarded as constant under the automated conditions of nine minutes' incubation with enzyme.

The dissociation constant of the enzyme-substrate complex, defined as Km (MICHAELIS constant) was graphically determined by applying the LINEWEAVER-BURK-plot of the reciprocal substrate concentration (l/s) against the reciprocal velocity (l/v) as shown in Figure 5. Km for AChE and ASCh was found to be 6.5 X 10-5 M.

From the experimental data so far presented in the graphs it is evident that the precision obtained with the automated procedure is quite satisfac­tory. For a better demonstration of this precision, standard deviations of the means were calculated from an experiment during which two concentrations of ASCh were ten times alternately pumped through the automatic system. With 10-3 MASCh an absorbance recording of 0.387 -+- 0.004 was obtained; with 2 X 10-4 MASCh it was 0.319 -+- 0.005. Coefficients of variation were therefore 1.0 and 1.4 percent, respectively.

2. ChE.~In contrast to AChE this enzyme was not inhibited by excess of substrate (Fig. 6). BSCh was hydrolysed twice as fast as ASCh. From the BSCh-values, Km was calculated to be 1 X 10-4 M whereas from the ASCh figures the LINEWEAVER-BURK plot did not give a straight line. This prob-

-2

1 -Km

1

Cholinesterase kinetics

·-1

l/v 6

4

2

X 104

lIS

81

2

Fig. 5. ;UNEWEAVER-BURK plot of l/v against l/s for the graphical determination of Km (MICHAELIS constant). Reaction velocity v is expressed as absorbance; s indi­

cates substrate concentration

ably indicates the presence of at least two enzymes hydrolyzing this thio­choline ester, a phenomenon which will be discussed later. When l/v was related to l/s for the three highest substrate concentrations shown in Figure 6 a Km value for ChE and ASCh of 1.4 X 10-4 M was obtained.

0.4

BSCh 0.3

.;:-·u 0 c;; >

0.2 c:

~ ~ <> c ., 0:

0.1

4 3 pS

Fig. 6. Relationship between ASCh- and BSCh concentration (pS) and ChE reaction velocity

d) Energy of activation (see Fig. 7)

Tubing No. I: water No. II: AChE or ChE, pH 8 No. III: buffer pH 8 No. IV: ASCh/DTNB

First incubation coil omitted Automated procedure repeated with heating bath at 22°, 27°, 32°, and 37"C.

82 GiiNTHER Voss

Although the activation energy of the enzymatic hydrolysis of ASCh cannot be regarded as an important factor in cholinesterase inhibition analy­sis, some workers in this field may be interested in the data obtained with the automated procedure. The temperature range tested was between 22 ° and 37°C. with a substrate concentration of 10-3 M. The ARRHENIUS plot (Fig. 7) of l/T, T being the absolute temperature, against log reaction velocity was found to result in a straight line for AChE only. The energy of activation was calculated to be 2.900 cal/mole. With ChE a bent curve was obtained, but the activation energy could be determined for the 27° -to-3rc. range. It was 4.900 cal./mole.

1:' 0.20 'u o q; > 0.15 c: o ti o Q)

a:: 0.101-'--..--------.--------.

3.2 3.3 (l/T1X 103

3.4

Fig. 7. ARRHENIUS plot of reaction velocity (log scale) against liT for the tem­perature range 22° to 37°C. Final ASCh concentration: 10-3 M. Enzyme: AChE.

T = temperature (oK.)

e) Enzyme activity and hydrogen ion concentration (see Fig. 8)

Tubing No. I: buffer pH 6.6-8.0 No. II: buffer pH 6.6-8.0 No. III: AChE or ehE in water No. IV: ASCh/DTNB

First incubation coil omitted

Soerensen phosphate buffer solutions, 1/15 M, were prepared with the lowest pH of 6.6 and the highest of 8.0, the intervals being 0.2 pH unit (Fig. 8). With both enzymes, hydrolysis rates increased with increasing pH. The pH-activity curve of ChE is steeper in its left part than that of AChE. With both enzymes a flattening effect, which started at pH 7.6, was noted. From the shape of the curves it is assumed that the optimal pH must be slightly higher than pH 8.0; unfortunately, this could not be experimentally checked, because Soerensen phosphate buffer has its highest pH value at 8.0 and other buffer compositions might not have been comparable with the standard buffer used for all other experiments. Furthermore, it was noted with the manual ASCh/D1NB method that self-hydrolysis of ASCh in­creased rapidly with pH values higher than 8.0. Therefore, from a method­ological viewpoint, pH 8.0 was considered the most convenient value for the automated ASCh/D1NB method.

-... ~

100

>-~ 80 u o ... ~ 60 o q; 0::

40

Cholinesterase kinetics

6.8 7.2 7.6 8.0 pH

83

Fig. 8. pH dependency of the ASCh (10-3 M) hydrolysis by AChE and ChE. Enzy­matic hydrolysis rates at pH 8.0 were taken as 100 percent

f) Pre-inhibition experiments (see Figs. 9 and 10)

Tubing No. I: inhibitor, from sampler No. II: AChE or OhE, pH 8 No. III: buffer pH 8 No. IV: ASCh/DTNB or BSCh/DTNB

First incubation coil necessary for preinhibition

The automated ASCh/DTNB method was originally developed for the determination of small amounts of organophosphate residues and for fast ho measurements of anticholinesterases. Both problems require the inhibitor to be added to the enzyme prior to the substrate to avoid protection of the enzyme by the thiocholine ester4 •

From the kinetic results obtained by ALDRIDGE (1950), which have been many times confirmed by other authors, it is evident that the reaction be­tween an irreversible inhibitor, such as an organophosphate, and cho­linesterases may normally be described as a simple bimolecular reaction. In most of the experimental cases the inhibitor concentration is much higher than the enzyme concentration and under these conditions pseudo-first-order reaction kinetics apply. Thus, the bimolecular rate constant, k2' may be ob­tained by using the formula shown.

2.303 tI

a X log­

at

4 Competitive inhibition was briefly studied by adding inhibitors and ASCh simultaneously to the enzyme AChE. LINEWEAVER-BURK plots of the experimental values showed a common intersection on the vertical axis. The intersections with the abscissa were used for estimating the inhibitor constants (K1 ) in the presence of substrate by applying the procedure described by DIXON and WEBB (1965) for the purely competetive type of inhibition. The results obtained with four compounds are shown later in Table III.

84 GiiNrnBR Voss

In this formula I is the inhibitor concentration in. the reaction mixture, t is the preinhibition time (16 minutes under the automated conditions), a describes the enzyme activity before inhibition, and at is the enzyme activity after incubation with inhibitor for t minutes.

The reaction between inhibitors and cholinesterase is not always ex­pressed in terms of bimolecular rate constants. Thus, for most practical purposes the inhibitory potency of a compound is presented as an Il';o-value. The presentation of I50-data should always include a precise description of the method used, and particularly the preinhibition time should be given. If this is known, the terms k2 and 150 are interconvertible, provided that the inhibition process follows pseudo-first-order kinetics.

As GAGE (1961) outlined in his publication on cholinesterase inhibition residue methods, the most convenient procedure for demonstrating this type of reaction kinetics is a plot of log percent residual enzyme ac­tivity against inhibitor concentration or against pre-inhibition time. Since with the automated method described in this paper inhibition time is constant, only the first type of graph could be obtained. It is shown in Figure 9 for AChE and the organophosphate monocrotophos. The straight line relationship indicates pseudo-first-order kinetics, the I50-value being 2.45 X 10-5 M and k2 = 1.8 X 10-3 min.-1 1 mol.-1

100

~ 50 ----------------u •

j:: I 2 4XjO-5M

Monocrotophos

Fig. 9. Plot of log percent residual AChE activity against inhibitor con­centration. The straight line indicates

pseudo-first-order reaction kinetics

100

~ 50 u +­c:: ., ~ {f 20

10

2XIQ-6 M

Monocrotqphos

Fig. 10. Plot of log percent residual ChE activity against inhibitor concentra­tion. Two thiocholine esters were used

as substrates

The situation with ChE is different from that described above for AChE. Figure 10 shows that with ASCh and BSCh a bent curve is obtained with monocrotophos. This phenomenon is probably due to inhibitor-insensitive esterases in crude plasma, which are also capable of hydrolyzing the two thio­choline-esters. The I50-values obtained with ASCh and BSCh differ from each other, being 6.2 X 10-7 and 5.1 X 10-7 M. Although a determina­tion of k2 for ChE is not fully justified one may obtain a theoretical value from the left hand part of the curve, where the slope is steepest. With BSCh

Cholinesterase kinetics 8S

as a substrate, k2 was found to be 9 X 104 min.-1 1 mol.-1 In addition to these figures, the ho-value of eserine with ChE as enzyme and BSCh as substrate was determined to be 1.6 X 10-8 M.

IV. Practical considerations for residue analyses

There are two problems connected with the use of thiocholine esters and DTNB in an automated method which have to be briefly treated for practical reasons.

First, with the extraction procedure used in this laboratory for residue analyses of enolphosphates in plants (voss and GEISSBUHLER 1967) only slight interferences caused by untreated samples of many plant species were observed. These usually result in a low degree of enzyme inhibition, which is of the same order of magnitude known from other cholinesterase inhibi­tion techniques. However, extracts of certain plants (e.g., tea, potatoes, and tobacco) require modified extractions or further cleanup steps because of the presence of cholinesterase inhibiting substances such as alkaloids. Significant interferences from plant substances containing sulfhydryl groups, which could react with DTNB, were not observed with this particular extraction method.

The second problem is of purely technical importance and concerns the contamination of the inner walls of the pump tubes by certain insecticides. The proper tube material has to be selected by performing preliminary ex­periments. With the water-soluble enolphosphates, such as phosphamidon, monocrotophos, or dicrotophos, "Solvaflex"2 tubes may be used and the sampler can be operated at a speed of 40 samples/hour with a sample aspiration period of one minute and interwash period of 30 seconds. Also dichlorvos and chlorfenvinphos may be pumped through "Solvaflex",2 but the wash period between samples should be increased to two minutes. The organophosphates paraoxon and C-ll042, however, are strongly absorbed by this tube material and therefore "Acidflex"2 is recommended. In order completely to exclude absorption of all test materials in the tubes a gradient dilution system was developed in which the insecticide solution flows only through a whole-glass system. This device was successfully applied for ho­determinations in this laboratory.

V. Discussion

As already outlined in the introduction, the investigations on cho­linesterase kinetics by the automated ASCh/DTNB method were under­taken for two reasons: first it was necessary to study certain kinetics for the development of an automated standard procedure, which is now used for many analytical purposes in this laboratory, and secondly it was done to demonstrate that many kinetic data may be obtained with high speed and excellent accuracy with only minor modifications of the standard flow system. The advantages of the ASCh/DTNB method in general have been sum-

86 GUNTHER VOSS

marized in the introduction part of the present paper and the automation leads to a very reliable and precise cholinesterase technique. This is particu­larly true for inhibition studies on large series of compounds for compara­tive purposes, where standardization is of the utmost importance.

In the following sections the kinetic figures obtained during the course of the present investigations will be compared with data from the literature. Since it cannot be the aim of this paper to present and discuss all kinetic data on cholinesterases available, the author restricted himself to certain values given in a few well-known review articles. Table III summarizes these surveys in a comparative manner. All data will be discussed in the same sequence as they were presented under "Results".

With AChE as an enzyme and ASCh as a substrate an optimal substrate concentration was found between 8 X 10-4 M and 1.6 X 10-3 M. These data confirm the results of HEILBRONN (1959), who described an optimal ASCh concentration for eel AChE of approximately 10-3 M. Concentration optima for ACh in the presence of various concentrations of sodium chloride were found to be between 8 X 10-4 M and 3.7 X 10-<) M (MYERS 1952). Thus the ionic strength of the buffer solution affects the optimal substrate concentration.

The MICHAELIS constants described in the literature for the reaction between cholinesterases and ACh or ASCh differ more or less from each other. The reason for this might be sought in the different techniques applied by different authors and in varying enzyme sources with changing degrees of purities. With ACh and AChE (eel, erythrocytes), for instance, K",­values of 4.5 X 10-4 M (WILSON and BERGMANN 1950, c/. COHEN and OOSTERBAAN 1963), 2.3 X 10-4 M (HEATH 1961), and 1 X 10-4 M (ENGELHARD et at. 1967) were found. If one considers this variation, the value of 6.5 X 10-5 M for ASCh and AChE (this paper) is very well com­parable with 1.2 X 10-4 M for the same enzyme-substrate mixture (WILSON

and BERGMANN 1950, c/. COHEN and OOSTERBAAN 1963). The low Km found in the present studies also confirms the findings of BERGMANN et at. ( 1958) that ASCh is hydrolyzed at a rate five times faster than ACh.

With respect to ChE, the experiment with BSCh will be considered here because a straight LINEWEAVER-BURK plot for the determination of Km was obtained only with this substrate. The Km value found was 10-4 M but this figure is not in accordance with that of HEILBRONN (1959), given as 8 X 10-4 M. The present author feels that HEILBRONN'S value must be too high, because with ACh, which is not a very good substrate for ChE, HEATH (1961) described a Km of 1.64 X 10-3 M. The difference between these latter two data is only two-fold, but should be much higher because ChE hydrolyzes thiocholine esters faster than choline esters and butyrates faster than acetates.

Studies on the effect of temperature on the hydrolysis of ASCh by AChE and ChE revealed that with the former enzyme a straight ARRHENIUS plot was obtained over the whole temperature range investigated (22° to 37°C.).

Cholinesterase kinetics 87

The experimental data with ChE, however, resulted in a bent curve, so that only the temperature range between 27° and 37°C. was taken for the cal­culation of activation energy. Unfortunately there are no values available in the literature on the enzymatic ASCh hydrolysis, so that activation energies (E) for ACh must serve for comparative purposes. E was determined to be 1,700 -+- 400 caL/mole for eel AChE and ACh by WILSON and CABIB

(1956, ct. COHEN and OOSTERBAAN 1963) and 3,700 cal./mole for erythro­cyte AChE and ACh by SHUKUYA (1953, ct. COHEN and OOSTERBAAN

1963). An E-value of 2,900 cal./mole for bovine erythrocyte AChE and ASCh obtained with the automated technique is well within the range ex­pected from the figures presented above for ACh. For ChE and ASCh, E was 4,900 cal./mole (27° to 37°C.) which is in the same magnitude of 4,200 to 4,600 cal./mole for a purified sample of horse plasma (DAVIES

1955, ct. CHADWICK 1963). It is evident from the pH studies that the optimal pH-value for the

enzymatic hydrolysis of ASCh by both types of cholinesterases must be slightly higher than 8.0. Many authors (ct. COHEN and OOSTERBAAN 1963, ENGELHARD et ai. 1967) found that the most likely value for this optimum is 8.25, at least for ACh. According to BERGMANN et ai. (1958, ct. COHEN

and OOSTERBAAN 1963) with ASCh a definite pH optimum (i.e., a bell­shaped curve) is not obtained. Maximal hydrolysis occurs at the same pH as for ACh (8.25), but with ASCh there is no decrease of enzymatic activity with increasing pH values higher than 8.25.

A few inhibition experiments served for the determination of inhibitor constants. The Kr-value of eserine with AChE as enzyme was measured under the automated conditions and was found to be 6.9 X 10-8 M. This figure very closely resembles that found by other authors (ct. COHEN and OOSTERBAAN (1963) who present a figure of 6.1 X 10-8 M.) When ASCh and ACh were simultaneously added to AChE, the latter substrate acted as competitive inhibitor, the Krvalue being 1.1 X 10-4 M. Theo­retically one would expect this figure to be close to the MICHAELIS constant for ACh and AChE, and this is indeed the case when a comparison is made. The best agreement is found with the Km-value of 1 X 10-4 M, presented by ENGELHARD et ai. (1967). With ChE, in contrast to AChE, no straight inhibition curves were obtained with monocrotophos in preinhibition ex­periments, when plots of log percent residual enzyme activity against in­hibitor concentration were prepared. The most likely explanation for this phenomenon is the presence of a second enzyme in human plasma, the so­called A -esterase (ALDRIDGE 1953), which is insensitive towards organo­phosphates (and eserine) and hydrolyzes acetates faster than butyrates. This latter property might explain, why in Figure 10 the BSCh-curve shows a smaller deviation from linearity than the ASCh-curve, where inter­ference by the A-esterase is expected to be higher.

It is obvious from the results of certain kinetic experiments that com­mercial AChE is a relatively pure product and that with crude plasma inter-

Enz

yme

AC

hE

AC

hE

AC

hE

AC

hE

ChE

ChE

AC

hE

AC

hEa

AC

hE

ChE

ChE

Tab

le I

II.

Com

pari

son

betw

een

the

kine

tic d

ata

obta

ined

by

the

auto

mat

ed A

SCh /

DT

NB

met

hod

and

thos

e de

scri

bed

in t

he l

itera

ture

Kin

etic

dat

a

Subs

trat

e In

hibi

tor

Kin

etic

par

amet

er

Thi

s pa

per

I F

rom

lit

erat

ure

ASC

h -

Opt

. su

bst.

cone

. 0.

8-1.

6 X

10

-3 M

,.

.,1

0-3

M

(H

EIL

BR

ON

N 1

959)

AC

h -

Opt

. su

bst.

cone

. -

0.8-

3.7

X 1

0-3

M

(MY

ER

S

1952

)

ASC

h -

Km

6.

5 X

10

-5 M

1.

2 X

1

0-4

M

(W

ILS

ON

an

d B

ER

G-

MA

NN

19

50,

cf.

CO

HE

N

and

OO

STE

R-

BA

AN

19

63)

AC

h -

Km

-

1.2

X 1

0-4

M

(W

ILS

ON

an

d B

ER

G-

MA

NN

19

50,

cf

CO

HE

N

and

OO

ST

ER

-B

AA

N 19

63)

2.3

X 1

0-4

M

(H

EA

TH

196

1)

10

-4 M

(E

NG

EL

HA

RD

et

al.

1967

)

BSC

h -

Km

1

0-4

M

8 X

10

-4

M

(HE

ILB

RO

NN

19

59)

AC

h -

Km

-

1.64

X 1

0-3

M

(HE

AT

H 1

961)

ASC

h -

Act

. en

ergy

2,

900

cal.

/mol

e -

AC

h -

Act

. en

ergy

-

1,70

0 ±

400

ca

l./m

ole

(WIL

SO

N

and

CA

BIB

19

56,

cf.

CO

HE

N

and

OO

ST

ER

-B

AA

N

1963

)

AC

h -

Act

. en

ergy

-

3,70

0 ca

l./m

ole

(SH

UK

UY

A

1958

, cf

. C

OH

EN

and

OO

ST

ER

BA

AN

196

3)

ASC

h -

Act

. en

ergy

4,

900

cal.

/mol

e -

AC

h -

Act

. en

ergy

-

4,20

0-4,

600

cal.

/mol

e (D

AV

IES

19

55,

d.

CH

AD

WIC

K

1963

)

00

0

0

C) ~: ~ ~

Enz

yme

Subs

trat

e In

hibi

tor

AC

hE

ASC

h -

AC

hE

AC

h -

ChE

A

SCh

-A

ChE

A

SCh

Ese

rine

AC

hE

AC

h E

seri

ne

AC

hE

ASC

h A

Ch

AC

hE

ASC

h M

onoc

roto

phos

AC

hE

ASC

h D

icro

toph

os

AC

hE

ASC

h M

onoc

roto

phos

AC

hE

ASC

h M

onoc

roto

phos

ChE

A

SCh

Mon

ocro

toph

os

ChE

B

SCh

Mon

ocro

toph

os

ChE

B

SCh

Mon

ocro

toph

os

ChE

B

SCh

Ese

rine

a E

el.

Talb

le I

II.

(con

tinu

ed)

Kin

etic

dat

a

Kin

etic

par

amet

er

Thi

s pa

per

I F

rom

lit

erar

ure

Opt

. p

H

>8

.0

8.25

(B

ER

GM

AN

N

et

at.

1958

, d

. C

OH

EN

and

OO

ST

ER

BA

AN

196

3)

Opt

. p

H

-8.

25

(BE

RG

MA

NN

et

ai

. 19

58,

d.

CO

HE

N a

nd O

OS

TE

RB

AA

N 1

963)

Opt

. p

H

>8

.0

-KI

6.

9 X

10

-8 M

-

KI

-6

X 1

0-8

M

(HE

AT

H

1961

) 6.

1 X

10

-8

M

(d.

CO

HE

N

and

oos-

TE

RB

AA

N 19

63)

KI

10

-4 M

-

KI

6.7

X 1

0-5

M

-

KI

3.2

X 1

0-5

M

-

150

2.45

X 1

0-5

M

-k2

1.

8 X

103

min

. -1

1 m

ol.

-1

-15

0 6.

2 X

10

-7 M

-

Iso

5.1

X 1

0-7

M

-

k2

9 X

104

min

.-II

mo

l.-1

-

150

1.6

X 1

0-8

M

-

(", f ~ I>i""

~. S· 0

0

'-0

90 GUNTHER VOSS

ferences by other esterases occur. LINEWEAVER-BURK plots, the ARRHENIUS

plot and the preinhibition curve of log percent residual enzyme ac­tivity against inhibitor concentration were all straight when AChE was the enzyme. Thus, whenever possible, this product should be used for analytical purposes. However, in many practical cases, ChE from human plasma must be used since it is more sensitive to many organophosphates. This sensitivity makes this particular cholinesterase an important tool in residue analysis. According to the author's experience organophosphates like phosphamidon, monocrotophos, dicrotophos, dichlorvos, and chlorfenvinfos inhibit ChE to a greater extent than AChE and the same is known for DFP, mipafox, and iso-OMPA (AUGUSTINSSON 1963). On the other hand, para­oxon inhibits the "true cholinesterase" to a greater extent than the plasma cholinesterase and methyldemeton-O-sulfoxide has about an equal effect on both enzyme types (SCHRADER 1963). Also many carbamates preferably in­hibit AChE. Thus, before a residue program is started, the analyst should determine the ho-values of the particular compound under investigation for both choHnesterases and then select the more sensitive type for his work.

In kinetic studies investigators deal with final concentrations in reac­tion mixtures, which they express in terms of molarity. However, for auto­mated enzyme inhibition analysis it is recommended that concentrations of references and unknowns be expressed in terms of p.p.m. present in the solution which is aspirated from the sampler. This allows a quick and simple calculation of the residues present in the extracted plant. material or in formulations. For convenience, the references which should always accom­pany the unknowns when analyses are being performed may be kept as frozen water solutions in the two-mi. sample cups. The concentrations of active compound should approximately cover the range between 150 (p.p.m.) /3 and 150 (p.p.m.) X3 and whenever the unknowns are found to contain higher concentrations they should be diluted for a repeated analysis. The dilution of the blood plasma used for the automated procedure depends on the sample obtained from the blood bank. From our experience the dilution factor may be given as 250-+-50. Another problem is the decrease in sensi­tivity under storage conditions, even when frozen, and it is recommended that the plasma sample be discarded after one or two months. Reliance should not be placed on a single standard inhibition curve prepared with the fresh blood sample, but inhibitor reference solutions should always be run simu­taneously with the unknowns. This automatically corrects for changes in enzyme sensitivity.

With the technique described in the present paper many different problems concerned with cholinesterase inhibiting insecticides were studied in this laboratory (Table IV). Standardization through automation resulted in a high degree of reliability, and it is the authors' opinion, that a world­wide use of a standardized, automated cholinesterase procedure would lead to much better possibilities of data comparisions in the fields of basic and applied research on cholinesterases and anticholinesterases.

Cholinesterase kinetics 91

Table IV. Problems which have successfully been studied by the automated ASChjDTNB procedure in the analytical laboratories of CIBA

Cholinesterase kinetics

Normal variation of human plasma cholinesterase activity

Determination of enolphosphate residues, also in connection with thin-layer chromatography

Hydrolysis rates of cholinesterase inhibiting insecticides (continuously and discontinuously)

Stability of active ingredients (carbamates, organophosphates) in different formulations

Evaporation rates of anticholinesterase insecticides from glass surfaces

Disappearance of insecticides from plant surfaces

Iso-determinations

Table V. Common, trade, and chemical names of insecticides mentioned in text

Common name Trade name

dicrotophos BIDRIN® (Shell) CARBICRON® (CIBA)

monocrotophosa AZODRIN® (Shell) NUVACRON@ (CIBA)

phosphamidon DIMECRON® (CIBA)

dichlorvos VAPONA® (Shell) NUVAN® (CIBA)

chlorfenvinphos BIRLANE® (Shell) SAPECRON® (CIBA)

DFP

mipafox

iso-OMPA

paraoxon

C-l1042b

PESTOX XV® ISOPESTOX®

a proposed common name b CIBA code no.

Chemical name

O,O-dimethyl-O- (2-dimethyl­carbamoyl-I-methyl) vinyl phosphate

O,O-dimethyl-O- (2-methyl­carbamoyl-I-methyl) vinyl phosphate

O,O-dimethyl-O- (I-methyl-2-chloro-2-diethyl-carbamoyl) vinyl phosphate

° ,O-dimethyl-O- (2 ,2-dichlorovinyl) phosphate

diethyl-I- (2,4-dichlorophenyl-2-chlorovinyl phosphate

Diisopropyl phosphorofluoridate

N,N'-Diisopropylphosphoro-diamidic fluoride

Tetramonoisopropyl pyrophosphoro­tetramide

O,O-diethyl-O- (p-nitrophenyl) phos­phate

O,O-dimethyl-O- (4-iodo-2,5-dichloro­phenyl) phosphate

92 GiiNTHER voss

Summary

The present paper describes investigations on the kinetics of cholin­esterases of bovine erythrocytes and human plasma by means of an automated procedure which was originally developed for the quantitative determina­tion of residues of certain insecticidal organophosphates. Some knowledge of enzyme kinetics is essential not only for developing adequate analytical methods but also for the interpretation of the results obtained particularly with cholinesterase inhibition experiments. Several examples demonstrate that the standard flow system developed for routine tests may also be used for kinetic studies by applying minor modifications. Thus, relationships be­tween enzyme activity on the one hand, and enzyme concentration, substrate concentration, temperature, pH-conditions, and the effect of inhibitors under competitive and non-competitive conditions on the other hand, were studied. The experimental data are compared with kinetic figures from the literature. The precision of the automated ASCh/DTNB method was also measured by determining standard deviations of the peak height obtained with AChE and two concentrations of ASCh. It is assumed that constant inhibition as well as incubation periods are the reason for the high accuracy of the auto­mated technique.

Resume * Le present article decrit des experiences concernant la cinetique de la

cholinesterase des bematies de bovins et du plasma humain a l'aide d'un systeme automatique. Celui-ci a ete primitivement developpe pour Ie dosage de residus de certains insecticides organophosphores. Quelques connaissances sur la cinetique des cholinesterases sont essentielles aussi bien pour la mise au point de methodes analytiques que pour l'interpretation des resultats acquis au cours d'experiences sur l'inhibition de ces enzymes. Les exemples decrits montrent que Ie systeme standard de deroulement conc;u pour les analyses residuelles peut etre facilement adapte aux besoins des experiences cinetiques moyennant quelques petites modifications. En outre, les relations entre l'activite de l'enzyme d'une part et la concentration de l'enzyme, la concentration du substrat, la temperature, les conditions de pH et l'effet inhibiteur (competitif et non-competitif) d'autre part, ont ete examines.

Les resultats obtenus au cours de ces recherches ont ete compares avec les donnees cinetiques citees dans la litterature. En plus, la precision de la methode automatique ASCh/DTNB a ete egalement evaluee par la deter­mination du coefficient de variation des deflexions de l'enregistreur en pres­ence de deux concentrations differentes de substrat. II semble que la constance des periodes d'inhibition et d'incubation soit a l'origine de la precision elevee de la technique automatique .

.. Traduit par H. GEISSBUHLER.

Cholinesterase kinetics

Zusammenfassung*

Die vorliegende Publikation beschreibt Untersuchungen zur Kinetik der Cholinesterasen von Rindererythrocyten und Humanblutplasma mit Hilfe eines automatischen Verfahrens, das urspriinglich zur Bestimmung von Riickstanden bestimmter insektizider Phosphorsaure-ester entwickelt wurde. Die Kennrnis der Enzyrnkinetik ist nicht nur fiir die EntwickIung analytischer Verfahren auf enzymatischer Basis von Bedeutung, sondero auch fiir die richtige Interpretation der Ergebnisse, was besonders fiir Hemmungsversuche gilt. Wie einige Beispiele zeigen, kann das automatische Fliess-System nach geringfiigigen Modifikationen auf kinetische Untersuchungen angewandt werden. 1m einzelnen wurden Beziehungen zwischen Enzymaktivitat einer­seits und Enzymkonzentration, Substratkonzentration, Temperatur, pH­Werten und der Wirkung von Inhibitoren unter kompetitiven und nicht­kompetitiven Bedingungen andererseits gepriift. Die experimentell ermittel­ten Werte wurden mit Daten aus der literatur verglichen. Die Genauigkeit der automatischen ASCh/DTNB-Methode wurde ebenfalls bestimmt durch Ermittlung der Standardabweichung der Ausschlaghohen auf dem Schreib­gemt. Es wird angenommen, dass die hohe Genauigkeit der automatischen Methode sich auf die konstanten Inhibitions- und Inkubationszeiten zuriick­fiihren lasst.

References

ALDRIDGE, W. N.: Some properties of specific cholinesterase with particular reference to the mechanism of inhibition by diethyl p-nitrophenyl thiophosphate (E 605) and analogues. Biochem. J. 46, 451 (1950).

- Serum esterases. 1. Two types of esterases (A and B) hydrolysing p-nitrophenyl­acetate. propionate and butyrate and a method for their determination. Biochem. J. 53, 110 (1953).

AUGUSTINSSON, K. B.: The normal variation of human blood cholinesterase activity. Acta physio!. scand. 35, 40 (1955).

- Assay methods for cholinesterases. In: Methods of biochemical analysis. Vol. 5, D. Glick (ed.), p. 1. New York: Interscience (1957).

- Classification and comparative enzymology of the cholinesterases and methods for their determination. In: Handbuch der experimentellen Pharmakol. Erganzungs­werk XV, p. 89. Berlin, Gottingen, Heidelberg: Springer.Verlag (1963).

BERGMANN, F., S. RIMON, and R. SEGAL: Effect of pH on the activity of eel esterase towards different substrates. Biochem. J. 68, 493 (1958).

CHADWICK, 1. E.: Actions on insects and other invertebrates. In: Handbuch der experimentellen Pharmako!. Erganzungswerk XV, p. 741. Berlin, Gottingen, Heidelberg : Springer-Verlag (1963).

COHEN, ]. A., and R. A. OOSTERBAAN: The active site of acetylcholinesterase and related esterases and its reactivity towards substrates and inhibitors. In: Handbuch der experimentellen Pharmako!. Erganzungswerk XV, p. 297. Berlin, Gottingen, Heidelberg: Springer-Verlag (1963).

" Dbersetzt vom Autor.

94 GUNTHER VOSS

DAVIES, ]. H.: The kinetics of butyrocholine esterase. Ph.D. Thesis, Univ. Bristol, England (1955).

DIXON, M., and E. C. WEBB: Enzymes. London: Longmans (1965). ELLMAN, G., D. COURTNEY, V. ANDRES, and R. M. FEATHERSTONE: A new and

rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88 (1961).

ENGELHARD, N., K. PRCHAL, and M. NENNER: Acetylcholinesterase. Angew. Chem. 79, 604 (1967).

GAGE, ]. c.: Residue determination by cholinesterase inhibition analysis. Adv. Pest Control Research 4, 183 (1961).

- The significance of blood cholinesterase activity measurements. Residue Reviews 18, 159 (1967).

GUNTHER, F. A., and D. E. OTT: Automated pesticide residue analysis and screening. Residue Reviews 14, 12 (1966).

HI:\ATH, D. F.: Organophosphorus poisons. Anticholinesterases and related com­pounds. Oxford, London, New York, Paris: Pergamon Press (1961).

HEILBRONN, E.: Hydrolysis of carboxylic acid esters of thiocholine and its analogs. 3. Hydrolysis catalysed by acetylcholine esterase and butyrylcholine esterase. Acta chem. scand. 13, 1547 (1959).

HOBBIGER, F.: Reactivation of phosphorylated acetylcholinesterase. In: Handbuch der experimentellen Pharmakol. Erganzungswerk XV, p. 921. Berlin, Giittingen, Heidelberg: Springer-Verlag (1963).

HOLMSTEDT, B.: Structure-activity relationship of the organophosphorus anticho­linesterase agents. In: Handbuch der experimentellen Pharmakol. Erganzungs­werk XV, p. 428. Berlin, Giittingen, Heidelberg: Springer-Verlag (1963).

LEVINE, J. B., R. A. SCHEIDT. and V. A. NELSON: An automated micro determina­tion of serum cholinesterase. Technicon Symposium, "Automation in Analytical Chemistry", New Yark, p. 582 (1965).

LONG, J. P.: Structure-activity relationships of the reversible anticholinesterase agents. In: Handbuch der experimentellen Pharmakol. Erganzungswerk XV, p. 374. Ber­lin, Giittingen, Heidelberg: Springer-Verlag (1963).

MOUNTER, L. A. : Metabolism of organophosphorus anticholinesterase agents. In: Handbuch der experimentellen Pharmakol. Erganzungswerk XV, p. 486. Berlin, Giittingen, Heidelberg: Springer-Verlag (1963).

MYERS, D. K.: Effect of salt on the hydrolysis of acetylcholine by cholinesterases. Arch. Biochem. 37, 469 (1952).

O'BRIEN, R. D.: Toxic phosphorus esters. Chemistry, metabolism, and biological effects. New York and London: Academic Press (1960).

SCHRADER, G.: Die Entwicklung neuer insektizider Phosphorsaureester. Weinheim: Verlag Chemie (1963).

SERRONE, D. M., A. A. STEIN, E. A. MENEGAUX, M. A. GALLO, and F. COULSTON: Continuous analysis of whole blood cholinesterase in monkeys. Technicon Sym­posium, "Automation in Analytical Chemistry", New York, p. 586 (1965).

SHUKUYA, R.: Kinetics of human blood cholinesterase. II. The temperature effect upon cholinesterase activity. ]. Biochem. (Tokyo) 40,315 (1953).

VOSS, G.: Automated determination of activity and inhibition of cholinesterase with acetylthiocholine and dithiobisnitrobenzoic acid. ]. Econ. Entomol. 59, 1288 (1966). Substituierte Phenyl-N-methylcarbamate. Cholinesterase-Hemmung und insekti­zide Wirkung. Anz. Schadlingskunde 40, 73 (1967).

-, and H. GEISSBUHLER: Automated residue determinations of insecticidal enolphos­phates. 19th Intemat. Symposium Crop Prot. Gent 1967. Mededelingen Rijks­faculteit Landbouwwetenschappen Gent XXXII, No. 3/4, 877 (1967).

WILSON, 1. B., and F. BERGMANN: Acetylcholinesterase. VIII. Dissociation constants of the active groups. ]. BioI. Chem. 186, 683 (1950).

Cholinesterase kinetics 95

-, and E. CABIB: Acetylcholinesterase. Enthalpies and entropies of activation. J. Amer. Chern. Soc. 78, 202 (1956).

WINTER, G. D.: Automated enzymatic assay of organic phosphate pesticide residues. Ann. N.Y. Acad. Sci. 87, 875 (1960).

-, and A. FERRARI: Automatic wet chemical analysis as applied to pesticide resi­dues. Residue Reviews 5, 139 (1964).

WITTER, R. F.: Measurement of blood cholinesterase. Arch. Environ. Health 6, 537 (1963).

The dipyridylium herbicides, paraquat and diquatt:

By

A. A. AKHAVEIN** and D. 1. LINscorr***

Contents

I. Introduction . 98 II. Properties and characteristics 99

III. Relationship of herbicidal activity and structure 100 a) 2,2'-Dipyridyl series 100 b) 4,4'-Dipyridyl series 101 c) 2,4'-Dipyridyl, 3,3'-dipyridyl, and 2,3'-dipyridyl series 101 d) Common characteristics of dipyridylium quaternary salts possessing

herbicidal action 102 e) Conclusion 104

IV. Mode of action, physiological and biochemical behavior 105 a) Photosynthesis 105 b) General studies on herbicidal action 107 c) Specific physiological and biochemical studies 109

V. Factors influencing absorption, movement, and activity 1 B a) Introduction . 113 b) Influence of surfactants 114 c) Light and darkness . 114 d) Humidity and temperature 116 e) Effect of rainfall 116 f) Effect of oxygen 117 g) Synergistic and antagonistic effects of other herbicides on paraquat 117 h) Discussion 118

VI. Losses, persistence, and inactivation 118 a) Adsorption 119 b) Degradation . 119 c) Photochemical decomposition 120

VII. Residues, degradation products, and toxicity 121 a) Residues in soils. 121 b) Residues in crops 121

" Contribution from the department of Agronomy, Cornell University in co­operation with the Crops Research Division, Agricultural Research Service, U. S. De­partment of Agriculture. Department of Agronomy paper no. 785.

"" Research Assistant, Department of Agronomy, Cornell University. " "" Research Agronomist, Crops Research Division, Agricultural Research Service,

U. S. Department of Agriculture, Ithaca, New York.

98 A. A. AKHAVEIN and D. 1. llNSCOTr

c) Residues in water 122 d) Residues in milk .......... 123 e) Degradation and absorption of paraquat and diquat by animals . . 123 f) Toxicity of the photodecomposition products of paraquat and diquat to

animals . . . . . . . . 124 g) Toxicity of paraquat and diquat to animals and man 124 h) Determination of paraquat and diquat residues 125

VIII. Uses of paraquat and diquat 125 a) Desiccation, curing, defoliation . 125 b) Uses based on selectivity 126 c) Control of vegetation before planting a new crop 129 d) Control of Agropyrons repens . 134 e) Control of weeds in an aquatic environment 134

Summary 136 Resume 137 Zusammenfassung 138 References . 140

I. Introduction

Since the discovery of their herbicidal potential in the mid-1950's, the dipyridylium herbicides 1,1'-dimethyl-4,4'-bipyridinium salts (paraquat) and 6,7-dihydrodipyrido[l,2-a:2', l'-c}pyrazinediium salts (diquat) have been used extensively for control and management of terrestrial and aquatic vege­tation. Crop desiccation, pasture renovation, crop production with limited or no tillage, and selective weed control are only a few of the basic agricultural operations for which these chemicals have proved useful.

Paraquat and diquat have properties which make them unique among herbicides. Upon contact with soil they are rendered biologically inactive. Both herbicides are rapidly absorbed by plants and are extremely fast-acting, even at low concentrations. The mechanisms of activity, the factors influencing absorption and movement, and the structural relationship to activity of para­quat and diquat are complex. The realization of the full potential of these herbicides requires an integrated concept of the information available, and that is why this review was prepared. Earlier reviews of CRONSHEY (1961) and FUNDERBURK (1967) are directed to the reader's attention.

The objective of this review is to collect and present the pertinent and significant findings published since the discovery of the herbicidal potential of paraquat and diquat. On points of controversy, all views known to us have been stated. We have endeavored to present a concise, integrated interpreta­tion and conclusion. We consider this to be an extensive review, but not a substitute for the original articles. The review was restricted principally to those articles in established scientific publications. Only limited reference is made to regional research reports, progress reports, and popular articles. No reference is made to anonymous reports, or to publications of industrial firms. Though these sources were not quoted, we wish to point out that they contain

Paraquat and diquat 99

much valuable information, particularly on the application and use of paraquat and diquat.

II. Properties and characteristics

Paraquat and diquat, two heterocyclic organic herbicides, are members of a much larger group of organic compounds, commonly referred to as di­pyridylium (or bipyridylium) quaternary ammonium salts.

Diquat, C12H12NzBr2, has been designated by various chemical names as: 6,7 -dihydrodipyrido ( 1,2-a: 2',1' -c) pyrazidiinium dibromide, 9: 10-di­hydro-8 ( a ) , lO ( a) -diazonia phenanthrene dibromide, or 1,1' -ethylene-2,2'­dipyridylium dibromide (BRIAN et at. 1958):

0-0 \ / 2Br-

H2C-CH2

Diquat dibromide

The empirical formula of paraquat cation (see structure) is C12H14N2 + +. Its chemical name has been stated as: 1,1'-dimethyl-4,4'-bipyridinium bis (methylsulfate); or 1,1'-dimethyl-4,4'-dipyridylium diiodide (HOMER et at. 1960). In older literature and to some extent in recent biochemical journals the term "methyl viologen" or the chemical name N,N'dimethyl-y,y'-dipy­ridylium dichloride is used for paraquat:

Paraquat diiodide

Hereafter, the common names 'diquat' and 'paraquat' are used to designate either the cations or the salts of the herbicides, unless the full chemical name of the compound is necessary for clarity.

The divalent cation of paraquat or diquat consists of two quaternized pyridine rings bonded together. Quaternization is effected by the addition of an organic group to bridge over the two nitrogen nuclei (diquat), or by the addition of a methyl radical to each of the two nitrogen nuclei in the pyridine rings (paraquat). The anions associated with paraquat or diquat are usually bromine, chlorine, iodine, methy lsulfate, or sulfate. The active part of these herbicides is the cation and the associated anion has no effect on the herbicidal activity (HOMER et al. 1960).

Diquat forms a pale yellow monohydrate when crystallized from aqueous

100 A. A. AKHAVEIN and D. L. LINseon'

solution (BRIAN et al. 1958) and, since it is used in aqueous solution, it is normally obtained in the form of monohydrate (CRONSHEY 1961). Pure paraquat is a colorless, shiny, crystalline substance (MICHAELIS and HILL 1933 a). Both diquat and parquat are stable in aqueous acid or neutral solutions. In alkaline solution, however, these herbicides will eventually decompose-a process due to uptake of a single mblecule of alkali/molecule of herbicide, resulting in the rupture of one of the pyridine rings. Diquat and paraquat are water soluble to the extent of 70 g./lOO ml. of water at 20°C. (BRIAN

et al. 1958). These chemicals are insoluble in most organic solvents, but they are slightly soluble in alcohol. Aqueous solutions of paraquat and diquat are nonflammable, nonexplosive, and nonvolatile.

Diquat was originally synthesized by R. ]. FIELDEN in the laboratories of the Dyestuffs Division of the Imperial Chemical Industries, Blackley, England (BRIAN et al. 1958, BOON 1965). In a series of investigations at lealott's Hill Research Station, Bracknell, Berks, England, on the herbicidal activity of a group of quaternary ammonium compounds, BRIAN et al. (1958) discovered the exceptional herbicidal potential of diquat early in 1955. This finding led HOMER et al. (1960) into an extensive program for synthesis of many quater­nary ammonium salts of isomeric dipyridylium compounds and investigation of their herbicidal activity.

Paraquat and a few other related compounds, such as benzyl viologen, have been collectively known as "viologen dyes" or "viologens" for some time (MICHAELIS and HILL 1933 a and b). They have been used as oxidation­reduction indicators and electron carriers in chemical and biological reactions (MICHAELIS and HILL 1933 a, HOROWITZ 1952, CALDERBANK 1961). HOMER

et al. (1960) discovered the phytotoxicity of paraquat.

III. Relationship of herbicidal activity and structure

a) 2,2'-Dipyridyl series

After the discovery of the herbicidal activity of diquat by BRIAN et al. (1958), a more detailed investigation into the herbicidal properties of the 2,2'-dipyridyl compounds showed a certain relationship between activity and structure of the compounds (HOMER et al. 1960). For example, herbicidal activity was present only when the quaternizing group formed a bridge between the two nitrogen nuclei, as in diquat (Fig. 1: I, n = 2). The un­abridged 2,2'-dipyridyl dimethyliodide was completely lacking in activity.

Next, by varying the number of carbon atoms in the quaternizing bridge, HOMER et al. (1960) noticed that the highest activity was present in the com­pound with the shortest bridge (I, n = 2, diquat). The activity was much reduced when trimethylene (I, n = 3) was the bridge and completely lost for a tetramethylene (I, n = 4) bridge in the 2,2'-dipyridyl series.

2,2'-Dipyridyl I

Paraquat and diquat

10

101

Ib

D-QY ~ D-O. 0=0 R-N+ J. :-. +N-R R-N :-. +N-R-R-N N-R elc ~ II \\ /; --.:e _ \\ /; _ /;' .

2)(- • Y

4,4'-Dipyridyl IT

Q-ON-R

I 2.-R

2,4'·- Dipyridyl III

ITa

00 ~~!I I I 2.-R R

3,3'-Dipyridyl l'l

ITb

Q--O+/; ~ +/; N N I I 2x-R R

2,3'- Dipyridyl Y.

Fig. 1. Chemical structures of some of the dipyridyls

b) 4,4' -Dipyridyl series

During the examination of a wide range of the quaternary derivatives of 4,4'-dipyridyl (II), HOMER et al. (1960) detected phytotoxic activity in many of the derivatives in this series. The activity was very similar to the activity of diquat and was present in these salts for many different substituted quater­nizing groups (II, R = an organic radical, Y = H). The highest activity was found in 1,1'-dimethyl-4,4'-dipyridyl salts, paraquat (II, R = CRa, Y = H), where the quaternizing group uR" is methyl (BOON 1965). Complete loss of activity occurred whenever one or more of the four ring-positions adjacent to the inter-ring bond was substituted by a radical rather than a hydrogen (HOMER et al. 1960). For example, 3,Y-dimethyl-1,1'-R-4,4'­dipyridyl (II, Y = CRa) compounds were inactive (CALDERBANK 1961).

c) 2,4'-Dipyridyl, 3,3'dipyridyl, and 2,3'-dipyridyl series

The 2,4'-dipyridyl series (III) show some degree of phytotoxic activity, though at a much reduced level. The other two series, 3,3'-dipyridyl (IV) and 2,3'-dipyridyl (V) compounds, lack any degree of herbicidal activity (HOMER et al. 1960).

102 A. A. AKHAVEIN and D. 1. LINscott

d) Common characteristics of dipyridylium quaternary salts possessing herbicidal action

1. Coplanarity of the pyridine rings. - HOMER et al. (1960) suggest that a dipyridylium quaternary salt should be coplanar or being capable of assuming a coplanar configuration to possess herbicidal activity. With the help of molecular models, they deducted that diquat has a flat configuration and is the most active in the 2,2' -dipyridyl series. On the other hand, 1,1' -tri­methylene-2,2'-dipyridyl compound (I, n = 3) could be forced to assume a flat configuration, but the molecule will be under some strain. It is less active than diquat. The tetramethylene compound (I, n = 4) could not assume coplanar configuration and is inactive.

In the 4,4'-dipyridyl series (II), activity is present as long as steric hindrance ( II, Y = H) does not prevent the molecule from assuming a flat configuration. The activity is lost, as in 1,3,1'3'-tetramethyl-4,4'-di­pyridylium di-iodide (II, R = Y = CHa), once a hindering substitute is introduced into the 3,3' -positions.

2. Free radical formation upon reduction. - Coplanarity of the mole­cule, though a necessary criterion for herbicidal activity, is not sufficient to explain the inactivity of 2,3'-dipyridal and 3,3'-dipyridal derivatives which could easily assume coplanar configuration. Clearly, a more general criterion was necessary to include the behavior of both active and inactive dipyridyl quaternary salts.

MICHAELIS and HILL (1933 a and b) had demonstrated that the reduc­tion of quaternary salts of 4,4'-dipyridyl (the "viologens") proceeds through two steps. The first step is the formation of relatively stable, intensely colored, water-soluble free radicals through the addition of an electron to each mole­cule of the divalent cation (lla of Fig. 1). The divalent cation (II) is colorless, whereas the partially reduced monovalent cation (IIa) is intensely colored and contains an odd electron. The odd electron is shared by all the nuclear carbon positions in the rings (CALDERBANK 1961). This step is completely reversible. The second step in the complete reduction sequence of these compounds is the addition of a second electron to the molecule. It could be effected under drastic chemical reducing conditions unattainable in a bio­logical system. The resulting fully reduced species is colorless.

The work of MICHAELIS and HILL (1933 a and b) provided a clue for HOMER and TOMBLINSON (1959) and HOMER et al. (1960) to investigate further the oxidation-reduction behavior of the dipyridyl quaternary salts. They reduced an aqueous solution of diquat with sodium dithionite to form an intense green free radical. In this process, the redox potential of diquat was found to be -349 -+- 3 mY. This value is very close to the value of redox potentials for the "viologens" recorded by MICHAELIS and HILL

(1933 a and b). It was also noticed that the reduction of diquat, as in the case of paraquat, is completely reversible and independent of pH and the

Paraquat and diquat 103

nature of the associated anion (HOMER and TOMBLINSON 1959). From these results, HOMER (from CALDERBANK 1960) deduced that, "herbicidal activity depended on the ability of the active compounds to form free radicals by uptake of one electron". KRUMHOLTZ (1951) had previously shown that the 1,1'-dimethyl isomer of the 2,4'-dipyridyl series (III, R = CHs) could be reduced with zinc dust to a purple-colored species and he speculated that the compound forms free radicals upon reduction. CALDERBANK (1961) synthe­sized the compound and showed that it was also reduced to free radicals the same way 2,2'- and 4,4' -dipyridyl c01Il!Pounds are reduced. However, its herbi­cidal activity was lower and its redox potential (-640 + 40mV) was more negative than that of diquat or paraquat (HOMER et at. 1960).

3 .. Stability of the free radical, coplanarity, and the structure of the molecule in relation to phytotoxicity. - The location of the unpaired electron in a free radical is not fixed. Hypothetically, this electron, without charge separation, has as many opportunities for delocalization as there are available positive centers in the two pyridine rings. Delocalization of the unpaired electron gives a considerable resonance stabilization to the free radical. As the result, the greater the number of positive centers available to an odd electron, the greater are the number of resonance structures and the more stable is the free radical.

Eighteen resonance structures could be written for the free radicals of either paraquat (IIa, lIb, etc., Y = H, R = CHs of Fig. 1) or diquat (la, Ib, etc., n = 2). In both cases, some of the possible resonance structures in­volve a double bond between the two pyridine rings (Ia and lIb) (HOMER

et at. 1960). If due to steric hindrance, the molecule cannot assume a coplanar configuration, the formation of a double bond between the two rings is im­peded, and the number of the possible structures contributing to the resonance stabilization is reduced. Under such a condition, the free radical is less stable. Therefore, HOMER et at. (1960) concluded that, "in a non-planar molecule the number of possible resonance forms is decreased and the radical becomes less stable".

Besides coplanarity of the molecule, the relative positions of the two nitrogen atoms with respect to the inter-ring bond determine the number of possible resonance structures of the free radical. The greatest number of resonance formulae (18) will result if the two nitrogen atoms occupy either 2,2'- (as in diquat: I, n= 2) or 4,4'- (as in paraquat: II, Y=H, R= CHs) or 2,4'- (III, R = CHs) positions in relation to the inter-ring bond (HOMER et at. 1960, CALDERBANK 1961). Under either circumstance, the odd electron is completely delocalized and able to occupy any of the nuclear positions in the two pyridine rings. This increases the number of resonance structures contributing to the stability of the free radical. The lower herbicidal activity of the diquaternary salts of 2,4'-dipyridyl (III, R = CHs) is, how­ever, thought to be "due to steric hindrance between the N-methyl group of the 2-linked ring and the O'-hydrogen atom of the 4-linked ring" (HOMER

104 A. A. AKHAVEIN and D. 1. LINSCOTI

et al. 1961) even though the locations of the two nitrogen atoms are favorable for resonance stabilization of the free radical.

The nitrogen atoms of 2,3'- or 3,3'-dipyridyl series do not occupy the above-mentioned favorable positions. The odd electron, therefore, is excluded from certain nuclear positions, resulting in a fewer number of resonance struc­mres for these two series of compounds-fifteen structures for 2,3'- and twelve strucmres for 3,3'-dipyridals (HOMER and TOMBLIN SON 1959). Quaternary ammonium salts of 2,3'- and 3,3'-dipyridyl series, therefore, cannot be reduced to free radicals in a aqueous solution and are herbicidally inactive (HOMER et al. 1960).

e) Conclusion

From their investigation on the phytotoxicity of dipyridylium quaternary salts, HOMER et al. (1960) postulated a number of requirements for herbicidal activity of these compounds. The molecule should have the ability to accept one electron and form a stable free radical upon reduction. Reduction should be easily attainable and freely reversible. With a few exceptions, the redox potentials of paraquat (-446 mV) and diquat (-340 mV) are the highest among the dipyridylium compounds investigated and hence they are the more phytotoxic. Some of the dipyridylium quaternary salts, especially in 4,4'­dipyridyl series, have redox potentials higher than diquat or paraquat, but they are either devoid of activity or possess a very low degree of phytotoxicity (HOMER et al. 1960, BOON 1965). MERKLE and ISBELL (1965) have tried to synthesize 1,2-di (4-pyridine methiodide ethane) (VI of Fig. 1) which theo­retically should produce a free radical more stable than paraquat or di­quat. They have not been able to synthesize such a compound but suggested that if reduction and free radical formation requirements for phytotoxicity are correct, then this compound should be more phytotoxic than paraquat or diquat.

As HOMER et al. (1960) mentioned themselves, they had no evidence that the free radicals are toxic. Furthermore, the formation of dipyridyl free radi­cals in vivo had not been shown at that time. However, HOMER et at. (1960) showed that the redox property of paraquat and diquat and their capacity to form stable free radicals are closely related to their herbicidal activity. Several authors have used the terms "free radical formation," "toxic free radicals," or "reduction to free radicals" loosely to indicate the mode of action of paraquat and diquat. These are general terms indicating, for the most part, requirements for activity and should not be confused with the acmal mode of action of these herbicides.

Paraquat and diquat

IV. Mode of action, physiological and biochemical behavior

a) Photosynthesis

105

Evidence to date indicates that the physiological and biochemical behavior of dipyridylium herbicides is intimately associated with photosynthesis and in particular with the light reactions. To facilitate future discussion, the main processes and reactions of photosynthesis (Fig. 2) pertinent to diquat and paraquat mode of action are reviewed briefly.

Volts 1.0

0.8

0.6

0.4

0.2

0.0

-0.2

-0.4

-0.6

-0.8

Z H O---~O

2 I 2

light:stem ""IP700

1 //~~P AlP

Y ,/ A,DP light system (CYCliC photophosphorylation

I ADP

N I· 1 /NADP~NADPH2 on-cyc IC photophosphorylation / Fd

X

Fig.2. light ( ... ) and dark (---) reactions during photosynthesis

It is now generally accepted that the light phase of photosynthesis en­compasses two distinct light systems (commonly called "System 1" and "Sys­tem II") acting in conjunction. Light system IT, which primarily involves the oxygen evolution reaction (s) (HILL reaction), is the least understood photo­act. It has been suggested that the illuminated accessory pigments (chloro­phyll b in green plants) of system II absorb light and transfer the energy to specialized chlorophyll a molecules which act as reaction centers (GOVIND JEE

1967). Trapping of the light energy in system II results primarily in the generation of a strong oxidant (Z) with a potential of about +800mV and formation of a weak reductant (Y) with a potential of approximately zero. The nature of the reduced or oxidized form of Z and Y moieties and the mode of electron ( hydrogen) transfer from the donor (Z ) to acceptor ( Y) are not known (KOK 1965). In green plants, the strong oxidant of light system II is involved in oxidation of water and the result is the evolution of molecular oxygen.

106 A. A. AKHAVEIN and D. L. lINSCOTr

In light system I, absorption of a quantum of far-red light by chlorophyll a and the subsequent transfer of absorbed energy to P700 chlorophyll mole­cules (reaction sites of system 1) results in the formation of excited P700 molecules. The primary light reaction associated with light system I may be the removal of an electron from P700, and the formation of photooxidized P700 with a normal potential of about +400mV. In addition, an associated electron acceptor (unidentified, X in Fig. 2) receives the electrons, thus a strong reductant with a normal potential of -600m V or lower is formed (KOK 1965).

In a series of "dark" reactions, the electron gained by the weak reductant of system II (Y) is passed to oxidized P700 of system I through a group of chloroplast electron carriers such as plastoquinone, cytochrome b, cytochrome i, and plastocyanine (KOK 1965, GOVIND]EE 1967).

The end products of the two light reactions in green plants are: oxygen, adenosine triphosphate (A TP), and reduced nicotinamide adenine dinucleo­tide phosphate (NADPH2 ). Oxygen is released to the atmosphere and in the "dark" phase of photosynthesis, A TP and NADPH2 are used in the synthesis of carbohydrate from carbon dioxide. The electron flow established in the "light" phase of photosynthesis is coupled with a NADP reduction system to generate NADPH2 and with a photophosphorylation system to produce ATP.

The electrons generated in light system I are transferred to the electron acceptor ferredoxin to form reduced ferredoxin (TAGAW A and ARNON 1962). The reduction of NADP in chloroplasts is ferredoxin dependant. Reduced ferredoxin is oxidized by the flavoprotein ferredoxin-NADP reductase, gen­erating oxidized ferredoxin and reduced ferredoxin-NADP reductase. In turn, the reductase is oxidized by reducing NADP to NADPH2 (ARNON 1965, BLACK and MEYERS 1966).

Two forms of photophosphorylation, "cyclic" and "non-cyclic", have been shown to occur in illuminated chloroplasts (ARNON 1959 and 1965, ARNON

et al. 1967). Isolated chloroplasts have been shown to support "cyclic" photo­phosphorylation which depends on the back reaction of the strong reductant (electron or X) with the weak oxidant generated in system I (equation 1) (ARNON 1965):

Light system I ADP + Pi ) ATP (1)

Fd

In addition to orthophosphate (Pi), Mg+ +, and ADP, an appropriate elec­tron carrier is needed to link the reductant and oxidant to establish a cyclic flow of electrons from chlorophyll molecules through the electron carrier and back to the chlorophyll. Many physiological and non-physiological electron carriers such as ferricyanide, menadione, flavin mononucleotide (FMN), ferredoxin, and phenazine methosulfate (PMS) are capable of supporting

Paraquat and diquat 107

"cyclic" as well as "non-cyclic" photophosphorylation (ARNON 1959 and 1965, MAHLER and CORDES 1966, ARNON et al. 1967). In cyclic phosphoryla­tion, oxygen is neither formed nor required. In fact, to determine the rate of cyclic phosphorylation, reoxidation of the reduced electron carrier by evolved oxygen from system II must be prevented. Usually, an inhibitor of oxygen evolution such as 3- (3,4-dichlorophenyl) -1-methoxy-1-methylurea (linuron) is used and the experiment is carried out under anaerobic conditions (ARNON

1965, ZWEIG et al. 1965). A TP is the only product of cyclic photophos­phorylation.

Non-cyclic photophosphorylation is an anaerobic, stoichiometric (Pi/2e­= 1) reaction catalyzed by ferredoxin or other electron carriers. This reac­tion depends on both light systems of chloroplasts. The products of non-cyclic photophosphorylation are ATP, reduced ferredoxin, and molecular oxygen (equation 2) (ARNON 1965):

Light systems

(2)

4Fdox. + 2ADP + 2Pi + 2H20 --~~ 4Fdred. + ATP + O2 + 4H+ I & II

Reduced ferredoxin serves as an electron donor for NADP reduction. To prevent reoxidation of reduced ferredoxin (or other reduced electron carriers) by photoproduced oxygen, determination of non-cyclic phosphorylation is carried out under conditions which impede the rapid reoxidation of reduced electron carrier with oxygen (equation 3) (ARNON 1965, ARNON et al. 1967, ZWEIG et al. 1965) :

(3)

Under normal conditions, oxygen is produced in chloroplasts. Therefore. a summation of reactions 2 and 3 results in equation 4 to indicate the aerobic. oxygen linked non-cyclic, or "pseudo-cyclic", phosphorylation (ARNON

1965) :

Light systems I & II ADP + Pi ATP (4)

b) General studies on herbicidal action

MEES (1960) working with several plant species, especially Vicia faba, observed a number of phytotoxic characteristics of diquat which later were proved to be characteristic also for paraquat. The death of green plant tissues was much more rapid in light than in darkness. A much longer time was re-

108 A. A. AKHAVEIN and D. L. LINscott

quired to kill diquat-treated plant material which lacked chlorophyll (roots, etiolated seedlings). Lack of chlorophyll and darkness seemed to have the same effect, that is, prolonging the period between diquat treatment and the death of the plant. Increasing the incident light intensity increased the rate of damage to chlorophyllous tissues. These results suggested the involvement of photosynthesis in the rapid damage to plant tissues.

MEES (1960) investigated next the interaction between diquat and N ( 4-chlorophenyl) -N,N' -dimethyl urea (monuron), an inhibitor of the HILL re­action, on a number of plant species. In general, monuron greatly delayed the rate of diquat action in light. Potassium cyanide, an inhibitor of carbon dioxide uptake, had no inhibitory effect on diquat, and presence or absence of carbon dioxide caused no change in diquat action. Oxygen was shown to be necessary for the rapid action of diquat in light. The rapid effect of diquat was almost completely inhibited in a nitrogen atmosphere. These results demonstrated clearly the necessity of active photosynthesis for the rapid and extensive damage by diquat. Since carbon dioxide was not essential to diquat action, it was argued that the light reactions of photosynthesis are involved in the process rather than carbon assimilation reactions. MEES (1960) sup­posed that diquat is reduced in the light as well as, or in place of, the normal photosynthetic reductants, and that death is more rapid in the dark because this more intense reducing potential increases the rate of radical formation. Since photolysis of water generates a reducing potential in plant cells, MEES

( 1960) surmised "that the energy for reduction comes from the photolysis of water in the light". Photolysis of water was not inhibited by diquat. There­fore, in the light the condition for free radical formation was favorable in the presence of oxygen as well as in its absence, but the absence of oxygen caused inhibition of diquat action. MEES (1960) posmlated that free radicals formed in the light were "detoxified" in the absence of oxygen. Consequently, it was concluded that, although formation of free radicals is necessary for manifestation of toxic effect, free-radical formation alone is not sufficient to kill the plant.

FUNDERBURK and LAWRENCE (1964) reported a decrease in oxygen pro­duction of diquat-treated or paraquat-treated Lemna minor but neither herbi­cide was as effective as atrazine (2-chloro-4-ethylamine-6-isopropylamino-s­triazine) or monuron in inhibiting the HILL reaction (oxygen evolution path­way). The effects of a diquat-monuron combination in reducing oxygen pro­duction were additive. Inhibition of photosynthetic carbon dioxide-fixation by diquat has also been reported by VAN OORSCHOT (1964) and COUCH and DAVIS (1966) for several plant species.

BARNES and LYND (1967) found that reduced chlorophyll content in Phaseolus leaf disks was closely related to increased paraquat concentration and levels of light. Magnimde of leaf chlorosis was related to time, tempera­ture, light intensity, and rate of applied paraquat.

MEES' (1960) smdies of the effect of diquat on plant respiration re-

Paraquat and diquat 109

vealed that diquat-treated plants died rapidly in the light, whereas it took four to five days for the plant issues with or without chlorophyll to die in the dark. Light greatly enhanced the effect of diquat, but it was not essential for phytotoxicity. If reduction of herbicide to the free radical is essential for the toxic effect, then, in darkness a 'reducing power' aside from photosynthesis is needed for free radical formation. MEES (1960) studied the effect of diquat on the respiration of plant tissues and found that diquat stimulated oxygen uptake in the beginning of the experiment and inhibited it later. It has been suggested that respiration supplied the necessary energy for the free-radical formation in darkness (MEES 1960).

FUNDERBURK and LAWRENCE (1964) reported stimulation of plant res­piration (oxygen uptake) by diquat and paraquat, but did not detect inhibi­tion during their short term studies (eight hours). DAVIES and SEAMAN

( 1964) also reported an increase in dark respiration (oxygen uptake) of Elodia canadensis and Potamogeton pectinatus leaves. They give no explana­tion for the effect of paraquat and diquat on plant respiration. Pretreatment of aquatic plants with inhibitors of respiration, sodium azide or potassium cyanide, reduced the uptake of diquat 38 to 48 percent (SEAMAN and THOMAS

1966); they concluded that diquat uptake by the plants depended in part on energy of respiration and in part on physical adsorption of the cation of the plant surfaces.

c) SPecific physiological and biochemical studies

Dipyridyl compounds are capable of accepting a single electron and thus are reduced to their respective free radicals (MICHAELIS and HILL 1933 a and b, HOMER and TOMBLINSON 1959, HOMER et al. 1960). In turn, the reduced species is easily reoxidized to the original cation in the presence of molecular oxygen. Based on this property, it was postulated that the mode of action depends on the ability of dipyridyl cations to undergo reversible oxidation­reduction in the plant system and that the reduced free radicals thus formed are detrimental to the plants. MEES (1960) supported this idea and showed the intimate association of the mode of action of these herbicides with the light phase of photosynthesis.

In addition to the studies reviewed above, more exacting and fundamental physiological and biochemical investigations were needed in order to elucidate several aspects of the mode of action of these herbicides. It was necessary to ( 1) show that plants possess a system capable of reducing dipyridyl herbi­cides to their free radicals within the plant, (2) demonstrate that free radicals were actually formed within the plant, (3) show whether the free radicals were the toxic agent or whether toxicity was brought about through their participation in other biological reactions, and (4) elucidate the site (s) of action and the mechanism ( s) of phytotoxicity. The evidence relating to these questions is as follows:

110 A. A. AKHAVEIN and D. L. LINSCOlT

1. Pre~nce of necessary reducing power within plants capable of reduction of dipyridyl compounds.-The exact redox potential of the strong reducing entity (electron), generated within the illuminated choloro­plasts, has not been established definitely. TAGAWA and ARNON (1962) isolated ferredoxin (E'o = --432 mY) from Spinacia oleracea chloroplasts and demonstrated its photoreduction by the illuminated chloroplasts. Recently, ZWEIG and AVRON (1965) showed that some low potential dipyridyl com­pounds, including triquat (l,l'-trimethylene, 2,2'-dipyridylium dibromide; E'o = -550 mV), are photoreduced by the illuminated chloroplasts from Beta vulgare var. Cicla.

In an attempt to determine the value of the reducing entity of photoact I, KOK et al. (1965) studied photoreduction of several quaternary salts of 2,2'­and 4,4'-dipyridyl series with isolated chloroplasts. The dipyridyls used had a range of E'o values from -318 to -739 mY. All were photoreduced with illuminated chloroplasts. They concluded that "the normal potential of the strong reductant, generated in the long-wave photoact of photosynthesis is as low or lower than -0.7V." The strong reductant, therefore, has the potential to reduce diquat and paraquat completely and quantitatively (ZWEIG and AVRON 1965, ZWEIG et al. 1965, KOK et al. 1965, BLACK 1966).

2. Detection of the free radicals of dipyridyls in plant chloroplasts. - In order to detect the formation and accumulation of the free radicals in a chloroplast mixture, oxygen must be excluded from the mixture and its evolu­tion impeded. Otherwise, detection of free radicals is hampered because of reoxidation.

By the use of a method that gave reasonable assurance of oxygen exclusion from the reaction mixture, KOK et al (1965) followed the time-course photo­reduction of a range of dipyridyl compounds. Formation of the reduced free radicals was revealed through spectrophotometric measurement. The intensity of light, and the nature and the concentration of the dipyridyls, affected the rate of reduction and the total amount of the reduced species accumulated. Both the initial rate of reduction and the accumulation of reduced radicals increased with increasing concentration. The compounds with lower negative redox potential (E'o = -550 mY) were harder to reduce than those with higher potential (E'o = -440mV).

BLACK (1966) also investigated photoreduction of five dipyridyl com­pounds, including diquat and paraquat, with a range of redox potential values from -342 to -656 mY. The percentage of photoreduced species when no further reduction was observed was: 100 percent for both diquat (E'o = -342 mY) and paraquat (E'o = -425 mY), 50 percent for triquat (E'o = -521 mY), seven percent for the compound with E'o = -636mV, and only three percent for the dipyridyl with E'o = -656 mY. Both in light and in darkness, in the absence of air, the photoreduced dipyridyls were stable for about 30 minutes. Admission of air into the mixture immediately reoxidized the reduced species.

Paraquat and diquat 111

3. The effects of dipyridyl herbicides on several reactions of chloro­plasts. - a) Cyclic and non-cyclic photophosphorylation. By exclusion of oxygen and complete photoreduction of diquat prior to the onset of the ex­periment, ZWEIG et al. (1965) showed that diquat has the capacity to support cyclic photophosphorylation. Since cyclic photophosphorylation depends on light system I, diuron (a potent inhibitor of oxygen evolution did not inhibit the "cyclic" ATP formation.

]AGENDORF and AVRON (1958) and HILL and WALKER (1959) have reported the ability of benzyl and methyl viologen to catalyze aerobic (oxygen-linked) photophosphorylation in illuminated chloroplasts. DAVEN­

PORT (1963) also has shown diquat-mediated aerobic photophosphorylation at rates comparable with FMN or menadion-dependent photophosphorylation.

ZWEIG et al. (1965) investigated various aspects of diquat-catalyzed photophosphorylation. Aerobic, oxygen-linked, diquat-catalyzed photophos­phorylation in chloroplasts was believed to occur for the following reasons: ( 1) the rate of ATP formation was much higher in the air than in nitrogen atmosphere, (2) diuron drastically inhibited diquat or ferricyanide activity, but had very little effect on PMS-mediated A TP formation, (3) catalytic amounts of diquat were sufficient for its activity, and (4) the P / diquat ratio was higher than one. They also showed noncyclic photophosphorylation to occur.

Diquat or PMS individually can support aerobic photophosphorylation, but the effect of the two compounds together was found to be strongly inhibi­tory rather than additive (ZWEIG et al. 1966). Diquat, in the absence of ascorbate and under aerobic conditions, competitively inhibits PMS-dependent photophosphorylation. On the other hand, diquat and ferricyanide had no inhibitory effect on each other's activity, but the ATP yield was not additive. Diquat and paraquat-catalyzed ATP production is neither enhanced nor in­hibited in the presence of ferredoxin (BLACK 1966).

(3) Inhibition of NADPH2 formation. The photoreduced ferredoxin is the electron donor for reduction of NADP to NADPH2 (equation 5) (AR­NON 1965, BLACK and MEYERS 1966):

NADP - reductase Fdred. + NADP ) NADPH2 (5)

ZWEIG et al. (1965) and BLACK (1966) showed that paraquat and diquat completely inhibited photoreduction of NADP (equation 5) by ferredoxin. Furthermore, in presence of NADPH2, NADP and NADP-reductase (or TPNH diaphorase), paraquat and diquat were quantitatively reduced by pulling electrons from NADPH2 (DAVENPORT 1963, ZWEIG et al. 1965, BLACK 1966).

4. The site and mechanism of action of diquat and paraquat. -Based on the experimental results from diquat and its interactions with some

112 A. A. AKHAVEIN and D. 1. LINSCOTT

of the inhibitors and cofactors of photophosphorylation, reviewed previously, ZWEIG et al. (1965) suggested that diquat occupies "the same site as phena­zine methosulfate in the electron-transfer path of chloroplasts." As it is evident from their proposed site, diquat is in a position to shunt the electrons from ferredoxin and to link the reducing power of the photoact I of photosyn­thesis to the electron transport system that is present. BLACK and MEYERS

(1966) have proposed a similar site and elaborated further on a possible mechanism of action for paraquat and diquat. Reduced ferredoxin can transfer electrons to several substances causing reduction. NADP reduction is the most important ferredoxin-dependent reaction. When a dipyridyl is substi­tuted for ferredoxin, the dipyridyl will accept electrons from two sources, in the light from photosynthesis (system I) or in the light or darkness from NADPH2 (BLACK and MEYERS 1966). Under both light and darkness, plants lose a major source of their energy, NADPH2 • This accounts for the toxicity of diquat or paraquat in light as well as in darkness.

Reduced ferredoxin has a much greater affinity for NADP-reductase and NADP than for oxygen as a means for its reoxidation (BLACK 1966, ARNON

et al. 1967). Dipyridyl herbicides, on the other hand, are rapidly reoxidized by oxygen. In the presence of oxygen, therefore, reduced dipyridyl is quickly autooxidized forming hydrogen peroxide and oxidized dipyridyL Oxidized dipyridyl is not used up and will go through the same oxidation reduction sequence. As the result, only catalytic amounts of the herbicide are needed in the plant system to bring about its death.

In considering the mechanism of action of dipyridyl herbicides, production of hydrogen peroxide presents a paradox. Paraquat and diquat are extremely fast-acting herbicides. Any mechanism of action should account for this property. Short circuiting the electron flow or inhibition of NADPH2

formation alone will bring about a slow death of the plant. Hydrogen peroxide formation during reoxidation of paraquat or diquat has been sug­gested by several investigators (MEES 1960, CALDERBANK and CROWDEY

1962, BLACK and MEYERS 1966) to account for the rapid action of these herbicides. But investigators who have suggested such a possibility have also mentioned the fact that catalases and peroxidases are common in plant systems and should dismute and inactivate hydrogen peroxide. However, in­direct evidence in the work of GOOD and HILL (1955) and of DAVENPORT

( 1963) indicates the formation of a hydrogen peroxide or metmyoglobin­peroxide complex in illuminated chloroplasts in the presence of dipyridyl compounds. It is probable that the excess hydrogen peroxide, formed during auto oxidation of the dipyridyl compounds in living tissues, will flood the plant catalase and peroxidase enzyme systems beyond the normal capacity.

Certain points need clarification. MEES (1960) suggested that diquat did not inhibit photolysis of water and implied that this reaction in light provides the necessary reducing potential for reduction of diquat within the plant cells. Other experimental evidence (ZWEIG et al. 1965, BLACK 1966 and

Paraquat and diquat 113

BLACK et al. 1966) showed that although the HILL reaction is not inhibited by diquat or paraquat, this reaction is not the direct source of reducing power for reduction of the dipyridyls in green plants in the light.

VAN OVERBEEK (1962) postulated that a diquat or paraquat molecule picks up a single electron emitted from excited chlorophyll and forms a stable free radical, while monuron and simazine (2-chloro-4,6-bis (ethyl-amino)­s-triazine] inhibit the oxygen production pathway and, in doing so, block the return of electrons to chlorophyll. On the basis of results of MEES (1960) that monuron delayed the activity of diquat, and using the above argument, VAN OVERBEEK (1962) suggested that simazine as well as monuron should inhibit diquat activity. Contrary to the above suggestion, in long-term studies of the effect of paraquat in combination with simazine or diuron (3- (3,4-dichlorophenyl) -l,l-dimethyl urea} an inhibitor of the HILL reaction, for control of Agropyron repens, PUTNAM (1966) concluded that: "paraquat at 1/2 pound per acre (lb/ A) with simazine or diuron at 3-4 lb/ A provided long term phytotoxicity greater than that obtained from either herbicide alone." In fact, a synergistic effect of diquat (or paraquat) and monuron, not suggested by PUTNAM (1966), fits VAN OVERBEEK'S argument and PUTNAM'S results better than a possible inhibitory interaction of these herbi­cides. FUNDERBURK and LAWRENCE (1964) used the same argument to account for the additive effect of diquat and monuron combinations on inhibition of oxygen production. These investigators and COUCH and DAVIS

(1966) conveyed the notion that dipyridyl herbicides are inhibitors of the HILL reaction. However, in view of the new findings on the mode of action of diquat and paraquat, discussed before, dipyridyl herbicides should not be considered inhibitors of the HILL reaction in the sense that urea compounds are thought to be.

V. Factors influencing absorption, movement, and activity

a) Introduction

To show effect, a foliar applied herbicide must be retained on the surfaces and penetrate living and non-living barriers to reach the sites of actions. Physical and chemical forces affect each step of the so-called "entry" and "movement" processes of a herbicide (HARTLEY 1966) SARGENT 1966). The first barrier to the entry of a herbicide into leaves is the cuticle, present on many plant species. The membrane of epidermal cells comes next. Once entry is gained, systemic herbicides must move further to become partially or totally spread through the plant organ (s) .

Natural factors (light, humidity) and unnatural factors (surfactants) influence uptake and movement of herbicides. Interactions make the isolation of single factor effects difficult. 1n this section factors reported to influence

114 A. A. AKHA VEIN and D. L. LINSCOTI'

absorption, movement, and toxicity of paraquat and diquat will be dealt with separately.

b) Influence of surfactants

EVANS and ECKERT (1965) used 22 different surfactants in combination with various rates of paraquat for control of Bromus teetorum. Paraquat at the rates of 0.09 to 0.72 lb./ A, without surfactant, failed to control this Bromus species. Seventeen out of 22 surfactants tested increased the effective­ness of paraquat at these rates. One of the most effective was X-77 (alkylaryl polyoxylethylene glycols; free fatty acids; isopropanol). In general, the most effective surfactants were high in water solubility and the least effective ones were slightly water soluble, but highly lipid soluble. Surfactant concentrations of 0.60 and 0.12 percent were more effective than higher or lower amounts.

SMITH and FOY (1967) studied the interactions of paraquat with a group of two cationic, four anionic, and four nonionic surfactants as they affected surface tension, pH, and toxicity. No significant correlations were observed among the three measured criteria. In general, cationic and nonionic surfac­tants enhanced paraquat toxicity to Zea mays more consistently than the anionic surfactants.

Specific evidence regarding the mode of entry of paraquat and diquat into leaves is lacking. Indirect evidence indicates that a site of entry for paraquat and diquat may be through the stomatal openings and the sub­stomatal chambers. Leaf surfaces are more pervious to fat-soluble than to water-soluble substances and both paraquat and diquat are water-soluble. Plant species hav}ng a more extensive waxy layer deposit on the leaf surface are more resistant to diquat (THROWER et at. 1965). Only a low concentra­tion (about 0.1 percent v Iv) of surfactant in the solution is sufficient to bring about the maximum activity of the herbicide. These findings tend to favor stomatal entry. However, only minute quantities of herbicides are necessary at the active sites to kill the plant. Therefore, direct penetration of the cuticle and cell wall cannot be discounted.

c) Light and darkness

Diquat and paraquat, generally considered potent contact herbicides, move within the plant system under certain conditions. Light and darkness probably influence the movement of these herbicides more than other factors.

BALDWIN (1963) treated single leaflets of Lyeopersieon eseulentum with a droplet of radioactive diquat and kept the plants in total darkness for 24 hours. No movement of radioactive material took place in this period, whereas labeled material was detected throughout the aerial portion of the plants when they were given five hours of light after the 24 hours of darkness. On

Paraquat and diquat 115

the other hand, plants treated and left in the light showed extensive local damage without appreciable diquat movement out of the treated leaflet. Destruction of the phloem of the treated leaflet petiole with steam did not stop outward movement of diquat. It was concluded that diquat movement takes place primarily in the xylem and depends on local reversals of transpi­ration stream created after localized tissue damage. The failure of diquat to move out of the treated leaflets in light was attributed to the rapid death of the leaf tissues, which prevented the herbicide from reaching the leaf vascular system for extensive translocation. SMITH and SAGAR (1966) and SLADE (1965) generally confirmed the observations and conclusions of BALDWIN (1963).

SLADE and BELL (1966) showed that paraquat movement is identical with that of diquat.

Submersed plants are subjected to less intensive light than terrestrial plants. FUNDERBURK and LAWRENCE (1963) showed that C14-labeled para­quat and diquat when applied to the shoots of a submersed weed, Heter­anthera dubia, did not move into the roots or upper shoots of the treated plant. Also, labeled herbicides applied to roots did not move into the stems and the shoots of the plant, but radioactive diquat and paraquat moved from the roots into the stems and to a lesser degree into the leaves of Alternanthera philoxeroides.

Despite variation among species, the quantitative studies of BRIAN

(1967) on the rate and amount of diquat and paraquat uptake generally support other investigators' conclusions (BALDWIN 1963, THROWER et al. 1965, SMITH and SAGAR 1966). BRIAN (1967) found that four hours of darkness after herbicide treatment doubled the amount of uptake. The rate of uptake was shown to be very fast in the beginning of the dark period, gradually tapering off as the time progressed. The rapid phase of uptake (in dark) was during the first seven hours following the treatment. Under continuous illumination, movement of paraquat and diquat out of the treated leaf was found (SLADE and BELL 1966); the dark period after treatment only enhanced the herbicides' entry into the plant system and increased their systemic effects. BRIAN and WARD (1967) observed that diquat uptake was twice as great at 2,000 lux light intensity as it was at 18,000 lux.

The effects of darkness and light on the movement of paraquat and diquat can be summarized as follows: (1) a period of darkness immediately after treatment permits sufficient herbicide to penetrate the internal leaf tissues during the dark period, (2) the absorbed herbicide will not move out of the treated area due to extremely low rate of transpiration, (3) upon illumi­nation and resumption of normal transpiration long-distance transport of herbicide within the plan vascular system will take place, and (4) as the treated leaf begins to wilt and desiccate, local reversal of transpiration flow moves the herbicide to other parts of the plant.

116 A. A. AKHAVEIN and D. L. LINSCOTI'

d) Humidity and temperature

BRIAN (1966) tried to determine the change in paraquat and diquat activity caused by various air and soil moisture conditions imposed on several species of higher plants. In one experiment four levels of relative humidity (RH) were used in combination with various periods of darkness before and after treatment. It was noticed that only post-treatment high humidity in­creased diquat activity. Uptake of diquat and paraquat, whether in light or in darkness, was two to five times greater at 93 percent RH than the uptake at 55 to 65 percent. The results of BRIAN'S (1966) experiment with five levels of soil moisture (30 to 100 percent water-holding capacity), under two levels of air humidity (70 and 90 percent RH) and with labeled-and­unlaheled-paraquat-treated Triticum aestivum, showed (1) paraquat uptake was not affected by soil moisture regimes, (2) movement of paraquat was lower at 70 than at 90 percent air RH under each of the five soil moisture levels, and (3) at both air humidities labeled paraquat movement in T. aestivum plants gradually increased with reduction of soil moisture content (highest movement was at 30 percent water-holding capacity). However, analytical determination of paraquat content of the plants (treated leaf excluded) showed significant reduction in paraquat movement only at the highest level of soil moisture content (100 percent water-holding capacity). All relative humidities reported by BRIAN'S (1966) were at 21°C. BRIAN

and WARD (1967) noted a two-fold increase in diquat uptake by Solanum tuberosum when atmospheric humidity was increased from 50 to 94 percent.

Assuming that a decrease in electrical resistance of a solution containing paraquat and plant leaves is a measure of the damage of cell membrane, MERKLE et al. (1965) reported that paraquat is less effective in damaging leaf cell membrane at low temperatures. BOVEY and DAVIS (1966) grew several plant species at various temperature levels and assessed paraquat damage on them. The rapidity of paraquat damage was lower at lower tempertures. No extensive experiment has been conducted to elucidate the combined effect of temperature and humidity on paraquat and diquat herbi­cidal activity. THROWER et al. (1965), however, reported more extensive damage to diquat-treated plants at 85 percent RH and 15°C. than to those grown at 28 percent RH and 35°C.

e) Effect of rainfall

Diquat, since its discovery as a herbicide, was known to be a "rainfast" compound. BRIAN et al. (1958) stated: "Rain falling shortly after application has no effect on the herbicidal activity of diquat." To simulate rainfall, BOVEY and DAVIS (1966) washed paraquat treated flex vomitoria immedi­ately, 1/2, 1, and 24 hours after spraying. They detected an increase in para-

Paraquat and diquat 117

quat damage as the period between spraying and washing increased. Some paraquat uptake, however, has been noticed by BRIAN (1967) to occur within 30 seconds after spraying.

f) Effect of oxygen

Though oxygen is essential for rapid phytotoxic effect of paraquat and diquat, MERKLE et al. (1965) have shown that it is not necessary for damage to leaf cell membrane. Leaf cell membrane was damaged to the same extent in the presence or absence of oxygen. .

g) Synergistic and antagonistic effects of other herbicides on paraquat

A combination of two or more herbicides may have a toxic effect equal to, greater than, or less than the effect of each component applied individually. Such information has very definite practical application. But as COLBY (1967) pointed out, "the words 'synergistic' and 'antagonistic' have been largely avoided in publication of results". The task of showing the synergistic or the antagonistic effect of a herbicide combination is difficult, but the expla­nation of such a phenomenon is even more difficult. Due to a scarcity of infor.rn,ation on herbicide combinations, this area of chemical weed control merits much closer attention by investigators.

COLBY and WARREN (1963) noticed that the combination of paraquat and 3'-chloro-2-methyl-p-valerotoluidide (solan) was less toxic to Lycoper­sicon esculentum plants than either herbicide applied singly. However, the above combination was more toxic to Digitaria sanguinalis or Solanum melongena than each herbicide alone. WEISE (1966) reported a combination of paraquat (0.2 lb./ A) with diuron (0.5 lb.! A) provided a better and longer lasting control of Digitaria sanguinalis than either herbicide alone. LEWIS and MARTIN (1967) also obtained a more complete killing of sod with a mixture of paraquat and atrazine. For control of Agropyron repens, PUTNAM (1966) found that a combination of paraquat with simazine or diuron applied at the same time or the application of 3-arnino-1,2,4-triazole (amitrole) plus ammonium thiocyanate, seven days prior to paraquat appli­cation, gave better results than the application of each chemical alone.

In Malaya, for control of Paspalum conjugatum, a stoloniferous grass in rubber plantations, HEADFORD (1966) used paraquat, amitrole plus ammo­nium thiocyanate, and disodium methanearsonate (DSMA) singly and in combination. Mixing paraquat with either amitrole or DSMA caused an antagonistic effect, but when amitrole was applied (0.375 lb./A) two weeks prior to paraquat application (0.5 Ib.! A), excellent control of the weeds was achieved. This synergistic effect was not observed for DSMA and paraquat.

118 A. A. AKHAVEIN and D. L. LINSCOTI

h) Discussion

Soil and atmospheric humidity affect plant processes that depend on water balance. Furthermore, light, temperature, and humidity effects are inter­related. These factors influence herbicide uptake and movement both directly and indirectly through their interactions. Usually, an experiment is designed to investigate the effects of only one or two variables on the experimental material. The results of a simple experiment may not support a complex interpretation of the data; and many times the interpretations of the inter­action between factors investigated and those not included in the experiment are either totally disregarded or oversimplified.

Paraquat and diquat are believed to move in the xylem. Consequently, movement depends on transpiration of the plant and it should be more extensive at higher rates of transpiration. CLOR et at. (1962) stated that transpiration of water from leaves into a saturated atmosphere cannot be stopped completely while the plant is in light. It is also known that the rate of transpiration decreases at high atmospheric humidity. CLOR et at. (1962 and 1963), in their experiments with other herbicides, acknowledged that part of the enhanced movement of those herbicides, in highly humid atmos­phere, was due to condensation of water vapor on the treated leaf; that factor provided a continuous supply of herbicide for absorption and movement. The increased activity of paraquat and diquat under high atmospheric hu­midity seems to be due more to availability of the herbicides for absorption than to a change in physiological response of the plants kept in humid atmosphere (BRIAN 1966).

A period of darkness after treatment enhances paraquat and diquat uptake. Under field conditions, uptake and movement of these herbicides should be greater following late afternoon application. At night, due to darkness and higher humidity, the absorption of paraquat or diquat would increase. As a day begins, both leaf temperature and transpiration rise, providing favorable conditions for increasing the movement of these herbicides. The increased activity of diquat and paraquat at higher temperature levels could be due to two factors: increased rate of transpiration or increased rate of biological reactions, or both. Photoreactions are known to be insensitive to temperature variation. Therefore, on the basis of available information, we conclude that temperature exerts its effect through more extensive movement of absorbed herbicide to sites of action brought by increased rate of transpiration.

VI. Losses, persistence, and inactivation

A herbicide coming into contact with soil or plant material may be altered, lost, or rendered inactive through several chemical or physical means. Leaching, adsorption, photodecomposition, volatilization, and biodegradation (by plants or soil micro-organisms) are some of these processes.

Paraquat and diquat 119

a) Adsorption

1. Adsorption to plant surfaces. - BRIAN (1967) applied aqueous solutions of diquat and paraquat to aerial parts of three plant species and within 30 seconds washed the treated parts with distilled water. In this short period of time, the amount of herbicide uptake was assumed to be negligible. Thus the portion of the applied herbicide which resisted washing was con­sidered as the amount of herbicide adsorbed to the plant surfaces. It was found that paraquat and diquat are rapidly and strongly adsorbed to leaf surfaces. Approximately 33 per~ent of the total amount of diquat applied to Beta vulgaris leaves was adsorbed. The percent adsorbed, however, de­pended on the concentration of herbicide sprayed: the higher the concentra­tion the lower the percentage of adsorbed herbicide. From the rapidity of adsorption and characteristics of the adsorption curves, BRIAN (1967) con­cluded that physical forces (ionic attraction between cations of herbicide and negative centers on the leaf surfaces) rather than chemical reactions are responsible for the adsorption process.

2. Adsorption to soil. - Paraquat and diquat, being two highly water­soluble organic cations, are tightly and strongly adsorbed to particles with cation-exchange properties. Adsorption and desorption characteristics of these herbicides in aquatic and terrestrial environments have been investigated extensively. HARRIS and WARREN (1964), COATS et al. (1966), WEBER et al. ( 1965 ), and CORBIN et at. (1965) have studied the effect of pH, tempera­ture, and exposure time on adsorption of paraquat and diquat to various soils, clay minerals, charcoal, and ion-exchange resins.

A few general conclusions arising from the studies listed above follow: (1) Both paraquat and diquat are tightly adsorbed by montmorillonite, kaolinite, cation exchanger, and soil organic matter. Sandy and sandy loam soils are less effective in adsorbing these herbicides. Adsorption by charcoal and anion exchangers is insignificant. Paraquat and diquat are much more easily desorbed from sandy soils than from soils high in clay (especially montmorillonite) or organic matter. (2) The adsorption mechanism is physical in nature and it seems to be an ion exchange process. (3) The amount of either organic cation adsorbed is not dependent on pH, temperature, or exposure-time.

b) Degradation

1. Degradation by soil micro-organisms. - BOZARTH et at. (1965) studied microbial degradations of paraquat and diquat and their effects on soil micro-organisms. Among several micro-organisms, a fungus Cephalo­sperium species and an unidentified bacterium which showed a high degree of paraquat tolerance were selected and grown in a liquid medium containing 20 p.p.m. of labeled paraquat. At the end of the incubation period, silica

120 A. A. AKHAVEIN and D. L. LINSCOTt

gel plates were spotted with the medium and later the plates were radio­autogramed. Two degradation products (unidentified) were produced by the bacterium and none by the fungus. No paraquat breakdown was detected within two days after incubation. Maximum degradation occurred after four days of incubation.

2. Degradation by higher plants. - The work of FUNDERBURK and LAWRENCE (1964), with labeled paraquat and diquat. indicated that neither herbicide is degraded by Alternanthera philoxeriodes or Phaseolus vulgaris.

SLADE (1966) applied methyl-labeled and ring-labeled C14 paraquat in aqueous droplets to the leaves of Lycopersicon esculentum, Vicia jaba, and Zea mays plants. The labeled materials used were degraded under conditions which were indicative of photochemical decomposition of paraquat rather than its metabolic degradation by the plants; e.g., the herbicide was not degraded in dark or in a lighted greenhouse.

To date, no investigator has shown conclusive evidence for paraquat or diquat metabolism by higher plants, and it has been generally agreed upon that such degradation does not take place.

c) Photochemical decomposition

By irradiating an aqueous solution of paraquat with ultraviolet light, SLADE (1965) was able to demonstrate rapid paraquat photochemical de­composition. At different intervals, chromatography papers were spotted with the irradiated paraquat solution. From the autoradiographs of these papers, SLADE (1965) showed that two decomposition products are formed, one being a transitional compound which itself is eventually photodecomposed to form the second product. Aqueous solutions of paraquat, exposed to sun­light, did not show decomposition of the herbicide to any appreciable extent. But, under sunlight or ultraviolet light, paraquat adsorbed to a filter paper or a silica gel layer was photodecomposed· and the same degradation products were formed. Later SLADE (1966) found that paraquat adsorbed to leaf surfaces was decomposed, also. FUNDERBURK et al. (1966) studied photo­chemical decomposition of diquat and paraquat by ultraviolet light (240 to 260mp. range), and their results generally are in agreement with those of SLADE (1966).

In a more extensive study of the effect of sunlight on the photodecompo­sition of paraquat applied to the leaves of Lycopersicon esculentum, Vicia jaba, and Zea mays plants, SLADE (1966) was able to isolate and identify two photodegradation products from paraquat: namely, 4-carboxyl-1-methyl­pyridinium chloride and methylamine hydrochloride. Though the treated plants were dead in a few days, the amount of the degraded products on the leaf surface increased with the length of exposure to sunlight during a three­week period. Paraquat applied to the plants grown in a greenhouse was degraded to a limited extent in the summertime but not in the wintertime.

Paraquat and diquat 121

When paraquat-treated plants were kept in darkness no degradation took place. Approximately 45 percent of the total amount of applied paraquat was photodecomposed within three weeks on treated plants grown under natural sunlight.

VII. Residues, degradation products, and toxicity

a) Residues in soils

A large quantity of paraquat or diquat can be adsorbed by most soils moderate in clay minerals and organic matter content without harm to the growing crop. Diquat or paraquat, when applied to a sandy loam soil at 0.1 mg./g. of soil (200 lb./two million pounds of soil), did not reduce the dry weight of two-week-old plants of Triticum aestivum (COATS et al. 1966). Soils with higher clay or organic matter content can adsorb and inactivate greater quantities of these herbicides.

Degradation of paraquat and diquat by soil micro-organisms under natural conditions has not been proved (BOZARTH et al. 1965). For the present time, we should assume that these herbicides are not broken down and will remain or accumulate in the soil as residue over the years. For agricultural purposes, however, the amount of paraquat or diquat used is in the order of Yz to two lb./ AI treatment. Diquat and paraquat are normally used on growing and established vegetation; thus, much of the sprayed chemical is intercepted by the growing vegetation and a large portion of it is eventually photodecomposed. Based on the present available information it is not expected that the proper use of these herbicides will pose danger to crops grown on a field which has received several applications of diquat or paraquat. In fact, the rapid adsorption and inactivation of these herbicides by soils is one of their unique properties which permits safe application of diquat and paraquat prior to, immediately after, or during many crop seeding operations.

b) Residues in crops

Since paraquat and diquat are not metabolized by higher plants, the amount of these herbicides adsorbed and absorbed by higher plants should be considered as the persistent residues, but several factors help to reduce the level of herbicidal residues in treated crops.

Light, temperature, and humidity have a great influence on adsorption, movement, and degradation of these chemicals. Therefore, by managing the time of application of paraquat or diquat it should be possible to vary the amount of adsorption and photochemical degradation. For example, if the herbicide is used for Solanium tuberosum haulm destruction, spraying on a hot, sunny, and dry day may help reduce potential residues in the tuber.

HEADFORD and DOUGLAS (1967) investigated the factors that influence

122 A. A. AKHAVEIN and D. L. LlNscorr

the level of diquat residue in Solanum tuberosum tubers after chemical treat­ment of the haulm. They observed the highest level of diquat residue in tubers "when plants were subjected to drought and a period of darkness, following spraying at a time when senescence of the foliage had commenced and when the tubers were still increasing in fresh weight."

Photochemical decomposition of diquat and paraquat is the most impor­tant factor in reducing the level of the herbicides residue in the sprayed crop. BLACK et al. (1966) sprayed herbage in the field with a theoretical rate of 80 p.p.m. of diquat (based on the dry matter weight of the herbage) and analyzed the sprayed vegetation at various intervals. One day after spraying, 12 to 48 p.p.m. of diquat was detected and die residue level sharply dropped to 1.0 to 5.7 p.p.m. seven days after treatment. After initial degradation of diquat in the field, ensiling the treated herbage for five months did not reduce the level of residue further. Under natural summer sunlight, within three weeks approximately 50 percent of the applied paraquat (SLADE 1966) and within one week 90 percent of applied diquat (BLACK et al. 1966) were photodecomposed. The rate and the amount of photochemical degradation of diquat is evidently greater than that of paraquat for a given period. This difference between the two herbicides might determine which herbicide should be used depending on the intended use of the crop.

When paraquat or diquat are used for weed control in such a way that the herbicide does not come into contact with the crop or processed com­modity, no residue has been detected (SPRINGETT 1965, CALDERBANK and SLADE 1966). A few such situations are: weed control in orchards or vine­yards, pre-harvest desiccation, and pre-emergence (to crop) application for control of weeds in many crops and post-emergence weed control in cereals, root crops, and vegetable crops. Pre-harvest desiccation leaves less than 0.1 p.p.m. of paraquat in Solanum tuberosum tubers, Gossypium seeds, and Dau­cus carota roots, less than 0.2 p.p.m. in Zea mays, and less than 0.5 p.p.m. in Allium cepa (CALDERBANK and SLADE 1966).

c) Residues in water

Diquat and paraquat concentration in treated water quickly diminish through adsorption to soil particles (COATS et al. 1964), plants, and plant materials suspended or living in the body of the water (SEAMAN and THOMAS

1966). Leaves of three species of aquatic plants, immersed in dilute diquat solution, accumulated a concentration of diquat several times greater than the concentration of the external solution (SEAMAN and THOMAS 1966). YEO

(1967) found that after applying diquat to reservoirs, the initial concentration (1.0 p.p.m.) was reduced to 0.06, 0.02, and 0.009 p.p.m. after 4, 8, and 12 days, respectively. The rate of paraquat disappearance in similar tests was lower than that of diquat. Similarly, FRANK et al. (1966) could not detect paraquat 12 days after its application to several ponds. However, analysis

Paraquat and diquat 123

of the top one-inch layer of the submersed soil in the ponds showed consider­able paraquat accumulation. As paraquat and diquat applied to pond water decreased in concentration the hydrosoil concentration correspondingly in­creased (FRANK and COMES 1967). After fixation to the hydrosoil complex, the rate of disappearance was slow.

d) Residues in milk

STEVENS and WALLEY (1966) administered orally a single sub-lethal dose, five or eight p.p.m. of animal body weight, of methyl- or ring-labeled paraquat or ethylerie- or ring-labeled diquat to lactating cows and analyzed their milk for seven consecutive days after the treatment. Measurements of the radioactivity levels of the milk samples, when graphed against time, showed two high peaks 24 hours and 70 hours after the treatment. However, colorimetric determination of diquat within the two peaks of activity revealed that the level of actual diquat residue was less than 0.002 p.p.m. Therefore, it was concluded that the high level of radioactivity in the milk samples was due to a labeled metabolite rather than to unchanged diquat. Measurements of radioactivity (metabolite + unchanged diquat), however, showed that 0.001 to 0.015 percent of ingested diquat or paraquat was excreted in the milk in seven days. Mter carrying out the same type of experiments with paraquat, STEVENS and WALLY (1966) suggested that most of the radio­activity in the milk of paraquat-fed cows was unchanged paraquat. The level of radioactivity in butter, casein, or whey fractions of the whole milk was found to be a third of the total activity, indicating equal distribution of the residue among the processed milk products.

In a long-term feeding trial, two lactating cows were fed on a silage containing 3.6 p.p.m. of diquat (based on dry matter) for 30 days and the milk from the 15th to the 30th day was analyzed on alternate days for diquat residue. Using a procedure which could detect 0.003 p.p.m., BLACK et al. (1966) did not detect a diquat residue in the milk.

e) Degradation and absorption of paraquat and diquat by animals

DANIEL and GAGE (1966), using ethylene-labeled diquat and methyl­labeled paraquat, showed that 70 percent of diquat and 30 percent of para­quat, administered orally to rats, were broken down. Further experiments indicated that the breakdown was due to microbial degradation of the herbi­cides in the gut. BLACK et al. (1966) fed silage, containing six to 13 p.p.m. of diquat (based on dry matter) to sheep and determined the amount of diquat ingested and excreted. They could not account for 54 to 60 percent of the ingested diquat and attributed a great portion of this loss to microbial degradation. Incubation of diquat with sheep feces caused 35 percent loss of diquat after two days. With sheep (BLACK et al. 1966) and with rats

124 A. A. AKHAVEIN and D. L. LIN5COlT

(DANIEL and GAGE 1966), approximately 10 percent of the ingested diquat or paraquat was found to be absorbed through the gut, and the rest was excreted. The total amount of the absorbed portion was also excreted in the urine within two days. In the literature cited above there is no mention of the identity of the degradation product (s) .

f) Toxicity of the photodecomposition products of paraquat and diquat to animals

Since methylamine (one of the photodegradation products of paraquat) is a naturally occurring substance in some plants, CALDERBANK and SLADE

(1966) assume it to be a non-harmful compound. They found also a second degradation product, 4-carboxy-1-methyl.pyridinium chloride, to have a very low order of mammalian toxicity, LD50 greater than 5,000 mg./kg. for rats.

g) Toxicity of paraquat and diquat to animals and man

HOWE and WRIGHT (1965) have summarized the results of several toxi­cological experiments conducted in Great Britain, Australia, and the United States on a single-dose and long-term toxic effects of diquat and paraquat on many animal species including fish and wild life. In general, the studies show ( 1) paraquat is more toxic to animal life than diquat, (2) cattle, sheep, guinea pig, and cat are among the most susceptible species (LD50: 40 to 75 mg./kg.), (3) mostly, respiratory and nervous systems of animals seem to be impaired by these herbicides, (4) rabbit eyes are not affected by 10 percent solutions of diquat, but 40 percent solutions are irritant to the eye, and paraquat at the same concentration is more irritant than diquat, and (5) LD50 for a single application of paraquat to rabbit skin is in the order of 500 mg./kg.

In a long-term feeding trial, silage with six and 13 p.p.m. of diquat (based on dry matter) was fed to group of sheep for 50 days. No diquat was detected in the brain, liver, and kidney tissues (BLACK et al. 1966). Also, no diquat could be detected in the meat, lung, liver, and kidney tissues of a steer feed on silage containing 3.6 p.p.m. of diquat for 30 days. HOWE

and WRIGHT (1965) supplied eight-month-old calves and sheep with diquat­or paraquat-treated drinking water (20 p.p.m.) for 30 days; no ill-effects were noticed.

HOWE and WRIGHT (1965) point out that although no oral dosages have been administered to man for long periods, the effects observed in animals would probably be observed in man. However, they state that because of no detectable or very limited quantities of residue «0.01 p.p.m.) in edible crops, the dipyridylium compounds are unlikely to be of chronic toxicity to man. HOWE and WRIGHT (1965) state that the lethal dosage to man has not been determined exactly but based on data from accidental poisonings

Paraquat and diquat 125

and suicides they suggest that the value will be above 25 mg./kg. Doses over 400 mg./kg. have resulted in death. Careless handling of the pesticide and failure to wash off spills can result in white patches on, and cracking of, the fingernails. Imprudent inhalation of dipyridyl dusts or mists, on occasion, resulted in short-term nose bleeding (HOWE and WRIGHT 1965). However, no harmful effects were observed when the chemicals were used properly.

h) Determination of paraquat and diquat residues

CALDERBANK et al. (1961) described a method for extraction, separation, and determination of diquat residues from Solanum tuberosum tubers. Later, CALDERBANK and YUEN (1966) and KIRSTEN (1966) improved the accuracy of the method and adapted it for determination of paraquat residues in fruits and food crops. The methods employed in the above articles have been used for the determination of paraquat or diquat residues in fruits, food crops (CALDERBANK and SLADE 1966), feed crops (BLACK et al. 1966), milk, animal tissues and excreta, and water (STEVENS and WALLEY 1966).

The basic principles for determination of either paraquat or diquat resi­dues are the same. Since paraquat and diquat are strong cations, they adsorb to plant materials and constituents (starch, cell fragments, etc.). To extract the herbicide, therefore, the material is boiled in aqueous sulfuric acid solution and filtered. The acidic solution thus obtained is neutralized, diluted, and then passed through a cation-exchange resin column. The divalent cations of either paraquat or diquat are retained by the resin and can be eluted from it with a saturated solution of a strong inorganic cation (e.g., H +, Na +, or Ca + + ). These cations of paraquat or diquat in the eluted solution are then reduced with sodium dithionite and the concentration of reduced monovalent cations is determined spectrophotometrically. The reduced diquat solution (green) exhibits a well-defined absorption peak at 378 m"". Reduced paraquat (blue) absorbs at 392 to 401 m"".

VIII. Uses of paraquat and diquat1

a) Desiccation, curing, defoliation

STUBBS (1958 and 1960) was the first to demonstrate the use of diquat for desiccation of Solanum vines prior to harvesting. He and BUTLER (1960) reported that the application of 0.5 to twO lb./ A of diquat in 20 to 100 gallons of solution killed haulms as effectively as either sulfuric acid or sodium

1 The discussion which follows reflects results of research and does not imply legality of such use in commercial practice. Restrictions and limitations on use of pesticides vary among states and nations. Readers interested in acceptable co=ercial pesticide application practices should consult their local authorities. For the United States of America, readers are referred to the "Summary of Registered Agricultural Pesticide Chemical Uses" published by the Pesticides Regulation Division, Agricultural Research Service, U. S. Department of Agriculture.

126 A. A. AKHAVEIN and D. L. LINscott

arsenite. The viability and the flavor of the diquat-treated Solanum tubers were not affected. SAWYER et al. (1959) also used diquat for Solanum vine destruction and obtained satisfactory results. BRIEN et al. (1958) showed the feasibility of using diquat to desiccate Trifolium pratense foliage prior to its harvest for seed. Diquat has been used for Gossypium defoliation prior to mechanical harvesting and has proved to be a satisfactory desiccant for this purpose (CRONSHEY 1961).

To minimize the loss of nutrients during field curing of hay, SHEPHERED

( 1959) applied diquat to the vegetation prior to cutting. He showed that diquat-treated forage cured faster than untreated hay, but crushing of cut hay reduced the curing time to a greater extent than diquat treatment.

b) Uses based on selectivity

1. Selective weed control in cereal grains. - Paraquat and diquat are considered to be non-selective herbicides. However, at low dosage, under certain conditions and toward some species of plants, these herbicides are selective. STUBBS (1958) and BRIAN et al. (1958) showed that the cereal most resistant to diquat is Avena sativa followed by Tfiticum aestivum, Zea mays, and Hordeum vulgare in the order of decreasing resistance. JACKSON

(1959) obtained good control of Spergula arvensis with diquat application to a Zea field, but the Zea plants were also damaged. RICHARDSON (1959) and CHIASSON (1961) reported that diquat successfully controlled Spefgula arvensis in Avena sativa. Diquat initially scorched the Avena plants, but they soon recovered. The yields of the diquat-treated plots were comparable to those obtained from the hand-weeded plots. However, it should be noted that selective weed control of diquat in cereal grains is erratic and risky (CRONSHEY 1961). Paraquat is much more active against grasses than diquat, and consequently it is not used extensively for this purpose.

2. Selective weed control in established legumes. - JONES (1962) applied paraquat at the rate of four oz./ A to an established field of Medicago sativa for control of Poa trivialis and concluded that: (1) P. trivialis and Stellaria media were successfully controlled, (2) in the treated plots, the total yield of M. sativa increased, but the total yield of Medicago plus grasses in the untreated plots were equal to the yield of Medicago in the treated plots, (3) P hleum pratense, F estuca elatior, and Dactylis glomefata were sup­pressed for a short time but they reestablished quickly, and (4) the best time to apply paraquat was early in the growing season during the dormant period of M. sativa.

Application of eight oz./ A or less paraquat to a weedy mature stand of M. sativa was reported by LEONARD (1964) a) to give satisfactory results for the control of Poa annua, Erodium moschatum, Cerastium glomefatttm, Stelleria media, and Veronica persica when they are young, but their toler-

Paraquat and diquat 127

ances increased rapidly as they matured. Taraxacum officinale, Crepis taraxa­cifolia, and Agrostis tenuis were temporarily scorched and recovered easily.

LEONARD (1964 c) investigated the possibility of using paraquat to sup­press Lotium spp. in Trifolium rep ens grown for seed crop. He found that an application of three to four oz./ A paraquat, when the Trifolium plants were two to four inches tall, suppressed Lotium and resulted in a higher yield of Trifolium seed.

3. Pasture improvement through selective weed control. - Various species of grasses and broad-leaved plants show different degrees of sus­ceptibility to paraquat. JONES (1962), applying low rates of paraquat on established pastures, noticed that Lotium perenne and Dactylis glomerata are fairly resistant to paraquat, while Agrostis stolonifera and Poa trivialis were completely killed. However, DOUGLAS and MCILVENNY (1962) showed that the selectivity of paraquat in a mixed pasture decreases rapidly with an increase in the rate of paraquat application. BLOOD (1964) applied six oz./ A paraquat to a permanent pasture and observed that the population of Poa trivialis was reduced, the proportion of Lolium perenne was increased, and the number of Trifolium rep ens remained unchanged in the sward. Col­chium autumnale was controlled with 0.25 lb./ A paraquat.

The results of a series of trials conducted by THOMPSON (1962) on mature swards for determination of grass species tolerance to various rates of paraquat indicated that (1) Lotium perenne, Festuca rttbra, and Dactylis glomerata were killed by 0.45 lb./ A paraquat, (2) a rate of 3.6 lb./ A paraquat did not control Agrostis tenuis and Paspalum dilatatum and they recovered quickly, and (3) Paspalum distichum was resistant to 1.8 lb./ A of either paraquat or diquat (THOMPSON 1962).

DOUGLAS et al. (1965) studied the effect of paraquat and diquat on the equilibrium of vegetation in some of the hill-pasture communities of Great Britain. Prior to the application of paraquat or diquat, 80 percent of the plant population of those pastures was composed of three grasses, Festuca ovina, Nardus stricta, and Molinia caerulea. Application of two lb./ A. of para­quat reduced the proportion of the three grasses to four percent of the total population in the sward at the end of the first growing season. The same amount of diquat did not alter the proportion of the grasses. At the end of the second growing season, the proportion of the three grasses was 19 and 60 percent of the total population for paraquat and diquat treated swards, respectively. Nardus and Molinia were quite susceptible to paraquat. In the second year, the Festuca population in diquat-treated swards was higher than in the original population. To some extent, the growth stage of each species affected the amount of the sprayed herbicide intercepted. A species with a rank growth not only receives a greater proportion of the sprayed herbicide, but also protects the low-growing species from the spray droplets.

In California, KAY (1964 b) tested paraquat on rangelands to determine

128 A. A. AKHAVEIN and D. 1. LINscorr

its effectiveness in removing annual grasses from the mixed vegetation. One­third of the rangeland was covered by some annual grasses and the rest by Trifolium subterraneum, T. hirtum, Phalaris tuberosa, and Dactylis glomerata. One-half lb./ A of paraquat successfully removed the annual grasses without an adverse effect on the four desirable perennial species. Paraquat applied in early or late spring was ineffective in removing the annual grasses from the mixed vegetation. Best results were obtained by mid-spring paraquat application.

In the Mediterranean climate rangelands of California, KAY (1964 a) compared the effect of paraquat spray, clipping of the vegetation, and the combination of both on reduction of the population of annual grasses. It was found that the three treatments reduced the population of annual grasses in i:he pasture to the same extent.

Paraquat concentration, composition of the sward, and nutrient status of the soil supporting the vegetation are thought to influence the selectivity of paraquat when applied to a mixed vegetation. GRIFFITHS (1966) studied the influence of those factors on the selectivity of paraquat on a pure and mixed stand of Holcus lanatus and Lotium perenne grown in boxes under high and low nitrogen levels. Eleven weeks after planting, various rates of paraquat were applied to the growing plants. The number of tillers, in the pure stands of both species, decreased as the concentration of paraquat in­creased. At the high nitrogen level, paraquat selectivity was the same re­gardless of the sward composition. At the low nitrogen level, paraquat was very effective against H. lanatus when it was the dominant species of the sward, but Holcus was resistant to paraquat when it was the minor species of the sward in which L. perenne was dominant.

The seasonal susceptibility of mixed swards to paraquat was studied by HEDDLE and YOUNG (1964). At different times during the growing season, they applied various rates of paraquat to a Nardus stricta-Festuca ovina mixed hill-pasture in Great Britain. It was found that the July and September appli­cations were the most effective for the control of Nardus and the March application did not control this species. Festuca population increased after the July application, while the December application reduced its population. Paraquat at the rate of one lb./ A, applied in July, gave the best control of Nardus. Trifolium rep ens and Poa species were not damaged by paraquat.

KENT (1964) investigated the influence of shading on the effect of para­quat applied to pure and mixed stands of Lolium perenne and Festuca rubra. Shading before or after application increased the effect of paraquat on the pure stand of Festuca and the mixed stand of both species. For pure stands of L. perenne, shading after the treatment increased the effect of paraquat. In New Zealand, LEONARD (1964 b) found that suppression of L. perenne with paraquat was excellent, whereas Hordium murimum control was poor.

In Great Britain, ALLEN (1965) showed that an application of 1.5 lb./ A

Paraquat and diquat 129

paraquat completely killed Agrostis stolonifera, Poa trivialis, Lolium perenne, Holcus lanatus, Phleum pratense, and Trifolium repens.

c) Control of vegett:ttion before planting a new crop

1. Introduction. - Since their introduction to the field of agriculture, herbicides have been used by many investigators (ARNOTI' and CLEMENT

1966, BARRONS and FITZGERALD 1952, BLACKMORE 1957, DAVIDSON and BARRONS 1954, DOUGLAS 1965, SPRAGUE 1952, VENGRIS 1964) as substitutes for part or all of the conventional tillage practices for establishing and grow­ing crops. SPRAGUE (1952) tested several herbicides in place of tillage to renovate permanent pastures. BARRONS and FITZGERALD (1952), after kill­ing a Trifolium repens sod with 2,4-D and planting the desired crops, ob­tained satisfactory yield of Triticum aestivum, Zea mays, Linum usitatissimum, and Glycine max. DAVIDSON and BARRON'S (1954) trials in Michigan demon­strated that the soil structure was better and moisture content was higher in an undisturbed, chemically killed sod planted to crops than in a conven­tionally cultivated, fitted, and seeded field. However, as ELLIOTI' (1958) pointed out, some failure and even contradictory results are to be expected due to a great variation among locations, methods, crops, climatic conditions, type of herbicides, and experimental procedures employed.

2. Planting without tillage: no-tillage methods. - a) Reseeding per­manent pasture. Renovation of pastures by drilling with the desired species without the aid of an herbicide or cultivation generally has been unsuccessful (HAMMERTON and JOHNSON 1962). The "ideal" herbicide for no-tillage methods of crop growing should eliminate the competition of a wide range of undesirable vegetation, without harming the intended crop by leaving toxic residues in the soil.

ELLIOTI' (1960) recognized three ecological phases in a chemically treated pasture: the immediate phase during which the direct effect of the herbicide is manifested; the static phase, when the residual activity of the herbicide gradually disappears; and the regeneration phase through which the growth of new species and the recolonization of the old species take place. For establishment of annual crops, probably the first and the second phases are of prime concern. But to assess the merits of a herbicide for permanent pasture establishment under no-tillage or minimum tillage methods, the entire sequence of ecological changes brought about by the application of a herbicide should be considered.

ALLEN (1966) compared the effects of paraquat, 2,2-dichloropropionic acid (dalapon), and amitrole applied in November to a mixed-sward. The initial effect of paraquat on the pasture was quick. Dalapon and amitrole were much slower in their effect. Twenty days after spraying, the amounts of green vegetation on the paraquat- treated plots were only one-tenth those

130 A. A. AKHAVEIN and D. L. LINSCOTt

on the untreated plots. Dalapon and amitrole did not reduce the amount of green vegetation after three months as much as paraquat did after 20 days. Plots treated with paraquat and dalapon showed some signs of regrowth by four months after spraying. Control of grasses was very good with both paraquat and dalapon. However, none of the three herbicides reduced the population of broadleaved species to less than one-half the population of the check plots. Amitrole was less effective than either paraquat or dalapon.

In a four-year period EVANS et at. (1964 and 1967) applied paraquat to some of the rangelands of northeastern California and Nevada, and reseeded them to the perennial wheat grasses Agropyron desertorum, A. trichophorum, or A. intermedium. Among the results were ( 1) 0.5 lb.! A or more of paraquat plus a surfactant gave consistent and satisfac­tory control of Bromus tectorum, (2) the stand of the sown perennial grasses in paraquat-treated plots was the same as that of conventionally cultivated and seeded plots, (3) spraying prior to seeding gave better results than after seeding, (4) the soils of the fields used in the experiments were found to have a great influence on the productivity of the newly established grasses and less influence on the seedling establishment, and (5) the success or failure of the no-tillage method was thought to be greatly dependent on the amount and distribution of precipitation in the seedling year.

BLACKMORE (1964) conducted a series of pasture improvement trials on low, medium, and high fertility pastures in New Zealand. Several methods of spraying, seeding, and application of different herbicides in combination with lime and fertilizer at various growth stages of the vegetation were employed. Broadcasting the seeds was found to be the best method of planting unless the amount of rainfall was inadequate. In this situation, over­drilling the seeds gave better results. Lime application proved unnecessary for pastures with a high level of fertility. Since the killed sad provided an abundant energy source, increased soil microbial activity temporarily tied up soil nutrients and caused an acute nutrient shortage in some cases. Paraquat was more effective in killing a sad which had been mowed to l;:4-inch height than to one-inch height. The sad was more susceptible to paraquat during its recovery from the dry summer period. Paraquat was not always consistent in effect. This variability was attributed to differing weather conditions, growth stage and vigor of the plants, and the predominant species in the sward.

In South Australia, with a Mediterranean climate, ROSS and COCKS (1964) applied none, ~ or one oz.! A paraquat to weeds, 14 and 35 days after their germination. On the same day of herbicide application, the plots were seeded to Lolium perenne, Phalaris tuberosa, and Dactylis glomerata with a sad seeder. Early application (14 days after weed seed germination) and a higher rate of paraquat resulted in better control of the undesirable broad-leaved weeds and annual grasses. The establishment of L. pere1zne was more suc­cessful than that of the other three species sown. Cryptoslemma calendula

Paraquat and diquat 131

reinfested the plots rapidly unless the growth of sown species was early and fast.

KAY (1964 a) carried out the following trials, in several locations in the Mediterranean climate region of California, to eliminate the competition of annual weeds, mostly T aeniatherum asperum, during the reseeding and the establishment of some ,permanent pastures. In November, paraquat was applied at 0.5 lb./ A in bands or over-all, ahead of the drill openers. Phalaris tuberosa var. stenoptera and Trifolium subterraneum seeds were drilled. At the end of the first growing season vigor and tillering of Phalaris in paraquat­treated plots were much higher than in the untreated plots. Most of the Phalaris seedlings in unsprayed plots died during the dry summer months, while those in sprayed plots survived through the summer. The establishment of Trifolium was the same in the check and sprayed plots and the number of plants was much higher at the end of the second growing year. The establishment of the species sown was the same, whether paraquat was applied on a five-to-eight-inch band over the row, or used as an overall spray.

{3) Direct drilling of crops into a chemically killed sod. Since 1961, a program has been conducted at Jealott's Hill, Great Britain, to investigate various aspects of growing cereal grains and a few other crops, such as Brassica oleracea var. acephala, with no cultivation. The method involves the killing of existing vegetation with paraquat and drilling the desired crop directly into the sprayed field. HOOD et al. (1963 and 1964) and JEATER and McILVENNY (1965) drilled Triticum aestivum and Brassica oleraceae var. acephala seeds into a Lotium perenne dominant pasture or into weedy stubbles killed by paraquat, or into stubbles plowed and cultivated prior to drilling. They reported that the yields of crops on chemically treated plots were comparable to those obtained from conventionally cultivated plots pro­vided adequate fertilizer was used. The plots which received two lb./ A of paraquat outyielded those receiving one lb./ A. Spraying, prior to, or on, the day of seeding produced the same yield. In addition it was noted that the standard grain drills were not well adapted for direct-drilling into trashy and uncultivated fields. In recent years, several types of equipment have been either designed or modified for the purpose of direct drilling of various crop seeds into uncultivated sods (ARNOTT and CLEMENT 1966, EVANS 1966, JEATER 1966).

To determine the optimum rate of paraquat application, JEATER and LAURIE (1966) applied various rates of paraquat to undisturbed sod plots at two different times. Generally yields of winter wheat or spring barley were not significantly different after a one or two lb./ A paraquat treatment. The yield of the plots sprayed in December was equal to the yield of the plots plowed and cultivated prior to seeding, but the February sprayed plots yielded less than the conventionally prepared plots. Recently, JEATER (1966) has summarized and tabulated the results of several such experiments.

y) Planting Zea mays without tillage. The works of MOODY et al. (1961)

132 A. A. AKHA VEIN and D. L. lINsCOTl'

and SHEAR et al. (1961) in Virginia showed that the no-tillage method of growing Zea mays in a sod killed with herbicides, herbicide combinations or black plastic cover was a feasible practice. Later, SHEAR (1962) used several herbicides including paraquat, singly or in combination, to kill a Festuca rubra, a Dactylis glomerata-Medicago sativa, or a small grain sod and planted Zea mays directly. A combination of paraquat and atrazine was found to kill the sod the fastest.

TREANOR and ANDREWS (1965), and ANDREWS and BRYANT (1966) planted Zea mays on chemically killed sods with no tillage in several locations in Tennessee. The mixed sods were treated with a combination of paraquat plus attazine, to ensure quick and long lasting control of the vegetation prior to corn planting. Their results revealed that (1) the sod should be killed quickly and completely to ensure the elimination of the vegetation competition for water, nutrients, and light, (2) when this ,end was not met by the chemicals, the Zea yield was reduced, and (3) Yz to one lb./ A of paraquat, plus two lb./ A of attazine was found to be a good combination for killing sod.

3. Planting with minimum tillage. - In England, DOUGLAS et al. ( 1965) killed a mixed sward with 0.25 Ib./ A of paraquat applied in October. During the winter months, ground limestone and basic slag were. applied to the dead litter to enhance its breakdown and the field was worked by a spiked harrow to loosen the root mat. In May of the following year, the field received a complete fertilizer and was seeded subsequently to a mixture of Lotium perenne, Phleum pratense, Trifolium repens, and Brassica napus. Two months after reseeding, 77 percent of the ground was covered with the sown species and 23 percent of it was bare. By the end of the second growing season, the regrowth of the original vegetation of the sward was still in­significant. DOUGLAS and McILVENNY (1962) observed that the use of para­quat for killing sod should be combined with some kind of cultivation to bring the seeds to a more intimate contact with the soil. EVANS (1966) mentions the name and characteristics of three seed drills, available in Great Britain, which have been designed to place the seeds in a favorable environ­ment in uncultivated grasslands.

DOUGLAS (1965) applied one to two lb./ A of paraquat to some low-land permanent pastures either in spring or in late summer. After the desiccation of the vegetation, some plots were burnt prior to cultivation. The burnt and the unburnt plots were then worked by different harrows or a rotary cul­tivator to open the dead litter. Seed mixtures of Trifolium rep ens and grasses were broadcasted and covered by harrowing. For purposes of comparison part of the field was plowed, cultivated, and seeded. The following species of the lowland pastures, Agropyron repens, Agrostis stolonifera, Alapecurtts pratensis, Dactytis glomerata, Festuca rubra, and Lotium perenne were found to recover from paraquat spray when no cultivation was practiced, whereas

Paraquat and diquat

Deschampsia caespitosa, Holcus lanatus, Phleum pratense, Poa annua, and P. trivialis were completely killed. A. stoloni/era recovered more quickly after spring application of paraquat than after summer application. The dead litter on the soil surface interferred with the seeding operation. Burning helped to get rid of the surface litter and made it easier to work the seed into the soil. Rotary cultivation was more consistent in producing a better seed bed than drag and disk harrows.

4. Delayed seeding. - For some crops the seedbed is plowed, fertilized, and fitted several weeks or months before planting. In the period from the last fitting of the field to planting time, the majority of the weed seeds in the top layer of soil will germinate. Under these conditions, it is possible to kill growing weeds with a herbicide and then plant with the least dis­turbance of the seedbed. This practice is sometimes called "stale seedbed technique."

Lalium multiflarum is a cultivated seed crop in Oregon, but it is an undesirable contaminant species in the fields intended for growing other seed crops. In Oregon, LEE (1965) used the stale seed bed technique for control of Lalium and other weeds germinating during the mild fall and winter months. During October, the field was over-sown with Lalium seed, fitted, and left undisturbed. In the subsequent rainy months, the sown species and weeds germinated. In December, February, and March the prepared plots were sprayed with paraquat, diquat, and the combinations of both, as well as several other herbicides and combinations. At the end of March, all the plots were seeded to the desired grass species. Most of the herbicides and combinations applied in December resulted in good control of Lalium multi­flarum. As application was delayed to February only few treatments, includ­ing paraquat and diquat, gave a satisfactory control of Lalium. Only paraquat, at rates higher than one lb./ A, controlled Latium in the March application. When the germinated weeds were killed by a 'herbicide and the soil was left undisturbed, very few additional weeds germinated during the estab­lishment period of the desired grasses.

With minor variations, in subsequent trials LEE (1965) found that not only Lotium, but several other volunteer crops planted prior to herbicide applications, could be eliminated successfully and thus prevent contamination of the intended seed crops. The seed yield of herbicide-treated plots was equal to that obtained from mechanically prepared plots. Some of the plots, treated previously with paraquat or other herbicides, received Yz lb./ A addi­tional paraquat at the time of planting to control volunteer crops and weeds germinating late. This retreatment with paraquat resulted in a much higher seed yield and lower contamination of the harvested seeds. The increased yield of the treated plots over the mechanically prepared plots was more than enough to offset the cost of the herbicide.

134 A. A. AKHAVBIN and D. L LINscOTT

d) Control of Agropyrons repens

Agropyron repens, one of the most persistent noxious weeds, is wide­spread over much of the coo~ humid regions of the United States, Canada, and Western Europe. Many investigators have studied the physiology, de­velopment, dormancy, and the effects of natural and induced factors on the growth pattern of A. repens shoots, roots, and rhizomes (JOHNSON 1958, JOHNSON and BUCHHOLTZ 1961 and 1962, MEYER 1961, MEYER and BUCH­HOLTZ 1963, PALMER 1958). The movement of several herbicides within the plant and its rhizome system has also been investigated ( PUTNAM 1966, SAGAR 1960, WAX and BEHRENS 1965). Many herbicides, various rates of application, and different times and methods of spraying, with or without fertilizer and in combination with light or repeated cultivations, have been employed for eradication or control of A. repens (LE BARON and FERTIG 1962, PUTNAM 1966, SHIRMAN and BUCHHOLTZ 1966, VENGRIS 1964). Though much valuable information on the life cycle and seasonal control of Agropyron has been gained through these and other works, still the means for a complete and thorough control of this weed are lacking. The capacity of Agropyron to regenerate new shoots and rhizomes from its numerous dormant rizome buds is its major protection against eradication by chemical and mechanical means. Under normal growing conditions in the field, most of the rhizome buds are dormant. All the dormant buds are theoretically capable of starting a new plant each. To be effective, any tillage method or herbicide used for the elimination of this weed should kill or inhibit the growth potential of every rhizome bud in a field.

JEATER and McILVENNY (1960) applied diquat, paraquat, dalapon, and amitrole, individually or in combination, to A. repens plants in the green­house or in the field. In the greenhouse, a mixture of diquat or paraquat plus dalapon proved the best treatment of all. More Agropyron was killed and less regenerated in the greenhouse than in the. field. In the field, regeneration of Agropyron was the same whether dalapon was applied alone or in combina­tion with paraquat or diquat.

PUTNAM (1966) studied the toxic effect of several herbicides and herbi­cide combinations on Agropyron. As it was mentioned previously, two herbi­cide combinations were successful in providing season-long control of A. repens: the first, paraquat (~ lb./ A) plus simazine or diuron (three to four lb./ A), and the second, amitrole plus ammonium thiocyanate additive applied seven days prior to paraquat application. In the greenhouse, plants with a more extensively developed rhizome system showed more regenerative capacity after spraying than those having a less developed rhizome system.

e) Control of weeds in an aquatic environment

Soon after their introduction, diquat and paraquat were tested and used for both terrestrial and aquatic weed control. Diquat and paraquat behavior

Paraquat and diquat 135

in an aquatic environment is essentially the same as in a terrestrial habitat. They are fast-acting phytotoxic chemicals on a wide range of aquatic plant species. Diquat and paraquat cations are tightly adsorbed by clay minerals, organic materia1, and plant surfaces present in water. This greatly speeds up the rate of disappearance of diquat and paraquat in naturally ocCUtring bodies of water and helps to reduce their hazard to aquatic animal life.

WELDON et al. (1961) tested the effectiveness of several quaternary am­monium compounds for control of Najas guadalupensis. Diquat was one of the most effective herbicides for control of this weed as well as of Pistia stratiotes, and it was moderately effective against Eichhomia crasspipes. BLACKBURN and WELDON (1964) observed that diquat and paraquat at 1.0 to 2.5 p.p.m.w. completely controlled N. guadalupensis in drainage and irri­gation channels. Diquat had the longest lasting effect among all the chemicals used. Regrowth of Najas started 13 to 15 months after treatment. More re­cently, WELDON and BLACKBURN (1967) have shown that diquat at 1.0 to 1.5 lb./ A successfully controlled Pistia stratiotes and addition of a surfactant did not increase the activity.

Paraquat and diquat at one p.p.m.w. were noted by BLACKBURN (1963) to be quite effective against such submersed weeds as Elodea densa, Najas guadalupensis, and Ceratophyllum demersum. One to two lb./ A effectively controlled Pistia stratiotes and Salvinia rotundi/olia, but Eichhomia crasspipes control was not satisfactory. The top of Alternanthera philoxeroides, an im­mersed plant, was killed quickly by paraquat and diquat, but regrowth took place within eight weeks.

In New Zealand, LEONARD and GREENLAND (1965) injected diquat and paraquat into the flowing water of irrigation channels at a concentration of two p.p.m. Both herbicides controlled Elodea canadensis, but only paraquat controlled Myriophyllum elatinides. After the elimination of these two species of plants from the irrigation channels, Nitella hookeri, which proved resistant to diquat and paraquat, colonized the water ways. FISH (1966) also reported that Nitella spp. became abundant after Lagarosiphon major was controlled by diquat in a lake in New Zealand. BArrEN (1965) noted that paraquat rapidly knocked back Glyceria maxima in drainage ditches. The effect, how­ever, did not persist and the weed grew back.

BLACKBURN and WELDON (1965) investigated the susceptibility of sev­eral aquatic weeds to diquat and paraquat. The order of susceptibility to both herbicides (from the most to the least susceptible) was: Spirodela polyrhyza, Lemna minor, W olffiella floridana, Azolla caroliniana, and W olffia columbiana.

HILTIBRAN (1964) reported effective control of several Potamogeton species, Myriophyllum exalbescens, Ceratophyllum demersum, and Ranunculus trichophyllus with diquat at 0.5 to 1.0 p.p.m.w., but Potamogeton nodosus was resistant. Emergent weed species such as Sagittaria tati/olia, Jussiaea repens var. glabrescens, Justicia americana, Typha lati/olia, and T. augus-

136 A. A. AKHAVEIN and D. L. LINSCOlT

tifolia were controlled when their foliage was sprayed with a solution of 50 mI. of diquat diluted to one gallon with water. Diquat, at 0.17 to 0.73 p.p.m.w., has been reported by RIEMER (1964) to control Utricularia spp., SParganium spp., and Lemna spp. in several small lakes in southern New Jersey. BLACKBURN et al. (1966) satisfactorily controlled Hydrochloa caro­liniensis with paraquat at two lb./ A.

WHITE (1967) evaluated diquat and paraquat on several groups of aquatic weeds. Both herbicides seem to be equally effective against floating and susceptible submersed aquatic weeds, but paraquat is more active on rooted emergent weeds.

Rapid and thorough killing of aquatic plants and decomposition of the dead vegetation usually causes a drastic reduction in oxygen content of the body of the water. NEWMAN and WAY (1966) observed severe deoxygena­tion in a lake where a single dose of paraquat killed all the weeds. By treat­ing various portions of the lake at different times, severe deoxygenation was prevented.

BLACKBURN and WELDON (1967) found that a combination of diquat plus copper sulfate, or diquat plus the mono(N,N-dimethylalkylamine) salt of endothall, was more effective against Elodea canadensis than either com­ponent alone.

Summary

Since the discovery of their herbicidal activity in the 1950's, paraquat and diquat have been used for selective and nonselective terrestrial and aquatic weed control and as an aid to some of the basic agricultural opera­tions, such as crop desiccation, pasture renovation, seedbed preparation, and crop production with limited or no tillage.

Paraquat and diquat are the most herbicidally active of the dipyridylium quaternary ammonium group. For this activity, dipyridylium quaternary am­monium compounds must have (or be capable of assuming) a coplanar con­figuration. The molecule must accept electrons and form a stable free radical upon reduction. Redox potential is related to the degree of phytotoxicity, and in general the higher the potential the greater the phytotoxicity of the dipyridyls.

By substituting for ferredoxin, both paraquat and diquat inhibit produc­tion of NADPH2 in a plant system and are capable of irreversibly removing electrons from previously formed NADPH2• NADP reduction is the most important ferredoxin-dependent reaction. When a dipyridyl is substituted for ferredoxin, it will accept electrons from photosynthetic electron flow (in light) or NADPH2 (in light or dark). In either case plants lose a major source of energy, NADPH2• In the presence of oxygen, reduced dipyridyl is auto-oxidized to form hydrogen peroxide and oxidized dipyridyl. Oxidized dipyridyl will continue through the oxidation-reduction cycle. As a result,

Paraquat and diquat 137

only catalytic amounts of the herbicide are necessary to kill most chloro­phyllous tissues.

Light intensity, temperature, and humidity affect the extent of adsorp­tion, absorption, translocation, phytotoxicity, and residual properties of para­quat and diquat. Low light intensity, high humidity, and high temperatures have been associated with increased dipyridyl uptake and activity. Presence of cuticular waxes interfere with absorption of the herbicide. Cationic or non­ionic surfactants generally enhance absorption and phytotoxicity.

Upon contact with soil, clay minerals and organic matter, paraquat and diquat cations are tightly adsorbed and are rendered biologically inactive. The adsorption mechanism is a physical ion-exchange process and does not depend on pH, temperature, or time-exposure.

Photochemical decomposition is the most important factor in reducing residues of the dipyridals on a sprayed crop. Paraquat and diquat are not metabolized or degraded by plant action. Microbial degradation in cultures and in the gut of animals has been shown. However, degradation by soil microoorganisms under natural conditions has not been proved. Paraquat and diquat applied to bodies of water quickly diminish in concentration through adsorption to soil particles, or plant materials suspended or living in water.

In this general survey of the published papers pertinent to paraquat and diquat we have emphasized chemistry, mode of action, adsorption, absorp­tion, degradation, and residues of these dipyridylium herbicides. Their uses in terrestrial and aquatic environments are discussed.

Resume*

Les herbicides du dipyridyl, paraquat et diquat

Depuis la decouverte dans les annees 1950 de leur activite herbicide, Ie paraquat et Ie diquat ont ete utilises pour la destruction selective et non selective des mauvaises herbes terrestres et aquatiques et pour faciliter cer­taines operations agricoles de base, telies que Ie fanage des cultures, la renovation des paturages, la preparation do lit de semences et la production de cultures avec labour reduit ou sans labour.

Le paraquat et Ie diquat sont les herbicides les plus actifs du groupe des arnmoniums quaternaires du dipyridyl. Pour avoir une activite herbicide, les composes de l'ammonium quaternaire doivent posseder (ou etre capables d'adopter) une configuration coplanaire. La molecule doit capter des electrons et former des radicaux libres, stables par reduction. Le potentiel redox est lie au degre de phytotoxicite; en general, la phytotoxicite des composes du dipyridyl est d'autant plus grande que Ie potentiel redox est plus eleve.

En se substituant a la ferredoxine, Ie paraquat et Ie diquat inhibent la production de NADPH2 dans Ie systeme vegetal et sont susceptibles

.. Traduit par S. DORMAL-VAN DEN BRUEL.

138 A. A. AKHAVEIN and D. L. LINscorr

d'eliminer d'une fas,:on irreversible les electrons du NADPH2 prealablement forme. La reduction du NADP est la reaction la plus importante qui depend de la ferredoxine. Lorsqu'un dipyridyl se substitue a la ferredoxine, il capte les electrons du flux photosynthetique (dependant de la lumiere) ou du NADPH2 (lumiere ou obscurite). Dans chaque cas, les plantes perdent une source importante d'energie, Ie NADPH2 • En presence d'oxygene, Ie dipyridyl reduit est auto-oxyde et forme un peroxyde d'hydrogene et un dipyridyl oxyde. Le dipyridyl oxyde est repris dans Ie cycle d'oxydo-reduction. 11 en J:esuite que des doses catalytiques de l'herbicide suffisent pour tuer la plupart des tissus chlorophylliens.

L'intensite lumineuse, la temperature et l'humidite affectent Ie degre d'adsorption, d'absorption, de translocation, de phytotoxicite et les proprietes residuelles du paraquat et du diquat. Une intensite lumineuse faible, une humidite elevee et de fortes temperatures ont ete associees a un accroissement de l'absorption et de l'activite du dipyridyl. La presence de cires cuticulaires interfere avec l'absorption de l'herbicide. Les tensio-actifs cationiques et non-ioniques inhibent generalement l'absorption et la phytotoxicite.

Les cations paraquat et diquat sont fortement adsorbes et rendus bio­logiquement inactifs par contact avec Ie sol, les mineraux argileux et la matiere organique. Le mecanisme d'adsorption est un processus physique d'echange d'ions; il ne depend pas du pH, de la temperature ou de la duree d' exposition.

La decomposition photochimique est Ie facteur Ie plus important dans la reduction des residus de dipyridyl sur les cultures traitees. La paraquat et Ie diquat ne sont pas metabolises ou degrades par l'action de la plante. La degradation microbienne dans les cultures et dans l'intestin de l'animal a ete demontree. Cependant, la degradation par les micro-organismes du sol, dans les conditions naturelIes, n'a pas ete prouvee. Le paraquat et Ie diquat appliques sur des pieces d'eau diminuent rapidement en concentration en raison de l' adsorption par les particules de terre ou les matieres vegetales en suspension ou vivant dans l'eau.

Dans cet examen general des articles publi~s au sujet du paraquat et du diquat, nous avons attire l'attention sur la chimie, Ie mode d'action, l'adsorp­rion, l'absorption, la degradation et les residus des herbicides du dipyridyl. Leur utilisation en milieu terrestre et aquatique est discutee.

Zusammenfassung*

Die Herbizide Paraquat und Diquat

Die herbizide Wirkung von Diquat und Paraquat wurde in den 50er Jahren entdeckt und wird seither zur selektiven und totalen, terrestrischen und aquatischen Bekampfung von Unkrautern ausgenutzt. Daneben dienen die Wirkstoffe als Hilfsmittel bei einer Reihe von landwirtschaftlichen

.. Dbersetzt von H. GEISSBUHLER.

Paraquat and diquat 139

Arbeitsmethoden wie Abbrennen von Stauden, Griinflachenerneuerung, Saat­beetbestellung und Kultur ohne oder mit beschrankter Bodenbearbeitung.

Diquat und Paraquat sind die herbizid-wirksamsten Vertreter aus der Reihe der Dipyridyl quaterniiren Ammonium-Verbindungen. Um aktiv zu sein, benotigen diese Stoffe eine koplanare Konfiguration oder miissen in der Lage sein, eine soIche einzunehmen. Die Molekeln nehmen Elektronen auf und bilden nach Reduktion stabile, freie Radikale. Je grosser das Redoxpotential der Dipyridyle desto grosser ist normalerweise ihre Aktivitat.

1m Mechanismus der pflanzlichen Photosynthese unterbinden Diquat und Paraquat durch Substitution des Ferredoxins die Bildung von reduziertem Nikotinamid-adenin-dinucleotidphosphat (NADPH2 ) oder entziehen dem bereits vorhandenen NADPH2 Elektronen. Dadurch wird nicht nur der licht­abhangige Elektronenfluss der Photosynthese unterbrochen sondern auch NADPH2 im Dunkeln zerstort. Nachdem die Reduktion von NADP die wichtigste Ferredoxin-gesteuerte Reaktion der Photosynthese darstellt, ver­lieren die Pflanzen eine ihrer Hauptenergiequellen. In Gegenwart von Sauerstoff wird reduziertes Dipyridyl autooxydiert unter Bildung von Wasserstoffperoxyd und oxydiertem Dipyridyl. Die letztere Substanz wird erneut in den Oxydations-Reduktionszyklus eingeschaltet. Dementsprechend sind nur katalytische Mengen des Herbizids notwendig, um die meisten chlorophyllhaltigen Gewebe abzutoten.

Das Verhalten von Diquat und Paraquat in den Pflanzen (Adsorption, Absorption, Translokation, Phytotoxizitat, Residualwirkung) wird von einer Reihe von Aussenfaktoren, wie Lichtintensitat, Temperatur und Luft­feuchtigkeit beeinflusst. Die vorhandenen Unterlagen weisen darauf hin, dass schwaches Licht, hohe Luftfeuchtigkeit und hohe Temperatur die Aufnahme und Aktivitat der Dipyridyle begiinstigen. Ihre Absorption wird durch das V orhandensein stark wachshaltiger Kutikularschichten der Blatter erniedrigt, durch Beimischung kationischer und nicht-ionischer Netzmitte1 erhOht.

1m Boden werden Diquat und Paraquat durch Tonpartikel und orga­nische Bestandteile so stark adsorbiert, dass sie biologisch unwirksam sind. Dieser Adsorptionsmechanismus beruht auf einem physikalischen Ionen­austausch und ist vom pH, von der Temperatur und der Expositionszeit unter Feldbedingungen praktisch unabhangig.

Die Riickstande der Dipyridyle auf behandelten Pflanzen werden in erster Linie durch photochemischen Zerfall reduziert. Ein biochemisch­enzymatischer Abbau der Wirkstoffe in der Pflanze wurde bis jetzt nicht nachgewiesen. Obwohl ein mikrobieller Stoffwechsel der Substanzen in Kulturlosungen und Tiermagen gezeigt werden konnte, fehlt bis jetzt der Nachweis eines Abbaus durch Bodenorganismen unter Feldbedingungen. Bei der Anwendung von Diquat und Paraquat im Wasser wird ihre Konzen­tration durch Adsorption an Bodenteile oder suspendierte Pflanzenmaterialien rasch erniedrigt.

140 A. A. AKHAVEIN and D. L. LINSCOTr

In der vorliegenden Uebersicht tiber die Dipyridyl-Literatur werden folgende Punkte herausgehoben: Chemie der Wirkstoffe, Wirkungsme­chanismus, Verhalten in der Pflanze, Abbau und Rtickstande. 1m weitern werden die Anwendungsmoglichkeiten dieser Wirkstoffklasse diskutiert.

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Subject Index

Adulteration of Articles of Food and Drug Act 38

A-esterase 87 Agrosan 2 Aldrin 67 - in animal feed 62-64 Alfalfa 56, 64 Alkaloids, inhibition of cholinesterase 85 Almonds 6, 40 Amitrole 117, 129, 130, 134 Animal feed, insecticides in 61 ff., 66 - feed sampling 56, 57 Anticholinesterases, literature review 73 Apple leaves, bark, and wood, mercury in

17, 18, 27, 28 Apples 6, 11-15, 40 - mercury in 16 ff., 27, 28, 30 Apricots 6, 40 Arrhenius plots 82, 86, 90 Arsenic 38 Atrazine 108, 117, 132 Automated cholinesterase assays 71 ff. Azodrin, see Monocrotophos

Barley 5, 13, 14, 61, 64 - mercury in 21, 22 Bean plants, mercury in 28, 29 Beans 8, 40 Beer 14 - mercury in 27 Beets 7, 40, 61, 64, 65 Benzyl viologen 100 BHC in animal feed 62-64 - in milk and butter 58-60, 65, 67 Bidrin, see Dicrotophos Birds (see also specific kinds) 2 - mercury in 22 ff. Bread, mercury in 27 Bream, mercury in 26 Broccoli 40 - mercury in 28 Brussels sprouts 8, 40 Bulbs (see also specific kinds) 4, 8 iI. Burbot, mercury in 26 Butter 55 iI., 123

- mercury in 27 - sampling 56

Cabbages 8, 40 Canada, Food and Drugs Act 37 ff. - legal action against residues 46 - pesticide residue authority 39 iI. - residue enforcement program 46 - residue monitoring program 46 - residue requirements 41 iI. - residue surveillance program 46 - residues in 37 ff. Canary seed 5 Cantaloupes 40 Carbicron, see Dicrotophos Carp, mercury in 26 Carrots 7, 40, 57, 122 - mercury in 28 Cats 2, 124 Cattle (see also Cows) 42, 124 Cauliflower 8, 40 Celery 7, 40 Cereal grains 2 iI. Ceresan 2 Cheese, mercury in 27 Cherries 6, 40 Chickens, mercury in 23 Chlordane 65 Chlorfenvinphos 85, 90 Cholinesterase inhibition, automated assay

71 ff. - reactions, fundamental kinetics 71 ff. Cholinesterases, literature review 73 Coconut expellers 63 Cod, mercury in 26 Coffee beans 12 - foliage, mercury in 27 - mercuty in 27 Copper 38 Corn 129, 131, 132 Corvids, mercury in 23 Cotton 5 Cottonseed cakes and expellers 63 Cows 61, 123 Cucumbers 6, 40 Curing by paraquat and diquat 125

148 Subject Index 2,4-D 129 Dalapon 129, 130, 134 Danish milk and butter, insecticides in

55 ff. Dazomet, decomposition in soil 49 ft. -- metabolites 49 ft. - properties 49, 50 DDE in animal feed 62-64 - in milk and butter 58-61, 65, 66 DDT in animal feed 62-64 - in grass and silage 65 - in milk and butter 58-61, 65-67 Defoliation by paraquat and diquat 98,

125 Demeton 40 Desiccation by paraquat and diquat 98,

122, 125 DFP 90 Dichlorvos 85, 90 Dicrotophos 77 ff. Dieldrin in animal feed 62-64 - in milk and butter 58, 59, 65-67 Dipyridylium herbicides 97 ff. Dipyridyls, chemical structures 101 - common characteristics 102-104 - coplanarity 102 - free radical formation 102 ft. - stability 103 - steric hindrance 102, 103 Diquat (see also Dipyridyls) 97 ft. - absorption, movement, and activity

113 ft. - adsorption 119 - and humidity 116 - and light and dark 114 - and oxygen 117 - and photosynthesis 105 ft., 124 - and rainfall 116 - and soil flora and fauna 119 - and surfactants 114 - and temperarure 116 - behavior 105 if. - degradation 119 - in crops 121 - in milk 123 - animal metabolism 123 - in soils 121 - in water 122 - lethal dosage to man 124 - metabolism 120 - mode of action 105 ff. - mode of entry 114 if. - persistence 118 - photochemical decomposition 122 - photochemistry 120

- properties 99 - reduction 102 if. - residue methods 125 - residues and products 121 ft. -- resonance structures 103, 104 - site of action 111 ff. -- toxicity 121 if., 124 - uses 125 if. - water solubility 100 Disodium methanearsonate 117 Dithizone 11 ft. Dimon 111, 113, 117, 134 Drift 67

Eggs 14 - mercuty in 22 if., 27 Endothall 136 Energy of activation 81, 82, 86, 87 Enzyme activity and pH 82, 83, 87 - concentration and reaction velocity

78, 79

Ferredoxin 106, 107, 110-112 Finches, mercury in 23 Fish (see also specific kinds) 2, 11, 42,

124 - mercury in (see also specific kinds)

22, 24, 25, 27, 30 Flax 5, 14 Fluorine 38 Food and Drugs Act and Regulations,

Canadian 38 Food and Druzs Act of Canada 37 ff. Free radicals 102-104, 108-110

Gas chromatography 10, 57 ff. Gladiolus 8 Glutathione 76 ff. Good agriculrural practice, Canadian 38 Grains (see also specific kinds) 13, 14 - mercury in 21 if. Grapefruit 40 Grapes 8, 40 Grass 64, 65, 127, 128, 130 Groundnuts, see Peanuts Guillemot, mercury in 24 Guinea pigs 124

Haddock, mercury in 24, 26 Halibut, mercury in 26 Hay 64, 126 Hazelnut expellers 63 Herbicidal activity and redox properties

104

Subject Index 149

Herbicide structure-actiVity relationships 100 if.

- translocation 113 if. Herbicides, adsorption to soil 119, 121 if. - and humidity 113, 116 - and light 113-115 Herring, mercury in 24 Hops 8 Hydrogen peroxide from paraquat and

diquat 112 Hydrolysis rate constants 91

160 determinations 91 Inhibitor constants 87 Ion-exchange resins 119 iso-OMPA 90

Kittiwake, mercury in 24

Lead 38 Lemons 40 Lettuce 40 - mercury in 28, 29 Light and dark reactions 105 if. Lindane in animal feed 62-64 - in milk and butter 58--60, 65 Lineweaver-Burk plots 81, 83, 86, 90 Linseed 5 - cakes 63, 64 Linuron 107

Maize 5 Maleic hydrazide 40 Meat, mercury in 11, 14, 27 Melons, see specific kinds Mercury bulb dips 9 if.

compounds, extent of use 2, 9 if. compounds, formulations 9 if. compounds, groups 3 compounds, residue analysis 10 if. compounds, translocation 27 if. compounds used in agriculture (see also specific trade names) 1 if. contamination of environment 2 ft. controlled plant diseases 4 if. glasshouse aerosols 9 if.

- lawn fungicides 9 if. - movement in plants 27 if. - orchard canker paints 9 if. - orchard sprays 9 if. - residues, hazardous narure 2 if. - residues in agriculrure 1 if. - residues in crops and tissues 15 if. - residues, naturally occurring 15 if. - residu€s, regulations 29 if.

- seed dressings 9 - seed potato dips 9 if. - soil fungicides 9 if. - sugarcane dips 9 if. - tolerances 29 if. Methyl demeton sulfoxide 90 - mustard oil 49 if. - viologen (see also Paraquat) 99,

111 Milk 14, 46, 55 if., 122, 125 - mercury in 27 - residue analytical methods 57 ff. - sampling 56 Millet 5 Mipafox 90 Monitoring program, Canadian 46 Monocrotophos 77 if. Monuron 108, 113 Muskmelons 40

Narcissus 8 Nectarines 40 Neutron-activation analysis, mercury com-

pounds 2, 15, 22 No-effect level 45 No-residue basis 45 Nuclear magnetic resonance 51 Nuvacron, see Monocrotophos

Oats 5, 14 Oilseed cakes 57, 61 Onions 40 Oranges 40 Organochlorine insecticides in Danish

milk and butter 55 if. Owls, mercury in 23 Ox meat, mercury in 24, 25

Paper chromatography 15, 55 ff., 120 Paraoxon 85, 90 Paraquat (see also Dipyridyls) 97 ff. - absorption, movement, and activity

113 ff. -- adsorption 119 - and humidity 116 - and light and dark 114 - and oxygen 117 - and photosynthesis 105 ff., 124 - and rainfall 116 - and soil flora and fauna 119 if. - and surfactants 114

and temperature 116 animal metabolism 123 antagonism 117 behavior 105 if.

150

- degradation 119

Subject Index

Pumpkins 6 - in crops 121 - in milk 123 - in soils 121 - in water 122 - lethal dosage to man 124 - metabolism 120 - mode of action 105 ff. - mode of entry 114 ff. - persistence 118 - photochemical decomposition 122 - photochemistry 120 - properties 99 - reduction 102 ff. - residue methods 125 - residues and products 121 ff. - resonance structures 103 - site of action 111 ff. - synergism 117 - toxicity 121 ff., 124 - uses 125 ff. - water solubility 100 Parsnips 7 Partridges, mercury in 23, 24 Pasture improvement 127 - renovation 98 Peaches 6, 40 Peanut cakes and expellers 63 Peanuts 7 Pears 6, 40 - mercury in 19, 30 Peas 8, 40 Pecans 40 Peppers 40 Perch, mercury in 26 Pesticide degradation products in Canada

41 Pesticides in Canada 41 ff. Pheasants, mercury in 23, 24 Phosphamidon 85, 90 Phosphorylation 106, 107, 111 Photodecomposition 118 Photophosphorylation 106, 107, 111, 112 Photosynthesis 105 ff., 108, 112, 124 Pigeons, mercury in 23, 24 Pike, mercury in 25, 26 Pineapples 7, 40 Plaice, mercury in 26 Plasma ChE activity 71 ff. Plums 40 Pork products, mercury in 24, 25 Potatoes 7, 40, 85, 121, 122, 125, 126 - mercury in 20 ff., 28, 29 Poultry meat, mercury in 23 Prunes 40

Rabbits 43, 124 Radishes 8 Rapeseed cakes and expellers 63 Rats 123, 124 Regulation of mercury residues 29 ff. Reindeer meat, mercury in 24, 25 Reproduction requirements, Canada 44 Residue analysis, confirmatory methods

41 - analysis, mercury compounds 10 ff. - analytical methods, Canada 41 Residues in Canada 37 ff. - in soils in Canada 42, 46 - in water in Canada 42, 46 Respiration inhibitors 109 Rice 5, 13, 15 - mercury in 22 Roach, mercury in 26 Rockmelons 6 Rodents, mercury in 25 Rutabagas 40 Rye 5, 14

Salmon, mercury in 26 Schiiniger flask combustions, mercury 14

ff. Seeds 15 Sheep 123, 124 Shellfish, mercury in 2, 30 Silage 56, 64, 65, 123, 124 Simazine 113, 117, 134 Slime control 4 Soils 13, 14, 121, 123 - adsorption of herbicides to 119 - decomposition of Dazomet in 49 ff. - mercury in 26 - microorganisms 119 ff. Solan 117 SOPP 40 Sorghum 5 Soyabean cakes and meal 63 - expellers 63 Soyabeans 63 Squash 6 Stabilities of formulations 91 Stale seedbed technique 133 Straw 56, 64 Strawberries 6, 40 Substrate concentration and reaction ve-

locity 79-81, 86 Sugar beets 7 - mercury in 27 Sugarcane 7, 9 ff.

Subject Index 151

Surfactants 113, 114, 130, 135 Surveillance program, Canadian 46 Swedes 61, 65 Sweet potatoes 7, 40

Tea 85 - mercury in 27 Thin-layer chromatography 10, 51, 52,

91, 120 Thiocarbazone methods, mercury 10-14 Thiocholine esters 71 ff. Tobacco 85 Tolerances, Canadian 38 ff., 45 - mercury 29 ff. Tomatoes 8, 11, 12, 40 - mercury in 19 ff., 29, 30 Toxaphene 65 Toxicity, Canadian viewpoint 42 ff. - diquat and paraquat 121 ff., 124 Triquat 110 Turnips 8 - mercury in 28

Ultraviolet spectrometry 51, 52

Urea herbicides 113 Uspulun 2

Veal, mercury in 24, 25 Vegetation control 129-134 Vinegar 14 Viologen dyes 100 Viologens 102, 111

Walnuts 6, 40 Water 121 - mercury in 25 ff., 30 - photolysis 108, 112 - residues in, Canada 42, 46 Watermelons 6 Weed control 98, 126, 129 ff. Weeds in aquatic environments 134 ff. Wheat 5, 13, 14 - mercury in 21, 22 Whitefish, mercury in 26 Whiting, mercury in 26 Wood pulp 4, 13 - pulp, mercury in 4

Zinc 38

Manuscripts in Press

The safety of flavoring substances. By R. 1. Hall and B. 1. Oser.

Pesticides in blood. By M. 1. Schafer.

Analytical control of polycyclic aromatic hydrocarbons in food and food additives. By E. O. Haenni.

Problems and results of residue studies after application of molluscicides. By R. Strufe.

Pesticide regulations and residue problems in Japan. By K. Fukunaga and Y. Tsukano.

A specific gas chromatographic method for residues of organic nitrogen pesticides. By C. C. Cassi~ R. P. Stanovick, and R. F. Cook.

Experimental approaches to pesticide metabolism, degradation, and mode of action: United States-Japan seminar. Special volume (No. 25).