145
907

 

DEGRADATION 0F ALACI-ILOR
[2 - CHLORO - 2' ,6' - DIET HYI. - N ~
(METHOXYMETHYIJ ACETANILIDEI
IN SOILS AND BY MICRWRGANISMS

Thesis for the Degree of IL .3“
MICHIGAN STATE UNIVERSITY
ROBERT W. TAYLOR;

1 9 72

 

  
 
  

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ABSTRACT
DEGRADATION OF ALACHLOR

[2-CHLORO-2',6'-DIETHYL-N-(METHOXYMETHYL)ACETANILIDEI
IN SOILS AND BY MICROORGANISMS

By
Robert W. Taylor

Alachlor was not used as a sole source of carbon and
energy for growth by soil or river water microorganisms as
judged by the absence of growth in enrichment cultures.

Metabolism of 100 ppm alachlor by growing and resting
(replacement) fungal mats was studied, Replacement mats of
Aspergillus niger metabolized only 12.7% of the herbicide
in a one week period. However, growing mats of Chaetomium
sp.metabolized as much as 97.2% of the alachlor during a
17 day growth period while growing mats of A. niger
metabolized only 18%. The gas-liquid chromatographic
~analysis of the Chaetomium medium also showed two additional
peaks that could be alachlor metabolites. Analysis of the

fate of the 14

C-ring labeled alachlor in the experiment

using Chaetomium showed that 85.7% of the label was extracted
in ethyl acetate and that, of this, only 43% could be
attributed to alachlor. A minor quantity (8.9%) of the

label remained in the aqueous phase and no label was trapped

Robert W. Taylor
as C02. Thus a large portion of the label (42.7%) appeared
to be contained in non-polar metabolites.

When 100 ppm (w/w) of 14

C-alachlor was incubated in a
Barry loam soil, only 16% of the original alachlor, as
determined by gas chromatographic analysis, was recovered
by acetone-water extraction after 21 days in both non-
sterilized and gamma sterilized soils. In contrast 44-50%
of the label was extracted by the same solvent; however,
in the non-sterilized soil one-quarter of this label was
in polar components while in the sterilized soil only one-
twelfth remained in the aqueous fraction. None of the

14

label was found as CO2 after 21 days.

DEGRADATION OF ALACHLOR
[2-CHLORO-2',6'-DIETHYL-N-(METHOXYMETHYL)ACETANILIDE]
IN SOILS AND BY MICROORGANISMS

By
Robert Wf Taylor

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Crop and Soil Sciences

1972

Dedication
To my parents, brother and sister whose memory,
encouragement, and never ending sacrifice
gave me the inspiration to

finish this work

ii

ACKNOWLEDGEMENTS

To Dr. J. M. Tiedje, my heartfelt gratitude is due for
his encouragement, patience and indispensable guidance.

To Dr. W. G. Fields, the author is indebted for his
cooperation in supplying pure cultures of the fungi used in
this study.

To Dr. D. Penner and Dr. M. J. Zabik, special thanks
is also due for their constructive criticisms of the
manuscript.

The financial assistance from the Environmental Pro-

tection Agency, Grant no. 8RDIEP00801-06, is acknowledged.

iii

TABLE OF CONTENTS

Page

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1
MATERIALS AND METHODS . 4
Fungi. 4
Enrichment on acylanilides 4
Replacement cultures 5
Growing fungal mats. 6
Growing fungal mats---14C-alachlor added . 7
Extraction of alachlor from aqueous media. 8

Soil experiment. . . . . . . . . . 8
Gas-liquid chromatographic (GLC) analysis. 9
Radioactivity assay. 10
Chemicals. . 10
RESULTS AND DISCUSSION. 11
REFERENCES. 25

iv

Table

LIST OF TABLES

Growth in enrichment cultures containing
acylanilides as the sole source of carbon
and energy . . . . . . . . . . . .

Metabolism of alachlor by a resting mat
of Aspergillus niger . . . . .

Chloride released from alachlor by resting
mats of Aspergillus niger. . . . . .

Metabolism of alachlor after a 17 day
incubation period by growing mats of
Aspergillus niger and Chaetomium 8p.

Metabolism of alachlor after a 21 day
incubation period by growing mats of
Chaetomium 8p. . . . . . . . . . . . .

14

Fate of the C-label of alachlor following

metabolism by growing mats of Chaetomium 3p.

after a 21 day incubation period .
Recovery of alachlor from soil

Fate of 14
with soil.

C-label from alachlor incubated

Page

12

13

14

15

17

20
22

22

LIST OF FIGURES
Figure Page

1 Gas chromatography traces showing alachlor
and possible metabolites . . . . . . . . . . . 19

vi

INTRODUCTION

Herbicides have become almost indispensable to large-
sacle agriculture. They are easy to apply, are less
damaging to economic plants than most mechanical weed con-
trol methods and their use is economically favorable.
However, a concern of herbicide use is the possibility of
environment pollution. Therefore, the biodegradability and
non-toxicity of the degradation products of any herbicide
should be known before it can be considered safe for use in
the environment.

Anilide herbicides are relatively new and show promise
for weed control in turf and a variety of economically
important crops including rice, cotton, soybeans, corn,
and tomatoes (Bartha, 1968). Among the attractive features
of this class of herbicides are their effectiveness,
selectivity, biodegradability and low mammalian toxicity
(Bartha, 1968). Alachlor [2-chloro-2',6'-diethy1§N-
(methoxymethyl)acetanilide] (commercially sold as Lasso)
is an anilide herbicide which recently has become popular
as a weed control agent.

Little information has been published on the detection
and degradation of alachlor with no information available
on microbial degradation. Hargrove and Merkle (1971) found
that chemical degradation of alachlor in the soil, under

1

2
conditions of low relative humidity and high temperature,
resulted in the formation of 2-ch10ro-2',6-'diethy1
acetanilide. Since the same product resulted from acid
catalysis in 5 N aqueous HCl, the degradative mechanism in
soil was attributed to acidic soil water films on mineral
surfaces. In this case cleavage resulted in the loss of
the N-substituted ether group with the aromatic system and
amide linkage preserved. Beestman and Deming (Agronomy
Abstract, 1972) suggest that microbial decomposition was
the major route of alachlor degradation in the soil with
a half-life of between 2 to 14 days. In addition, informa-
tion from the manufacturer states that 40% and possibly
as much as 90% of alachlor degradation in soil is carried
out by the microflora.

In studies on alachlor metabolism in plants Armstrong
(1972) concluded that alachlor was metabolized rapidly by
yellow nutsedge to at least one water-soluble metabolite.
Lamaureux, Stafford, and Tanaka (1971) studying plant
metabolism of a related herbicide, propachlor, (Z-chloro-
N-isopropylacetanilide) found at least three water-soluble
metabolites, one of which was identified as the glutathione
conjugate of propachlor. Based on these few reports,
metabolism of alachlor could be expected to produce water-
soluble metabolites.

Bartha and Pramer (1967) demonstrated that the micro-
bial degradation of propanil (3',4'-dichloropropionanilide)
produced 3,4-dichloroaniline and propionic acid as the

products, which showed that enzymatic cleavage of the parent

3

molecule occurred at the amide linkage. The same site of
enzymatic attack could be postulated for the microbial
degradation of alachlor.

Isolation of an organism capable of using alachlor as
a sole source of carbon and energy for growth has not been
reported. This suggests that if there is bio-degradation
in nature it might be accomplished by a cometabolic mechanism.
Evidence is available indicating that cometabolism may be
an important phenomenon in the breakdown of pesticides
(Horvath and Alexander, 1970).

A need for further studies on the degradation of
alachlor is evident and this study was initiated to find
out if alachlor is microbially degraded and to detect any

degradation product(s).

MATERIALS AND METHODS

Fungi

Aspergillus niger and Chaetomium sp., two soil fungi,
were used in the study. Pure cultures of these fungi were
obtained from Dr. William G. Fields, Department of Botany
and Plant Pathology, Michigan State University. The cultures
were maintained by aseptic transfers on potato dextrose

agar slants.

Enrichment on acylanilides

 

 

The enrichment media were made from a basal salts solu-

tion consisting of 1.6 g KZHPO4; 0.4 g KHZPO 0.2 g

4:

NH NO 0.0014 g FeSO

4 3' 3‘ 4’
made up to 1 liter with deionized water. The following

0.2 g MgSO4, and 0.017 g CaCO

chemicals were added to 100 ml portions of the salts solu-
tion to give a final concentration of 0.05%: l) propachlor;
2) CDAA; 3) alachlor; 4) propanil; 5) 3'-chloro-acetanilide;
6) propionanilide; 7) meethyl acetanilide; 8) acetanilide;
9) oxanilic acid. Fungal enrichment media were prepared
similarly except that KZHPO4 was omitted and the pH was
adjusted to 4.6 with1.N H3PO4. The inoculum was 1 ml of
water from the Red Cedar River for aquatic organisms and

1 ml of a soil suspension from a turf covered Conover loam

from the Michigan State University campus for soil organisms.

5
Growth was recorded from visual observations, showing the
appearance of turbidity or an increase in biomass, after

two weeks.

Replacement cultures

 

One liter of malt extract broth was made by dissolving
the dried broth in the basal salts solution; 100 ml portions
were autoclaved in 300 ml Erlenmeyer flasks, then inoculated
with pieces of A. niger mycelium. Fungal mats developed
on this medium during a 14 day incubation period. Five of
the fungal mats were freed of the malt extract broth by
aseptically pouring out the spent medium. The mats were
washed twice with sterilized deionized water to remove the
excess fungal debris and to dilute the chloride which
remained from the malt extract. During the second wash,
the mats were allowed to sit on the deionized water for
16 hr. This wash was used to establish the level of back-
ground chloride. Inorganic chloride released from alachlor
was determined colorimetrically according to the method of
Bergmann and Sanik (1957). The other five mats remained on
the malt extract broth and were used as controls for sub-
stances secreted by the mats. After the second wash the
fungal mat was resuspended on 100 ml of a solution contain-
ing 10 mg alachlor (100 ppm) and the basal salts solution
previously described.

Both the alachlor media and the malt extract broth in
the controls were separated from the mats and extracted with

two 50 ml portions of redistilled ethyl acetate at 0 time,

6
5 hr, 10 hr, 24 hr and 168 hr following incubation at room
temperature. Simultaneously, the sterile controls which
consisted of five 100 ml portions of the same 100 ppm
alachlor solutions, were extracted. Each of the fungal
mats was homogenized in 100 ml of redistilled ethyl
acetate, the homogenate filtered, and the cell debris
discarded. The ethyl acetate extracts were dried by addi-
tion of anhydrous NaZSO4 prior to gas chromatographic
analysis. Inorganic chloride was determined on all the
alachlor containing aqueous solutions after extraction with

ethyl acetate.

Growing fungal mats

 

Two liters of 100 ppm alachlor malt extract broth solu-
tion were made up by adding 0.2 g of alachlor and 30 g of
malt extract. broth. to the basal salts solution. From.the
above solution nine 100 ml portions were filter-sterilized
through a 0.20 micron Millipore filter into presterile
300 m1 Erlenmeyer flasks. Two flasks were inoculated with
mycelial pieces of each of the following fungi: A. niger;
Chaetomium sp.; A. niger plus acetanilide (0.01 g per flask).
Three flasks which were not inoculated served as zero time
controls. Two flasks of 100 ml containing only malt extract
broth media were made up separately, autoclaved and inoculated
with A. niger and Chaetomium sp.; these flasks served as the
controls for substances secreted by the mats. Fungal mats
were allowed to grow for 17 days at room temperature, after

which the media and mats were extracted as described in the

7
replacement mat experiment above. The ethyl acetate extracts

were assayed for alachlor by gas chromatography.

14

Growing fungal mats--- C-alachlor added

 

 

A 100 ppm alachlor malt extract solution was made up as
described above. Two-tenths milliliter of uniformly ring-
labelled 14C-alachlor (specific activity--1.02 u Ci/umole)
in 95% ethanol was added to 500 m1 of this solution (110 dps
per 1 ml of media). As described above 100 m1 portions were
filter sterilized and placed into four sterilized biometer
flasks. Twenty milliliters of sterilized l N NaOH was
aseptically transferred to the side arm of each biometer
flask. Two of the flasks were inoculated with Chaetomium
sp. The flasks were sealed and the fungi allowed to grow
for 21 days at room temperature. One of the other two
inoculated flasks was extracted with redistilled ethyl
acetate at zero time while the other was extracted after 21
days. After termination of the experiment the fungal mats
were extracted by immersing in ethyl acetate for 24 hr; a
dry weight of the residual was determined after the extrac-
tion. The aqueous solutions were extracted as described
in the replacement mat experiment. Radioactivity of the
aqueous residue and the ethyl acetate extract was determined
by liquid scintillation counting. Following extraction,
the aqueous solutions were acidified to pH 2 and re-extracted
with ethyl acetate. The quantity of radioactivity in this
second ethyl acetate extract was also determined. The

aqueous solutions were then readjusted to approximately pH 6

8
before the radioactivity assay. The ethyl acetate extracts

were assayed for alachlor by gas chromatography.

Extraction of alachlor from aqueous media

 

 

Alachlor was extracted from the aqueous media with two
50 ml portions of redistilled ethyl acetate. The aqueous
and organic phases were shaken for one minute in a 250 ml
separatory funnel and the two phases were separated after
12 hr. The two ethyl acetate fractions were combined and
dried over anhydrous NaZSO4 prior to gas chromatographic

analysis.

Soil experiment

 

 

The metabolism of alachlor was studied in a Barry loam
obtained from a plot at the Michigan State University Soils
Experimental Farm which had never been treated with alachlor.
Five grams of soil was placed in small glass bottles (4
inches high x 1 3/4 inches in internal diameter). A 2 ml

14

plastic vial containing 1 m1 of] N NaOH to trap CO2 was

suspended from the plastic cap. Fifty ul of an alachlor—
14C-alachlor solution (2915 dps and a final concentration
of 100 ppm) was distributed dropwise from a 28 gauge needle
on the soil in each bottle. The soils were moistened with
1.5 m1 of tap water and the bottles sealed. The sterile
control was obtained by subjecting one of the bottles to

3 M rads of 60

Co-radiation (McLaren and Peterson, 1967).
The soils were extracted with two 20 ml portions of an
acetone-water solution (80:20) at zero time and after 2,

7, and 21 days incubation. The radioactivity of the

9
acetone-water extract (0.1 ml) was assayed prior to evapora-
tion of the acetone in a water bath at 60 C under a stream
of nitrogen. The remaining aqueous portion was extracted
with 20 ml of ethyl acetate. The ethyl acetate phase was
removed, assayed for radioactivity (1 m1) and analyzed for
alachlor by gas-liquid chromatography. The radioactivity

remaining in the aqueous phase was also assayed.

Gas-liquid chromatographic (GLC) analysis

 

 

Quantitation of alachlor was by GLC employing a flame
ionization detector. Triphenylmethane, which was well
separated from alachlor, was added as an internal standard;
1 m1 of a 100 ppm solution (in ethyl acetate) was added to
10 ml of the ethyl acetate extract. The same procedure was
followed for preparation of the standard curve, which
included values from 5 to 100 ppm alachlor. The injection
volume of all samples was 2 ul. Gas chromatographic condi-
tions were as follows:

Perkin-Elmer Model 900 gas chromatograph

Carrier gas (Helium) flow-40 ml/min

Injector temperature ~210 C

Detector temperature ~250 C

Column over temperature -198 C

Column - 182 cm x 3 mm 0. D. stainless steel

packed with 4% SE-30/6% QF-l (Thompson,
Walker and Moseman, 1969) on 60/80 gas
Chrom Q

10

Radioactivity assay
14

 

The C-label was followed by liquid scintillation
counting using a Packard Tri-Carb Liquid Scintillation
Spectrometer, Model 8310. A11 aqueous samples were counted
in Bray's solution (Bray, 1960) while samples in ethyl
acetate were counted in a toluene based liquid scintillator
of POP and POPOP. The CO2 trap of 1 N NaOH was counted in
Bray's solution plus 4% Cab-O-Sil. All counts were corrected

for quenching by external standardization and for machine

efficiency.

Chemicals

 

Analytical grade alachlor, propachlor, CDAA (Z-chloro~
N,N-dially1acetamide) and propanil were supplied by the
Monsanto Company, St. Louis, Missouri. The purity of
alachlor was reported to be 99.8%. 3'-Chloroacetanilide,
propionanilide, N-methylacetanilide, acetanilide and
oxanilic acid were obtained from Eastman Organic Chemicals,
Rochester, New York. The malt extract broth was purchased
from Difco Laboratories, Detroit, Michigan. Uniformly
l4C-ring labelled alachlor was a gift of the Monsanto

Company. The radioactive purity was 99.7% as determined

by thin-layer chromatography.

RESULTS AND DISCUSSION

Enrichment media containing either propachlor, CDAA
or alachlor as the sole source of carbon and energy showed
no visible growth of microorganisms (Table 1). Growth was
observed on all the other acylanilides used in this study.
There appeared to be no difference between the inoculum
source with regard to range of substrate degraded. The
media at neutral pH were dominated by bacteria while those
at the acidic pH contained mycelial and single cell (yeast)
fungi. The results of the enrichment experiment explain
why an organism capable of using alachlor as the sole source
of carbon and energy has not been reported. Rather, come~
tabolism is a more likely mechanism for alachlor biodegraw
daticn in nature.

Aspergéllus was used in this study because this genus
is capable of utilizing an enormous variety of substances
for food owing to the large number of enzymes produced
(Alexopoulos, 1962). The Chaetomiaceae are referred to as
cellulosemdestroying fungi which grow on paper, fabrics,
dung and straw (Alexopoulous, 1952).

As shown in Table 2, alachlor was not metabolized by
a replacement mat of A. niger until 168 hr of incubation at
which time 12.7% of the alachlor was lost. This quantity of
degradation compares with the value 18% inorganic chloride

11

12

Table 1 - Growth in enrichment cultures containing
acylanilides as the sole source of carbon
and energy

 

 

 

 

Media at pH 7.1 Media at pH 4.5
Substrate Soil ’Water Soil ’Water
Propachlor *- — - _
CDAA - - - _
Alachlor — - - s
Propanil + + + +
3'-Chloroacetanilide + + + +
Propionanilide + + + +
N-Methyl acetanilide + + + +
Acetanilide + + + +
Oxanilic acid + + + +

 

* -, no growth; +, growth.

released in the aqueous medium at the same time (Table 3),
suggesting that the chlorine atom of the parent molecule was
removed during metabolism by A. niger. The relatively small
amount of alachlor degraded in this experiment (12.7%) could
be due to the fact that the fungus was not growing during
metabolism. No metabolites were detected by GLC in this
experiment.

Growing mats of A. niger showed a small increase in
alachlor degradation with approximately 18% unaccounted for
(Table 4). Again no metabolites were detected by GLC.

Acetanilide was added to A. niger mat media in an attempt to

13

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induce enzymes capable of breaking the amide linkage of
alachlor. The results showed little difference in the
amount of alachlor metabolized (17.5%) due to the presence
of acetanilide.

Growing mats of Chaetomium sp., however, metabolized
an average of 95.4% of the alachlor added but again no
metabolites were detected (Table 4). In a second growing
mat experiment (Table 5), replicate flasks containing
Chaetomium sp. degraded 69.1% and 43.9% of the alachlor.
In this experiment two possible metabolites were detected
(Figure 1), both of which were shoulder peaks on the solvent
tail. The presence of these peaks in this experiment and
not in the previous appeared to be due to slightly earlier
elution of the solvent in this case, which was not the case
in the previous experiment and thus masked small peaks of
this retention time. Since no mat control was included in
this experiment it is not certain that the peaks are from
the herbicide. No attempt was made to identify the possible
metabolites. The difference in the amount of alachlor
metabolized by the replicates of Chaetomium sp. could be
attributed to the difference in fungal mat masses, Chaetomium
(l) weighing 0.053 g and Chaetomium (2) weighing 0.041 g.
Extraction of the aqueous media after adjusting the pH to
2 did not yield any extractable metabolites detectable by
gas chromatography.

Table 6 provides information on the fate of the 14C-
labelled alachlor used in this same growing mat experiment.

Essentially none (0.08% and 0.06%) of the radioactivity was

l7

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18

Figure 1. Gas chromatography traces showing alachlor
and possible metabolites.

A and B = possible metabolites

Tpm = triphenylmethane (internal standard)

 

 

 

:Alachlor x 4

 

 

 

Tpm
Sterile control
Tpm
Alachlor x 4
Chaetomium (2)
Tpm

 

 

Alachlor x 4

Chaetomium (l)

 

 

Figure l

 

 

20

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21
present in the NaOH trap showing that the ring was not

14

cleaved and degraded to CO Almost all (81.4% and 90.1%)

2°
of the radioactivity was extracted by the ethyl acetate.
Since the GLC analysis showed that Chaetomium (1) and (2)
did not metabolize 30.9% and 56.1%, respectively, of the
alachlor in the medium, 50.1% and 46.0% of the radioactivity
in the respective ethyl acetate extracts was not alachlor
and this supports the suggestion that the two products
detected are alachlor metabolites. Also, the finding of
most of the radioactivity in the ethyl acetate extract sug-
gests that the majority (3/4) of the metabolites from
cometabolism of alachlor by Chaeto0ium sp. are non-polar.
The remaining radioactivity (11.4% and 6.4%) found in the
aqueous media suggests that there may be some water soluble
metabolites also. This finding appears to be contrary to
work on alachlor metabolism in plants in which the majority
of metabolites are water soluble (Armstrong, 1972). The
amount of radioactivity extracted by ethyl acetate from

the acidified solution was minor.

Acetone-water (80:20) was used to extract alachlor from
the soil because of its high efficiency of extraction for
non-polar molecules, such as alachlor, which possess
functional groups capable of forming hydrogen bonds with
metal ions 00 the clay surfaces via water bridges.

The results of the experiment on alachlor degradation
in soil are shown in Tables 7 and 8. At zero time, the GLC
analysis (Table 7) indicated that 72.1% of the alachlor was

recovered in the ethyl acetate extract. After 21 days only

22

Table 7 - Recovery of alachlor* from soil

 

 

Alachlor
Incubation Time extracted
Treatment (days) (ppm)
Soil 0 72
Sterile soil 21 1600
Soil 1 21 1601
Soil 2 21 1605

 

* 100 ppm of alachlor was added to each soil.

Table 8 - Fate of 14C-label from alachlor* incubated

with soil

 

Percent of total counts added

 

 

 

Incubation Acetone Ethyl
Treat- time NaOH water acetate Aqueous
ment (days) trap extract extract phase
Soil 0 - 9100 9303 490
Soil 2 002 93t7 - -
Soil 7 092 9008 - -
Soil 21 007 4309 3208 1205
Soil 21 0.9 44.1 30:8 1403
Sterile
Soil 21 0.7 49.9 4209 306

14

* 100 ppm containing 0.078 uCi C-alachlor was added to

each soil.

23

16% of the original alachlor could be recovered from the
sterile control and both soil replicates. The low recovery
in the case of the sterile control could be due to destruc-
tion of the alachlor parent molecule by gamma radiation,
chemical degradation or degradation by soil enzymes which
are not usually destroyed by the dosage of radiation used
in this experiment (Peterson, 1962). Thus because of the
uncertainty of explanation for the poor alachlor recovery
from the sterilized soil it is not possible to make any
conclusion on the extent of its biodegradation in soil.
Gas-liquid chromatography analysis of all four soil extracts
showed another peak as a shoulder on the tail of the alachlor
peak. No attempt was made to identify this peak. The
possibility exists that this shoulder peak is a degradation
product of alachlor or it may be a compound extracted from
the soil and unrelated to the alachlor addition.

The 72% of the original alachlor present in the ethyl
acetate extract as determined by GLC analysis (Table 7)

14c-1abe1 in

does not appear to agree with the 93.3% of the
the same ethyl acetate extract (Table 8). This discrepancy
could be due to experimental error or to rapid catalysis of
alachlor in soil. Liquid scintillation counting showed that

14C~1abel was extracted at zero time in the

91% of the
acetone-water extract and approximately the same amount was
extracted in the ethyl acetate extract. After 21 days,

however, the soil replicates and sterile control contained.

14

only 43.9%, 44.1% and 49.9% of the C~1abe1, respectively,

in the acetone-water extract. The apparent loss of alachlor.

24
here could be due to microbial or chemical degradation or
irreversible adsorption to the surface of the clay particles.
Degradation by the acidic soil water film surfaces (Hargrove
and Merkle, 1971) was apparently not important here since
the soil was very moist throughout the experiment.
The ethyl acetate extracts of the soil and sterile

control contained 32.8%, 30.8% and 42.9% of the 14

C-label,
respectively. This also did not agree with the GLC analysis
of the same solutions. This discrepancy could be explained

by the possibility of a non-polar metabolite(s) in the
extract. Radioactive analysis of the water extract (Table

8) indicates that there may also be a water-soluble metabolite

14C-label remained in the

since 12.5% and 14.3% of the
aqueous portion of the soil replicates. In this case, how-
ever, both the zero time and sterile controls showed less
radioactivity in the water extracts.

The aromatic ring of alachlor was not metabolized to
14CO2 (Table 8) in the soil, suggesting that the apparent
degradation would have resulted in the transformation or
removal of at least one functional group.

In summary, the herbicide alachlor was cometabolized
by pure cultures of A. niger and Chaetomium sp.i The latter
fungus produced two possible non-polar metabolites. No
conclusion could be made as to the extent of the biodegraw
dation of alachlor in soil, though apparent disappearance

of the herbicide occurred with the production of both non~

polar and polar metabolites indicated.

REFERENCES

REFERENCES

Alexopoulos, J. C. 1952. Introductory mycology. John
Wiley and Sons, Inc., New York. pp. 271-303.

Armstrong, T. F. 1972. Factors affecting the response of
yellow nutsedge (Cyperus esculentus L.) to 2-chloro-
2',6'-diethy1-N-(methoxymethyl)acetanilide. Ph.D.
thesis, Michigan State University.

Bartha, R. 1968. Biochemical transformation of anilide
herbicides in soil. J. Agr. and Food Chem. 16:
602-604.

Bartha, R., and D. Pramer. 1967. Pesticide transformation
to aniline and azocompounds in soil. Science 156:
1617-1618.

Bergman, J. G., and J. Sanik. 1957. Determination of
trace amounts of chlorine in naphtha. Anal. Chem.
29: 241-243.

Bray, G. A. 1960. A simple efficient liquid scintillation
counter. Analyt. Biochem. 1: 279-285.

Hargrove, R. S., and M. G. Merkle. 1971. The loss of
alachlor from soil. Weed Sci. 19: 652-654.

Hovarth, R. S., and M. Alexander. 1970. Cometabolism of
m-chlorobenzoate by an Arthrobacter. Appl. Micro-
biol. 20: 254-257.

Lamaureux, L. G., E. L. Stafford, and S. F. Tanaka. 1971.
Metabolism of 2-chloro-N-isopropylacetanilide
(propachlor) in leaves of corn, sorghum, sugar cane
and barley. J. Agr. and Food Chem. 19: 346-350.

McLaren, A. D., and G. H. Peterson. 1967. Soil Biochemistry.
Marcel Dekker, Inc., New York. pp. 377-380.

Peterson, G. H. 1962. Respiration of soil sterilized by
ionizing radiation. Soil Sci. 94: 71-74.

Thompson, J. F., A. C. Walker, and R. F. Moseman. 1969.
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25

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