Chapter 8
Enhanced Degradation of S-Ethyl N,N-Dipropylcarbamothioate in Soil and by an Isolated Soil Microorganism 1
2
Enhanced Biodegradation of Pesticides in the Environment Downloaded from pubs.acs.org by COLUMBIA UNIV on 12/03/18. For personal use only.
W. A. Dick, R. O. Ankumah , G. McClung , and N. Abou-Assaf The Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691
Enhanced degradation of EPTC occurs in soil after repeat applications of EPTC. Studies were conducted to evaluate the mechanisms of enhanced degradation in soil and by an isolated soil microorganism (Rhodococcus sp.). Inoculation of a soil without a history of EPTC treatment with 1.0% (w/w) of a soil with enhanced EPTC degradation capabilities increased the rate of EPTC degradation in the previously untreated s o i l . Degradation of C-labelled (1-propyl position) and unlabelled EPTC by the microbial isolate yielded N-depropyl EPTC (a product of α-propyl hydroxylation) and EPTC-sulfoxide (sulfur oxidation). It is proposed that i n i t i a l reactions of soil microorganisms involve both hydroxylation and sulfoxidation, resulting in products that are further metabolized to CO . The hydroxylation reaction is thought to be dominant when degradation of EPTC occurs at enhanced rates. 14
2
Enhanced
pesticide
pesticide treated
is
with
the
compared
to
To
more
date,
reported
to
including
degradation in
its
than
have
an
refers
increased or
a
different potential
as
volume).
carbamothioate failed
applications.
to
the
in
phenomenon
in
a
soil
similar
pesticide adequate
that
was
EPTC
initial
have in
insecticides,
The e a r l i e s t
report that
by
of
weed c o n t r o l one
as soil.
been
soil and
enhanced
Rahman e t
(s-ethyl
report,
a
structure, untreated
degradation
herbicides,
whereby
previously
comparable
enhanced
herbicide
give
Since
the
agricultural pesticides for
classified
this
to
rate
compound o f
degradation
New Z e a l a n d w h e r e
carbamothioate) repeat
a
of
25
the
(Racke, of
at
pesticide, rate
pesticides
fungicides (1)
degradation
degraded
al.
tf,tf-dipropyl in
fields
of
the
after
most
Current address: Department of Agricultural Science, Tuskegee University, Tuskegee, A L 36088 2
Current address: U.S. Department of Agriculture, Agricultural Research Station, Room 100, Building 050, Beltsville Agricultural Research Center—West, Beltsville, MD 20705 0097-6156/90/0426-0098$06.00/0
© 1990 American Chemical Society
8. DICK ET A L
Degradation of S-Ethyl Ν,Ν-Dipropykarbamothioate in Soil
widely soil.
enhanced
studied
Enhanced
EPTC
microorganisms capabilities pesticide
a
been
for
in
before
theory
additions,
Wliile
possible
reason
is in
substrate
to
how a
the
longer
microorganisms.
may a l s o
act
on
another
of
other
degradative
genes
mechanism whereby
s y s t e m may c o n t i n u e isolated
from
contained (9).
within
that
the one
pesticide
are
active
soil
maintain
pesticide
their
"steady-state"
of
soil
in
ingredients
obtain
is
not
always
Possibly thus
EPTC
degradative to
enzymes
maintaining
exploit
EPTC
as
a
untreated Equally
pesticide
the
although
rates.
of
a
suitable
explain,
degradation
the
(i.e.
and a
s i m i l a r but
to
of
another
repeatedly may a l s o in
serve
the
substrate
(7).
Some
soil
for
to the
periods,
unrelated
of
maintenance
extracellular
commonly
extracellular
long
is
EPTC,
degradation
in
Many
groups
by
degradative
the
result
as
induced
A microorganism,
exposed
soil.
component
EPTC (8).
mediated
functional
enzyme
is
potential.
to
which
molecules.
activity
pesticide
phase
enzyme
degradation
the
adaptive
the
degradation
against
The
survival year
one
required for
The l a g
of
normally
induced by
the
presence
among m i c r o o r g a n i s m s
had been
plasmids,
the
addition
on p l a s m i d s
E x t r a c e l l u l a r enzymes
enhanced
enzymes
soil
four
from
enhanced
the
level.
persistence
rapidly
theories
in
to
substrates
to
Two
period
time
contains
soil
enhanced
difficult
baseline
for
in
microorganism,
been
the
ability
lag
necessary
by
in
mechanisms
postulates
adaptive
enhanced
has
for
Transfer
of
the
inability
account no
the
enzymes,
the
(6),
the
that
The
EPTC
caused
involvement
theory
of
degradative
dominant w i t h
is
adaptable
establish
to
soil
microorganisms'
EPTC
contain
is
the
an a p p r e c i a b l e
develop
an
supposedly
microorganisms.
observation
soils
puzzling
fully
condition)
This
begins
adaptive
produced by
pesticide,
observed.
mutation
to
that
remain unresolved.
competition.
build
many s o i l s
environmental
(control)
of
been
involvement
microbial
w h i c h become
postulates
are
susceptible
EPTC
to
r e q u i r e d to
ability
explain
degradation
enzyme
is
has
additional
recognized,
phenomenon
to
absence
population
soil
acquire
been
The chance
mutant
one
has
microorganisms
substrate
observed
this
(5).
in
to
systems
Although microbial
proposed
mutant
period
appear
(2-4).
degradation of
degradation
which
degradation
responsible have
degradation
contained enzymes
yielding
to
current
a microbial
proliferation. To
sort
hypotheses is the
out
for
important
that
biochemical
Although
degrade been
in
current
in
further EPTC to
studies
hydrolysis
and
(15).
2
animal
degradation
involves C0
i n v o l v e d be they
plants,
Plant
these,
the
in
mammals
undergoes was
the
also
determined
ester
linkage
EPTC be
is
conversion the
that
based
EPTC
form
to
carbamoyl
on
a bond
hydroxylation
carbamate
that to
it
and
(10-12).
undergo
by
soil,
determined.
information
at
to
followed
in
identified for
involves
cleavage
additional
proposed
systems
hypothesized
sulfur,
have at
soil,
and p l a n t
or
degradation
have
(13) . (14) .
of
EPTC
pathways
which
In
microorganisms
degradation
carbon alpha
importance enhanced
by which
sulfoxide the
the
of
different
conducted
Carbamothioate
relative
pathway(s)
several
carbamothioate studies
the
explanation
cleavage
degradation
mercaptan,
amine
at
99
100
ENHANCED BIODEGRADATION OF PESTICIDES IN T H E ENVIRONMENT
Lack
of
mechanisms limited and
information concerning
involved
attempts
other
initiated
rapid
enhanced
1)
evaluate
soils,
2)
and
the
isolate
used
collected
that
had previously years
were
breakdown o f in
soil.
and
this
This
of
microorganism(s)
active
biochemical
three are
from a
field
located
been
treated
or
surface given
with
from s i t e s
(0
to
15 I.
near
Canal
of
I.
Soil
(clay
1,
Chemicals. (specific
EPTC a n d
Chemical 97% a s
loam)
Company.
quantities
of
were
Purification
was
Ohio
and
4
Dothan
Carolina,
butylate
Characteristics
35
carbon
PH
Argiaquoll
7.5
4.5
Typic
Argiudoll
5.9
3.8
5.5
3.0
mCi/mole)
Paleudult
were
thin
layer and
1 4
synthesized
C-l-propyl
U
the
C-EPTC
chromatography the
method
by TLC with
of
(%)
EPTC
the
Stauffer
was
greater
(TLC).
than
Millimole
C-labelled EPTC-sulfoxide by
accomplished
1 4
and
s u p p l i e d by
The r a d i o p u r i t y o f
unlabelled
EPTC-sulfone
and
Typic
Plinthic
by
3,
use.
T e c h n i c a l EPTC a n d b u t y l a t e
determined
Brookston
Organic
loam)
activity,
2,
The P i a n o
Subgroup
loam)
(sandy
sample
The
Soil
Dothan
EPTC
Winchester,
Eradicane for
carbamothioate)
(texture)
(silt
of
cm
i n W i s c o n s i n and South
which had h i s t o r i e s
Table
Piano
and
in
pathway(s)
in Table
had remained untreated.
(s-ethyl-N,N-diisobutyl
Brookston
was
field
microbes.
the
this
collected
respectively,
Series
herbicide,
study in
the
the
seriously
EPTC d e g r a d a t i o n
study
in
was
consecutive
microorganisms
Methods
Characteristics soils
soil
determine
isolated
soil
soils
the
to
E P T C a n d 3)
Materials Soils.
control
the
EPTC d e g r a d a t i o n h a s
pesticides,
degradation by
depth)
to
enhanced
carbamothioate
laboratory degrading
in
Casida
and
et
ultraviolet
al.
(16.) .
radiation
(UV)
detection. Analytical and
Procedures.
metabolites
carbowax mesh).
A Varian
Gas obtained Center
Gas c h r o m a t o g r a p h i c
performed using
glass
20M o n C h r o m WHP a n d 3% O V - 1 7 o n
thermoionic
and
were
at
the
using
separation
a
Ohio
of
Thin-layer was
with
hexane-ethyl
were by
spectrometry
University
F i n n i g a n 4021 compounds
EPTC 5%
(100/200
a nitrogen
GC/MS
specific
accomplished hexane-acetone (3:2).
results
Both electron
samples
(TLC) f o r silica
(6:1),
5% c a r b o w a x separation gel
60
hexane-ether
Detection
of
impact
following
chromatograph equipped
using
were
Instrumentation
instrument.
3% O V - 1 7 o r
chromatography
(GC/MS)
Chemical
performed on
a gas
containing
acetate
Supelcoport
chromatograph with
of
containing
used.
State
ionization
columns
metabolites developed
gas was
chromatographic/mass
chemical
capillary
3700
detector
(GC) a n a l y s e s columns
F 4 2 5
with
20M. of
EPTC
(4:1)
unlabelled
and
chromatoplates and
or or
8. DICK ET AL.
Degradation of S-Ethyl ^^-Dipropylcarbamothioate in Soil
radiolabelled
pesticide
autoradiography, accomplished sections
by
of
liquid
was
b y UV a d s o r p t i o n
Quantitation
scintillation
U
of
counting
and
C-activity
(LSC) of
was
scraped
gel.
Non-protein
thiol
content
precipitation
of
assayed
Ellman's
a
and m e t a b o l i t e s
respectively.
using
protein
in
using
the
culture
media,
0.2M t r i c h l o r o a c e t i c
reagent
(12) , w i t h
after acid,
was
2-mercaptomethanol
used
as
standard.
Microbial
Isolate.
as
carbon and energy
a
sole
soil
(Typic
Argiaquoll;
enrichment added 200
to
batch
50
mg L "
1
EPTC
at
250
for
as in
based a
its
ability sp.
on
fresh
at
to
the
basis
1 ml
on
a
an
flasks
were
action
shaker
were were
went
through
degrade
for
all
EPTC
its
morphology
plated
plates
their to
and
with
three ability
utilize cell
to
studies EPTC
identified and
out
purified
subsequent
and
tentatively
or
was
The
Isolates for
100
inoculum
agar
tested
was
with
rotary
isolates
been
of
of
salt
then
EPTC loam
soil
supplemented
ml d i l u t i o n s
retained
rapidly
on
by
on b a s a l
(BSAE). and were
J E 1 has
7.5)
media.
27°C
0.1
grow
J E 1 was
source.
(18)
Individual
to
source
Isolate
carbon
Rhodococcus
plates.
pH,
intervals,
transfers,
BSAE p l a t e s
BSME.
10-day
ability
carbon
on
on
sole
(NA)
their
the
transfers grow
agar
(BSM)
incubated
four
4.5%;
growing
from Jimtown
One g r a m f i e l d - m o i s t
containing
and
of
isolated
carbon,
medium
At
flask
After
on n u t r i e n t EPTC
a
cotton
rpm.
screened
salt
capable
was
technique.
(BSME) .
into
with
isolate
source
organic
culture
ml b a s a l
transferred plugged
A microbial
as
as
wall
composition. Metabolic soil, kg'
1
of
EPTC
various or
Studies.
aliquots or
butylate
GC. 1
and
The results
average
of
soil.
The soil
with
at
rate (10
measured as The soils
was
analyses
at
layer
was
level
level
added
in
an
EPTC
a
incubator
for
4
mg
At and
EPTC
10:3
EPTC
or
butylate
butylate
was
0.05
mg
1.0,
10
90%.
EPTC h i s t o r y
rate
in
with
dark.
with
or
evaluated
time
the
the
analyzed
Brookston
at
various
from
exceeded
of
history
25°C
treated
extracted of
was
degradation
were
by
soil
of
soil
to
a
4 mg k g "
intervals
which
adding
was
0.1,
non-history 1
soil
and
measured
the
as
described.
effect
of
various
by
of
100
mg k g "
1
after
0,
3,
assays by
experimental a of
separations
1
soil.
and and
the
variance were
The
samples
as
were
for
using
on
were
EPTC
EPTC
at
the
or
Brookston
cycloheximide
treated
extracted
with
and
described. and b u t y l a t e
history
(19.) ·
experiments with
degradation
history then
E P T C was
Singh
all
design
performed
determined
3-yr
previously
T a b a t a b a i and design
enhanced the
chloramphenicol,
conducted
randomized block
of
remaining
7 days
were
on
aliquots
streptomycin,
mg k g " soil)
described
antibiotics
treating
kanamycin,
Rhodanese soils
3-yr
after
investigated a
was
degradation
a
soil
removed
soil
recovery
E P T C was
remaining
previously
EPTC
toluene
enhanced
(w/w)
Brookston
was
were
the
minimum i n o c u l u m in
100%
amount
the
and b u t y l a t e
incubated
The minimum d e t e c t a b l e
soil
and
The
in
EPTC
Brookston
and
samples
remaining
toluene :water. kg"
air-dried
intervals,
butylate
by
To measure
of
three
each Least
conducted
replicates.
sampling
time.
Significant
on
the
Separate Mean
Difference
101
102
ENHANCED BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT
(LSD)
test
only
when
the
F-test
was
significant
at
the
0.05
level
of
significance. Microbial rotary An
aliquot
a
For
swept
the
final
the
EPTC
culture
had
samples
were
were
of
the
was
with
The aqueous and a n a l y z e d
hexane-acetone,
6:1)
the
0.5
1 A
headspace
with
the
to
At
100
8 h
of
ml under TLC and
used
the by LSC.
EPTC
exception 1
being
after
the
for
The
32
nitrogen
h,
supernatants
undegraded
toluene
liquid
was
above
mg L "
for
the
EPTC
C-l-propyl
intervals,
analyzed
and
aliquot
measured
and c e n t r i f u g e d .
f r a c t i o n was by
An
formed d u r i n g
then
GC o r
mg L " ) ,
with
a
0.06-0.08).
microbiol cells
treated
used
grown on
6 0 0 >
1
(50
C - a c t i v i t y was
phase.
and
O.D.
intervals.
no
The remainder
using
counting.
2 h
adjusted
removed
approximately
methanol,
u
was
was
toluene,
and m e t a b o l i t e s
EPTC
intervals,
midlog
GC a n d T L C . to
with
also
the
L " ) was
days;
metabolites
asceptically
by
at
1
mg E P T C
(2-3
EPTC b u t
same p r o c e d u r e
reached
concentrated EPTC
8 h
concentration
extracted
remaining
of
NaOH a n d
identification
degradation,
treated
culture
at
into
(100
measured
amount
A midlog
mg L " ) a n d ,
was
was
was
similar
BSME
m i d l o g phase
culture
1
(100
culture
to
solution
control.
EPTC
JE1 i n
27°C
the
in
containing a
at
of
remaining as
isolate
shaker
EPTC
extract
was
and a n a l y z e d
for
scintillation
lyophilized,
dissolved
GC, TLC (hexane-ether,
9:1
or
of
EPTC
in
and L S C .
Results Metabolic
Studies
Brookston
soil
EPTC
(Table
year
of
degraded
a
Enhanced degradation
was
after
rate not
of
rapidly
with of
at
the
Table
II.
treated
no
in
prior
treated with
field
degradation for
of
EPTC was
in
different 2,
3,
or
soil
to
from
EPTC,
than
1).
that
of
4
mg E P T C 1,
2,
Days use
0
kg"
3 or after
3
- - - EPTC
1
4
rates
years. was
than
in
The of
in
EPTC
Brookston
consecutive
soil years
application 7
10
remaining,
mg k g "
1
14
soil-
- -
0
3.86
3 . .63
2.76
2. 41
1. . 3 1
1
3.87
1. ,82
0.64
0. 44
0, . 3 4
2
3.84
1. , 7 3
0.78
0 . 33
0. . 3 0
3
3.82
1. , 8 7
0.55
0 . 27
0, . 2 2
4
3.86
1. , 7 8 0. . 4 9
0.74
0 . 36
0, . 2 9
0.44
0 . 12
0, . 4 5
LSD(0.05) LSD = F-test
least was
+
significant significant
NS
difference at
Ρ
