WATER POLLUTION CONTROL RESEARCH SERIES • I6O60 OMP 03/71
INTERACTION OF HERBICIDES
AND SOIL MICROORGANISMS
U.S. ENVIRONMENTAL PROTECTION AGENCY
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WATER POL
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INTERACTION OF HERBICIDES AND SOIL MICROORGANISMS
Boyce Thompson Institute for Plant Research, Inc.
1086 North Broadway
Yonkers, New York 10701
for the
OFFICE OF RESEARCH AND MONITORING
ENVIRONMENTAL PROTECTION AGENCY
Project #16060 DMP
March 1971
For gale by the Superintendent of Documents, U.S. Government Printing Office
Washington, D.C., 20402 - Price 78 cents
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Review Notice
This report has been reviewed by the EPA
and approved for publication. Approval
does not signify that the contents neces-
sarily reflect the views and policies of
the Environmental Protection Agency, nor
does mention of trade names or commercial
products constitute endorsement or recom-
mendation for use.
ii
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ABSTRACT
In pure culture and in soils the addition of 2,3,5,6-tetrachloro-
terephthalate (DCPA) had little or no effect upon bacterial growth,
and several microorganisms appeared to utilize the herbicide as a
carbon source, Methyl-2,3,5,6-tetrachloroterephthalate and
2,3,5,6-tetrachloroterephthalic acid were identified as degrada-
tion products,
A fungus, Penicillium paraherquei Abe, was isolated from soil that
had been treated previously with 5-bromo-3-se c-butyl-6-methyluraoil
(bromacil) and found to degrade bromacil in culture. When added to
sterile bromacil treated soil, the fungus resulted in enhanced
bromacil degradation. When added to non-sterile soil, probably due
to competition from other organisms, the fungus was ineffective in
hastening the degradation of bromacil.
The encouragement of the soil microflora by the addition of nutrient
broths resulted in a reduction of toxicity to plants of a number of
herbicides. These results indicate that the decontamination of soil
by the degradative activities of the natural microflora may be
accelerated by the addition of suitable nutrient sources.
A mixture of organisms cultured on isopropyl N-phenylcarbamate (IPC)
as the sole carbon source was used to study the influence of ring
chlorine on the degradation of a series of anilide herbicides. The
retarding effect of ring chlorine on the rate of ring degradation,
and for the most part, on microbial respiration, increased according
to the configuration sequence: 0 > 2,4 > 2,4,5 > 3 > 4 > 3,4. The
implications of this and the effects of other structural features on
degradation are discussed.
The IPC-degrading organisms were used in a series of experiments which
demonstrated that when added to soil they accelerated the degradation
of IPC and related compounds. A membrane •'biological filter" device
for reducing waterborne biodegradable pollutants was also demonstrated
using these organisms.
This report was submitted in fulfillment of Project No. 16060 EMP,
under partial sponsorship of the Environmental Protection Agency.
iii
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CONTENTS
Section
I Conclusions 1
II Be commendations 3
III Introduction 5
IV Interactions of Soil-borne Microorganisms and 7
Dimethyl Tetrachloroterephthalate
V Study of the Degradation of Dimethyl Tetrachloro 15
terephthalate
VI Microbial Degradation of Bromacil 19
VII Promotion of Herbicide Degradation in Soil by the 21
Application of Microbial Nutrient Broths
VIII Accelerated Degradation of Phenylcarbamates in Soil 29
by the Application of a Mixed Suspension of IPC-
cultured Microorganisms
IX Influence of Ring Chlorine on the Degradation of 37
Some Anilide Herbicides and Related Compounds by
IPC-cultured Microorganisms
X A Membrane Biological Filter Device for Reducing 49
Waterborne Biodegradable Pollutants
XI Relationship of Chemical Structure of Herbicides to 61
Degradation by Microorganisms
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Page
XII Acknowledgments ^
XIII References £o
XIV Publications
vi
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FIGURES
Page
The effect of residence time in the soil on the 36
ability of applied microorganisms to degrade IPC
as measured by corn yield.
Influence of various anilide substrates on the 46
respiration of IPC-cultured suspensions of
microorganisms.
Influence of ring chlorine on the degradation of 47
IPC»s and Anilines. Shown are UV scans of samples
taken from culture flasks at the indicated number
of days after compound addition. Figures at
lower right of each group are the percent ring
remaining after 9 days.
Influence of differing structures on rates of 48
hydrolysis and ring degradation. Shown are UV
scans of samples taken from culture flasks at
the indicated number of days after compound addition.
View of membrane biological filter device. Insert 59
at upper left shows detail of channels in PlexLglas
plates. At far right is bacterial bath and pump.
At left coming from pump is effluent line and
reservoir. At far left the feed stream line leads
in from reservoir which is not shown.
Dependence of effluent IPC concentration on flow rate 60
vii
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and type of ultrafilter material in the Plexiglas
model. Comparison is also made to the theoretical
maximum calculated for these conditions from
Equation 5.
viii
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TABLES
No.
1 Distribution of microorganisms in selected soils from 9
New York and Colorado
2 The effect of DCPA on the population of microorganisms 9
in the soil as measured by dilution plate technique
and expressed as colonies per sq cm
3 The effect of DCPA on growth of microorganisms as 10
measured by the percentage of 20 different isolates
which grew on media containing various quantities
of DCPA
4 The ability of microorganisms to utilize DCPA as a 10
sole carbon source as measured by the percentage
of different isolates which grew on the respective
media from the 20 tested
5 The degradation of DCPA by microorganisms as measured 11
by the recovery of the parent compound and the
monomethyl ester by gas liquid chromatography
6 The degradation of DCPA using ^°C1-labeled herbicide 12
as measured by the radioactivity of the various
extracts
7 Experiment !• Effect of broth addition and incubation 24
time on the yield of test plants grown in soil flats
treated with different herbicides
ix
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Page
8 Experiment II. Effect of broth addition and incubation 25
time on yield of test plants grown on soil flats
treated with different herbicides
9 Per cent difference between the mean foliage yield of 26
broth treated and no-broth control flats in Experi-
ment I and Experiment II
10 The effect of an IPC grown mixture of microorganisms on 32
IPC toxicity to test plants
11 The effect of an IPC grown mixture of microorganisms on 33
IPC, CIPC, and Swep toxicity to test plants
12 Summary of identification of eight isolates taken from 35
an unsterile suspension of IPC-cultured microorganisms
13 Degradation of IPC, Aniline and five of their chloro- 43
analogs
14 Degradation of eight compounds by four isolates 44
15 Rate of IPC degradation (|ig IPC/ng microorganism/hr) 51
at three levels of IPC and microbial concentrations
16 Influence of Mineral Salts concentration on a quantity 51
of IPC degraded over a two day period
17 Dialysis Tubing Model, Influence of flow rate and 52
IPC concentration of the feed stream on IPC
concentration of the the effluent
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SECTION I
CONCLUSIONS
The soil contains a rich and varied population of microorganisms
capable of degrading a vast number of natural and synthetic chemicals.
But many man-made compounds such as the herbicides studied are new to
the environment, and the natural soil microorganisms may not have the
capabilities required to degrade them. However, by continually
exposing a population of soil microorganisms to a particular molecule,
organisms can frequently be derived by selection or adaptation that
have such capabilities. These organisms can then be cultured and
used to rid soil or water of the chemical and in some cases structurally
related chemicals as well. The methods used here in demonstrating
the potential of such organisms for decontaminating soil and water
are probably not at this time ready for practical application, but we
believe may point the way to the future development of more practical
methodology.
The studies reported here, as well as those done by others on the
relationship of chemical structure to persistence in the soil in a
series of isomers and homologs, should be useful to chemists seeking
to develop molecules that combine herbicidal potency with minimum
persistence potential. Only with such information available can
synthesis chemists intelligently design the types of pesticides
needed in the years ahead if agriculture is to continue to produce
the food and fiber required by an expanding population but without
adverse environmental effects.
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SECTION II
RECOMMENDATIONS
The studies reported here and elsewhere have demonstrated that
microorganisms that are capable of degrading pesticides and other
synthetic organic molecules can be obtained from the soil. Ws
recommend that, since such organisms can be found or developed,
studies on their possible use in decontaminating soils and water
should be encouraged. Likewise, the continued development of
information on the relationship of chemical structure to environ-
mental persistence should be encouraged since chemists will then
have the information necessary for the synthesis of biodegradable
nonpersistent pesticides.
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SECTION III
IMrRODUCTION
The use of herbicidal chemicals to control plant growth is increasing
at a rapid rate. They have become an integral part of cultural
practices on farms, home gardens, parks, and roadways. With the ever-
increasing use of herbicides and other pesticidal chemicals, it has
become obvious that there are many problems associated with the intro-
duction of such large volumes of toxic chemicals into the environment.
Herbicide residues in the soils may be picked up by plants which when
used as food or fodder represent a hazard to man and animals. Rain
may wash them into streams where they may contaminate our surface water
supplies or they may leach downward into underground water sources.
Although they may be present only in sub-toxic quantities, the long-
term effects of such materials are not known and their presence can
be considered only as undesirable. Microbiological degradation of
herbicidal chemicals in the soil appears to be one of the prime routes
for their dissipation from the environment. The rate and ease of
their degradation is known to vary, but the influence of environmental
factors and the chemistry of these processes is poorly understood.
A study of some of these processes was therefore undertaken with the
aim of giving us a better understanding of the problem. A better
understanding of the effects of chemical structure on decomposition
kinetics should be useful to those involved in the designing of new,
active, but more biodegradable, herbicides. The study of some of the
organisms involved indicated that it may be possible to use cultural
practices that will favor their activity and thus decrease the amount
of herbicide residues in the soil. The introduction of microorganisms
with specific degradative capacities into the soil was shown to be
a possible means of ridding the soil of contaminating chemicals.
Using these same organisms a "biological filter" system effective in
removing a herbicidal chemical from water was developed. The procedures
involved in these studies, the results obtained, and their possible
significance are discussed in Sections IV-XI.
An investigation of the interactions of soil microorganisms and several
groups of herbicidal compounds, primarily chlorinated derivatives, was
made. Specific objectives were: 1) to isolate and characterize
microbial species responsible for complete or partial herbicide degrada-
tion; 2) to characterize the rates of degradation of structurally re-
lated herbicides; 3) to study the influence of environmental factors
such as supplemental substrates, previous adaptation of microorganisms,
etc., upon herbicide degradation; 4) to identify the routes of degrada-
tion and fate of degradation products; and 5) to determine if micro-
organisms found capable of rapidly degrading a herbicide can be used
to decontaminate soil and water.
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INTERACTIONS OF SOIL-BORNE MICROORGANISMS AND
DIMETHYL TETRACHLOROTEREPHTHAUTE
Since the discovery at Boyce Thompson Institute (Limpel, et al., 1959)
of the pre-emergence herbicidal activity of dimethyl-2,3,5,6-tetra-
chloroterephthalate (DCPA or Dacthal), its use for the control of
crabgrass, other grass annuals and certain broadleaved weeds in a
number of crops has increased rapidly. Although its efficacy as a
herbicide has been the subject of many investigations, its inter-
actions with soil microorganisms had not been thoroughly investigated.
The latter area was, therefore, selected for study.
Methods
General. Soil samples were collected from the Boyce Thompson
Institute Farm to which DCPA had been applied previously at a rate of
19 Ib/A each year for five years and from similar plots that had not
been treated with DCPA, An additional soil sample was obtained from
a Colorado field where DCPA had been ineffective as a herbicide.
Untreated Colorado soil was not available.
Populations of microorganisms in soil samples were determined by
dilution plate techniques* Commercial nutrient agar was used for
these platings as well as for maintaining stock cultures, A minimal
medium used in many of the studies contained 0,05 M glucose, 5,0 X
10~3 M dibasic potassium phosphate, 1,0 X ICH* M magnesium sulfate
and 0,01 M sodium nitrate,
Effect of DCPA upon the populations of soil microflora. Determina-
tions of soil microbial populations were made immediately after the
soil samples had been collected. Subsequently, samples of these soils
were mixed with DCPA at the rate of 20 Ib/A and incubated at 37° C in
nearly air-tight containers to maintain moisture content. Samples
treated in a similar manner but without herbicide treatment served as
controls. Sub-samples of each soil treatment were removed periodi-
cally and the populations of bacteria and actinomycetes determined.
Each treatment was replicated five times.
The DCPA tolerance of microorganisms in pure culture was determined
by incorporating DCPA into the minimal medium defined above at 1,
10, 100 and 1000 ppm. The agar used for these experiments had been
extracted previously with 80% ethanol by refluxing for 16 hours to
free the agar of extraneous carbon sources. Cultures of bacteria
and actinomycetes were transferred to plates containing the media of
varying DCPA concentrations using the replicate plate technique
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(Lederberg and Lederberg, 1952),
The ability of selected microorganisms to utilize DCPA as a sole
carbon source was investigated by substituting various concentrations
of DCPA for glucose in the minimum medium. Studies on solid media
(1.8$ agar) were supplemented by using a liquid medium of a similar
constitution. In the case of the liquid medium, aliquots were
subsequently removed and plated to determine any change in numbers
of viable cells.
Degradation studies. Selections were made from the bacterial
isolates based upon their ability to utilize DCPA as a sole carbon
source. Cultures were incubated for 96 hours and extracted as
follows. The microbial cells were removed and the ambient solution
adjusted to pH 12 with 5 N sodium hydroxide in order to form the
sodium salt of the acid forms of DCPA. The cells and the supernatant
were each extracted with dichloromethane. The aqueous solution was
then adjusted to approximately pH 3 with concentrated hydrochloric
acid and extracted with ethyl acetate. The quantities of DCPA and
methyl-2,3,5,6-tetrachloroterephthalate were analyzed by gas liquid
chromatography (GLC).
For GLC determinations, the dichloromethane and ethyl acetate
solutions were evaporated to dryness and the residues dissolved in
acetone. A Wilkins Gas Chromatograph Model 204 equipped with an
electron capture detector was used for the GLC determinations. The
following column conditions were used: 5$ Dow 11 on 60 to 80-mesh
Chromosorb W (HMDS), oven temperature 180° C, injector temperature
for dichlororaethane fractions 250° C, for ethyl acetate fractions
325° C, and the nitrogen flow rate was 40 ral/min.
To study the removal of chlorine from the ring ^Cl-labeled DCPA
(0.1 nc/100 ml of solution) was used. Two bacterial selections
which tolerated high concentrations of DCPA and appeared to degrade
it were used for these studies. The concentration of cells when
the labeled herbicide was added was approximately 1 x 10? cells/ml
After incubation the cells and the ambient solution were extracted
separately with dichloromethane and ethyl acetate. After extraction
with ethyl acetate, the aqueous solutions were adjusted to pH 12
with sodium hydroxide to prevent the loss during drying of any
chlorine present. Aliquots of each of the extracts were evaporated
to dryness at room temperature in a hood. Radioactivity was
determined with a low background (2 cmp) gas flow counter.
Results
Interaction studies, Plate counts showed that the ratio of acting
mycetes to bacteria was higher in soils previously treated with DCPA
8
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than in untreated soils (Table l). In New York soils, the percentage
of actinomycetes was three times greater than in untreated soils* In
Colorado soil, the percentage and population of actinomycetes were
much higher than in the New York soil. Gram-negative rod bacteria
were absent in the New York soil treated with DCPA, but comprised a
high percentage of the microbial population in untreated soils. How-
ever, Gram-negative rods were prevalent in the previously treated
Colorado soils.
Table 1. Distribution of microorganisms in selected soils from New York
and Colorado
— New York
Microorganism Treated Untreated Colorado
Bacteria, Gram + coccoid
Bacteria, Gram + rods
Bacteria, Gram - rods
Actinomycetes
29
41
0
30
27
45
27
9
6
19
19
56
The initial population of microorganisms in previously treated New
York soil was slightly lower than in untreated soil (Table 2). The
addition of DCPA to previously treated soil caused a depression in the
rate of microbial increase, whereas, treatment of previously untreated
soil resulted in a slower increase in microbial population than for
the control. These results indicate that, although DCPA has little
effect upon the total microbial population, it may select the
organisms which predominate in the soil.
Table 2. The effect of DCPA on the populations of microorganisms in
the soil as measured by dilution plate technique and
expressed as colonies per sq cm
Time in days
Soil sample
Colorado
Colorado
New Yorka
New York.
New York
New York
Dilution
105
105
icA
Iff*
icA
icA
Treated
Control
Treated
Control
Treated
Control
0
72
70
69
69
59
59
1
172
64
98
150
120
134
4
00
70
198
348
406
475
7
00
90
340
522
750
890
10
00
150
520
853
805
00
14
00
200
650
GO
00
00
a No previous DCPA treatment. b Previous DCPA treatment.
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When the herbicide was incorporated into a minimal medium containing
a carbon source, it appeared to have little detrimental effect upon
the growth of the Gram-positive and Gram-negative rod bacteria at
concentrations of 100 ppm (Table 3)» DCPA had no effect upon any of
the organisms at 1 and 10 ppm, thus those values are not reported*
Fewer of the coccoid bacteria grew on media containing DCPA at 100
and 1000 ppm than at 1 and 10 ppm. Slightly more selections of the
actinomycetes grew on the media containing 100 and 1000 ppm than at
concentrations below 100 ppm.
Table 3« The effect of DCPA on growth of microorganisms as measured
by the percentage of 20 different isolates which grew on
media containing various quantities of DCPA
MM +MM +Water Water agar
Microorganism MMa 100 ppm 1000 ppm agar extracted b
Actinomycetes
Bacteria,
Bacteria,
Bacteria,
Gram +
Gram -
Gram +
rods
rods
coccoid
90
90
75
65
100
85
65
25
100
30
45
25
85
35
25
25
5
15
6
5
a Minimal medium.
b Extracted with 80$ ethanol by refluxing for IB hours before
preparation of agar medium.
When the isolates were grown on an agar medium containing DCPA as
the sole carbon source, few bacteria grew (Table 4). More bacterial
isolates grew at 1000 ppm than at 100 ppm or less. However, several
of the actinomycetes grew at 100 ppm and nearly all actinomycetes
selections grew at 1000 ppm. These data were confirmed by growing
the organisms in liquid media and subsequently plating an aliquot to
determine the increase in cell population.
Table 4« The ability of microorganisms to utilize DCPA as a sole
carbon source as measured by the percentage of different
isolates which grew on the respective media from the 20 tested
Microorganism
Actinomycetes
Bacteria
MM4
95
80
MM minus
carbon
source
15
5
MM without fllucose
100 DTO 1000 mm
25 90
5 15
10.000 ircra
90
15
Minimal medium.
10
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Degradation studies« Less than one-third of the DCPA was recovered as
the parent compound or the monomethyl ester (Table 5)« Most of the
monomethyl ester was recovered from the cells, and only a trace was
recovered from the ambient solution, thus it appears that degradation
occurs intracellularly. All selections of microorganisms tested
appeared to be equally capable of degrading DCPA*
Table 5« The degradation of DCPA by microorganisms as measured by the
recovery of the parent compound and the monomethyl ester by
gas liquid chromatography
DCPA Recove
Microorganism
ry
% Re-
Monomethyl eater Recovery
Liq- Bac- % Re- Liq-Bac- % Re- Total
uid terial covery uid terial covery % Re-
cells cells covery
Bacteria, Gram +
coccoid
Bacteria, Gram +
rod
Bacteria, Gram
variable rod
Actinomycete
Bacteria, Gram +
rod
Bacteria, Gram +
rod
Actinomycete
Control
21
14
20
14
29
15
12
188
13
8
5
2
trace
18
3
trace
17
11
13
8
15
17
15
94
0
1
1
0
trace
trace
trace
trace
10
15
1
3
5
1
5
*•
5
8
1
2
3
1
-
0
22
26
14
10
18
18
10
94
When 36ci-labeled DCPA was incubated with two bacterial selections,
very little radioactivity was found in the aqueous solution (Table 6)
which indicated that little if any chlorine was freed from the ring.
It is probable that the activity remaining in aqueous solution was a
result of incomplete extraction with dichloromethane or ethyl acetate
rather than a dechlorination of the ring moiety.
More than 80$ of the radioactivity was found in the ethyl acetate
fraction for both selections. This fraction would contain both the
monomethyl ester and acid fragments of DCPA, Nearly all of the radio-
activity was associated with the microbial cells, thus confirming the
previous supposition that DCPA is degraded intracellularly.
11
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Table 6. The degradation of DCPA using 36ci-labeled herbicide as
measured by the radioactivity of the various extracts
Ambient solution Bacterial cells
Dichloro- Ethyl Water Dichloro- Ethyl Water
methane acetate methane acetate
Bacterium,
coccoid
Bacterium,
variable
Gram •*•
Gram
rod
1200s
1800
1400
1700
58
48
670
1580
10200
14110
413
256
a Counts per minute.
Discussion
The data show that DCPA has little or no adverse effects upon the
population of soil-borne microorganisms. The data also indicate that
growth of some of the actinomycetes is enhanced by the presence of high
concentrations of DCPA, which possibly serves as a carbon source* When
the herbicide was added to a liquid medium and its disappearance studied
little or no difference could be found between the ability of the micro-
organisms to degrade DCPA, All cultures appeared to degrade DCPA to
about the same extent; however, it is doubtful that this herbicide is
an important carbon source for microorganisms. At the low concentra-
tions which usually would be encountered in the soils, there was al-
most no stimulatory effect and the amount of carbon being supplied
would be negligible.
The stimulatory effect on growth when high concentrations of DCPA were
used was unexpected in view of the fact that its solubility is approxi-
mately 0,5 ppnu Thus, at concentrations above 0,5 ppm, aqueous solu-
tions would be saturated. It is possible that the bacteria set up a
microenvironment around the herbicide particles, and concentrations of
100 ppm and above are required to supply sufficient carbon to result
in measurable growth. Another possibility is that, at these high
concentrations, the quantity of DCPA dissolves more quickly as it is
being removed from the microenvironment by the microorganisms.
The data (Tables 5 and 6) indicate that DCPA is being degraded by
microorganisms and two degradation products are monomethy1-2,3,5,6-
tetrachloroterephthalate and 2,3|5,6-tetrachloroterephthalic acid.
These two degradation products have been found to occur under field
conditions by Skinner et al.. (1964), who proposed that the hydrolysis
of the methyl esters occurs mainly in the soil and very little hy-
drolysis occurs in plants and animals,
12
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Fields et a^L. (196?) found that several groups of organisms including
paramecia, fungi, bacteria, and algae would tolerate DCPA and a
stimulatory effect often was observed. They also found no buildup of
DCPA when applied over a 7-year period.
Unfortunately, a quantitative method could not be employed succesfully
to determine the quantity of 2,3*5>6-tetrachloroterephthalic acid in
these studies, and the method for determining monomethy1-2,3,5»6-tetra-
chloroterephthalate was only semi-quantitative* Mien the injector
oven was raised to a temperature sufficiently high to decarboxylate this
compound to methyl tetrachlorobenzoate, a constant temperature for the
column oven could not be maintained. "When known amounts of the mono-
methyl compound were analyzed, decarboxylation varied greatly with
temperature.
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SECTION V
STUDY OF THE DEGRADATION OF DIMETHYL TETRACHLOROTEREPHTHALATE
It is frequently possible, upon examination of the structure of a
herbicide molecule, to hypothesize a plausible series of degradative
steps. Thus a rational approach for the study of the decomposition
of such compounds as herbicides in a biological environment can be
developed. Identification of specific intermediates may allow for the
reconstruction of a possible degradative pathway.
The proposed degradation path of dimethyl tetrachloroterephthalate (l)
to 1,2,4,5-tetrachlorobenzene (VI) is shown in Scheme I below. Likely
points of attack on the dimethyl tetrachloroterephthalate (I) (DCPA)
molecule are the ester linkages which may be cleaved singly or simul-
taneously, resulting in monomethyl tetrachloroterephthalate (II) and
tetrachloroterephthalic acid (III), respectively, II may be decarbox-
ylated to yield methyl 2,3,5,6-tetrachlorobenaoic acid (IV), and III
may also be mono or didecarboxylated to yield 2,3,5.6-tetrachloro-
benzoic acid (V) and 1,2,4,5-tetrachlorobenzene (VI), respectively.
The disappearance of VI from soil may be partly due to its high
volatility, according to Josephs et al. (1957), Hydrolysis of the
chlorine atoms followed by ring opening with subsequent degradation
to innocuous small molecules should also be considered.
Previous work on the presence of degradation products of I in plants
grown in I-treated soil was carried out by Skinner et al. (1964)• II
and III were found in plant tissues; but compounds I, IV, V and VI were
not detected. This may be due, in part, to the extraction procedure
employed in which the plant extract was partitioned between ether and
water at pH 6, The ether phase, which was discarded, could have
contained I, IV, and VI.
The work reported here was concerned with the development of an
analytical method for the detection and identification of compounds I
to VI, and some preliminary data on findings in I-treated soil,
Since, among the proposed degradation products, II, III, and V would
be susceptible to thermal decarboxylation in the gas chromatograph,
the products formed could be confused with proposed soil degradation
products. Consequently, all free carboxylic acids were converted to
the acid chlorides, by means of thionyl chloride, which in turn were
treated with propanol to form the propyl esters. These derivatives
would be stable, sufficiently volatile, and unlikely to be found in
soil as such.
15
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COOCH3
COOCH3
COOCH3
VI
Scheme I. Proposed degradation pathways of dimethyl tetrachlorotereph-
thalate (l) to 1,2,4,5-tetrachlorobenzene (VI).
Methods and Materials
Compounds* I, II (as sodium salt), III, and tetrachloroterephthaloyl
chloride were obtained from Diamond Shamrock Company, Painesville,
Ohio; V and 2,3,5,6-tetrachlorobenzoyl chloride were obtained from
Hooker Chemical Corp., Niagara Falls, N. Y.; and VI was purchased from
Aldrich Chemical Company, Inc., Milwaukee, Wis. The remaining esters,
IV, propyl 2,3,5»6-tetrachlorobenzoate (VII), methyl propyl tetrachloro-
terephthalate (VIII), and dipropyl tetrachloroterephthalate (IX) were
prepared.
Methyl 2.3.5.6-tetrachlorobenzoate (IV). A mixture of 2.0 g. (0.0076
mole; of 2,3,5»6-tetrachlorobenzoic acid, 20 ml of thionyl chloride,
and 1 drop of pyridine was heated under reflux overnight. Excess
thionyl chloride was removed by vacuum evaporation, and to the residue
was added 40 ml of methanol. The solution was again heated overnight,
after which the solvent was flash evaporated. The yield of crude
product was 1.85 g (88$), m.p. 66° to 68° C. An analytical sample was
crystallized from aqueous ethanol, m.p. 68° to 69° C. A similar yield
of product was obtained from the preformed acid chloride.
Anal, calcd. for CgfyCl^: C, 35.08; H, 1.47; 01, 51.77. Found:
C, 35.36; H, 1.45; Cl, 51.33.
16
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Propyl 2.3 . 5 1 6-tetrachlorobenzoate (VII) was prepared from 2,3,5,6-
tetrachlorobenzoic acid, as well as from the preformed acid chloride
in 86 to 88% yield. A sample was crystallized from aqueous ethanol
for analysis, m.p. 74° to 75° C.
Anal, calcd. for CioHgCl/C^: C, 39.77; H, 2.6?; Cl, 46.96. Found:
C, 39.94; H, 2.45; Cl, 46.72.
Methyl propyl tetrachloroterephthalate (VIII) was prepared from sodium
methyl tetrachloroterephthalate in 95$ yield in the same manner as IV.
The sample for analysis was crystallized from aqueous ethanol, m.p.
65.5° to 66.5° C.
Anal, calcd. for CioHioCl^: C, 40.03; H, 2.80; Cl, 39.39. Found:
C, 40.26; H, 2.60; Cl, 39.09.
Dipropyl tetrachloroterephthalate (IX) was prepared from tetrachloro-
terephthalic acid, as well as from the preformed acid chloride in 84
to 86^ yield. An analytical sample was obtained by crystallization
from aqueous ethanol, m.p. 88.5° to 89° C.
Anal, calcd. for C^H^Cl^: C, 43.33; H, 3.64; Cl, 36.55. Found:
C, 43.38; H, 3.39; Cl, 36.65.
Analytical Procedure. All separations were carried out in an Aerograph
Model 204 gas chromatograph fitted with an electron capture detector.
The column employed was 5 ft X 1/8 in. o.d. stainless steel packed with
5,t hexamethyl disilazane on Chromosorb W (80/100 mesh) (QF-I). Reten-
tion data were obtained under isothermal conditions. For the standard
compounds the column temperature was kept at 193° C., and the detector
and injector temperatures were 199° C. and 247° C., respectively, with
a flow rate of nitrogen at 25 ml/min. The soil extract was chromato-
graphed at column, detector, and injector temperatures of 180° C.,
190° C., and 205° C., respectively, and the flow rate of nitrogen was
31.5 ml/min.
Preparation of Soil Extract. A 100-kg soil sample collected to a depth
of 24 in. was obtained from a plot of land which had been treated with
a cumulative total of 94 Ib/acre of I in five annual treatments, and
then remained untreated for three additional years. The sample was
twice extracted with acetone at pH < 2 overnight. The acetone extract
was evaporated to near dryness under vacuum, and residual water was
removed by azeotroping with benzene. After flash evaporation of the
benzene, the residue was dried over sulfuric acid for one week, and
83.5 g. of dry product resulted. For analysis, 5.0 g. of soil extract
was heated under reflux with 50 ml of thionyl chloride overnight. The
unreacted thionyl chloride was removed by vacuum distillation, and the
residue was refluxed with 50 ml of propanol for 48 hours until very
17
-------
little hydrogen chloride continued to evolve. The propanol was flash
evaporated and the residue dissolved in carbon tetrachloride. The
solution of esters was washed several times with water and dried by
azeotropic distillation. The carbon tetrachloride was flash distilled,
and the dry residue was dissolved in a known volume of acetone and
was chromatographed.
Results and Discussion
A mixture of I, IV, and VI to IX was resolved in the gas chroraatograph.
The feasibility of separating and identifying a mixture of compounds
related to the proposed degradation products of I was thus demonstrated.
When the extract of I-treated soil was chromatographed peaks 1, 2, and
3, on the basis of retention times were suspected as being due to the
presence of compounds, I, VIII, and IX, respectively. Since these peaks
were reasonably well separated from one another, small portions of
authentic samples of I, VIII, and IX were added to another portion of
extract, and the mixture chromatographed. Upon co-chromatographing
the extracted compounds in the presence of true samples, no separa-
tion was apparent. Thus peaks 1, 2, and 3, were tentatively identi-
fied as compounds I, VIII, and IX, which indicates the presence of
dimethyl tetrachloroterephthalate (l) as a residue in soil and
monomethyl tetrachloroterephthalate (II) and tetrachloroterephthalic
acid (III) as degradation products. Further work would be necessary
to establish whether or not the remaining proposed degradation
products of dimethyl tetrachloroterephthalate can be identified in
soil extracts.
18
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SECTION VI
MIGROBIAL DEGRADATION OF BROMACIL
Since bromacil, 5-bromo-3-sec-butyl-6-methyluracil, is recommended for
use in general weed control in noncropland areas at rates up the 24 lb/A,
its use, unless degraded readily by soil microorganisms, may present
an environmental hazard. Studies were, therefore, initiated on the
microbiological degradation of bromacil and its effects on soil micro-
organisms .
Soils having no history of exposure to bromacil were treated in the
laboratory with 40 ppm of bromacil in a soil perfusion system (Audus,
1946 and I960) or by mixing at rates up to 200 Ib/A and allowed to
incubate. Using dilution plate techniques (potato-dextrose agar for
fungi and nutrient agar for bacteria), fungi and bacteria were isolated
from the various treated soils. Fifty-five fungus and 73 bacterial
cultures were selected and their ability to degrade bromacil studied
by growing them in a Czapek-Dox broth containing 20 ppm of bromacil
and periodically determining the amount of bromacil present using a
bioassay system in which bromacil at concentrations down to less than
1 ppm can be detected using buckwheat as the test organism. None of
the bacterial cultures appeared to degrade bromacil, but four of the
fungus cultures exhibited this capability. One of these cultures
which has been identified by the Centraalbureau voor Schimmelcultures,
Baarn, The Netherlands, as Penicillium paraherquei Abe, was particu-
larly active and was selected for further study. No significant
amounts of bromacil could be detected 15-20 days after Czapek-Dox
broth containing 20 ppm of the herbicide had been inoculated with this
organism. Sterile soil treated with bromacil at a rate of 3»1 lb/A
was still toxic to buckwheat after 56 days but no herbicidal effects
could be detected 21 days after treatment with 6,2 lb/A or 28 days
after treatment with 12.5 lb/A in sterile soil inoculated with JP.
paraherquei. Herbicidal effectiveness of the 25 and 50 lb/A applica-
tions was reduced to 50 and 80$, respectively, after 56 days. This
organism was also able to degrade Terbacil (3-tert-butyl-5-chloro-6-
methyl uracil), a closely related compound. However, when non-sterile
soil was inoculated with P. paraherquei. no degradation of either
Bromacil or Terbacil was observed. Apparently this organism is not
sufficiently aggressive to compete with the natural soil microorganism
population.
When liquid and solid Czapek-Dox media containing Bromacil were
inoculated with P. paraherquei, the fungus grew normally at concentra-
tions up to 200 ppm, but at higher levels growth was slower and more
compact in appearance. Transfers from liquid media containing 800-
1000 ppm of Bromacil yielded variant cultures characterized by the
lack of the yellow pigment associated with the original culture. When
19
-------
inoculated into a liquid mineral salts medium containing Bromacil as
the sole carbon and nitrogen source, the variant grew slowly, whereas,
the original culture failed to grow. When included in the medium at
2,000 ppm, Bromacil crystals precipitated out of solution, and mycelia
of the variant fungus were observed growing on their surface. Addi-
tional studies on the degradative properties of the variant and the
ability of Bromacil to produce variants are underway.
Following continued maintenance in culture, our isolates of P.
paraherquei lost thejr ability to degrade Bromacil; By continual
growth on culture media containing Bromacil, this ability could be
regenerated but was soon lost again when grown on non-Bromacil contain-
ing media. Due to this instability and lack of aggressiveness in un-
sterile soil and because more interesting and promising organisms
were being isolated in other aspects of the program, work was discontin-
ued with P. paraherquei.
20
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SECTION VII
PROMOTION OF HERBICIDE DEGRADATION IN SOU BY THE APPLICATION OF MICRO-
BIAL NUTRIENT BROTHS
Pesticides applied to cropland often remain in the soil longer than is
desirable, and may find their way into water resources or other ecosys-
tems via groundwaters and harvested foliage. Herbicides may remain in
the soil until the next growing season and limit land use and crop
rotation options. To control the amounts and lifetimes of these sub-
stances in soil, a preventive approach, such as superior design and
practice, would be far more favorable to a corrective approach. How-
ever, several corrective approaches have been suggested including the
application to soil of absorbants, chemicals, and even UV light as well
as rotation, tillage and irrigation practices (Foy and Bingham, 1969;
Kearney, et al., 1969). Application of microbes having the capacity to
degrade herbicides has also been suggested (Audus, 1951) and attempted
(Kearney, et al., 1969; MacRae and Alexander, 1965). Stimulation of
the indigenous microflora to attack pollutants is yet another possi-
bility. For example, it has long been known that the decomposition
of resistant soil organic matter is increased by the addition of easily
degraded materials (Bartholomew, 1957). It has also been noted that
microbial degradation of certain herbicides and related compounds is
accelerated by the addition of supplementary nutrients such as those
found in ordinary microbial culture broths (Barbha, et al., 1967).
In this context, the application of nutrient materials to lands that
have been polluted may in some instances be a practical possibility.
The purpose of the research reported below was to seek data in support
of this suggestion.
Materials and Methods
The principal objective of our experimental approach was to contrast
residue levels on broth treated soils with those on no-broth treated
soils. Two major experiments were undertaken which were similar in
design and differed only in the herbicides studied, the kinds of
nutrient broths added, and the lengths of incubation time between
broth addition and measurement of residual herbicide. It was decided
that a bioassay rather than a chemical assay would be used to measure
residue level after treatment. This choice, although sacrificing
accuracy and precision, has the advantages of convenience and biolog-
ical significance.
The herbicides arbitrarily chosen for study were diphenaraid (N,N-
dimethyl-2,2-diphenylacetamide), monuron [ 3-(p-chlorophenyl)-!,!-
dimethylurea], atrazine (2-chloro-4-ethylaniino-6-isopropylamino-si-
triazine), dicamba (2-methoxy-3»6-dichlorobenzoic acid), CIPC [iso-
21
-------
propyl N-(3-chlorophenyl)carbamate], and amiben (3-amino-2,5-
dichlorobenzoic acid). These herbicides were applied to the surface
of a composted Norfolk dry loam which was contained in small rect-
angular flats (7.5 in. wide, 9 in. long, 4 in. deep). Rates of
application were selected to give an adequate response but not an
excessive one under these conditions. The rates chosen were calcu-
lated on a surface basis and were at the lower limit of the dosage
range recommended for field soils in the WSA handbook (Weed Society
of America, 196?) and were as follows in Ib/A: diphenamid 3, monuron
1.5, atrazine 1.5, dicamba 0,9, CIPC 1.8, and amiben 1.8. Test
plant species selected on the basis of their sensitivity to all the
herbicides used in the study were foxtail grass (Alopecurus pratensis
L.), Johnson grass [Sorghum halepense (L.) Pers.], crabgrass [Digitaria
sanguinalis (L.) Scop.1, oats (Avena sativa L.), velvet leaf (Abutilon
theophrasti Medic.), and mustard (Brassica sp.). Uncertainty over
which species was the most sensitive indicator for any particular
herbicide and the desirability of having comparable estimates of all
residue levels for all six herbicides led to the decision to use all
six species simultaneously in the bioassay. Therefore, the estimate
of residual activity was made by collectively harvesting any living
plant material remaining in the soil flat. The assumption was made
that under these conditions and for the rates used the yield was
acceptably proportional to the effective soil concentration and that
any abnormalities in growth were also manifested as a yield effect.
In Experiment I, each herbicide was applied to 12 soil flats by
spraying onto the moist soil surface 40 ml of a formulated suspension.
Immediately following treatment, 4 of the 12 flats were sprayed with
200 ml of water, 4 were sprayed with 200 ml of double strength Czapek
Dox broth, and 4 were sprayed with 200 ml of double strength Difco
nutrient broth. The Czapek Dox broth contained the following sub-
stances in grams per liter: sucrose 30, NaN03 3, K2HP04 1, MgS04 0.5,
KC1 0.5, FeS04 0.01. The Difco nutrient broth contained 3 g/1 of
beef extract and 5 g/1 of peptone. The flats were then incubated at
30° C« Two of the flats (replicates) in each group of 4 were planted
7 days after treatment (Group I); the remaining 2 (Group II) were
planted 21 days after treatment. Group II flats also received two
additional broth treatments 7 and 14 days after the original treatment.
Thus Group II flats represented a more intensive treatment and a longer
time interval through which the treatment might act. Three weeks after
planting, all green foliage was collectively harvested without regard
to species, dried and weighed.
Experiment II was designed identically to Experiment I with the follow-
ing exceptions: diuron [3-(3,4-dichlorophenyl)-l,l-diinethyl urea] was
tested at the rate of 1.5 lb/A in addition to diphenamid, monuron, and
atrazine at the same rates as in Experiment I. Instead of 2 groups
22
-------
there were four groups differing both in incubation time and in the
amount of nutrient broth subsequently added after the initial broth
treatment. Group I flats were incubated 2 weeks after the initial
broth treatment. Group II flats were incubated 4 weeks and received
one additional broth treatment after 2 weeks. Group III flats were
incubated 7 weeks and received additional broth treatments at 2, 4>
and 6 weeks. Group IV flats were incubated 15 weeks and received
additional broth treatments at 2, 4, 6, 8, and 10 weeks. Thus, as in
Experiment I, Groups II, III, and IV represented successively more
intensive broth treatment as well as longer incubation times. Finally,
instead of applying two different kinds of broth, as in Experiment I,
only one kind of broth was added. This was a combination of double
strength Czapek Dox broth and Difco nutrient broth in the proportion
of three parts to one, respectively. After the stated incubation
time, all flats were planted and harvested 4 weeks later.
Results and Discussion
The dry weight yields of plants in each flat for Experiments I and II
are recorded in Tables 7 and 8, respectively. The data at each time
period are representative of a bioassay at that time for the amount
of herbicide remaining in the soil after treatment. Yields of replicate
broth and no-broth treated flats are grouped in adjoining columns along
with their means for easy comparison. Although the quantitative re-
lation between residue levels and yield is not known, it may be
assumed nevertheless that the two are in some straightforward inverse
relationship. Conversely, the toxicity and the residue level are
directly related. Therefore, any reduction in herbicide toxicity
with time and/or broth treatment may be interpreted as a reduction
in residue level as a result of a particular rate of microbial degrada-
tion.
It is evident that there was an apparent beneficial effect of broth
treatment alone and also an effect due to variable greenhouse condi-
tions during the different bioassay periods. For this reason all the
data of Table 7 and 8 have been re-expressed in Table 9 as the percent-
age difference between the means of broth treated and no-broth treated
flats. Any percentage figure which does not substantially exceed
its appropriate no-herbicide control value is probably insignificant
as a measure of accelerated herbicide degradation by the broth.
Percentage differences preceded by a negative sign signify a lower
yield on the broth treated flats than on the no-broth treated flats.
Positive figures are a measure of the effectiveness of the broth
treatment relative to the level of toxicity in the soil after any
incubation time.
23
-------
Table 7. Experiment I, Effect of broth addition and incubation time
on the yield of test plants grown in soil flats treated with
different herbicides.
Yield of foliage after 3-week growing period
(g. dry wt/flat)
Incubation time and number of broth treatments
Herbicide Rate
applied Ib/A
Diphenamid 3
Monuron 1.5
Atrazine 1.5
Dicamba 0.9
CIPC 1.8
Amiben 1.8
No-herbicide -
control
Rl
R2
X
Rl
R2
X
Rl
Rg
X
Rl
Eg
X
R!
fig
X
R!
Rg
X
R!
Rg
X
1 week
No-broth
control
0.78
0.66
0772
0.01
0.06
0.04
0.17
0.12
OJA
0.51
0.40
(5746
1.33
1.09
1.21
0.46
0.50
0748
1.74
1.82
1778
Group I
- 1 treatment
Czapek
Dox
broth
0.63
1.00
0782
0.02
0.01
07(52
0.15
0.12
0.14
1.78
1.72
1.75
2.40
1.78
2709
0.84
0.92
0788
2.06
2.24
27l5
Difco
nutrient
broth
1.30
0,94
1.12
0.01
0.01
oToi
0.15
0.17
oTIo"
1.06
0.50
0.73
2.37
2.23
2730
0.50
0.63
0755
2.29
2.28
2728
3 weeks
No-broth
control
0.66
0.34
0.50
0.18
0.05
0.12
0.09
0.12
0.10
1.77
2.31
2.04
2.58
2.34
274^
0.57
1.19
0738
2.24
2.08
2.16
Group II
- 3 treatments
Czapek
Dox
broth
1.65
1.85
1.75
0.24
0.12
0713
0.20
0.09
0.14
3.14
3.10
3.12
3.56
3.89
3.72
1.50
1.86
1738
2.35
3.12
2.74
Difco
nutrient
broth
0.96
0.94
0.95
0.05
0.02
0.04
0.15
0.23
0.19
1.99
1.48
1.74
2.78
2.80
2779
0.55
0.78
0735
2.92
1.64
2728
24
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Table 8. Experiment II. Effect of broth addition and incubation time
on yield of test plants grown on soil flats treated with
different herbicides
Herbicide
applied
Diphenamid
Monuron
Atrazine
Diuron
No-herbicide
control
Yield of living foliage after 4-wk growing period
(g. dry wt/flat)
Incubation time and number of broth treatments
Group I Group II Group III Group IV
Rate 2wk- 4 wk - 7wk- 15 wk -
Ib/A 1 treatment 2 treatment 4 treatment 6 treatment
No- Com- No- Com- No- Com- No- Corn-
broth bined broth bined broth bined broth bined
con- broth con- broth con- broth con- broth
trol trol trol trol
3 HI 0.00 0.00 0.01 0.02 0.04 0.25 0.35 0.16
R2 0.00 0.00 0.01 0.08 0.16 0.89 0.31 0.49
x 0.00 0.00 0.01 0.05 0.10 0.57 0.33 0.32
1.5 RI 0.08 0.20 0.53 1.95 1.35 - - 2.88
R2 0.05 0.18 0.82 2.25 1.6? 2.68 2.11 2.55
x 0.06 0.19 0.68 2.10 1.51 2.72
1.5 RI 0.03 O.Q3 0.14 0.33 0.60 1.30 1.80 1.49
R2 0.03 0.03 0.10 0.23 0.37 2.41 1.94 1.24
x 0.03 0.03 0.12 0.18 b748 1^86 O? 136
1.5 RI 0.22 - 0.38 2.65 1.65 4.25 2.16 2.40
R2 0.23 0.26 0.14 3.38 2.05 4.56 2.60 1.98
x 0.22 0.26 3.01 1.85 4.40 2.38 2.19
- R! 2.34 3.80 1.60 3.96 3.23 5.69 3.48 4.84
R2 2.50 3.41 2.10 3.64 3.64 5.47 3.50 3.43
x 2.42 3.60 1.85 3.80 3.44 5.58 3.49 4.14
25
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Table 9. Percentage difference between the mean foliage yield of broth
treated and no-broth control flats in Experiment I and Experi-
ment II
Experiment^ _
Percentage difference
Herbicide applied
Diphenamid
Monuron
Atrazine
Dicamba
CIPC
Amiben
No-herbicide control
Czapek
Dox
broth
14
-50
0
280
73
83
21
Group I
Difco
nutrient
broth
56
-75
14
59
90
17
28
Group_
Czapek
Dox
broth
250
50
40
53
51
91
27
II
Difco
nutrient
broth
90
-67
90
15
13
-25
6
Experiment II
Percentage difference
Group I
Group II Group III Group IV
Diphenamid
Monuron
Atrazine
Diuron
No-herbicide control
0
122
0
78
48
400
210
50
1060
105
470
78
288
138
62
-30
29
-27
-8
19
Broth addition has clearly accelerated the rate of degradation of most
of the compounds. A study of the three tables reveals the following
highlights: Czapek Dox broth addition reduced dicamba toxicity by a
factor of about 4 after one week of incubation. Difco nutrient broth
was not as effective. Toxicity of CIPC was almost halved by both
broths after one week. Only with these two herbicides did degradation
appear to proceed to the point where the soil concentrations were so
low as to be stimulatory. Amiben toxicity was nearly halved after one
and three weeks by Czapek Dox broth but Difco nutrient broth was in-
effective. Only after more intensive time and broth treatment was
diphenamid toxicity reduced 3*5 fold by Czapek Dox broth. Again,
Difco nutrient broth was less effective. In Experiment II the toxicity
of diphenamid was apparently greater or else the combination of the
two broths was less effective than Czapek Dox broth alone. Neverthe-
less, there is a decided effect of the combined broth treatment on the
26
-------
degradation of diphenamid after 7 weeks* incubation and 4 broth treat-
ments. Monuron toxicity was significant]^ reduced by the combined
broth treatment after 4 weeks of incubation and 2 broth treatments,
but little thereafter. Atrazine toxicity was not affected by the
combined broth treatment until after 7 weeks of incubation and k broth
treatments. Finally, the addition of the combined broths so reduced
the toxicity of diuron after k weeks* incubation that the yield on
these flats approached 80$ of the no-herbicide controls compared to
14$ for no-broth treated flats. This was the most outstanding broth
effect of the experiment.
The data show Difco nutrient broth alone to be an apparently inferior
degradation accelerator compared with Czapek Dox broth alone. This
may be only an artifact of the method of measurement of herbicide
degradation as the yield of foliage. In the case of nutrient broth
the yield of foliage may have been reduced by bacterial fixation of
available mineral nutrients. Unlike Czapek Dox, Difco nutrient broth
contains no mineral nutrients to offset the loss to plants created by
bacterial fixation. This phenomenon is probably operating also in
Experiment II after 15 weeks of incubation and 6 broth additions as
evidenced by the predominance of negative percentage differences.
The addition of any large amount of carbonaceous or nitrogenous mat-
erial to soil probably requires a supplementary mineral nutrient
addition for fertility balance.
An incidental benefit that may accrue to the practice of adding micro-
bial nutrient broths to soil is the greatly improved soil structure
that was seen to result. Broth treated soils were more granular and
porous and showed less tendency to puddle than soil in no-broth treated
flats. This may be the principal reason why the yield on the broth
controls was, on the average, about 40$ greater than that on the no-
broth control. Presumably, the change in structure followed an in-
crease in the colloidal organic matter on the soil particles. Such
a change would be expected to increase the adsorptive capacity of the
soil and thus aid in its detoxification (Upchurch, 1966). The extent
to which this increased adsorptive capacity, if any, plays a role has
not been estimated but cannot be overlooked.
However, assuming that microbes are probably the principal agents of
herbicide degradation in soil (Audus, 1964; Foy and Bingham, 1969), it
follows that any factor that stimulates microbial activity will also
influence the duration of herbicide activity in soil. The total system
is surely very complex and one can speculate in a general way about
what the mechanism may be for accelerated herbicide degradation by
added broths. Most simply the additions of readily available carbon
sources may act to increase the populations of those organisms active
on the herbicide, thus accelerating its disappearance from soil. The
apparent effects of length of incubation and type of broth added would
be expected to operate through the control of these factors on both
the succession and the kinds of populations which proliferate in response
27
-------
to the added carbon sources. Also, it is conceivable that the metabol-
ism of any less preferred substrate, such as the herbicide, is depend-
ent on the concurrent cometabolism of the preferred substrate such as
the sugars and the amino acids in microbial broths*
These data indicate both the need and the promise of more work in this
area. The contamination of land by organic chemicals of various kinds
threatens to become more, not less, serious. The rapid destruction of
such contaminants may, in many instances, be both necessary and feasible
by methods suggested by this research. Studies employing a wider
combination of pesticides and liquid manures, or other economically
feasible materials, may yield information that would significantly
strengthen the case for this novel approach to the problem of pesticide
pollutants in soil*
28
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SECTION VIII
ACCELERATED DEGRADATION OF PHENYLCARBAMATES IN SOIL BY THE APPLICATION
OF A MIXED SUSPENSION OF IPC-CULTURED MICROORGANISMS
In the previous section the possibilities of stimulating the indigenous
soil microflora to attack herbicide residues by the application of
nutrient broths to soil were discussed. This section also deals with
the elimination of herbicide residues but differs in that microorganisms
known to degrade certain phenylcarbamates have been successfully applied
in macroquantities directly to soil containing these compounds.
The difficulties in establishing a functioning population of introduced
microorganisms in natural ecosystems are well known, MacRae and
Alexander (1965) attempted to protect alfalfa seedlings from 2,4-
dichlorophenoxy butyric acid by inoculating the seed with a Flavo-
bacterium known to degrade the latter. The bacterium afforded
protection to the plants only in sterile soil indicating the Flavo-
bacterium was not able to function in the presence of the native soil
population, Kaufman and Kearney (1965) isolated pure cultures of soil
bacteria which degraded and utilized IPC and CIPC as the sole carbon
source. Clark and Wright (1970) applied pure cultures of IPC and CIPC
degrading organisms to herbicide treated soil in Petri dishes and,
using a parley root assay technique, observed rates of degradation
exceeding controls.
The work reported here supports and adds to these findings. We have
isolated a number of species of microorganisms which degrade phenyl-
carbamates and acylanilides in pure culture on agar or in liquid media
and also in mixed cultures using unsterile agar, liquid, and soil
media. This section is concerned with the action of unsterile mixed
cultures in soil from the standpoint of their possible utility in
detoxifying polluted soils.
Materials and Methods
IPC (isopropyl N-phenylcarbamate) was added at concentrations of
1 rag/ml to a flask containing a dilute suspension of a composted
Gloucester sandy loam soil in a mineral salts solution (MSS) buffered
with phosphate at pH ?•!• The MSS contained the following salts in
grams per liter: K2HP04 1.6, KH2P04 .4, MgS04 .2, CaS04 .1, NaNQ3 .1,
NHAN03 .5, FeClj .002, After about two weeks on a rotary shaker, a
white turbidity was observed in the flask. Successive transfer of a
few drops of inoculum from this and succeeding flasks to fresh liquid
media resulted each time in a dense, complex microflora able to subsist
on IPC as the sole carbon source. The concentration of organisms
produced by this method was commonly in the neighborhood of ,5 mg/ml
dry weight of bacteria as measured after cheesecloth filtration. The
29
-------
studies to be described were carried out using this population in
unsterile media.
A trial experiment (Expt. I) was conducted to determine whether the
bacterial population would accelerate the degradation of IPC in soil
as determined by a plant bioassay. Rectangular wooden flats (7.5" X
9" X 4" deep) were filled with composted Gloucester sandy loam and
planted to 6 IPC-sensitive plant species. These were corn (Zea mays L.),
rye grass (Lolium perenne L.), oats (Avena sativa L.), foxtail grass
( Alopecurus pratensis L.). rice (Oryza sativa L.), and crabgrass
[Digitaria sanguinalis (L.) Scop.]. Rates of IPC equivalent to 4 and
12 Ib/A were homogenized in MSS and sprayed onto the soil surface in
a 40 ml volume. The mixed, unsterile bacterial suspension as described
above was sprayed onto the soil surface in a 60 ml volume. All treat-
ments were replicated twice. The flats were placed in the greenhouse
and the foliage, if any, was harvested, dried and weighed after two
weeks.
The promising results of this study led to a second greenhouse experi-
ment (Expt. II) having the same objective, but broadening the range
of herbicides employed. Five, 10 and 15 Ib/A of the herbicides IPC,
CIPC (isopropyl N-[3-chlorophenyl]carbamate), Swep (methyl N-[3,4-
dichlorophenyl]carbamate) and Fenuron (3-phenyl-l,1-dimethyl urea) were
formulated in 40 ml of 100 ppm Triton X155 (alkyl aryl polyether alcohol,
Rohm and Haas, Inc.) and sprayed onto soil flats described previously.
Sixty ml of bacterial suspension having a concentration of 0.3 mg dry
wt/ml was sprayed onto the flats. Half of the flats were sprayed with
a steam sterilized suspension of the same organisms to serve as a
control. The flats were incubated for 1 week at 30° C. They were
then planted to the same bioassay species used in Expt. I. After
two weeks* growing time all living foliage was harvested, dried and
weighed.
A third experiment was undertaken to determine how long the applied
microorganisms remain functional in the soil. Twenty-four soil flats
were sprayed with 100 ml of a suspension of IPC-grown microorganisms
at a concentration of 0.4 mg dry wt/ml. The flats were divided into
four groups. The first group was sprayed with IPC at a rate of 15
Ib/A and planted to corn. The second, third, and fourth groups were
treated the same way 3,7 and 36 days, respectively, after the bacter-
ial application. In the meantime, they remained together in the green-
house, and the soil surface was prevented from drying by periodic
wetting. In each group there were suitable no-bacteria and no-IPC
controls. Each treatment was replicated twice. Plants in each group
were harvested 15 to IS days after planting. In this way the effective-
ness of the applied population after four time intervals could be
estimated by measuring corn yields.
30
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Attempts were made to isolate and identify the major microbial
components of the mixed, unsterile population used in these studies.
IPC agar was prepared by homogenizing solid IPC on a rotary pestle
in about 50 ml of MSS. The homogenate was then added to the final
volume of MSS and agar such that the final IPC concentration was
.7 to 1 mg IPC/ml agar. The mixture was then autoclaved and trans-
ferred to Petri plates. At the higher levels of IPC, solid crystals
formed in the Petri plates. Isolations and transfers were made by
streaking or pipetting an inoculum onto fresh agar and incubating
three weeks at 30° C.
Results and Discussion
The data from Expt. I are presented and summarized in Table 10 as
the dry wt yields of foliage on each soil flat. The small grasses,
oats, rice, foxtail, crabgrass, and rye grass, were collectively
harvested as their individual response to the treatments appeared
comparable. Corn data are presented separately in the table because
of their larger biomass and different response patterns. It is
obvious that the application of IPC degrading bacteria to the soil
resulted in the near complete dissipation of IPC toxicity to the
test plants. It is interesting that the bacteria degraded the low
rate to a concentration apparently stimulatory to corn whose yield
is double that of the control value.
The results of Expt. II are presented in Table 11 and show a similar
striking effect of the applied microorganisms on the effectiveness
of both IPC and CIPC. Compared with a control of killed cells,
application of microorganisms reduced even high rates of herbicides
to levels at or approaching the vanishing point as indicated espe-
cially by the data for corn. Swep was not toxic to corn except at
the highest rate. This toxicity was neutralized by the microorgan-
isms which presumably did not eliminate Swep, but reduced it to a
level tolerable to the corn. That Swep was still present in the soil
flats was indicated by the more sensitive grasses, especially at the
higher rates. This suggests that Swep is more resistant to degrada-
tion by the microorganisms.
The flats treated with Fenuron were not harvested, as a careful
visual inspection revealed no differences between bacteria treated
flats and no-bacteria treated flats. Thus the applied microorgan-
ism appeared unable to attack Fenuron despite similar structural
aspects shared in common with IPC. This result is in agreement
with that of Kearney (1965) who found that an enzyme of bacterial
origin capable of hydrolyzing several phenylcarbamates was
ineffective on the corresponding dimethylphenylurea.
31
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Table 10, The effect of an IPC-grown mixture of microorganisms on
IPC toxicity to test plants
Experiment I
Yield K dry \fb/flat
Hate of IPC No organism applied Treated with organism % Increase
(ib/A) RI R2 x R! **2 x
Yield of grasses a
0
4
12
0
4
12
1.0
0.8
0.6
1.5
0.2
0.2
1.4 1.2
0.8 0.8
0.4 0.5
Yield of
1.9 1.7
0.9 0.6
0.1 0.2
1.7
1.9
1.1
corn
1.8
4.0
1.8
1.4
1.4
1.1
1.5
3.7
1.1
1.6
1.6
1.1
1.7
3.8
1.5
33
100
120
0
530
650
a Cumulative weight of oats, rice, foxtail, crabgrass, rye grass,
32
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Table 11. The effect of an IPC-grown mixture of microorganisms on IPC,
CIPC, and Swep toxicity to test plants
Experiment II
Yield K dry
Rate
(lb/A)
Killed cells
Rl R2
applied
X
wt/flat
Viable cells applied
R! R2 x
% Increase
IPC treated flats
Yield of grasses a
0
5
10
15
0
5
10
15
0.7
0.2
0.2
0.2
1.8
0.2
0.3
0.1
0.8
0.3
0.2
0.1
1.5
0.8
0.2
0.0
0.8
0.2
0.2
0.2
Yield of
1.6
0.5
0.2
0.1
0.7
0.5
0.6
0.4
corn
1.9
1.9
2.0
2.0
0.6
0.5
0.3
0.3
1.9
1.5
2.3
2.1
0.7
0.5
0.5
0.4
1.9
1.7
2.2
2.1
0
150
150
100
20
240
950
2000
CIPC treated flats
Yield of grasses a
0
5
10
15
0
5
10
15
0.8
0.2
0.1
0.1
1.4
1.6
0.6
0.5
0.8
0.1
0.1
0.0
1.5
0.7
1.0
0.8
0.8
0.2
0.1
0.1
Yield of
1.5
1.1
0.8
0.7
0.7
0.5
0.8
0.9
corn
1.8
1.7
1.7
1.4
0.6
0.6
0.7
0.7
1.8
1.4
1.8
2.0
0.6
0.6
0.7
0.8
1.8
1.5
1.8
1.7
0'
200
600
700
20
36
125
143
Swep treated flats
Yield of grasses a
0
5
10
15
0
5
10
15
0.8
0.3
0.2
0.1
1.4
1.5
1.6
1.1
0.8
0.4
0.2
0.1
1.5
1.6
1.6
1.2
0.8
0.3
0.2
0.1
Yield of
1.5
1.5
1.6
1.1
0.7
0.7
0.4
0.4
corn
1.8
1.5
1.7
2.2
0.6
0,4
0.4
0.2
1.8
1.3
1.2
1.8
0.6
0.6
0.4
0.3
1.8
1.4
1.4
2.0
0
100
100
200
20
0
0
82
a Cumulative weight of oats, rice, foxtail, crabgrass, rye grass.
33
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The results of Expt. Ill are shown graphically in Figure 1 by express-
ing the mean yields of corn on the IPC-treated microorganism-treated
flats as a percentage of the mean yields of corn on the control flats
(no IPC applied, microorganisms applied) at each of the four sampling
periods. The effectiveness of the applied microorganisms clearly
decreases in a somewhat exponential fashion as their residence time
in the soil increases. By extrapolation of the curve the applied
population was estimated to have lost all of its effectiveness in
about 2.5 months. Presumably under these conditions, the less hardy,
more exacting species in the mixed population perish first when de-
prived for long of the carbon source to which they have been adapted,
followed by the remainder of the population. This is completely
fortuitous for agricultural practice since a return to the normal
biological balance in a short time is much to be preferred. However
it is apparent that this may be only one of a number of studies
necessary to determine to what extent and for how long an applied
population of microorganisms affects normal soil processes.
Isolation and Identification of Microorganisms. Streaks of the unsterile
IPC-degradruig organisms were made on sterile agar media containing
IPC as the sole carbon source. After incubating for three weeks at 30°
C, the dissolution of the IPC crystals in the vicinity of microbial
was apparent. Dilution plates on sterile IPC containing agar were
made and subcultures of representative types made. Eight isolates
which appeared to be distinctly different on IPC containing agar
were sent to three authorities for identification (Dr. Ruth E.
Gordon, Institute of Microbiology, Rutgers University, New Brunswick
N. J.; Dr. Norvel M. McClung, Dept. of Botany and Bacteriology '
University of Southern Florida, Tampa, Florida; and Mr. Gerard M.
Thomas, Dept. of Entomology, University of California, Berkeley
California). The results of the identifications were not consistent
and must be considered as tentative. Some of the isolates appeared
to be mixed cultures. In the areas of greatest divergence of opinion
C1'6'* Corynebacterium. Arthrobacter and M^cobacterium), the taxonomy
of these and similar forms has not been established (Gordon, 1966)
which makes identifications rather meaningless. A summary of the
identifications of the eight cultures is given in Table 12 with
divergence of opinion being indicated by the word "or" and a mixed
culture by the word "and".
The contamination of land by organic chemicals of various kinds
threatens to become more, not less, serious. Although the phenyl-
carbamates themselves are not notable for their residual longevity
in soil, the principle and implications are nonetheless clear* if
microorganisms can be found which attack and degrade particular
classes of compounds without threats to health or major disruptions in
S°;r effu S"' then 11> my be P°ssible to "mass inoculate" contaminated
soil with the results that residue levels are reduced. This report
34
-------
demonstrates that there is, at least, no theoretical reason for the
impossibility of such a practice. The suspensions of microorganisms
used in this study were easily cultured under nonsterile conditions,
were able to function in a soil environment, and became nonfunctional
in a relatively short time. The feasibility of this approach points
the way to yet more extended and complex uses of these microorganisms.
For example, their local application to the root zone or to seed
(as Rhizobium is now applied to legumes) may serve to protect crops
from a herbicide to which the crop is sensitive. Such a practice
would, therefore, reduce the selectivity requirements of a given
herbicide and extend the conditions under which it might be employed.
Table 12. Summary of identification of eight isolates taken from an
unsterile suspension of IPC-cultured microorganisms
Isolate number Identity
12 Fusarium solani
21 Nocardia sp. and an Arthrobacter sp.
22 Aspergillus sp. or Penicillium stoloniferum
and Corynebacterium or Arthrobacter or
Mycobacterium
23 Corynebacterium or Arthrobacter or Myco-
bacterium
24 Penicillium stoloniferum and Corynebacterium
or Arthrobacter _or Mycobacterium
32 Corynebacterium _or Arthrobacter or Myco-
bacterium
33 Corynebacterium or Arthrobacter and/or
Salmonella
34 Streptomyces sp.
35
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100
o
CC
Q
UJ
< O
UJ
DC li_
h- O
0
Q.
a
_j
UJ
ili 50
O
UJ
en
0 10 20 30 40
RESIDENCE TIME OF MICROORGANISMS IN SOIL (DAYS)
Figure 1. The Effect of Residence Time in the Soil on the Ability of
Applied Microorganisms to Degrade IPC as Measured by Corn
Yield.
-------
SECTION IX
INFLUENCE OF RING CHLORINE ON THE DEGRADATION OF SOME ANILIDE HERBICIDES
AND RELATED COMPOUNDS BY IPC-CULTURED MICROORGANISMS
In the previous section, the capacity of a group of microorganisms culti-
vated exclusively on a mineral salts medium containing IPC (isopropyl
N-phenylcarbamate) to degrade phenylcarbaraates when added to soil was
discussed. This mixture of microorganisms was found to contain the
gram positive coccoids of a Mycobacterium sp., an Arthrpbacter sp.,
and possibly a Corvnebacterium sp. in greatest abundance followed by
Fasarium sp., Nocardia sp., Streptomyces sp., Aspergillus sp., and
Penicillium sp. In the work reported below, the activities of these
organisms are described, especially as regards the influence of molec-
ular structure and ring chlorine on their ability to degrade compounds
structurally related to IPC.
Considerable interest has centered around the degradation of three
classes of anilide herbicides, namely, the dimethylphenylureas, phenyl-
carbamates, and acylanilides, because chloroaniline is produced as an
intermediate in their degradation (Bartha, et al., 196?; Dalton, et al..
1965; Kaufman, 1967; Kearney and Kaufman, 196*577 Not only are some
chloroanilines quite persistent in soil (Alexander and Lustigman, 1966),
but it appears that a substantial number of the chloroanilines may
condense to form chloroazobenzene (Bartha, 1968; Bartha and Pramer,
1967). The formation of chloroazobenzenes is of concern in view of
the carcinogenic activity of some of them. Bartha, et al. (1968)
studied this reaction further and found the formation of chloroazo-
benzenes to be markedly dependent on the chlorine configuration of the
aniline. For these reasons the influence of position of ring chlorine
on the degradation of anilines or of compounds degrading to anilines
is pertinent.
Methods and Materials
The microorganisms used in these studies were originally derived from
a mineral salts medium containing IPC which had been inoculated with a
small quantity of soil. The composition of mineral salts media in grams
per liter used throughout these studies was: KaHPO/^. 1.6, KHaPO^ .4,
MgS04 .2, CaSO^ .1, NaN03 .1, NH4N03 .5, and FeCl3 .002. IPC was added
to obtain concentrations usually ranging from 1 to 10 mg/ml. Fresh
cultures were easily generated by inoculating the mineral salts-IPC
containing medium in Erlenmeyer flasks with a few ml of suspension from
an older culture and incubating on a rotary shaker. After an incuba-
tion period of approximately two weeks, a conspicuous turbidity developed.
The final dry weight concentration of microorganisms normally ranged
from 0.3 to 1.0 mg/ml of suspension.
37
-------
The chemicals used in these studies were obtained from commercial
suppliers and donors. Except when of analytical grade they were
re crystallized from ethyl alcohol until a satisfactory melting point
was obtained. Because they were difficult to obtain, isopropyl N-
4-chlorophenylcarbamate (4-IPC) and isopropyl N-2,4-dichlorophenyl-
carbamate (2,4-IPC) were synthesized in this laboratory by refluxing
the appropriate chloroaniline with isopropylchloroformate in pyridine
and ether. The products were recrystallized from ethyl alcohol and
gave melting points in agreement with those in the literature. For
testing, compounds were formulated in 100 ppm of Triton X-155 sur-
factant (Rohm and Haas) as solutions or suspensions containing 50
Mmol/ml. One ml or less of this stock solution or suspension was
then added to 30 ml of basic mineral salts medium in duplicate 125 ml
Erlenmeyer flasks. Concentration of chemicals used ranged from
0.5 to 0.7 Hmol/ml (90 to 125 ppm). However, in one experiment a
concentration as high as 1.7 Hmol/ml was used. After being inoculated
the flasks were incubated at 30° C on a rotary shaker. Periodically,
1 ml samples were removed for assay and placed in an 8 ml screw-top
vial. Prior to sampling, each flask was weighed and sufficient water
added to replace that lost by evaporation. Samples were either extract-
ed immediately or stored in a freezer until extracted. The extraction
procedure consisted of adding directly to the sample vial 2 ml of a
spectrograde hexane:chloroform (2:1) mixture. The vials were then
agitated vigorously for 10 minutes on a reciprocating shaker. The
solvent layer was then poured from the vial into a silica cuvette
and scanned in the UV (ultraviolet) from 320 to 220 microns using a
Beckman DBG spectrophotometer. Extraction into solvent was found to
have advantages over direct scanning of the centrifuged aqueous
sample (Whiteside and Alexander, I960) because of the elimination of
water soluble background components and better peak definition.
The information sought in the scans was whether or not the compound
degraded, how fast it did so, and whether intermediates accumulated
in quantity. Intermediates were detected by changes in the absorp-
tion maximum and ring degradation was detected by decreases in absorp-
tion at the maximum. Rate of degradation was datermined by estimating
the amount of phenyl ring (a direct function of the UV absorption)
remaining after a period of time, usually one or two weeks. To
estimate rate, the final absorption at the maximum was expressed as
a percentage of the initial absorption at the maximum. This method
was applied even if the initial and final maxima were different, as
in transitions, since it was found the molar absorptions of parent
and intermediate in these studies were not appreciably different.
In this way ring degradation could be quantitated. Rate of transi-
tion to the intermediate was equivalent to rate of peak shift. But
this could be estimated in only a roughly qualitative way since the
relative contribution of parent and intermediate to the total absorp-
tion during the transition could not be determined by these methods.
38
-------
It was of some interest to know whether the composite effect of the
isolates together in the unsterile suspension would be similar to the
individual effect of any single species under sterile conditions.
Accordingly, four isolates which had been continuously cultured on
IPC agar (l mg IPC per ml of basic mineral salts agar) and selected
for their vigor, were transferred to 30 ml of a sterile medium
containing 1 mg IPC per ml of mineral salts solution. The four
isolates were a Fusarium sp., Mycobacterium sp., Arthrobacter sp.
and Penicilllum sp. that was later found to contain an Arthrobacter
sp. also. When the IPC was exhausted 3 ml of the sterile suspensions
were transferred to 50 ml flasks containing 12 ml of MSS. Compounds
dissolved in methyl alcohol were added giving a final concentration of
0.7 fimol/ml. Samples were taken at intervals and analyzed as described
above.
Warburg respirometer studies were conducted to determine the relative
effect of the chemicals on the respiration of the microorganisms and as
a further measure of their susceptibility to degradation. A 3*5 ml
sample of microbial suspension was added to a Warburg flask and .5 ml
of formulated compound suspension was added to the sidearm. The final
concentration of compound in the flask after tipping ranged from
4 jimol/ml in some experiments to .3 nmol/ml in others. Each compound
was assayed in triplicate at 30° C by measuring any ©2 uptake occurring
during the course of metabolism.
Results and Discussion
Throughout these studies the microorganisms could be cultured in liquid
or agar mineral salts media using unchlorinated anilides such as IPC,
aniline, formanilide or propionanilide as the sole carbon source. All
attempts to substitute chlorinated"analogs of these molecules resulted
in failure to produce visible cell masses of any kind even though
degradation of the substrate molecule did occur in most cases. One
interpretation is that the metabolism of the chlorinated ring is funda-
mentally different from that of the unchlorinated ring resulting in
bypass or blockage of some critical growth process. Another is that
the rate of metabolism of chlorinated substrates is simply too slow
to support sustained observable growth. A third possibility is that
chloroanilines or their derivatives are simply toxic at the concentra-
tions employed.
In several respirometry studies, the use of chlorinated anilide sub-
strates at a concentration of 4 Hmol/ml consistently resulted in ©2
uptake that were at or below that of endogenous controls. Some chlor-
inated substrates were less toxic at lower substrate levels. Unchlorin-
ated anilide substrates resulted in 02 uptake considerably above these
controls. The results of one experiment using a substrate concentra-
tion of .3 nmol/ml are shown in Figure 2. It is seen that the three
unchlorinated substrates IPC, propionanilide and aniline all exhibit
39
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high rates of uptake relative to the chlorinated substrates. The curve
for aniline falls off rapidly after a steep initial rise, presumably
because its molar carbon content is lower relative to IPC or propion-
anilide. Giving unexpectedly high rates of respiration was isopropyl
N-2,4-dichlorophenylcarbamate. The lowest respiration rates were
observed for the monochloroanilines and the monochloroisopropyl N-
phenylcarbamates•
Measurement of Degradation Rates, Shown in Figures 3 and 4 are groups
of UV scans of the hexane-chloroform extract of a particular culture
flask sampled at different times. Many of the scans show a leftward
shift of the UV absorption maximum from that of the parent compound
time to that of a degradative intermediate at later times. In Figure
2, the dotted line indicates an identity between the absorption maxi-
mum of the intermediate and that of authentic aniline or chloroaniline.
After initial independent verification by the method of Pease (1962),
this identity was always taken as sufficient evidence that degrada-
tion intermediate was aniline or chloroaniline. The initial attack
on molecules of this type is assumed to be a hydrolysis at the carbonyl
carbon followed by spontaneous decarboxylation resulting in aniline or
chloroaniline (Kearney and Kaufman, 1965).
The degradation of dimethylphenylurea and its chlorinated analogs was
little or none in this system. Typical is the unchlorinated herbicide,
dimethylphenylurea (fenuron), in which very little degradation was
observed within 60 days (Figure 4)« The failure of these organisms to
degrade fenuron in soil had been observed previously (see previous
section). Assumed intermediates of this degradation, formanilide and
phenylurea, however, degraded with relative ease, especially the former
(Figure 3), Thus it appears the two methyl groups are responsible for
the degradative recalcitrance of these compounds. This is in agreement
with Geisbuhler et al. (1963) who demonstrated that dealkylation of the
two methyl groups precedes hydrolysis of the urea linkage.
All phenylcarbamates and acylanilides tested were degraded, usually
with an accompanying production of aniline or chloroaniline. Whether
or not aniline or chloroaniline actually appeared is viewed as a
kinetic phenomenon involving the relative rates of two processes. The
first is hydrolysis of the side chain at the carbonyl carbon by
exogenous hydrolytic enzymes. The second is metabolic degradation of
the aniline or chloroaniline formed by hydrolysis. If the hydrolysis
is rapid or if the ring does not degrade, aniline or chloroaniline
appear in the medium. On the other hand, if the hydrolysis is slow,
the ring will degrade as soon as formed, and no intermediate appears.
The relative rates of both processes appeared to be influenced pri-
marily by the structure of the compound undergoing degradation, partic-
ularly its chlorine configuration.
40
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Effect of Chlorine Configuration on Side Chain Hydrolysis and Ring
Degradation. A simple structural effect of the side chain was noted in
the difference between the rate of hydrolysis of IPC and propionanilide.
Aniline never appeared during IPC degradation (Figure 3) but did appear
during the degradation of propionanilide (Figure 4). Aniline, itself,
unlike chloroaniline, was rapidly metabolized by the organisms (Figure 3)«
According to our kinetic hypothesis aniline was metabolized as fast as
it was formed from IPC hydrolysis, but propionanilide hydrolysis was so
much more rapid that aniline momentarily accumulated in the medium. Thus
side chain hydrolysis is rate limiting in IPC degradation but not in
propionanilide degradation. Also rapid was the hydrolysis of the
herbicide 3,4-dichloropropionanilide (3,4-Prop) and N-(3,4,-dichloro-
phenyl)-2-methylpentanamide (Karsil) as seen in Figure 4. These
repeated observations suggest that acyl bonds are more rapidly broken
than ester bonds in these systems. This idea finds parallel ex-
pression in the Warburg data (Figure 2).
The effect of ring chlorine on the rate of hydrolysis is less clear in
these studies. Slow transitions were noted for isopropyl N-3,4-dichloro-
phenyl carbamate and especially for isopropyl N-4-chloroisopropylcarba-
mate as shown in Figure 3. More rapid shifts were noted for the other
isopropyl phenylcarbamates. Kaufman (196?) also noted an effect of ring
chlorine on hydrolysis of phenylcarbamates. However, our data indicate
that the principal role of ring chlorine for most compounds tested was
its effect on the rate of degradation of the ring. Without exception
this effect was to greatly retard that rate.
The retarding effect of ring chlorine on ring degradation was not equal
for all configurations. Figure 3 shows scans of various chloro-IPC's
and chloroanilines which were subjected to microbial attack over a
period of 9 days (Expt. A). The percentage of ring remaining after 9
days is given in the figure at the lower right of each group of scans.
It is seen that chlorine configuration has the same effect on rate of
chloro-IPC degradation as it does on the rate of chloroaniline degrada-
tion. This is reasonable since chloroaniline is the principal degrada-
tion intermediate of chloro-IPC and the effect of ring chlorine on the
hydrolytic production of chloroanilines is relatively small (with the
possible exception of the 4-chloroanalog), Thus the chloroanilines
and chloro-IPC's may be ranked by chlorine configuration according to
their rapidity of degradation: 0 >2,4 > 2,4,5 >3 >4 >3,4.
This order has been arrived at by consideration of five additional
experiments as well as Experiment A shown in Figure 3. These are
summarized in Table 13. The percentage of compound remaining at the
indicated time increases according to the above order. Three kinds
of exceptions are noted: l) In 8 out of 11 instances, 2,4 degraded
faster than 2,4,5 according to the above order. However, in one
instance (Sxpt. Rj) the reverse was true, and in two instances little
41
-------
difference was noted. 2) In 8 out of 11 instances, 3 ranked fourth in
order of degradation according to the above sequence. However, in three
instances it ranked second after the 0 configuration (Expt. RI and AP3),
3) In 4 out of 11 instances, 4 degraded faster than 3>4 according to the
above order. However, in one instance the reverse was true (Expt, 112)*
and in 3 instances little difference was noted. In two instances
neither compound was degraded owing to a particularly inactive suspension
of microorganisms (Expt, R). In a final instance 3,4-dichloro IPC
hydrolyzed faster than the 4-chloroanalog but little ring degradation
was noted. One possible source of these differences may be that there
were differences in the behavior of the organisms themselves toward
a given compound because of unspecified environmental changes and/or
adaptive phenomena.
On balance, however, the evidence is strong that the indicated sequence
is the true one with some uncertainty attached to the relative position
of the 4-chloro and 3»4-dichloro configurations. It is noteworthy that
this order finds some parallel expression in the Warburg data, particu-
larly the primacy of 2,4-dichloro IPC among the chlorinated substrates.
The results of the degradation experiment using four selected isolates
under sterile conditions are shown in Table 14. The high percentage of
ring remaining in this experiment is due to the 5-fold dilution of
organisms involved in the experimental procedure. However, it is clear
that the isolates differ little from each other in their ability to
hydrolyze the substrate or degrade the resultant chloroaniline. Precise
comparisons are not possible as accurate estimates of the relative
cell concentrations in the suspensions were not made. Notable is the
degree to which the order of degradation of the substrates according
to chlorine configuration correspond to that already described for the
composite population. One exception was that no hydrolysis of Swep
was observed for any of the four isolates,
The Toxicity Hypothesis. The observed sequence of decreasing degrada-
tion rates as a function of ring chlorine configuration is difficult
to explain in terms of any uniform variation in physiochemical proper-
ties of the molecules themselves such as solubility, partition coef-
ficient, etc. Kearney (1965) was able to correlate enzymatic rates
of hydrolysis of several phenylcarbamates with steric properties and
electron density at the reactive site. However, no single approach
is likely to succeed with intact cells because of the greater complex-
ity of a system in which absorption, transport and concurrent effects
on related processes are likely to play a role. Nor is explanation
made easier by revising the sequence according to one or more of the
three discrepancies noted above. Kaufman (196?) observed the order of
degradation of chlorophenylcarbamates in perfused muck soil to be
42
-------
Table 13. Degradation of IPC, Aniline and five of their chloroanalogsa
Percent "ring" remaining after indicated no.
Compound
IPC
2,4-dichloro
2,4,5-trichloro
3-chloro
4-chloro
3,4-dichloro
Aniline
2,4-dichloro
2,4,5-trichloro
3-chloro
4-chloro
3,4-dichloro
Days 22
Expt. R
^b
62?
85b
100°
100°
100b
11
14
20
80
100
100
14
Rl
0(3 )c
27K
63b
11?
*%
100b
0(3)
B
3
0(14)
35
38
8
AP3
0(1)
33
39
0(1)
65
79
0(1)
0(4)
27
28
46
100
9
A
0(2)
12
14
33
60
58
0(2)
7
9
24
40
40
8
Bf
0(1)
56
73
80
100d
100d
e
—
—
—
—
"'
of days
14
R2
0(1)
24
29
44
64
58
_
12
19
31
44
54
a All experiments replicated twice except A, A?3 and B.
b Little, or no hydrolysis noted.
0 Figures in parenthesis are no. of days within which depletion occurred,
d Some hydrolysis but no ring degradation noted.
® -- not measured.
Mean values of 4 isolates. See Table 14.
43
-------
Table 14. Degradation of eight compounds by four isolates
Compound
IPC
2,4-dichloro
2,4, 5-trichloro
CIPC
4-chloro
3,4-dichloro
3,4-Prop
Swap
Isolate No.a
12
23
24
33
12
23
24
33
12
23
24
33
12
23
24
33
12
23
24
33
12
23
24
33
12
23
24
33
12
23
24
33
Percent remaining after B days
completely degraded
1 day
60
60
49 5c = 56
63
74
69
73 x - 73
76
73
slow hydrolysis
86
80 x =» 80
slow and incomplete
hydrolysis slow but
89
90
95 x « 92
95
little hydrolysis
after
hydrolysis
complete
a Identity of isolates as follows: 12, Fusarium sp.; 23. Mycobacterium
sp. ; 24, Arthrobacter sp.; 33 Penicillium sp. later found to be con-
taminated with Arthrobacter sp.
44
-------
0 >3 >4 > 2. Dichlorophenylcarbamates were not degraded, If the
soils were enriched with CIPC prior to treatment the order was 3,5>
4 >2 > 2,4 >3,4 >2,5. This sequence like the one found in our
studies for what is in essence an IPC adapted system, is also puzzling
and no explanation was offered.
A clue to the rationalization of such sequences was the discovery by
Bartha et al. (196?) that a substantial fraction of the chloroaniline
derived from the degradation of 3»4-dichloropropionanilide in soil
condensed to form a toxic residue that depressed soil respiration.
This was subsequently identified as 3,3r»4,4t-tetrachloroazobenzene
(Bartha and Pramer, 1967). Moreover, evidence was offered that the
condensation reaction was catalyzed by peroxidase but that the enzyme
was not active on all ohloroanilines (Bartha et al.. 1968). Among the
anilines not converted to chloroazobenzenes by peroxidase were those
having a 2,4-dichloro and a 2,4,5-trichloro configuration. However,
3-chloro and 4-chloro and 3,4-dichloroanilines, among others, were
converted to the corresponding chloroazobenzenes.
Unfortunately, we are not able to substantiate the presence of these
compounds in our systems using the extraction and UV assay techniques
described. However, conspicuous colorations ranging from yellow-orange
to red were observed in certain Experiment R suspensions. This was a
unique experiment because the substrate concentrations were high (1.7
Hmols/ml as opposed to about 0.6 in other experiments) and the suspen-
sion was particularly inactive indicating a greater degree of toxicity.
The 3-chloro and 4-chloroaniline suspensions were red colored and the
3,4-dichloroaniline suspensions were orange colored, but the 2,4-di-
chloro and 2,4,5-trichloroaniline suspensions were colorless. Since
the 3,3t|4,4»-tetrachloroazobenzene crystals isolated by Bartha and
Pramer (1967) were "orange colored" this would seem to provide some
presumptive evidence for the presence of such compounds in our systems.
These compounds are, apparently, especially evident and toxic at the
higher substrate concentrations. The possibility that a desirable
degradative sequence is being blocked by an intermediate whose toxicity
depends on the chlorine configuration of the parent molecule bears
further investigation.
45
-------
S
JO
as
D. CO
co (b
£ 3
CO H-
cn (b
ll
CO
O C+
a ct-
P. o>
o »
o 3
TO c«-
B8*
co
CD
CO
500 -
(PC
A CIPC
A 4 IPC
34 IPC
o Aniline
3 CA
©4 CA
v Prop.
34 Prop.
• Endogenous
TIME IN HOURS
-------
CHLORINE POSITION ON RING
H{? /H,
N-C-O-CH
^| \H.
3.4
Itopropyl J|.-Phinyl- 3
CarbumotM
Anlllnu
WAVELENGTH (MICRONS)
Figure 3. Influence of ring chlorine on the degradation of IPC»s and
Anilines. Shown are UV scans of samples taken from culture
flasks at the indicated number of days after compound addi-
tion. Figures at lower right of each group are the percent-
age ring remaining after 9 days.
47
-------
N-C-CH-(CH2)E-CH3
C Jn''^3 H 0 H 0 H 0 H 0 /
C| N-C-CH2-CH3 N-C-0-CH3 N-C-CH2-CH3 N-C'N
H9
H0
N-C-NH2 N-CH
U *«, fll fH
Cl Cl
Karsil Propanil Swep Propionanilide Fenuron -. Phenylurea Formonilide
1.0
.5
O
en
CQ
301
.3
320 280 320 280 320 280 320 280 320 280 320 280 320 280
WAVELENGTH (MICRONS)
Figure 4. Influence of differing structures on rates of hydrolysis and
ring degradation. Shown are UV scans of samples taken from
culture flasks at the indicated number of days after compound
addition.
48
-------
SECTION X
A MEMBRANE BIOLOGICAL FILTER DEVICE FOR REDUCING
MTERBORNE BIODEGRADABLE POLUJTANTS
The increasing use of pesticides and other synthetic organics together
with the known resistance of many of them to microbiological attack
(Alexander, 1965; Kearney, et al., 1969) indicates that the quantity
of these substances in the environment will increase. The ultimate
fate of most pollutants not fixed or degraded by soil is a water
resource. The problem of pollutants in water is aggravated because
many of the molecules which are more or less readily degraded by
microbes in soil, such as the phenylcarbamates and phenoxyacetic
acids, may be much more resistant to degradation in water (Schwartz,
1967).
As the result of work originally initiated as a study of the kinetics
of herbicide degradation, an interest was developed in studying the
possibility of using a "biological filter" system for the removal of
pesticides from water. The term "biological filter" is customarily
employed to designate the trickling film reactors common in sewage
treatment plants. A different type of biological filter was studied
in this laboratory in which the herbicide IPC (isopropyl N-phenyl-
carbamate) was removed from a flowing water source by a concentrated
suspension of microorganisms retained behind an ultrafilter barrier.
The fundamental concept of the membrane biological filter device is
that a pollutant substance in a feed stream is transferred by diffusion
or mass flow across a barrier behind which is a bath containing an
active suspension of microorganisms able to attack the pollutant. The
microorganisms may be a very general population or a highly specific
and selected one depending upon the resistance of the pollutant to
biodegradation. In any case the destruction of the pollutant by the
microorganisms results in an effluent containing a reduced pollutant
concentration. The fundamental requirements for a practical device
are as follows: l) a population of microorganisms able to rapidly
attack the molecule at hand; 2) a population relatively unfastidious
in its nutritive requirements; 3) an ultrafilter material which is
rugged, inexpensive and permits efficient exchange of pollutant from
feed stream to bath while protecting and preventing loss of the
population; and 4) a high flow rate output containing a significantly
reduced concentration of the pollutant. What follows is a discussion
of how a phenylcarbamate metabolizing population of microorganisms
contained in a laboratory ultrafilter device successfully modeled
these requirements.
Methods and Results
The derivation and constitution of the mixture of IPC-degrading micro-
organisms used in these studies were described in Sections VIII and IX.
49
-------
It was necessary to determine the rate at which a unit weight of the
microorganisms degraded a unit weight of IPC in order to have some basis
for predicting the relation between flow rate of the feed stream, popula-
tion size, and effluent concentrations. A population of microorganisms
was grown as usual by inoculating 1 liter of MSS containing a gram of IPC
(well over the solubility) with liquid inoculum from a previous culture.
The MSS had a pH of 7.1 and contained the following in mg/1: K2HP04, 400;
KH2P04, 100; MgS04, 50; CaSOA, 25; NaN03, 25; m^NOj, 125; and FeClT, 2.
When the medium became turbid after incubation on a rotary shaker at 30°
C, the suspension was filtered through cheesecloth, centrifuged and resus-
pended in fresh MSS. Amounts of suspension were added to one-liter beakers
containing various quantities of soluble IPC, such that the final micro-
bial concentrations determined gravimetrically were 30, 20, and 10 ng dry
wt/ml and the final IPC concentrations were 110, 75, and 40 Hg/ml in a
total volume of 600 ml of MSS. The beakers were aerated and the decrease
in IPC concentration was followed over a period of several hours at room
temperature. This was done by withdrawing 1 ml samples and extracting
them in 8 ml vials with 2 ml of hexane: chloroform (2:l). The UV absorb-
ance of the solvent was then read at 276 ran and related to a standard
curve derived by carrying through identical procedures on known IPC
concentrations.
After a short lag period of 3 to 4 hours the rate of destruction of IPC
was found to be linear over all concentrations of IPC and microorganisms
employed. The rates of IPC-degradation are shown in Table 15. The rates
of IPC metabolism per unit of bacteria vary somewhat depending on both
the concentration of microorganisms and IPC. The mean rate over all con-
centrations was .22 mg IPC detroyed/mg of microorganisms. The range was
from .13 at the lowest concentration of IPC and the highest concentration
of IPC and the highest concentration of microorganisms to .39 at the high-
est concentration of IPC and the lowest concentration of microorganisms.
The values may be expected to increase at higher IPC concentrations. On
the other hand, the values may be expected to decrease at high microbial
concentrations, but the rate of decrease outside the range tested here is
not known.
The mineral nutrition requirements of the microorganisms were not rigor-
ously determined but the ability of the organisms to function in a nutri-
ent environment of various strengths was estimated over a period of 2
days. This point is relevant because the mineral nutrient content of
the bath must soon come to some sort of equilibrium with that of the feed
stream. Therefore, the nutrient content of the feed stream must be
sufficient for the level of functioning expected in the bath. In the
event the bath is deficient in either mineral or organic nutrients, the
slow metered addition of supplementary materials to the bath to insure
adequate levels of cometabolism or maintenance cannot as yet be ruled out.
Five hundred ml of various dilutions and multiples of the MSS described
above were placed in aerated liter beakers, A microbial suspension pre-
pared as described above was added to give a final concentration of
50
-------
200 fig/ml. Five hundred mg of solid IPC was added to each beaker. After
2 days the solid IPC remaining was harvested, washed and weighed and the
percentage metabolized determined. The results are shown in Table 16.
The data described a skewed convex curve with a maximum in the vicinty of
the standard MSS in which the organisms were originally cultured. The
significance of the data is that a depleted or exhausted mineral nutrient
environment is not without some effect but, on the other hand, it is not
by any means disastrous for the destruction of IPC over a limited period
of time.
Table 15. Rate of IPC degradation (|ig IPC/ng microorganism/hr) at three
levels of IPC and microbial concentrations.
Microbial concentration
UK/ml
10
20
30
X
IPC concentration M-g/ml
40 75 110
.20 .24 .39
.17 .24 .29
.13 .20 .24
.15 .22 .28
X
.27
.23
.19
.22
Table 16. Influence of Mineral Salts concentration on a quantity of IPC
degraded over a two-day period.
Concentration Factor
4
1
0 (dist. H20)
Percent IPC degraded
48
88
80
74
50
The first and simplest laboratory "biological filter" system used
was what may be called the dialysis tubing model. Mineral salts solu-
tion containing IPC was pumped through 65 feed of £ inch dialysis
tubing. The tubing was loosely wound on a lattice spool and immersed
in an 8-liter bath containing a stirred suspension of microorganisms
at a concentration of .3 mg/ml. Feed stream IPC concentrations were
.12 and .20 mg/ml and flow rates, achieved by a Technicon proportioning
pump were .25 and 1.50 1/hr. Effluent was sampled two or three times
51
-------
after each change in feed stream flow rate or IPC concentration to be
certain the device had reached an equilibrium, IPC concentration of
the samples was measured as described above. The device was operated
2 days at room temperature.
The dependence of IPC concentration of the effluent on the flow rate
of the feed stream and its IPC content is shown in Table 17. The
relation was so predictable over the ranges employed that a constant
was evaluated for the equation:
[IPC]E.= K'[IPC]F.S.(FR) (Eq. l
where: [lPC]g.= IPC concentration of effluent in mg/ml
[lPC]jpws. = IPC concentration of feed stream mg/ml
FR = flow rate in l/nr
KT = a constant
Constant variation did not exceed 6% and was attributable to random
errors. IPC in the effluent was reduced 94$ at the lowest flow rate
and about 66% at the highest. Presumably higher flow rates would
result in even less efficient reductions unless the tubing lengths
were increased. Effluent concentration was naturally increased at
higher flow rates because the time and, therefore, the chances for
any given molecule to contact the membrane surface were reduced.
Therefore, it is clear that the physical rates of transfer of IPC
across the membrane surface were the limiting factors in this model
and not the rate of the microbial destruction of IPC*
Table 17. Dialysis Tubing Model—Influence of flow rate and IPC
concentration of the feed stream on IPC concentration of
the effluent.
FR 1/hr
.25
.50
.75
1.00
1.25
1.50
.25
.50
.75
1.00
1.25
1.50
Feed Stream
LIPC]F.S. mg/ml a
.12
.12
.12
.12
.12
.12
.20
.20
.20
.20
.20
.20
Effluent Stream
[IPC]E. mg/ml a
.007
.014
.021
.029
.032
.042
.011
.023
.034
.044
.057
.066
K»
See Eq. 1
.234
.234
.234
.242
.214
.234
.220
.230
.226
.220
.228
.220
FR « flow rate in l/hr
[IPC]F.S. mg/ml = mg/ml of IPC in feed stream
mg/ml = mg/ml of IPC in effluent stream
52
-------
A second model was developed with the aim of achieving greater durability
and versatility as well as higher IPC transfer rates at increased feed
stream velocities. The efficiency of such a system should hopefully be
limited only by the capacity of the microbes to destroy IPC. Such a
system would require 100$ transfer of pollutant across the ultrafilter
barrier. An equation describing such a system is derived below.
To meet the condition of 100$ transfer, assume a feed stream containing
IPC is emptied directly into the bath. Further assume that effluent
leaving the bath at a rate equal to the input, is filtered in some way.
Thus the volume and microbial concentration remain constant during the
operation. It is clear that, as the feed stream rate increases, a point
will be reached where the destructive action of the microbes can no
longer keep pace with the input so that the IPC concentration of the bath
begins to increase. At this point IPC appears in the effluent and is
identical from then on to the bath concentration. We may write:
IPC input » IPC metabolized + IPC not metabolized (Eq. 2)
furthermore: FR[lPC]F.s. = K(v)(C) + FR[lPC]E. (Eq. 3)
where FR = flow rate of feed stream » flow rate of effluent in ml/hr
PClp.S. - IPC concentration of the feed stream
PCJE. = IPC concentration of the effluent
C = concentration of microorganisms in mg/ml
V = volume of microorganism bath
K = a constant for given conditions in mg IPC/mg microbes/hr
collecting and rearranging: [lPC]Et = [IPC]F.S. - K(V)Cc) (Eq. 4)
FR
Dividing by iPCp.g. and multiplying by 100 gives another useful form:
% IPC remaining - 100 - K(y)(C)lOO (Eq. 5)
This equation then defines the relation between the major variables of
any filter device in which complete transfer of pollutant from the feed
stream to the bath occurs. The term C would tend to slowly increase
over a period of days if the. pollutant is of a type that can be used
for growth of new microbial cells. The term K is seen to be identical
in meaning to that evaluated earlier at a mean of .22 mg IPC/mg microbes/
hr.
The second membrane biological filter device which may be called the
Plexiglas model is shown in Figure 5. ^'he heart of the model consisted
of 3 one-foot square, £ inch thick Plexiglas plates. Parallel £ inch
wide channels were cut zigzag fashion into the plates (see inset) so
that, when the plates were stacked, the channels all coincided. The
total channel length of each plate was approximately 14 feet and the
53
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total channel area 40$. An entrance and exit hole was cut into the
wide dimension of each plate so that liquids could enter and exit from
the channels through tygon tubing. Membrane filter sheets were placed
between the plates, thus separating the channels of the center plate
from those of the two outer plates. Solid g inch thick plates were
stacked above and below the three channeled plates and the entire device
was squeezed together with throat clamps. The feed stream containing
IPC at a concentration of .2 mg/ml was pumped through the center plate
channels and the bath stream containing .28 mg/ml of microbes was
pumped in countercurrent flow from a bath through the two outer plate
channels and back into the bath. Bath stream rates were held between
5 and 6 1/hr by regulating the flow from a centrifugal circulating
pump located in the bath. Total bath volume was 2700 ml. Feed stream
rates through the device were achieved by a Harvard proportioning pump
and varied from .25 to 3*5 1/hr. A peculiar feature of the device
was the rerouting of the effluent tubing back through the proportion-
ing pump next to the feed stream tubing. This was necessary in view
of the unavoidably different pressures between the feed stream channels
and the microbial bath channels. In this way, effluent and feed
stream rates were kept equal as were channel pressures.
Results for the Plexiglas unit are shown in Figure 6. Each point on
the graph is the mean of two determinations. Data are effluent con-
centration expressed as a percentage of the feed stream concentration.
As predicted by Equation 5, all data show a decreasing percentage of
IPC remaining as the flow rate decreases. Where membrane parchment
was used as the barrier between feed stream and bath stream the
effluent concentration dropped below 50$ feed stream concentration
at about .2 liter/hr. Where Millipore Ultrafilter material of pore
size .45 micron was employed, effluent concentration dropped below
50$ feed stream concentration at approximately .8 liter/hr. Thus
a higher transfer rate was achieved using the higher porosity materials.
Two feed stream center plate widths were tested, one .25 inch and the
other .10 inch. It is seen that virtually no difference in efficiency
resulted from using the narrower channels under these conditions.
The performance of this model may be evaluated to some extent by
calculating the theoretical maximum efficiency using Equation 5. For
the conditions of run V - 2700 ml, C •» .28 mg/ml and [ IPC]F-S =
.20 mg/ml. The value of K is taken at .22 mg IPC/mg microbes/hr
which is the mean value found earlier for a limited range of microbial
and IPC concentrations. Since the microbial and IPC concentrations
employed in the filter device were different than those for which
the constant was evaluated, the resulting curve can only be an
approximation. This curve is plotted in Figure 6 as the theoretical
maximum for these conditions and shows that the effluent concentration
drops below 50$ of the feed stream concentration at a flow rate of
approximately 1.7 liters/hr. At low flow rates the rate of IPC
54
-------
transfer through the material appears to be limiting. But at higher
flow rates the efficiency approaches the maximum. One explanation for
this is that the amount of turbulence in the feed stream channels
increases at higher velocity causing a higher transfer of IPC between
the feed and bath streams.
Discussion
Improving the Plexiglas model. Although the transfer rates at higher
velocities approach 100$, the percent IPC remaining in the effluent at
these velocities is unacceptably high. Clearly then, the unit must
operate at lower feed stream velocities. At low velocities, however,
the transfer efficiency of IPC to the bath stream is poor and indicates
that we have not yet succeeded in building the efficient model desired.
Several possibilities for improvement exist. 1) The data show an increase
in transfer as the porosity of the ultrafilter increases. Since the size
of Mycpbacterium and Arthrobacter cocci is not under 1 micron, the pore
size diameter could be safely increased from its current .45 micron.
2) The feed stream channels could be made more tortuous to increase
channel mixing. They could be lengthened to increase the time in which
a unit of volume resides in the device. They could also be greatly
narrowed below .1 inch to increase the S/V ratio and thus the likelihood
of transfer. 3) A pressure differential might be imposed to facilitate
transfer. This could be done by applying a positive pressure to the
feed stream or by manipulating the relative velocities of feed and bath
streams. However, any device causing the mass flow of liquid volumes
between the feed stream and bath stream would require a net transfer of
zero within the device. Otherwise the bath volume would fluctuate.
The volume output of the filter can be increased by increasing the
number of feed stream plates alternating with bath stream plates. For
example a single unit of three plates operating at a feed stream
velocity of 6.3 1/hr has reduced the IPC concentration by 70$ accord-
ing to Figure 6. The same reduction can be achieved at a flow rate
of 3 1/hr by operating 10 feed stream plates and 11 bath stream
plates simultaneously from common manifolds. Under such conditions
the quantity of microbes must be increased to handle the enlarged
input. Equation 5 permits prediction of the bath volume necessary
for specified percentage reductions, flow rates, and concentrations.
In the example above, the new volume required is calculated to be
6.8 liters. Assuming the ability to construct less unwieldy units
having high transfer efficiencies, we may ask what bath volume would
be required to achieve 70$ reduction of IPC at total flow rates
approaching pilot capacity such as 2000 I/hr. The equation predicts
a bath volume of 4500 liters, roughly the dimensions of a tank
2 meters tall and 2 meters in diameter.
55
-------
The "Separated" and '"Mixed" Options. The models just described are
examples of what may be termed the "separated" option. In this
approach feed stream and bath are kept separated at all,times by an
ultrafilter barrier. An alternative is the "mixed" option in which
the feed stream is emptied directly into the bath followed by ultra-
filtration of the entire mix at a rate equal to the input. This
approach was foreshadowed during the derivation of Equation 5» Both
approaches would require considerable prefiltration by sand or dia-
tomaceous beds. Whereas, separated systems would pass the bulk of
the remaining solids directly through the device, mixed systems would
face formidable filtration problems in that all solids down to bacterial
size must be retained if functional organisms are not to be lost from
the bath. The problem is aggravated if the solids content of the feed
stream is already high. In addition, the accumulation of solids in the
bath may appreciably interfere with the degradation of pollutant partic-
ularly if the population is in any way a selected one. At some point
the bath will approach the characteristics of a sludge which would
have to be removed following diversion of the feed stream to a fresh
microbial bath.
The separated approach would be indicated in situations where a high
solids content of the feed stream overtaxes existing filtration
capabilities or would interfere with bath functioning owing to a
selected or fastidious microbial population. It would also be an
advantage where it is clearly undesirable to remove all forms of life
from the effluent. The mixed approach would be called for if the
resource requirement associated with the achievement of high transfer
efficiencies of pollutant across the ultrafilter are prohibitive.
It would also be of advantage if a general microbial population were
not inhibited by accumulating solids and if the removal of such solids
from the effluent were also an objective of the treatment.
In connection with the concept of multiple usage, an interesting
variant is the potential adaptability of devices of this kind to the
removal of inorganic nutrients such as nitrogen and phosphorus from
water. The eutrophication of fresh water resources by algal and
bacterial growth has its origin in the increased levels of mineral
nutrients finding their way into water from fertilized field runoff
as well as wastewater effluents. The problem is so severe in some
sections that N and P, once innocuous, must be considered nothing less
than pollutants under these conditions. A question for which data
are needed is whether mineral nutrients could be removed from feed
streams upon transfer to a microbial bath by either the mixed or
separated approach. The nutrients would be immobilized in fast grow-
ing bacterial tissue by the addition to the bath, if necessary, of
readily assimilable carbon sources. The rate of addition could be
adjusted to keep cell proliferation populations expanding at a
56
-------
sufficient rate to maintain low levels of nutrients in the bath. Not
the least of the problems inherent in this approach is the periodic
disposal of the large biomass so produced. Various approaches to this
problem have been compared and reviewed by Eliassen and Tchobanoglous
(1969).
The Developing Maturity of Ultrafiltration Technology. Regardless of
approach, the purely physical aspects of Ultrafiltration of large volumes
at high rates would at first appear to be more formidable than is actu-
ally the case. The Battelle Memorial Institute (1969) on membranes
for industrial, biological and waste treatment processes made clear that
Ultrafiltration process technology is evolving rapidly. Sophisticated
Ultrafiltration systems now available from companies such as Amicon
Corporation (Lexington, Mass.) and Dorr Oliver Corporation (Stamford,
Conn.) employ ingenious methods of handling high flux rates. In one
process the feed stream under low pressure is pumped through thin (.25
to 2.5 mm), spiral channels parallel to the ultrafilter surface. The
channel configuration induces laminar flow that maximizes fluid shear
stresses at the membrane surface. Also minimizing the buildup of solids
is an ultrafilter microstructure that promotes separations at the
surface, avoiding the pore plugging that causes conventional filters to
lose permeability after long use. The adoption of these advanced systems
to model the biological filter process and to evaluate various system
configurations and operating parameters on a laboratory or pilot scale
would be within current capabilities.
The concepts developed here are in need of more exploration. As water
pollution problems intensify the evaluation of the claims of competing
technologies can be made only on the basis of a more adequate under-
standing of the more innovative approaches as well as the existing ones.
57
-------
Figure 5« View of Membrane Biological Filter Device. Insert at
upper right shows detail of channels in Plexiglas plates,
At far left is bacterial bath and pump. At right coming
from pump is effluent line and reservoir. At far right
the feed stream line leads in from reservoir which is
not shown.
58
-------
Z 100
Ul
UJ
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CD
Z
UJ
cc
o
0.
UJ
o
a:
UJ
a.
50
Membrane
Theoretical maximum
AA O.I Inch center plate
O 0.25 Inch center plate
0
1234
EFFLUENT RATE (LITERS/HOUR)
Figure 6. Dependence of Effluent IPC Concentration on Flow Rate and
Type of Ultrafilter Material in the Plexiglas Model.
Comparison is also made to the theoretical maximum calcu-
lated for these conditions from Equation 5.
59
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SECTION XI
RELATIONSHIP OF CHEMICAL STRUCTURE OF HERBICIDES TO
DEGRADATION BY MICROORGANISMS
As part of the overall objectives of this research program, an attempt
was made to develop a body of knowledge on the relationship of chemical
structure to persistence in the soil of a series of isorners and homologs
which should be helpful to chemists seeking to synthesize molecules
that combine herbicidal potency with minimum persistence potential.
For this study, sixty compounds were chosen. They were selected because
they: 1) were used commercially as herbicides (about half of those
selected), 2) were presumed degradation products of these pesticides,
or 3) contributed to a structurally homologous series between two or
more of the herbicides selected. All of the compounds selected had a
single aromatic nucleus with a side chain of varying complexity and
nearly all had varying numbers of chlorine atoms in different positions
on the aromatic ring. Among the classes of compounds represented were
the chlorinated phenols, benzoic acids, anilines, phenoxyacetic acids,
phenylureas and phenylcarbamates. The compounds were either purchased
from chemical supply houses or, in the case of proprietary herbicides,
were procured from the manufacturer. Compounds of questionable purity
were recrystallized prior to use. The compounds used in this study
are listed below in groups having similar structural features:
Phenols
Phenol
3-Chlorophenol
4-Chlorophenol
2,4-Dichlorophenol
3,4-Dichlorophenol
2,4,5-Trichlorophenol
2,3,4,5,6-Pentachlorophenol
Benzoic Acids
Benzoic acid
3-Chlorobenzoic acid
4-Chlorobenzoic acid
2,4-Dichlorobenzoic acid
3,4-Dichlorobenzoic acid
2,4,5-trichlorobenzoic acid
2,3,4-Trichlorobenzoic acid
2,3,6-Trichlorobenzoic acid
2,3,5,6-Tetrachlorobenzoic acid
61
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Anilines
Aniline
3-Chloroaniline
4-Chloroaniline
2,4-Dichloroaniline
3,4-Dichloroaniline
2,4,5-Trichloroaniline
Phenoxyacetic acids
Phenoxyacetic acid
3-Chlorophenoxyacetic acid
4-Chlorophenoxyacetic acid
2,4-Dichlorophenoxyacetic acid (2,4-D)
3,4-Dichlorophenoxyacetic acid
2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)
2,3»4>5,6-Pentachlorophenoxyacetic acid
Phenyl ureas
3-Phenyl-l,1-dimethylurea (f enuron)
3-(3-Chlorophenyl)-l,1-dimethylurea
3-(4-Chlorophenyl)-l,1-dimethylurea (monuron)
3-(2,4-Dichlorophenyl)-l,1-dimethylurea
3-(3 *4-Dichlorophenyl)-l.1-dimethylurea (diuron)
3-(2,4,5-Trichlorophenyl)-l»l-d;iraetnylurea
3- (3»4-Dichlorophenyl )-l-butyl-l-methylurea (neburon)
3-(3»4-Dichlorophenyl)-l-methoxy-l-methylurea (linuron)
Phenyl carbamates
Isopropyl-H-phenylcarbamate (IPC)
Is opropyl-N-(4-chlorophenyl)carbamate
Isopropyl-N-(3-chlorophenyl)carbamate (CIPC)
Is opropyl-N-(2,4-di chlorophenyl)ca rbamate
Isopropyl-N-(3,4-dichlorophenyl)carbamate
Is opropyl-N-(2,4,5-trichlorophenyl)carbamate
Methyl-N-(3,4-dichlorophenyl)carbamate
Phenoxybutyric acids
4-(2,4-Dichlorophenoxy)butyric acid (2,4-DB)
4-(2,4,5-Trichlorophenoxy)butyric acid
Fhenoxyethyl sulfates
Sodium(2,4-dichlorophenoxy)ethylsulfate (sesone)
Sodium(2,4,5-trichlorophenoxy)ethylsulfate (2,4,5-TES)
Anilides
Propionanilide
3,4-Dichloropropionanilide (propanil)
3,4-Dichloro-2-methylacrylanilide (dicryl)
N-(3,4-Dichlorophenyl)-2-methylpentamide (karsil)
62
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Miscellaneous Compounds
2,2-Dichloro-2-(2,4, 5-trichlorophenoxy)ethylester of propionic
acid (erbon)
0-(2,4-Dichlorophenyl)-0-methyl isopropylphosphoroamidothioate
(zytron)
Dimethyl-2,3,5,6-tetrachloroterephthalate (dacthal)
4-Amino-3,5,6-trichloro-picolinic acid (tordon)
2,3,6-Trichlorophenylacetic acid (fenac)
2,316-Trichlorobenzyloxypropanol (tritac)
3,6-Dichloro-2-methoxybenzoic acid (dicamba)
3,5,6-Trichloro-2-methoxybenzoic acid (tricamba)
Since all of the compounds of interest absorb in the ultraviolet region,
their rate of degradation by microorganisms was followed using UV spectro-
photometry. Basically the compounds were exposed in a medium contain-
ing a biological phase, samples were withdrawn from the medium at various
intervals, extracted with hexane-chloroform (2:1), and the quantity of
compound present determined. This rather simple assay technique has
provided a means of rapidly assaying a large number of the compounds.
The sixty compounds were exposed to soil suspensions in flasks on
rotary shakers. These experiments generally lasted three to four months
and were repeated with minor variations in measuring conditions, soil
inoculum, mineral salts composition, test compound concentration,
supplementary nutrients, type of controls, etc. In order to have a
diversity of microorganisms present, the flasks were usually inoculated
with a composite sample of soils obtained from various locations. The
aim of basic experiments such as these was not only to measure relative
rates of microbiological degradation under varying conditions but also
to create conditions of long-term exposure under which microorganisms
of unusual degradative capacities could develop or be selected over
a period of time and could be isolated for further study. Efforts in
this direction led to the consideration and trial of many methods and
combinations of methods which seemed to have some promise of inducing
microorganisms to attack these compounds. In one experiment the
inoculum consisted of soil samples taken from areas under long-term
exposure to chlorinated aromatic compounds in a chemical plant. In
other experiments, various supplementary nutrients such as broths,
sugars and yeast extract were added in order to stimulate degradation
of the compoxind of interest. A continuous culture technique in which
a mixed active population of microorganisms was slowly starved of
substrate in an effort to initiate metabolism of a herbicidal compound
of interest was tried. It appears, at least in theory, that the latter
technique properly pursued might yield results of great interest.
Because the termination date of this program was made earlier than had
originally been anticipated, it was not possible to obtain as complete
63
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data as necessary but several trends did emerge. Using the data at
hand to determine the effect of chemical structure on the rate of
degradation of these compounds, a regression line was computer-
calculated from the spectrophotometer measurements of the herbicide
cultures^over a period of time. The slope of the linear component
of the line was taken as a measure of the rapidity of degradation.
By ranking the slope values from greatest to least (most rapid to
least rapid degradations), several conclusions were made. One was
that the nature of the side chain or R-groups exerts a greater influence
on degradation than the number and position of chlorine on the benzene
ring nucleus. Slope values were distributed over a range of 126 units
for R-group configurations as compared with a range of 68 units for
chlorine configurations. The mean order for R-group configuration was:
phenols > anilines > phenylcarbamates > benzoic acids > phenoxyacetic
acids > phenyl ureas. The addition of supplementary nutrients to the
degrading media did not significantly change this order. The mean
order of degradation for chlorine configurations was: 4-chloro >
2,4-dichloro > 2,4,5-trichloro > 3-chloro > 3,4-dichloro. In this
case, however, supplementary nutrients caused marked changes in the
order of degradation with there being no current explanation of the
effect.
An attempt at constructing and testing a hypothesis that would explain
these observed differences in rates of degradation was initiated but,
although these studies were not completed for the reason mentioned
above, they are mentioned here as a matter of record. Conceivably
the rate at which a molecule is degraded is dependent on a number of
factors such as: 1) the ease with which the molecule can penetrate
the cell and reach the appropriate enzyme site, 2) the extent to which
steric effects interfere with enzyme bonding, and 3) the extent to
which electronic effects of molecular substituents either interfere
with enzyme bonding or alter the energy required to break the critical
bonds in the molecule. The first factor may be estimated by the lipid
solubility of the compounds. Lipid solubility in the form of the
partition coefficient can be determined or learned from the literature.
Electronic effects of a given functional group can be estimated by Haramet
functions, many of which are available from the literature. Steric
effects may be approximated by the Taft Steric Parameter. This approach
has been used quite successfully by toxicologists attempting to correlate
structure and toxicity in certain systems (Hansch, et al.. 1963- Hansch
and Deutsch, 1966). By using an equation of the type:—
log BR - kjUog Px)2 + k2(log Px) + po + k3Es -f k4
64
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where BR = biological response
Px = partition coefficient of compound x
P = the Hammet constant
a = the appropriate Hammet substituent value
Es = the appropriate Taft steric parameter
kj_, \H2» ^3, k^ = computed regression coefficients
it is possible to estimate the relative importance of the various factors
that contribute to the biological response as defined by the equation.
The our knowledge, no workers have attempted to use this approach to
explain the microbiological degradation of a series of homologous com-
pounds, but it appears to be an approach worthy of investigation. When
sufficient data are collected, it may be possible to use this approach
to estimate the susceptibility to degradation of other compounds.
65
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SECTION III
ACKNOWLEDGMENTS
The support and help of Boyce Thompson Institute and its Managing
Director, Dr. George L. McNew, and of the Environmental Protection
Agency are gratefully acknowledged.
The research reported here was conducted during the early part of
the program by Dr. G. B. Tweedy and Dr. Herman Gershon and more recently
primarily by Dr. G. W. McClure.
67
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SECTION XIII
REFERENCES
Alexander, M. 1965. Persistence and biological reactions of
pesticides in soils. Soil Sci. Soc. Amer. Proc. 29: 1-7.
Alexander, M., and B. K. Lustigman. 1966. The effect of chemical
structures on microbial degradation of substituted benzenes.
J. Agr. Fd. Chem. 14: 410-413.
Audus, L. J. 1946. A new soil perfusion apparatus. Nature 158: 419.
Audus, L. J. 1951. The biological detoadcation of hormone residues
in soil. Plant Soil 3: 170-192.
Audus, L. J. I960. Microbiological breakdown of herbicides in soils.
In Woodford, E. K. and G. R. Sager, ed. Herbicides and the soil.
Blackvell Scientific Publications, Oxford, pp. 1-19.
Audus, L. J. 1964.. Herbicide behaviour in the soil. II. Interactions
with soil microorganisms, pp. 163-206. In Audus, L. J., ed.
The physiology and biochemistry of herbicides. 555 pp. Academic
Press, New York.
Bartha, R. 1968. Biochemical transformations of anilide herbicides
in soil. J. Agr. Fd. Chem. 16: 602-604.
Bartha, R., and D. Pramer. 1967. Pesticides transformation to aniline
and azo compounds in soil. Science 156: 1617-1618.
Bartha, R., R. P. Lanzilotta, and D. Pramer. 1967. Stability and effects
of some pesticides in soil. Appl. Microbiol. 15: 67-75.
Bartha, R., H. A. B. Linke, and D. Pramer. 1968. Pesticide transforma-
tions. Production of chloroazobenzenes from chloroanilines.
Science 161: 582-583.
Bartholomew, W. V. 1957. Maintaining organic matter. U. S. Dept. Agr.
Yearb. Agr. 1957: 245-252.
Batelle Memorial Institute. 1969. Conference on membranes for industrial,
biological, and waste treatment processes. Oct. 20, 21, 1969.
Columbus, Ohio.
Clark, C. G., and S. J. L. Wright. 1970. Detoxication of IPO and CIPC
in soil, and isolation of IPO-metabolizing bacteria. Soil Biol.
Biochem, 2: 19-26.
69
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Hansch, C., and E. W. Deutsch. 1966. The use of substitution constants
in the study of structure-activity relationships in cholinesterase
inhibitors. Biochem. Biophys. Acta. 126: 117-128.
Hansch, C., R. M. Muir, T. Fujita, P. P. Maloney, F. Geiger, and M. Streich.
1963. The correlation of biological activity of plant growth regu-
lators and chloromycetin derivatives with Hammet constants and
partition coefficients. J. Amer. Chem. Soc. 85: 2817-2824.
Dalton, R. L., A. W. Evans, and R. C. Rhodes. 1965. Disappearance of
diuron in cotton field soils. Proc. Southern Weed Conf. 18: 72.
Eliassin, R., and G. Tchobanoglous. 1969. Removal of N and P from
waste water. Environ. Sci. Tech. 6: 536-541.
Fields, M. L., R. Der, and D. D. Hemphill. 1967. Influence of DCPA
on selected soil microorganisms. Weeds 15: 195-197.
Foy, C. L., and S. W. Bingham. 1969. Some research approaches toward
minimizing herbicidal residues in the environment. Residue Rev.
29: 105-135.
Geissbuhler, H., G. Haaelback, H. Aebi, and L. Ebner. 1963. The fate of
N-(4-chlorophenoxy)-phenyl-N,N-dimethylurea in soils and plants.
Weed Res. 3: 277.
Gordon, R. E. 1966. Some strains in search of a genus - Corynebacterium,
Mycobacterium, Hocardia or what? J. Gen. Microbiol. 43: 329-343.
Josephs, M. J., D. R. Mussell, J. K. Leasure, and R. L. Warden. 1957.
l,2,4*5-Tetrachlorobenzene for the control of wild oats. Proc.
North Central Weed Control Conf. 14th, pp. 37-38.
Kaufman, D. 1967. Degradation of carbamate herbicides in soil. J.
Agr. Fd. Chem. 15: 582-591.
Kaufman, D. D., and P. C. Kearney. 1965. Microbial degradation of
isopropyl-N-3-chlorophenylcarbamate and 2-chloroethyl-N-3-
chlorophenyicarbamate. Appl. Microbiol. 13: 443-446. ~
Kearney, P. C. 1965. Purification and properties of an enzyme responsible
for hydrolyzing phenylcarbamates. J. Agr. Fd. Chem. 13: 561-564.
Kearney, P. C., and D. D. Kaufman. 1965. Enzyme from soil bacterium
hydrolyzes phenylcarbamate herbicides. Science 147: 740-741.
Kearney, P. C0 E. A. Woolson, J. R. Plimmer, and A. R. Isensee. 1969.
Decontamination of pesticides in soils. Residue Rev. 29: 137-149.
Lederberg, J., and Ester M. Lederberg. 1952. Replica plating and indirect
selections of bacterial mutants. J. Baot. 63: 399-406.
70
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Limpel. L. E., P. H. Schuldt, and R. F. Lindemann. 1959. DAC-893, a
promising new pre-emergence herbicide for use on turf. Agron.
Abstr. p. 90.
MacRae, I. C., and M. Alexander. 1965. Microbial degradation of selected
herbicides in soil. J. Agr. Fd. Chem. 13: 72-76.
Pease, H. L. 1962. Separation and colorimetric determination of monuron
and diuron residues. J. Agr. Fd. Chem. 10: 279-281.
Schwartz, H. G. 1967. Microbial degradation of pesticides in aqueous
solutions. J. Water Poll. Control Fed. 39: 1701.
Skinner, W. A., D. E. Stallard, and W. E. Priddle. 1964. The isolation
and identification of the metabolites of Dacthal herbicide. Abstr.
of papers, 147th National Meeting, Amer. Chem. Soc., Philadelphia,
Pa., pp. 4A-5A.
Upchurch, R. P. 1966. Behavior of herbicides in soil. Residue Rev.
16: 47-85.
Weed Society of America. 1967. Herbicide Handbook, 1st ed. 293 pp.
Geneva, New York.
Whiteside, J. S., and M. Alexander. I960. Measurement of microbiological
effects of herbicides. Weeds 8: 204-213.
71
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SECTION XIV
PUBLICATIONS
Gershon, H., and G. W. McClure, Jr. 1967. Approach to the study of
the degradation of dimethyl tetrachloroterephthalate. Contrib.
Boyce Thompson Inst. 23: 291-294.
McClure, G. W. 1969. The dissipation of phenyl carbamate toxicity by
a bacterium. Proc. N. E. Weed Cont. Conf. 23: 419.
McClure, G. W. 1970. Accelerated degradation of herbicides in soil by
the application of microbial nutrient broths. Contrib. Boyce
Thompson Inst. 24: 235-240.
McClure, G. W. 1971. A membrane biological filter device for reducing
waterborne biodegradable pollutants. J. Water Pollution Control
Federation. In manuscript.
McClure, G. W. 1971. Influence of ring chlorine on the degradation of
some anilide herbicides and related compounds by IPC-cultured micro-
organisms. J. Agr. Fd. Cham. In manuscript.
McClure, G. W. 1972. Degradation of phenylcarbamates in soil by a
mixed suspension of IPC-cultured microorganisms. J. Environ.
Quality. In press.
Torgeson, D. C., and H. Mee. 1967. Microbial degradation of bromacil.
Proc. N. E, Weed Cont. Conf. 20: 584.
Torgeson, D. C. 1969. Microbial degradation of pesticides in soil.
p. 58-59. In,: Gunkel, James E., ed. Current Topics in Plant
Science. 461 pp. Academic Press, New Tork.
Tweedy, B. G., and Nikki Turner. 1965. Effect of Dacthal on micro-
organisms. Phytopathology 55: 1080.
Tweedy, B. G., Nikki Turner, and Miriam Achituv. 1968. The interactions
of soil-borne microorganisms and DCPA. Weed Science 16: 470-473.
73
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1
5
Accession Number
2
Subject Field &
05G
Or6antza"on BOYCE THOMPSON INSTITUTE
Group
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
FOR PLANT RESEARCH, INC.
Yonkers, New York
Title
INTERACTION OF HERBICIDES AND SOIL MICROORGANISMS
1 0 | Authors)
Torgeson, D. C.
16
Project Designation
16060DMP03/71
21 Note
22 Citation
Descriptors (Starred First)
Herbicides,* Soil Microorganism,* Biodegradation,*
Bacteria, Fungi, Soil Treatment, Biological Membranes,
Biological Filter.
25 I Identifiers (Starred First)
27
Abstract The herbicide 2,3,5,6-tetrachloroterephthalate (DCPA) had little effect on
soil microflora but was degraded by a number of bacteria and fungi. Methyl-2,3,5,6-
tetrachloroterephthalate and 2,3,5,6-tetrachloroterephthalic acid were identified as
degradation products. Penicillium paraherquei Abe isolated from soil previously
treated with bromacil (5-bromo-3-sec-butyl-6-methyluracil) was found to degrade
bromacil in culture. When added to sterile bromacil treated soil accelerated degrada-
tion was also obtained.
A mixture of microorganisms capable of degrading and utilizing IPC (isopropyl N-
phenylcarbamate) was used to study the influence of ring chlorine on the degradation
of a series of aniline herbicides. The retarding effect of ring chlorine on the rate
of degradation increased according to the configuration sequence: 0 > 2,4 > 2,4,5 >
3 > 4 > 3,4. When added to the soil these organisms accelerated the degradation of
IPC and related compounds. A membrane irbiological filter" device for reducing water-
borne biodegradable pollutants was also demonstrated using these organisms.
The addition of microbial nutrient broths to herbicide treated soils resulted in
increased degradation of some of the herbicides, presumably as the result of increased
microbial activity.
Abst
ractor
D. C. Torgeson
Boyce Thompson Institute for Pl^nt Researchf Inc.f Yonkera^ flTy,
JJJR:I02 (REV. JULY I9«9»
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 20240
* CPO! 18«8-3gs-339
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