Degradation of p32-Bromophosby Microorganisms and Seedlings By J. STENERSEN Norwegian Plant Protection Institute, VoIlebekk, Norway Biological degradation of bromophos has been studied by Rowlands (i) and Stiansi et al. (2).

They

conclude that among other products desmethylbromophos (mbPS) is formed in wheat grains and rats.

No bis-

desmethyl bromophos (bPS) was found in their studies. This paper describes the degradation of p32_ bromophos by microorganisms and some seedlings, using qualitative thin layer chromatography to detect the products. METHODS Materials p32-1abeled bromophos was commersially obtained from Amersham radiochemical centre, U.K.

The compound

was purified by elution through a column of activated silica gel by tetrachlorcarbon 0

Unlabeled bromophos

and methyl parathion were obtained as gifts from producers os these insecticides.

Methyl phenyl phos-

phorothionate

(m~PS) and sodium hydrogen phenyl phos-

phorothionate

(~PS) w e r e s y n t h e t i s e d

in o u r

laboratory.

The p o t a s s u m s a l t

as a gift

from Dr.

Princeton,

U.S.A.

tained

as a gift

Sutherland,

converted

u n d Sohn C h e m i s c h e F a b r i k ,

o f bPS c o u l d

determined.

The s p o t s ,

reaction

ing sprays. that

obtained

stable

n o t be d i r e c t l y

t o be bPS, and

was e x a c t l y

gave

phenyl

the

was u s e d a s t h e

was h i g h e r

DPS h a s a h i g h e r rather

therefore

detect-

same a s

w i t h ~PS when DQC ( 2 . 6 - d i b r o m o b e n z o e -

quinone-4-chloroimide) The RF v a l u e

Germany.

o f bPS was o b t a i n e d .

phosphate-

The c o l o u r

The

from C.H. B o e h r i n g e r

supposed

with

t o mPS.

Ingelheim,

substance

The i d e n t i t y

positive

o f m2PS, o b -

f r o m B a y e r C h e m i e AG, L e v e r k u s e n ,

o f mbPS was a g i f t

No r e f e r e n c e

C y a n a m i d e Company,

The ammonium s a l t

G e r m a n y , was s p o n t a n i o u s l y sodium salt

o f m2PS was o b t a i n e d

value

against

compound g a v e p r o d u c t s

than than

alkali,

the

spray

value

mDPS.

for

reagent.

mbPS, a s

A l t h o u g h mbPS i s

hydrolysis

w i t h RF v a l u e s

of this

and colour

r e a c t i o n s with DQC identical w i t h mPS and bPS. A s o l u t i o n of m2PS, mbPS and bromophos nol was found after several weeks,

in etha-

to contain two

more substances w h i c h was c h r o m a t o g r a p h i c a l l y tified as mPS and bPS.

iden-

This s o l u t i o n was u s e d as

r e f e r e n c e s o l u t i o n throughout the study.

105

Analysis Analysis

of the incubation mixture were done

by thin layer chromatography.

Activated silica

gel G layers with the thickness

0.5 mm or 0.25 mm

were used.

The chromatograms

were developed

in

acetonitril

: water

p32-1abeled

compounds were located on the chromato-

(85 : 15) or (80 : 20) v/v

(3).

gram by laying an X-ray film in close contact with the plate for 24 hours.

The reference substances

were localised by spraying with the DQC reagent (1% 2.6-dibromobenzoequinone-4-chlorimide acid).

This reagent

phosphorothionates

(3)

in acetic

gives red colours with aryl

(aPS), and m2PS, and yellow

colours with maPS and mPS. Microbiological

studies

Preliminary

studies with the fungi Alternaria

tenius and Trichoderma

li~norum had shown that all

the water solublemetabolites The mycel contained

were in the medium.

most of the unmetabolised

phos which was absorbed or adsorbed, soluble metabolite was detected. were macerated

but no water

After the myceles

in a mortar with quartz sand and

partioned between petrolether radioactivity

bromo-

and water,

all the

was found in the petrolether

106

phase.

Therefore

only the

medium was a n a l y s e d

in these

investigations. 50 strains of microorganisms from garden soil. extract agar

were isolated

They were isolated on soil

(4) (which contained

15 g agar,

1.0 g

glucose 0.5 g K2HPO 4, I00 ml soll extract and 900 ml water) with

and were grown in I ml of the same medium

no a g a r

added.

48 h o u r s

p32bromophos,

dissolved

48 h o u r s

after

the

solution

was a d d e d i n s u f f i c i e n t

a concentration the

medium.

the

in ethanol

of the

inoculation,

was a d d e d .

bromophos addition,

of 5 ~g/25

25 ~ 1

after

the

amounts to give

~1 of

its

compounds i n

medium was t h e n

on c h r o m a t o g r a m s o f 0 . 5 m m

standard

thickness

applied

and developed

as described. Laboratory Trichoderma solani the

strains

lignorum,

and Phizopus

Aspergillus

nigricans

tenius,

niger,

were also

Fusarium tested

using

same m e d i u m .

Studies

on s e e d l i n g s

Seeds of onion, with

of Alternaria

carrot

and wheat were treated

1 ~1 p32-bromophos solution

on a m o i s t e n e d

filter

paper.

107

pr.

seed and placed

A forthnight

after

the

treatment,

separated tely

the

roots,

and the

leaves

from the

seeds,

and the

parts

macerated

extracted solution already

quartz

in ethanol, added. described.

unmetabolised with

with

were separa-

in a mortar,

with

and then

some o f t h e

The e x t r a c t s I n s om e o f

standard

were then the

were

analysed

as

experiments,

the

b r o m o p h o s was r e m o v e d b y e x t r a c t i o n

petrolether.

RESULTS Microbial degradation All the microorganisms degraded bromophos to some extent, but the activity varied considerably. Some produced m2PS and mPS, and others produced mainly bPS.

It was not possible to detect mbPSo

Fig. i is a typical chromatogram of microorganisms, Other products, possibly the oxygen analoges of m2PS and mPS were sometimes detected.

A compound

with slightly less R F value than mbPS may be its oxygen analoge (mbPO).

T. li~norum and A. tenius

seem to be particularly active in the bromophos metabolism. Metabolism by the seedlings The seedlings also produced metabolites. was m o s t a c t i v e ,

and the

carrot 108

least

active.

Wheat The

main m e t a b o l i t e s compounds

are

inorganic

w i t h RFO.

phosphates

or other

The o n i o n s e e d l i n g s

produced

m o s t bPS, w h i c h was l o c a t e d Onion seeds

in the

which did not germinate,

metabolites.

In the

bPS a p p e a r e d excellent

mainly

leaves.

p r o d u c e d no

c h r o m a t o g r a m shown

as double spots,

coincidence

there

(fig.

2)

was,

however,

between the carrier

and the

radioactivety.

DISCUSSION The r e s u l t s directly

indicate

by b i o l o g i c a l

phosphorothionates.

that

degradation However,

I f f o r m e d a s an i n t e r m e d i a t e to accumulate transfer are

easily

pounds.

to detectable

reactions,

aPS c a n be f o r m e d of dimethyl

mbPS i s

one s h o u l d amounts.

mbPS a n d o t h e r

reagents~as amines react

(5)

dimethyl

h a s shown t h a t sulfide,

Also in biological

expect

it

By m e t h y l

dimethyl

com-

nucleophilic

thiourea

w i t h m2aPS t o g i v e

stable.

maPS c o m p o u n d s

formed from the analogous Hilgetag

rather

aryl

salts

and tertiary o f maPS.

system~ such methyl transfer

actions

occurs.

Fukami and Shishido

soluble

enzyme s y s t e m which t r a n s f e r r e d

group from methyl parathion

109

(6)

found a a methyl

to glutathione.

re-

C4.,J:,

9169169169

-%

1 F i ~ . 1.

2

3

4

5

6

TLC of p32-bromophos m e t a b o l i t e s formed

by some fungi.

The radioactive metabolites are

dotted and the non-radioactive are encirceled

reference substances

(from top downward: bPS, mbPS, m2PS

and mPS) i, 2, 4, 6: Unidentified soil fungi, 3: Trichoderma !ignorum,

5:

tion time was 9 days.

Alternaria tenius.

Incuba-

Unmetabolised p32-bromophos

was removed by petrolether extraction before chromatography.

110

I 0000o 0@O0o 2

I Fi~.

2.

stances

4

5

The r a d i o a c t i v e

and t h e n o n - r a d i o a c t i v e

reference

are sub-

( f r o m t h e t o p d o w n w a r d s : b r o m o p h o s , bPS,

of germinated wheat. 3: P u r i f i e d

of germinated seeds.

formed

metabolites

mbPS, m2PS a n d mPS) a r e e n c i r c e l e d . wheat.

6

TLC o f p 3 2 - b r o m o p h o s m e t a b o l i t e s

by s e e d l i n g s . dotted

3

2:

The l e a v e s

The s e e d o f g e r m i n a t e d

p32-bromophos.

onion,

1: 4:

The l e a v e s

and 6: u n g e r m i n a t e d

Growing t i m e was 14 d a y s .

111

onion

Hydrolytic degradation by biological systems seems, however, to give the bisdesmethyl compound directly. This may also be the case for other dimethyl aryl phosphorothionates.

REFERENCES 1.

ROWLANDS, D. G.

J.

2.

STIANSI, M. e t a l .

S t o r e d Prod. Res. 2, J. Agric.

1 (1966)

Fd. Chem. 15,

474 (1967) 3.

STENERSEN, J .

4.

ALLEN, O. N.

J.

In p r e s s .

Experiments in Soil Bacteriology

Burgess Publishing 5.

Chromatogr.

HILGETAG, G. e t a l .

Co. M i n n e a p o l i s , J.

prakt.

Minn. 1957.

Chem. 8 ,

207,

(1959). 6.

FUKAMI, J . and SHISHIDO, T. 5_99, 1138 ( 1 9 6 6 ) .

112

J.

econ. Entomol.