msrmsmon AND EFFECTS OF om mo ' ‘
2,4,5»? m moms meme-mm - *

Bisserfiation for the Segree of Ph. D.
MiCHiGAH SEATS UNWERSH‘Y
GARLAND F. macs, R.
1975

 

WWW

WWWWWW WW WWWWWWWWWWWW

63/2143

This is to certify that the

thesis entitled
Distribution and Effects of DDT and
2,4,S-T in Bacillus megaterium

presented by

Garland F. Hicks, Jr.

has been accepted towards fulfillment
of the requirements for

Ph. D. den greein Microbiology

films”

v

 

Major professor . W
ihomas R. Corner

Date July, 1975 I

04mg W

 

 

 

 

 

 

 

 

 

 

 

 

 

.Ililin'ljll

ABSTRACT

DISTRIBUTION AND EFFECTS OF DDT AND
2,4,5-T IN BACILLUS MEGATERIUM

BY
Garland F. Hicks, Jr.

DDT, a water insoluble insecticide, and 2,4,S-T, a
water soluble herbicide, exerted similar effects on cells
of Bacillus megaterium. ‘The viability of cells in non-
nutritive media was reduced after several h exposure to
10 ug/ml (S ug/mg cell dry weight) of either pesticide.
Respiration was not affected by either pesticide at 100
ug/ml. Growth was affected only at 100 ug 2,4,5-T/m1.

A small amount of DDT was degraded to a structurally
similar compound, DDD, but no other degradation products
were evident.

A sucrose gradient centrifugation procedure that
separated particulate DDT from cells in suspension was
developed and characterized to allow precise measurement
of DDT uptake by cells in vitro. Bach pesticide parti—
tioned into viable cells which retained 175 ug DDT or
0.5 ug 2,4,S-T/100 mg of cells (dry weight). About 75%

of the accumulated DDT or 60% of the accumulated 2,4,5-T

Garland F. Hicks, Jr.
was sequestered by the limiting membrane. The DDT
retained in the membrane represented about 2% of the
membrane dry weight. Chemical analyses indicated that
DDT but not 2,4,5-T replaced membrane lipids. After
about 1.5 h exposure, DDT and the generated DDD were
slowly eluted from the cells but DDD was eluted at a
faster rate. The eluted DDT also became much more gravi-
tationally stable in aqueous media, as measured by cen-
trifugation characteristics, probably due to its associa-
tion with membranes or other cellular material. These
elution patterns offer an attractive explanation of how
DDT can enter and move through lower trophic levels.
Residual 2,4,5-T in cells was not eluted but remained
associated with cells and membranes even after the via-
bility of the cell population was reduced. Thus it
appears that the limiting membrane is a major site of

interaction of these pesticides with bacteria.

DISTRIBUTION AND EFFECTS OF DDT AND

2,4,s-T IN BACILLUS MEGATERIUM

BY

. “of
Garland Ff Hicks, Jr.

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1975

DEDICATION
This dissertation is dedicated to my wife,

who through her support and infinitely
good humor made my doctoral program possible.

ii

ACKNOWLEDGMENTS

The author expresses his gratitude to Dr. Thomas R.
Corner, whose patient guidance in this investigation and
throughout my graduate program was most highly appreciated.
I thank guidance committee members Dr. James Tiedje,

Dr. Arthur Wolcott, Dr. Clarence Suelter and Dr. Charles
San Clemente for their interest and helpful criticisms.

Special acknowledgments are due to Alexander Filonow
for his instructions in pesticide analytical procedures
and to Dr. Donald DeZeeuw, whose initial encouragement

helped to make this investigation a reality.

iii

TABLE OF CONTENTS

Page
INTRODUCTION. . . . . . . . . . . . . . .W. . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . 4
Microbial Degradation of DDT . . . . . . . . 4
Photooxidation of DDT. . . . . . . . 14
Microbial Degradation of Chlorinated
Biphenyls. . . . . . . . 15
Uptake of DDT by Microbial Cells;
Methods Employed to Measure Uptake . . . . 16
Effects of DDT or DDT Metabolites on
Various Physiological Processes of
Microflora . . . . . . . . . . 25
Nitrogen Transformations. . . . . . . 25
Photosynthesis. . . . . . . . . . . . 27
Growth Effects. . . . . 27
Mode of Action of DDT or Its Metabolites
on Biological Systems. . . . . . . 31
Microbial Interactions with 2, 4, S- T. . . . . 33
Summary. . . . . . . . . . . . . . . . . . . 35
REFERENCES. . . . . . . . . . . . . . . . . . . . . 39
EXPERIMENTAL RESULTS. . . . . . . . . . . . . . . . 49
SECTION 1. THE SEPARATION OF DDT FROM
CELLS IN SUSPENSION AND THE CHANGES IN
SOLUBILITY OF DDT ON EXPOSURE TO CELLS . . . 49
SECTION 2 (ARTICLE 1) - LOCATION AND CON-
SEQUENCES OF 1,1,1-TRICHLORO-2, 2-BIS(p-
CHLOROPHENYL)ETHANE UPTAKE BY BACILLUS
MEGATERIUM . . . . . . . . . . . . . . . . . 59
SECTION 3 (ARTICLE 2) - DISTRIBUTION AND
EFFECTS OF 2,4,5-T IN CELLS OF BACILLUS
MEGATERIUM . . . . . . . . . . . . . . . . . 67
SECTION 4. MEMBRANE MODIFICATION INDUCED
BY DDT . . . . . . . . . . . . . . . . . . . 94
DISCUSSION. . . . . . . . . . . . . . . . . . . . . 103

iv

Table

LIST OF TABLES

Chemical names and abbreviations .

DDT concentrations in biological systems
from European and North American regions .

Changes in DDT sedimentation character-
istics during exposure to cells. . .

SECTION 2 (ARTICLE 1)

Effect of exposure to DDT on oxygen uptake
by intact cells of B. megaterium . . .

SECTION 3 (ARTICLE 2)

Uptake of 2 4, 5- T by cells of Bacillus
megaterium . . . . . . . . . .

Retention of 2,4,S-T by 100 mg of cells
of Bacillus megaterium or by membranes . .

SECTION 4
Summary of gross membrane composition.
Relative percent fatty acid and pesticide

in chloroformzmethanol extracts of puri'
fied membranes . . . . . . . . . . .

Page
13

23

S7

63

86

87

99

100

LIST OF FIGURES

Figure Page
1 Reported pathways for DDT degradation. . . . 12
2 Procedure developed to prepare intact
cells or membranes for DDT assay . . . . . . 51
3 Distribution of DDT in aqueous media,
phosphate buffer washes and sucrose density
gradients over a 24 h exposure period. . . . 55

SECTION 2 (ARTICLE 1)
1 Effect of DDT on the viability of B.

megaterium . . . . . . . . . . . 62
2 Relative amounts of DDT and protein

found in each of six fractions of the

sucrose gradients. . . . . . . . . . . . . . 64

3 Uptake of DDT by resting cells of B.
megaterium or by membranes isolated from
similar cells after exposure to 10 ug of

DDT/m1 o o o o o o o o o o o o o o o o o o o 64
4 DDD content of the total pesticide recovered
from whole cells of B. megaterium. . . . . . 65

SECTION 3 (ARTICLE 2)

1 Survival of cells of B. megaterium in
buffer containing 2,4,S-T. . . . . . . . . . 80

2 Effects of 2,4,5-T on cells of B. mega-
terium grown in buffered peptone . . . . . . 82

3 Oxygen uptake at 30 C by cells of B.
megaterium exposed to 2,4,S-T. . . . . . . . 35

vi

INTRODUCTION

Insecticide and herbicide use accelerated markedly
after World War II with the advent of the scientific dis-
coveries and the technology that led to the production of
these pesticides in large amounts at low cost. The pri-
mary use of pesticides has been in agriculturally related
areas. Scientists initiated investigations to determine
the effects of these compounds on both target and non-
target organisms_which resulted in a voluminous accumula-
tion of data. Many of these data concern the effect of
pesticides on basic soil processes or, more directly, on
the microbial inhabitants of soils and waters.

Of the hundreds of papers pertaining to pesticide
interaction with microflora, four general research goals
were apparent: (i) the characteristics of pesticides as
they are degraded and move through the biosphere, (ii) the
toxic effects on soil and water microflora and the ensuing
ecological effects, (iii) the role microorganisms play in
concentrating various pesticides and how microorganisms
introduce the pesticides to higher organisms through food
chains, and (iv) microorganisms as cellular models for
other organisms to determine the toxic effects of

pesticides.

2

DDT1

has been the most heavily and most widely used
of all the insecticides, and the relative number of
research publications pertaining wholly or in part to DDT
reflects this. The reported effects of DDT in higher
organisms, notably the more conspicuous birds of prey,
has created emotionally charged political issues that may
have accelerated this research. Moreover, the effects
that DDT may have on any living system are decidedly
subtle, may be of many years' duration and, by virtue of
the movement of DDT and its metabolites over the entire
surface of the earth (Harrison et al., 1970; Woodwell

et al., 1971), may be applicable to all organisms.

During the late 19605 many reports appeared in both
the scientific and lay press which indicated that DDT
could be metabolized or otherwise degraded by microorganisms.
This capacity seemed to lessen somewhat the controversy
over the widespread use of DDT. At the same time a group
of very recalcitrant compounds called the polychlorinated
biphenyls were being investigated extensively and found to
have vaguely similar toxicological and physical properties
as DDT and the slightly modified degradative products of
DDT (DDD and DDE). Recently a theoretical connection has

been proposed between photodegradation of DDT and the poly-

chlorinated biphenyls, and to some extent between

 

1See Table l and Figure l for full chemical names
and structures of DDT and degradation products discussed
herein.

3
photodegradation of DDT and several of the DDT degradation
products heretofore attributed to microbial breakdown.
Thus, the ecological fate of DDT is still uncertain.

As evidenced by many thousands of determinations on
higher organisms, DDT can and does become highly concen-
trated in the upper trophic levels of food chains. How-
ever, valid studies on DDT uptake at the bases of most
food chains, i.e., in microorganisms, are rare and not
illustrative, probably because of the techniques used for
evaluation. In addition, no papers report any data con-
cerned with intracellular DDT buildup or distribution.
Again due to techniques used as well as divergent experi-
mental conditions, data indicating the altering effects
that pesticides of any sort have on microorganisms are so
contradictory as to be meaningless.

The overall goal of this research report was to con-
trast the effects of two pesticides, DDT and 2,4,S-T, a
water soluble herbicide, on a model bacterium. These
effects include uptake by the whole cell and membrane of
the cell, the effects on growth and viability, the intra-
cellular distribution of the pesticides and other effects
which the pesticides may have on the whole cell or parts
of the cell. Implicit in this was a major program to
develop and test techniques critically needed to perform

and evaluate this research.

LITERATURE REVIEW

Microbial Degradation of DDT

DDT and its immediate degradative products, DDD and
DDE, are extremely recalcitrant in the natural environ-
ment (Alexander, 1965). This has led many investigators-
to look for additional evidence of microbial degradation
of these compounds and to look for the physiological and
biochemical circumstances associated with the conversion.

Apparently the only previous study ever undertaken
to elucidate the involvement of pesticides directly with
bacterial membranes was done by French and Hoopingarner
(1970). These authors were interested in finding if the
conversion of DDT to DDD was a biologically dependent
process and subsequently determining the intracellular
site of such a process. Membranes or cytoplasm isolated
from Escherichia coli were associated with some DDD forma-
tion but large quantities of DDD were produced by isolated
membranes only in the presence of reduced flavine adenine
dinucleotide (FAD). Conversely, cytoplasm alone or cyto-
plasm containing boiled membrane fractions produced little
DDD, which indicated that the process may be largely

enzymatically mediated. With the site of DDD production

5

established, it was noted that the absence of Kreb's
cycle activity, due to anaerobiosis, was correlated with
increased DDD production and that electrons from the
cytochrome system did not contribute significantly, if
at all, to DDD generation.

Wedemeyer (1966) concluded that reduced cytochrome
oxidase (cytochrome as) was the enzyme responsible for
DDD production in Aerobacter (Enterobacter) aerogenes.
French and Hoopingarner (1970) disagreed because an
active Kreb's cycle should contribute more electrons to
the cytochrome system and thus aerobic conditions would
facilitate rather than inhibit DDD generation. These
results may not be inconsistent; anaerobic conditions,
i.e., a block at the cytochrome oxidase level, may result
in a more highly reduced cytochrome oxidase than if the
cytochrome system were actively passing electrons to
oxygen. If this were the case, the electrons necessary
to produce DDD might come from sources other than the
cytochromes. In any event, French and Hoopingarner sug-
gest that FADH may be the active molecule associated with
the enzymatic conversion of DDT to DDD.

Production of DDD under natural conditions may not
be limited to functional biological systems. Iron por-
phyrins are oxidized by alkyl halides (Castro, 1954) and
reduced porphyrins can contribute electrons necessary for

the conversion (Miskus et al., 1965). Ecobichon and

6
Saschenbrecker (1967) described the appearance of DDD in
frozen blood, where presumably enzyme activity is minimal
or nonexistent, when only DDT was added.

The production of DDD from DDT commands more than an
isolated academic interest. Chacko et al. (1966) were
the first to illustrate this reduction by soil microor-
ganisms. Ko and Lockwood (1968b) reported that DDD may
be somewhat more resistant to degradation than DDT and
DDD also may be more toXic to soil microorganisms as
measured by growth inhibition on artificial media. Ko
and Lockwood also observed that DDD formation was accel-
erated under anaerobic conditions and further accelerated
on the addition of an organic substrate (alfalfa). Chacko
et al. (1966) found that, of the organisms tested, two
genera of actinomycetes, but no fungi, degraded DDT to
DDD in significant quantities. However, in an observa-
'tion that led to an apparent contradiction of conclusions,
maximal DDD production was attained during the active
growth phase of aerated cultures.

DDD production from DDT is a phenomenon that occurs
in many microbial environments (e.g., Cope, 1966; Pfaender
and Alexander, 1972) and may be chacteristic of many
bacteria (Chacko at al., 1966; Barker et al., 1965;
Braunberg and Beck, 1968; Johnson et al., 1967; Mendel
and Walters, 1966; Wedemeyer, 1966, 1967; Kokke, 1968;

Malone, 1970), some fungi (Kallman and Andrews, 1963;

7
Matsumura and Bousch, 1968; Anderson and Lichtenstein,
1971), and one alga (Kiel and Priester, 1969).

The biological conversion of DDT to DDD is probably
limited to microorganisms. Barker and Morrison (1964)
found DDD only in the alimentary tracts of mice fed DDT,
and there only in areas of high microbial activity. DDT
injected peritoneally was not significantly degraded,
suggesting that the liver had little to do With the
"detoxification reaction" (however, little evidence exists
to indicate that DDD is any more or less toxic to higher
organisms than DDT). Subsequently, Barker et al. (1965)
isolated from intestines of mice a Proteus vulgaris
strain that would generate DDD in vitro. The authors
implied that DDD found in mammalian and avian cadavers
may be a function of bacterial processes, as P. vulgaris
is a primary invader of deceased animals.

Less commonly reported are the microbial conversion
of DDT to DDE (e.g., Braunberg and Beck, 1968; Wedemeyer,
1967; Kokke, 1970a,b; Pfaender and Alexander, 1972;
Miyazaki and Thorstein, 1972) and the conversion of DDD
or DDE to other products by microbes. Wedemeyer (1967)
has shown, by means of sequential analysis, that many
other degradative products of DDT are generated by cell-
free extracts of E. aerogenes. A representation of his
analysis is included in Figure 1. In this model, DDE is

not an intermediate of DDD generation.

8

Sink et al. (1972) found-that DDD, DDE, and DDMU were
produced in ovine rumen fluid in vitro, but DDE was an end
product rather than an intermediate. Peterson and Robison
(1964) noted that rats contained DDD and DDE upon oral
administration of DDT. Wedemeyer speculated that metabo-
lites similar to those produced by E. aerogenes may also
be produced by the intestinal flora.

Guenzi and Beard (1968) found that DDT was degraded
to DDD (and possibly other unidentified products) by
anaerobic soil over a period of several weeks, whereas
sterile soil facilitated no DDD production. Unidentified
alfalfa volatiles tended to accelerate DDD generation.

Ko and Lockwood (1968b) reported similar results. Thus
DDD generation seemed dependent on viable organisms.
Burge (1971) indicated that autoclaved 5011 may have
undergone changes (e.g., free radicals may have been
quenched or other physical and chemical properties may
have been modified) sufficient to inhibit nonbiological
transformations of DDT. To test the conclusions of Ko
and Lockwood (1968b) and Guenzi and Beard (1968), Burge
added a very small inoculum of natural soil to autoclaved
soil containing DDT. The processes that converted DDT
to DDD were restored by this procedure, and all DDT
eventually disappeared. He too noted that alfalfa vola-
tiles accelerated the disappearance of the DDT, and that

2% oxygen contributed to the stability of the pesticide.

9

DDD was the only breakdown product detected, and by addi-
tion of DDD and DDE to other samples it was found these
compounds were somewhat more resistant to degradation than
DDT. It was concluded that unknown pathways of DDT
breakdown, not involving DDD as an intermediate, may exist.

Stenersen (1965) isolated Serratia marcescens, E.
coli and an unidentified bacterium from excrements of
DDT-resistant or -susceptible flies. Each bacterium, when
grown with an oxygen deficiency, converted about 90 per
cent of the DDT to DDD, and about 5 per cent to DDE. The
flies themselves were analyzed for pesticide content and
no difference in products was found in the two strains.
Stenersen concluded that the ability to degrade DDT, by
the flies proper or by associated microflora, was not a
factor that determined resistance to DDT. Dinamarca et
al. (1971) later suggested that the conversion of DDT to
DDE was a mechanism of resistance in houseflies.

Although the relative toxicity of DDT, DDE, and DDD
for microorganisms is unclear, Kiel and Priester (1969)
suggested that DDE, produced in vitro by a marine diatom,
may be less toxic to water microflora because it is much
more water soluble than DDT and thus would more easily be
leached from cells. This hypothesis apparently has not
received support.

Alexander and colleagues (1970, 1972, 1973) reported

that a Hydrogenomonas sp. degraded DDM to one or more

10
products and that cell-free extracts of the bacterium,
under anaerobic conditions, could transform DDT to DDD,
DDMS, and DBP. PCPA was found when oxygen was added to
the extract. An Arthrobacter sp. was shown to degrade
PCPA to PCPGA. Sewage and fresh water stream samples
degraded DDT to large quantities of DBP, DDD and to
smaller quantities of DDE, but no PCPA. Focht (1972)
isolated a Fusarium sp. that could degrade both DDM and
PCPA to H20, C02, and HCl. Thus, a plausible model has
been established to account for complete microbial degra-
dation of DDT (Figure l).

The enzymes involved in the DDT transformation are
constitutive in Hydrogenomonas and are associated with a
cometabolic process (Pfaender and Alexander, 1973), i.e.,

a degradative pathway in which the organism derives no
energy or elemental requirements. The concept of cometabo-
lism attained prominence largely from studies on pesticide
degradation (Horvath, 1972) and represents a wide and
curious aspect of microbiology.

Several reports claim that some animals converted
g,pf—DDT to p,p'-DDT (summarized in Bitman et al., 1971),
which prompted others to examine E. aerogenes for this
ability, but no evidence of the isomerization was found.
Bitman et al. (1971) refuted the original reports by
demonstrating that the original data were a result of

poor experimental technique.

11

Figure 1. Reported pathways for DDT degradation
(a composite diagram adapted from many authors' work--
see text).

Solid lines indicate proposed pathways of DDT degrada-
tion by individual microbial species, cell-free
extracts of specific miroorganisms or microbial eco-
systems. Broken lines indicate that degradative
products were reported but no sequence necessarily

was implied in original articles. Dotted lines
illustrate proposed pathways for photoconversion of
DDT. Specific investigators and organisms or eco-
systems studied, if applicable, are as follows:

1. Wedemeyer, 1967; E. aerogenes (cell-free extracts
and whole cells).

2. Focht and Alexander, 1972; Fusarium sp.

3a. Pfaender and Alexander, 1972; microbial communi-
ties of sewage and fresh water.

3b. Pfaender and Alexander, 1972; whole cells of
Hydrogenomonas sp. + 02'

3c. Pfaender and Alexander, 1972; cell-free extracts
Of Hydrogenomonas sp.

4. Sink et al., 1972; rumen fluid.

5. Focht and Alexander, 1970.

6. Maugh, 1973.

7. Plimmer et al., 1970.

8. Ahmed and Focht, 1973; Achromobacter sp.

9. Kaiser and Wong, 1974; unspecified organism(s).
10. Numerous other investigators (see text).

11. Bitman et al., 1971.

12

 

 

 

 

 

 

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13

Table 1. Chemical names and abbreviations

 

BA = benzoic acid
p7CBA = p—chlorobenzoic acid
CB 8 chlorinated biphenyl

DBP = 4,4'-dichloroben20phenone

DDA = 2,2-bis(p-chlorophenyl)acetate

DDD = l,1-dichloro-2,2-bis(p-chlorophenyl)ethane
DDE = l,l-dichloro-2,2-bis(p;chlorophenyl)ethylene

DF = 3,6-dichlorof1uorenone
DDOH = 2,2-bis(p-chlorophenyl)ethanol
DDM = bist-chlorophenyl)methane

DDMS = l-chloro-2,2-bis(pfchlorophenyl)ethane
DDMU = l-chloro-2,2-bis(pfchlorophenyl)ethylene
DDNU = UNSYM-bis(p-chlorophenyl)ethylenev

DDT = 1,1,l-trichloro-2,2-bis(pfchlorophenyl)ethane
PCB = polychlorinated biphenyl

PCPA = pfchlorophenylacetic acid

PCPGA = p-chlorophenylglycolaldehyde

R = pfchlorophenyl group

R1 = g-chlorophenyl group

 

l4
Photooxidation of DDT

Apart from DDT degradation associated with biological
materials, a considerable body of data emanating from in
vitro experiments indicates that DDT can be photooxidized
rather extensively. The vapor pressure of DDT is high
(Lichtenstein and Schulz, 1970; Spencer and Cliath, 1972)
and that of DDE is even higher (Maugh, 1973). Thus the
potential photooxidation of the compounds could have
important ecological significance.

Plimmer et al. (1970) described UV-induced photooxi-
dation of DDT to several products, including polychlori-
nated biphenyls (PCBs). Maugh (1973) condensed the
research of several workers and came to some guarded
conclusions which are summarized here. During the last
decade thePCBs were discovered to be as ubiquitous as
DDT in the biosphere (e.g., Reynolds, 1969; Risebrough,
1968). The PCBs were thought to have originated from
industrial processes. However, unlike DDT, PCBs are
relatively nonvolatile, and their appearance in the
arctic and in newly created artificial lakes was mysterious.
In addition, the fate of the large quantities of DDT used
in the past three decades was, and remains, unaccounted
for in view of its recalcitrance. This led to the finding
that radiation found in the lower atmosphere (290 to 310
nm) converted vaporized DDT to various PCBs, with inter-

mediates consisting of DDE, DDMU and DBP. DDD, however,

15

was formed slowly, as its formation is dependent on a
hydrogen donor, and was not further reduced or oxidized.

Maugh's description of DDT photooxidation may help
to clarify several longstanding uncertainties about DDT
degradation: (i) the ubiquity and quantity of PCBs in
the biosphere, (ii) the disappearance of at least some
DDT from the earth's surface, (iii) the fate of DDE, which
previously was unknown, and (iv) the failure of any
researcher to observe in situ the DDT degradation schemes
in their entirety proposed by Pfaender and Alexander
(1973), Wedemeyer (1967), and others; indeed, in no
papers reviewed here is there mention of intensively
controlled experiments to check specifically for photo-
conversion of DDT, either acting alone or in synergy
with microbial cometabolism. Radiation of wave lengths
greater than 286 nm will penetrate Pyrex glass (Su and
Zabik, 1972), which may give particular significance to

this latter point.

Microbial Degradation of Chlorinated Biphenyls

Only recently has evidence appeared describing
microbial degradation of some PCBs. 4-Chlorobiphenyl
was degraded by two Achromobacter spp. to benzoic acid
and parachlorobenzoic acid (Ahmed and Focht, 1973).
Although the literature pertaining to DDT degradation
does not account for a direct link to monochlorobiphenyl,

unidentified bacterial isolates degraded a mixture of

16
mono-, di-, tri-, and tetrachlorobiphenyls to 14 non-
chlorinated aliphatic and alkyl aromatic compounds, but
no chlorinated degradation products were found (Kaiser
and Wong, 1974).
Uptake of DDT by Microbial Cells: Methods
Employedfto Measure Uptake

Many investigators have attempted to determine
quantitatively the ability of microflora to accumulate
DDT from aqueous surroundings. This is difficult to do
with a compound that has a solubility in water of about
5 ng/ml, as defined by gravitational stability (Shin et
al., 1970) or about 1.2 ng/ml as defined by ultracentri-
fugation sedimentation characteristics (Bowman et al.,
1960). The problem of separating the particulate DDT
from suspended cells is critical to the attainment of
accurate uptake data; investigators have attacked this
problem in various ways.

DeKoning and Mortimer (1971) inoculated a growing

culture of Euglena gracilis with 14

C-DDT and periodically
took samples of the culture to determine the extent of
DDT uptake by the cells. Their method of removing the
DDT from the medium consisted of extracting the aliquots
with hexane. They assumed the DDT in the mediumor on
the cell surfaces would be removed with hexane but DDT

within the cell would be unaffected, an assumption not

checked experimentally. In any event, the data were

17
presented in terms of radioactive material within the
cells but no cell weights were given. Thus the actual
quantities of DDT accumulated by the cells were impossible
to determine. One unrelated conclusion given by the authors
was that DDT uptake could involve physiological control,
since a polychlorinated biphenyl accidentally introduced
into a Euglena culture was not taken up whereas DDT was.

A more popular way of separating the water-insoluble
DDT from suspended cells was simply to wash the cells
with water. Chacko and Lockwood (1967) washed several
bacteria-pesticide or fungi-pesticide mixtures through a
Millipore filter and analyzed the material on the filter
for pesticide content. No mention was made of checking
the filter for nonfilterable pesticide. The authors felt
this was a valid procedure because Streptomyces griseus
was found to contain 80 per cent of the pesticide to
which it was exposed after the mycelia were washed through
an 80-mesh wire sieve. The materials on the filter con-
tained from 38 to 100 per cent of the DDT originally
added. Similar experiments suggested that uptake was
independent of pH or cell viability.

Using a somewhat different procedure, Ko and Lockwood
(1968a) mixed fungal mycelia with soil treated with DDT.
The mycelia were removed from the soil and washed through
a wire sieve. The DDT concentration in the mycelia of

Rhizoctonia solani increased over 50 h, from about 3 to

18

60 pg DDT/gram mycelium, and at 24 h had accumulated about
50 pg. Ko and Lockwood concluded that the microorganisms
accumulated only about 10 per cent of the pesticide to which
they were exposed but under natural soil conditions the
uptake could be higher due to the greater surface area of
fungi actually growing in the soil. They also speculated
that uptake by soil microorganisms could contribute to
the persistence of DDT in soils and that pesticide
retention "...by microorganisms may also reduce the bio-
logical activity of [DDT] in soil."

To circumvent the problem of aqueous insolubility of
DDT, Johnson and Kennedy (1973) attempted to determine
quantitative uptake of DDT by E. aerogenes or B. subtilis
by exposing each organism to from 1 to 5 ng DDT/ml suspen-
sion and subsequently analyzing the DDT content of the
cell pellet after centrifugation. The pellets contained
between 13 and 17 per cent of the DDT to which the bacteria
were exposed. No correction factors were imposed to
account for interstitial space or void space within the
peptidoglycan. These percentages were reported to repre-
sent magnification factors between 2800 and 4200, which
were independent of pesticide concentrations in the
exposure medium. As the exposure time increased up to
24 h, the uptake of DDT increased only slightly. The
aqueous media in which the cells were suspended were com-

posed of, variously, distilled H20, 0.05 M phosphate

19
buffer or water containing salts that approximated those
found in pond water. The composition of the suspending
medium did not affect pesticide uptake.l Furthermore,
the amount of DDT taken up from each medium did not vary
as the DDT concentrations were increased from 1 ng/ml to
5 ng/ml.

Johnson and Kennedy (1973) apparently were oblivious
to the research of Sodergren (1968). Sodergren conducted
studies with DDT and came to a number of conclusions
about the behavior of the compound in the presence of a
Chlorella sp.: (i) 0.6 ng/ml was the maximum concentra-
tion of DDT that could be used in experiments where the
separation of DDT from cells in aqueous suspension by
centrifugation or filtration was necessary, since precipi-
tation could result at higher concentrations, (ii) 1.2
mg cells could accumulate 0.38 ug DDT from aqueous sur-
roundings, and the rate of uptake was identical to the
rate of diffusion of DDT in water and (iii) DDT uptake
is most probably a form of absorption and not adsorption,
as evidenced by the irreversible nature of the concen—
trating effect.

Vance and Drummond (1969) performed uptake experi-
ments by growing four algal species in the presence of
1 ug DDT/ml. The algae and DDT were separated by filtra-
tion. The results of these experiments suggested that

maximal uptake of DDT occurred after 30 min of exposure

20
and that DDT was concentrated in large but undetermined
quantities in the algae. Cell weights or numbers were
not given, thus precluding any quantitative estimate of
uptake. Similarly, Kiel and Priester (1969) found that
the diatom Cylindrotheca closterium concentrated the
pesticide 265 times above the ambient concentration when
exposed to 0.1 ng DDT/ml. Reportedly, the cells were
separated from the suspended DDT by filtration.

Lower concentrations of DDT (0.274 to 0.362 pg per
unknown amount of H20) were used by Cox (1970a) to illus-
trate that marine algae will accumulate 16 to 54 per cent
of the initial DDT levels from seawater. Filtration was
employed to remove residual DDT from the environment of
the cells. Using similar techniques, Gregory et al.
(1969) found that Anacystis nidulans (a blue-green alga),
Scenedesmus obliquus (a green alga), Euglena gracilis
and two Paramecium spp. contained from 99 to 953 times
the amount of DDT to which they were exposed (1.0 ug/ml);
again, filtration reportedly separated the DDT and cells.

An entirely different method was employed by Kokke
(1970a,b) to determine relative amounts of DDT concen-
trated by microorganisms. Cells isolated from various
microbial environments were grown on agar plates impreg-

nated with 0.8 pg 14

C-DDT/ml. After incubation, auto-
radiography was performed in the cold over the surface

of the plates. Colonies with heavy concentrations of

21
DDT showed as dark spots on the photographic film. Fewer

than 1 per cent of the colonies arising from a tap water
sample accumulated the DDT, but DDT was concentrated by
2 to 7, 60 to 70, or 80 to 90 per cent of the colonies
from, respectively, polluted surface water, garden soil
or nursery soil heavily enriched with organic matter.
Kokke also established a direct correlation between the
ability of the bacteria to concentrate pesticide and

the ability to grow on agar containing DDT. Other
effects were also observed: (i) the percentage of
organisms able to accumulate DDT increased when other
pesticides were sprayed on the soil prior to isolation,
(ii) microorganisms that were sensitive to 1 ug DDT/ml
of peptone agar could be induced to grow on the agar by
sequential transfer from other plates containing 0.6

ug and 0.8 ug DDT/ml. From these data Kokke suggested
that bacteria in nature may have adapted to pesticide-
contaminated environments, as the amount of DDT available
to microorganisms in water is less than that available
to bacteria in garden or nursery soil.

Apparently, Cox (1970a) performed the only in situ
assessment of long term accumulation of DDT by marine
phytoplankton. Levels of DDT or DDT metabolites found
within the marine algae increased from about 0.25 ppm
(1955) to about 0.55 ppm (1970). Controls indicated
that the increase was not an apparent one due only to

advancements in analytical technology.

22

The concentration of DDT at various trophic levels
and in various nonmicrobial elements of food chains has
been extensively investigated in situ. All reported
uptake values for bacteria and for most other microorganisms
are from in vitro experiments which only represent the
potential of the cells to concentrate DDT. The few in
situ determinations relating to DDT uptake by microorganisms
are listed in Table 2 along with DDT residues in other
organisms. The data in Table 2, representative of several
publications (Woodwell, 1967; Cade, 1971; Goodman, 1974;
Reichel et al., 1969; Cox, 1970b; Woodwell et al., 1971;
Burnett, 1971; Deichmann, 1972), are presented to relate
uptake by microorganisms to uptake by other organisms, and
to illustrate the biological magnification of the compound
as it moves through various trophic levels.

Though the data from the above papers vary in magni-
tude, it is evident that microorganisms in general will
accumulate and concentrate large amounts of DDT from
aqueous or soil environments. Apart from the study by
French and H00pingarner, little was known of the precise
interactions of microorganisms and DDT; is DDT absorbed
primarily on the surface of the cell or do large quanti-
ties of the insecticide actually enter the cell? Pfister

(1972) states:

Table 2.

23

DDT concentrations in biological systems

from European and North American regions
(data are representative of several reports)

 

Z DDT.(ppm)a

 

Woodwell (1967)
Goodman (1974)

Woodwell

(1971)

Goodman (1974)

Woodwell

(1971)

Woodwell (1967)

Woodwell (1971)
Goodman (1974)

Woodwell (1971)

Goodman (1974)
Cox (1970)
Goodman (1974)
Woodwell

(1971)

Woodwell (1967)
Woodwell (1967)
Woodwell (1967)

water

rain over United
Kingdom

biota of swamps and
marshes

untreated soils, remote
areas of U.K.

biota of lakes and
streams

plankton (mostly zoo-
plankton)

open ocean algae

fat of antarctic seals
and penguins

ocean protozoa, coel-
enterates, annelids,
nematodes, mollusks,
echinoderms (avg.)
human milk

marine algae (coastal)

lake trout fry (high
embryonic mortality)

continental shelf algae

flounder

estuary bottom samples

shore birds (gulls, terns,

herons, cormorants,
mergansers)

0.00001-0.00005

0.0001

0.001

0.001

0.01

0.04

0.1
0.55
1.0

1.0

1.28
1.28

1.48-1.75

24

Table 2 (continued)

 

Burnett (1971)

Deichmann (1972)

Deichmann (1972)

Reichel
-(1969)

Cade (1971)
Goodman (1974)

2 DDT (ppm)a

A

Emerita analoga--sand 3.62
crab (California coast)

--avg.

"normal" human cadaver 9.7
body fat

human cadaver body fat; 17.3-24.8
cause of death was (avg. 21.1)
atherosclerosis, leukemia,
carcinoma, or hypertension

pesticide poisoned bald
eagle (Florida)

carcass 33.5

liver 85.0

brain 31.6
peregrine eggs l67.0-899.0
herring gull with 1000.0

impaired reproduction

 

3: DDT = DDT + DDD + DDE. Data based from samples
taken in late 19605 and early 19705. PPM = ug/ml or ug/gm.

25
Whether this [DDT] storage ability is actually
inside the cell or merely the entrapment of
halogenated compound in polymer matrices on
the external cell surface remains to be seen.
In either case, [the] extensive surface area
that the microbial system presents for adsorp-

tion places them [sic] in an important position
with respect to a larger ecology.

Effects of DDT or DDT Metabolites on Various
PhysiologicalProcesses of Microflora

Nitrogen Transformations

One microgram DDT/ml is sufficient to inhibit nitri-
fication in Nitrobacter agilis (Garretson and San Clemente,
1968). However, nitrification of ammonium sulfate was
not affected in soil treated with DDT (Bollen et al.,
1954; Martin et al., 1959) at concentrations that approxi-
mate those found in soil immediately after application for
agricultural purposes (20 lbs/acre--Martin et al., 1959).
Eno and Everett (1958) demonstrated that DDT initially
will increase nitrification (12.5 ng DDT/g soil) in soil
planted with legumes, but after sixteen months nitrifica-
tion was decreased. Bollen (1961) reported a decrease
in clover root nodulation when DDT was applied to soil at
10 lbs/acre. Jones (1956) reported that DDT had few or
no effects on ammonifying organisms in culture unless
very high concentrations of DDT were used (0.1%, the
approximate equivalent of l ton/acre). Wilson and
Choudhri (1946) reported that up to 0.5 per cent DDT had

no effect on ammonification in soil, and had no effect

26

on the growth of leguminous bacteria in pure culture.
In the tradition of true sages, Wilson and Choudhri
stated in 1946 that more investigation involving DDT
"seemed useless."

In blatant disregard of this advice, Jones (1952)
found that nitrifiers in soil were affected only at a
DDT concentration of 0.1% (1000 ppm) as determined by
plate counts, whereas 1.0% DDT slightly stimulated growth
of nitrogen-fixing bacteria. Similarly, Bartha et al.
(1967) found no effect of 150 ppm DDT on nitrate produc—
tion in soil but 1500 ppm DDT increased nitrate production
somewhat. DDT did not inhibit nitrogen fixation or growth
of Azotobacter vinelandii, even at 50 times the DDT con-
centration normally applied to agricultural areas
(Mackenzie and MacRae, 1972).

DDD at 10 ug/ml interferes with growth and, thus,
with nitrite oxidation in pure cultures of N. agilis
(Winely and San Clemente, 1970), whereas nitrite consump-
tion was only halved in cells suspended in non-nutritive
medium containing 125 ug DDT/m1. These authors suggested
that growth studies may be a better indicator of some
pesticide effects. Nitrite oxidation studies show only
differences in energy assimilation, whereas growth studies

allow elucidation of biosynthetic reaction inhibition.

27

Photosynthesis

 

The effects of DDT on nitrogen transformations may be
sketchy, contradictory and inconclusive, but little doubt
exists that DDT dramatically reduces photosynthesis in
marine algae. DDT concentrations as low as 5 ng/ml
reduced carbon assimilation in pure algal cultures and,
significantly, produced similar results in a mixed phyto-
plankton culture taken directly from coastal regions
(Wurster, 1968; Ware and Roan, 1970). In another study
Stadnyk et al. (1971) found that 0.1 ug DDT/ml culture
medium decreased the photosynthetic rate and the biomass
of a freshwater alga. Studies on inhibitory actions of
DDT in microbial photosynthesis apparently are not
available. However, Rogers et al. (1971) indicate two
sites of inhibition of electron transport in barley
chloroplasts: one before photosystem II and the other
in the electron transport chain linking the two photo-
systems. Stadnyk et al. (1971) did suggest that inhibi-
tion of photosynthesis may be secondary to inhibition of
growth or acceleration of death in the alga studied.
Indeed, the most extensively investigated effect of DDT
on microorganisms has been that of possible growth

inhibition.

Growth Effects

 

Analysis of data from growth experiments is difficult

due to the nature of the experiments. As the biomass in

28

culture containing DDT increases, the ratio of pesticide
to biomass naturally changes, thus eliminating the possi-
bility of obtaining dose-response data. Ordinarily, one
would expect that the effective concentration of a compound
in solution would not exceed the solubility limit and the
precipitated or excess compound merely would replenish the
solute as it was removed from solution. If this were the
case, the volume of the culture medium would affect the
time in which the individual DDT molecules could be solu-
bilized and could reach the microbial cell. Adsorption
of DDT to surfaces presents yet another problem to be
dealt with, and this may be the key contributor to the
many discrepancies between in situ experiments conducted
in soil or gut and in vitro determinations involving only
glassware. Lastly, the solubility of DDT in aqueous
environments varies with the composition and the pH of
the suspending medium, the carrier solvent used to intro-
duce the DDT to the medium, and the surface area and
chemistry of particles to which DDT may be absorbed
(Shin et al., 1970). With all these factors considered,
it is of little wonder that the experimental results of
three decades are so varied, and in some cases appear to
be so contradictory and inconclusive. However, analysis
of all research reports reveals several most definite,
albeit only qualitative, trends.

Bollen et al. (1964) detected no increase in CO2

evolution or glucose decomposition in soil treated with

29
an unspecified amount of DDT. Another field study (Martin
et al., 1959) indicated that DDT had no effect on the
decomposition of organic matter as measured by C02 evolu-
tion, nor on the relative numbers or kinds of microflora
present, as measured by plate counts. Technical grade
DDT was used in this work and was applied to the soil at
a rate of 20 1bs/acre/6 inch soil surface, a concentra-
tiOn that would approximate maximal normal agricultural
use. Eno and Everett (1958) suggested that 12.5 to 100
ppm DDT did not appreciably affect numbers of fungi in
soil as determined by plate counts, but CO2 evolution was
increased. Varshney and Guar (1972) reported that many
species of fungi were inhibited by 1 ug DDD/g soil, but
noted that the addition of manure protected the fungi
against adverse effects.

Jones (1956) found that concentrations of DDT suf-
ficient to inhibit nitrogen transformations would increase
microbial numbers in soil. Dalton et al. (1971) found
that growth rates of aquatic hyphomycetes were enhanced
when the culture media contained DDT above concentrations
of 2 ug/ml. The authors suggested that the cause of
these increases may have been that DDT was metabolized,
was acting as a cofactor in metabolic processes, or had
altered the permeability of the cells. They further
implied that DDT may be beneficial ecologically because

decomposition of organic matter may be stimulated, leading

30
to a slowing of the eutrophication process. Wurster
(1968) indicated that 5 ng DDT/ml would affect growth of
photosynthetic organisms which in turn would increase
the eutrophication rate.

Ko and Lockwood (1968a) found that both DDT and DDD
were highly toxic to soil microorganisms in cultures
containing from 0.1 to 10 ug pesticide/ml, but DDD
appeared to affect a greater number of species. However,
neither DDT nor DDD at similar concentrations seemed
to have any effect on microorganisms in soil. Ko and
Lockwood suggested that these pesticides could have eco-
logical implications in areas of high microbial activity
such as the gut or the rhizosphere. However, Braunberg
and Beck (1968) found no differences in flora of the
alimentary canals of rats maintained on a constant diet
of normal food plus "100 ppm" DDT.

The growth of Pseudomonas fluorescens or Staphylo-
coccus aureus was inhibited at 50 ug DDT/ml trypticase
soy broth, but inhibition was detected only as the sta-
tionary growth phase was attained; no inhibition of similar
organisms in DDT treated skim milk was detected (Collins
and Langlois, 1968; Langlois and Collins, 1970).

Menzel et al. (1970) found that DDT would inhibit
cell division in two of the four species of marine algae
tested. One hundred nanograms DDT/m1 culture were added

each day to the algae in an attempt to maintain some degree

31

of dose-response relationship. The authors concluded that
DDT may "...exert a dramatic influence on the succession
and dominance of individual forms."

Several other researchers used very high DDT concen-
trations to screen the growth effects of this compound
on bacteria. Although growth inhibition at high DDT
levels would be somewhat academic, a lack of inhibition
would be significant. In the most extensive of these
experiments, Trudgill et al. (1971) found that DDT would
not inhibit growth of any organism tested (except Bacillus
megaterium) on agar containing a surface film of DDT of
unspecified concentration but probably 0.5 mg/cmz.
Daugherty et al. (1971) found that 0.01 M DDT
was the minimum inhibitory concentration for B. thurin-
giensis spores on agar.

Mode of Action of DDT or Its Metabolites
on Biological Systems

 

 

Winely and San Clemente (1970) found that low DDD con-
centrations interfere with nitrite oxidation in N. agilis
not from direct inhibition of nitrite oxidation but rather
through growth inhibition.

Apart from the above study little or no work has been
performed to elucidate the precise biochemical effects of
DDT or DDT analogs on prokaryotic cells. Janicki and
Kinter (1971) report that 1 to 50 ug DDT/m1 will partially

+ 2

inhibit the Na+, K , Mg +-ATPase in homogenates of gills

32
or intestinal mucosae of flounder. This enzyme is believed
to be instrumental in sodium transport across membranes,
and its inhibition would affect the osmotic differential
required in marine fish. Janicki and Kinter believe
that this may explain the sensitivity of many fish to
low environmental concentrations of DDT, but they add
that pesticide concentrations in in vitro studies are
essentially meaningless unless the uptake patterns of
the lipid-rich membrane homogenates are known.

Matsumura and Patil (1969) also observed DDT-induced
inhibition of a Na+, K+, Mg2+-ATPase but emphasized that
the enzyme may not be involved in a sodium-potassium pump
because DDT action on a neuron is quick (e.g., Hilton
et al., 1971) and inhibition of this enzyme likely would
produce a gradual effect on the neuron. Hence, the
authors suggested that the ATPase was a secondary target
of DDT.

Hilton et al. (1973) reported that the lethal action
of DDT in arthropods probably results from an overexcita-
tion of neurons caused by an extended increase in sodium
conductance. Using a lecithin bilayer (Tinsley et al.,
1971, had previously reported a strong association between
DDT and lecithin in vitro) with valinomycin added to

I

induce K+ conductivity across the layer, the authors

found K+ conductance was decreased by 10'6

6

M DDT, was

decreased less with 10' M DDE, and showed an uncertain

33
response with DDD. However, the effects did not correlate
with those observed in arthropod neurons. The authors
speculated that DDT may increase the fluidity of the

artificial membrane.

Microbial Interactions with 2,4,S-T

 

The herbicide 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T) has been used extensively throughout the world
to control broadleaf woody vegetation. Unlike most other
herbicides, 2,4,5-T is somewhat recalcitrant under natural
conditions. The compound may remain stable from three
weeks to 270 days, depending on the location and type of
microbial environment (DeRose and Newman, 1948; Walker
and Newman, 1957; Loos, 1969a). Technical grade and even
more highly purified preparations of 2,4,5-T may contain
varying amounts of chlorodibenzodioxin and chlorodi-
benzofuran isomers (Woolson et al., 1972). These sub-
stances have been implicated strongly as the cause of
various diseases in man and as teratogenic agents in
animals. One dioxin isomer, 2,3,6,7-tetrachlorodibenzo-
dioxin,1 is one of the most toxic compounds known (Kimbrough,
1972). Nearly all scientific investigations of 2,4,5-T
preparations have emphasized the effects of the contami-

nants and relatively little has been focused on the

 

1The designation Z,3,7,8-tetrachlorodibenzodioxin
also is used to describe an identical compound.

34
herbicide proper as compared to other previously used
pesticides. Moreover, the literature pertaining to
2,4,5-T studies on microorganisms is essentially restricted
to studies on metabolism or cometabolism of the compound
(e.g., Horvath, 1970, 1972; Loos, 1969, 1969b).

Several authors (e.g., Okey and Bogan, 1965) have
suggested that the molecular structure of 2,4,5-T is
responsible primarily for the recalcitrance of 2,4,5-T.
However, Horvath (1971) found that a Brevibacterium sp.
will degrade the molecule in vitro, by cometabolism, to
3,5-dichlorocatechol, an intermediate in the metabolism
of 2,4-D, a 2,4,5-T analog (e.g., Bollag et al., 1968;
Tiedje et al., 1969).

The effects of 2,4,5-T on microbial processes such
as photosynthesis, nitrogen transformations, and other
specific biochemical properties evidently are unknown.
Dougherty et al. (1971) reported that the minimum inhi-
bitory concentration for B. thuringiensis is 0.01 M (2.56
mg/ml). Loos (1969b) studied soil that originally
deactivated 2,4,5-T and found that inhibition of bio-
logical activity arose when the perfusing solution con-
tained more than 200 ppm herbicide. Bell (1960) reported
increased respiratory activity of an Achromobacter sp.
after exposure to 2,4,5-T, which was interpreted to
indicate substantial oxidation of the compound. Some-
time later, Horvath (1971) claimed that he was the first

to detect oxidation of 2,4,5-T.

35

In an earlier study, Bell (1957) found that 2,4,5-T
inhibited an unidentified soil bacterium growing in liquid
medium containing 3 mM 2,4,5-T. At lower concentrations,
the bacterium showed an increased endogenous respiratory
pattern.

There is no evidence to indicate that 2,4,5-T is
magnified within food chains (e.g., Leng, 1972; Southwick,
1974) and there is evidence suggesting that some ruminants
will not retain ingested 2,4,5-T within body tissues for
more than one week (Leng, 1972). No research has been
reported on the uptake of 2,4,5-T by microorganisms, nor
on the distribution of 2,4,5-T within prokaryotic or
eukaryotic cells.

Woolson (1972) has developed reliable and relatively
simple methods to extract contaminants from 2,4,5-T
preparations. This represents a critical advance in the
techniques needed to evaluate independently the biological

effects of 2,4,5-T, dioxin, or other contaminants.

Summary

From the many studies on higher organisms and from
the work of Kokke, it is evident that microflora can
concentrate DDT to an indeterminate but many-fold amount
over environmental concentrations. The results of studies
that attempted to elucidate uptake of the water-insoluble
compound by filtering, centrifuging, or directly extract-

ing DDT from pesticide-bacteria mixtures should be

36
interpreted carefully; the precise quantity of DDT a
bacterium is able to accumulate is entirely unknown.
The intracellular disposition of DDT similarly is unknown,
either in bacteria or in higher cells.

The potential of DDT to be metabolized or cometabolized
by microorganisms has been determined to some extent, but
the artificial conditions (e.g., cell-free extracts) under
which many experiments were conducted leads only to specu-
lation about the mechanisms of degradation under natural
conditions. Most researchers who studied DDT degradation
by whole cells reported detecting only DDE or DDD as
end products. However, a few workers observed rather
extensive DDT degradation in cultures that approached
in situ conditions, but reports of photoconversion cast
considerable doubt about the ecological importance of
microbially-mediated DDT degradation in the biosphere.

DDT severely affects photosynthesis in algae but
results from studies of nitrogen transformations are so
contradictory as to preclude even an intelligent guess
on the effect of DDT in nitrogen cycles. Similarly,
growth effects on microorganisms are as varied in in
vitro studies as they are in soil studies. Kokke's
work illustrates that microbial response to DDT may vary
and can be altered; other workers have shown that the
organic matter content of soil may at least temporarily

affect the response of soil organisms to DDT. Studies

37

involving dose-response effects of DDT on the growth of
microorganisms essentially are meaningless unless the
precise DDT content of the target cell is known; availa-
bility of DDT to the cell, affected by solubility of

DDT and adsorption to nonliving particles, most probably
will affect the uptake characteristics of the cell which
in turn will affect the response of the microbial
community.

In no paper reviewed here was there an attempt to
integrate these factors, either experimentally or
intellectually, in determining the gross interactions of
cells and DDT. It is evident that the results of DDT
interactions with microflora, reported over the last
decade, fit only into the broadest of patterns. The
diversified research techniques, good or bad, combine
with the uncertainties of DDT solubility, with the wide
range of organisms and with a wide range of microbial
environments to give widely scattered or conflicting
results.

The herbicide is not considered to be a recalcitrant
compound in the context of many other organochlorine
pesticides such as DDT. Possibly because of its rela-
tively short half-life or due to research emphasis placed
on contaminating molecules, 2,4,S-T has been largely
neglected by microbiologists or cytologists interested

in subcellular distribution or effects. Considerable

38
evidence indicates that 2,4,5-T does not become concen-
trated or magnified within food chains but as yet no
author has reported uptake characteristics of this com-
pound by any of the microflora.

A series of experiments performed under constant
conditions with one organism would provide some insight
on biologically mediated pesticide transformations, be
they physical or chemical, and on pesticide mediated
biological effects. First, however, techniques must be
developed to remove suspended DDT from mixtures of the
pesticide and cells, and to evaluate 2,4,5-T uptake as
well as retention of 2,4,5-T by cells. These experiments
and technique development have been done, some have been
published, and all are reported in this dissertation.

This dissertation is organized into two major sections
which include (i) a critical literature analysis of all
phases of DDT or 2,4,5-T interaction with microflora, and
(ii) experimental results. The latter incorporates a
published paper, a manuscript prepared for publication,
and other sections containing pertinent experimental

data.

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. r
A .. if

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'n.

, .
.
-lh"

 

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EXPERIMENTAL RESULTS

SECTION 1. THE SEPARATION OF DDT FROM CELLS IN
SUSPENSION AND THE CHANGES IN SOLUBILITY
OF DDT ON EXPOSURE TO CELLS

Introduction

 

A The various techniques previously reported to
ascertain uptake of insoluble pesticide by cells are
inadequate for quantitative determinations. Such tech-
niques include (i) autoradiography of microbial colonies
grown on medium containing DDT, (ii) direct extraction
with hexane of a suspension of DDT and cells, (iii) cen-
trifugation of the suspension prior to extraction, (iv)
filtering the suspension prior to extraction; (v) washing
mycelia with water prior to extraction; and (vi) a combi-
nation of (iii), (iv), and (v). The researchers who
performed these techniques apparently did not check the
validity or reliability of results stemming from their
use. Moreover, the aqueous solubility of the pesticide
‘has been reported to be S or less ng/ml. Interactions
of DDT with other biological materials in effect may
change the solubility of the compound, which in turn may
alter the efficiency of the technique used to separate
DDT and cells. The possible changes in the solubility of

49

50
the DDT after exposure to cells could have a profound
effect on the fundamental interactions of biological
systems with the pesticide. In addition, all suspended
and soluble DDT must be removed from the suspended cells
before any accurate estimates of uptake, by intact cells
or subcellular fractions, can be determined.

After initial trials it became apparent that both
differential centrifugation and filtration of pesticide-
cell mixtures were entirely unsuitable procedures for
achieving good separation. Microscopic examination of
the suspensions suggested that many DDT particles were as
large as, or larger than, the bacterial cells in the
immediate environment. Moreover, the density of DDT is
about 1.5, whereas the density of the bacterial cell is
about 1.1. Thus a program was launched to develop sucrose
density gradient techniques to achieve this separation.
By use of this procedure one could see, even without
chemical analysis, the degree of gross separation of DDT
and cells, a factor which was highly desirable in expedit-
ing the screening of initial parameters involved in the

separation.

Materials and Methods
Most methods used here are outlined in Article 1.
The procedures used to remove supernatant DDT from cells
are illustrated in Figure 2. When extra detail or

emphasis is appropriate, the methods are reiterated here.

51

Incubation mixture contained 500 ml phosphate buffer,
pH 7.0, 2 mg cells (dry wt)/ml and 10 ug DDT/ml.
Mixture was agitated continually at 30 C.

Aliquots of 50 ml were centrifuged at
10,400 x g for 10 min and washed with
50 ml phOSphate buffer, pH 7.0, to re-
move soluble DDT and small suspended
DDT particles. Cell pellet resuspended
in 0.8 ml phosphate buffer

DISCONTINUOUS SUCROSE GRADIENT, composed
of’20-70% sucrose, 4 ml per layer, was
layered with cell suspension and centri-
fuged at 4,470 x g for 4 min.

Removed cells with syringe

DDT ASSAY FOR MEMBRANE ISOLATION PROCEDURE

INTACT_CELLS Resuspended cells in 100 ml
phosphate buffer with lysozyme
and DNAse. Mixture was incu-

 

 

bated 2 hr at 30 C.

  

PESTICIDE IN

CELLS Suspension of cell walls and burst

 

  

protoplasts was centrifuged at

3,300 x g for 6 min to remove PHB,
debris. Decanted supernatant, which
contains membranes.

MEMBRANE SUSPENSION was
centrifugediat 19,600 x g
for 20 min to sediment

 

 

 

 

 

membranes 10 m1 used
for DRY WT
DETERMINATION
Removed and PESTICIDE
washed uppermost
part of membrane MEMBRANES
pellet. Resuspended 10 ml used
in 20 m1 H20 for DDT
ASSAY——

 

Figure 2. Procedure developed to prepare intact cells
or membranes for DDT assay.

52

Cellulose nitrate centrifuge tubes were used to hold
all gradients because they are very transparent and are
not susceptible to the actions of organic solvents used
to extract DDT that may have been adsorbed to the walls
of the tube. Gradients were prepared immediately before
use because the 70% sucrose solution in the bottom layer
tended to precipitate if stored more than about three
hours. Many trials consisting of different centrifuge
speeds or various cell preparation procedures were needed
before optimal separation of the DDT and cells was ob-
tained. In all sucrose phases of the gradient, these
were followed by extraction and and analysis over
24 h. After long exposure of cells to DDT these
analyses tested for efficiency of separation and also
elucidated the changing physical state of the DDT. In
addition, prior to sucrose gradient centrifugation, the
amounts of DDT in cell washes and in incubation mixture
supernatants were determined to evaluate total recovery

of the pesticide.

Results
The optimum relative centrifugal force and time were
found to be about 4 min at a force that would just exceed
the vibration point of the centrifuge (this is stated in
Article 1 to be 4,470 x g, but slightly smaller forces
were also satisfactory). The cells formed a wide band

in the 40% sucrose layer, whereas particulate DDT

53
sedimented into the lowermost (70%) sucrose layer. The
cells did not reach an equilibrium point in the 40%
sucrose; centrifugation for longer periods eventually
would drive the cells into the 70% sucrose. Total
recovery of pesticide, expressed as percent DDT plus
DDD extracted divided by the amount of DDT introduced
to the incubation medium, was always in excess of 88%.
Thus, little pesticide was lost through vaporization.
In addition, no evidence was found of any degradation
product other than DDD. Occasionally small but irrepro-
ducible extraneous peaks appeared during gas chromato-
graphic analysis.

Most of the pesticide in the upper five layers (e
plus f in Figure 3) was associated with the intact cells
(f) in the 40% sucrose layer. Successive washing of
cells With phosphate buffer prior to gradient centrifu-
gation reduced somewhat the amount of DDT found apart
from the cells within the gradient. Since the gradient
itself apparently duplicated this washing effect, pre-
liminary washes were eliminated from the procedure.

Good separation of cells and DDT was achieved over
the entire 24 h test period. Surprisingly, much of the
DDT became more soluble in the aqueous medium as the
exposure time increased (Figure 3). There was an initial
increase in cell associated DDT (up to 1.5 h) followed by

a marked decrease of pesticide in the gradient with a

54

Figure 3. Distribution of DDT in aqueous media,
phosphate buffer washes and sucrose density gradients
over a 24 h exposure period.

Closed circles denote total measured recovery of pesti-
cide from 500 ug in each sample. Pesticide unaccounted
for is indicated by a. Centrifugation removed increas-
ingly large amounts of free DDT (V) and washing the cells
continued the trend (I). The pesticide in the super-
natants (b) and cell washes (c) was discarded. Most
DDT sedimented to the 70% suc?ose of the gradient (d)
and remaining pesticide 03) was free in the other
sucrose layers (e) or was bound to the cells (A) in

the 40% sucrose Iayer (f). Data are composite averages
of many determinations.—

55

 

I

 

 

 

 

500

400?

300’

So o1

24

4
HOURS

Figure 3

S6

concomitant increase in unsedimentible pesticide in the
supernatant of the incubation mixture. The quantity of
pesticide in the cell washes also increased over 24 h.

To confirm this apparent increase in pesticide solubility,
supernatant fluids of the incubation mixture were sub-
jected to ultracentrifugation which removed 26 to 50% of
the DDT. Nevertheless, extended exposure caused more

DDT to remain in solution even at high centrifuge speeds
(Table 3).

Crystalline material, presumably pesticide, was
observed microscopically in the supernatants of the incu-
bation mixture, as well as in the buffer used to wash the
cells. Whereas the DDT in the 70% sucrose layer of the
gradient was primarily macroscopic, microscopic particles,
some of which were smaller than B. megaterium cells, were
also included within this layer. Thus, the gradient was
effective in separating relatively small as well as large

particles from the cells.

Discussion

 

It is clear that the centrifugation of a suspension
containing DDT plus cells does allow suspended free DDT
to enter the cell pellet. However, the described sucrose
gradient technique effectively and efficiently removes
the particulate DDT associated with the cell pellet after
initial centrifugation and washes. Thus, the technique

seemed appropriate for use in preparing cells for precise

57

Table 3. Changes in DDT sedimentation characteristics
during exposure to cells

 

Time (h)

 

 

pg 2 DDTa in cell 48 30 78 107 172
supernatant after

centrifugation at

10,400 x g for 10 min

pg 2 DDT sedimented 22 15 28 34 44
from supernatant by

ultracentrifugation

(100,000-280,000 x g;

1 h)

% Z DDT sedimented by 48 50 36 34 26
ultracentrifugation

 

a2 DDT = DDT + DDD.

58
measurements of pesticide uptake by intact cells and,
indirectly, subcellular parts.

Development and testing of the gradient technique
unexpectedly revealed that the apparent solubility of the
pesticide changed markedly upon continued exposure to the
bacterial cells. Consequently there exists no single
value to express DDT solubility during the course of the
experiments to be described below.

The B. megaterium strain used here forms particu-
larly large cells which tend to form chains containing
two to four bacteria. Therefore, the functional units
of this organism are considerably larger than many other
bacteria. As a sucrose gradient separates particles on
the basis of size as well as density, it would appear
that efficiency of separation of a somewhat smaller bac-
terium from DDT particles would be even greater; a
smaller bacterium most probably would not move as fast
through the gradient. Other microbial species or other
insoluble particles were not tested but, in light of
its practicability, the method seems entirely suitable

for other systems.

SECTION 2 (ARTICLE 1)

LOCATION AND CONSEQUENCES OF l,l,l-TRICHLORO-
2,2-BIS( -CHLOROPHENYL)ETHANE UPTAKE
B BACILLUS MEGATERIUM

BY

G. F. Hicks, Jr. and T. R. Corner

Reprinted from Applied Microbiology 25:381-387 (1973)

S9

Vol. 25, No.

>. .7'13‘P‘_Y;¥;‘
' . AmericanSociety for Microbiology Printed in U.S.A

Location and Consequences of
l ,l', l-Trichloro- 2, 2- -bis(p-Chlorophenyl)
Ethane Uptake by Bacillus megaterium

G. F. HICKS, JR., ANDT R. CORNER

...'a‘

 

APPLIED MICROBIOLOGY. Mar. 1973. p. 381—387
Copyright © 1973 American Society for Microbiology

Location and Consequences Of
1 , l, l-Trichloro-Z , 2-bis(p-Chlorophenyl)
Ethane Uptake by Bacillus megaterium

Vol. 25, No. 3
Printed in U.S.A.

G. F. HICKS, JR., AND T. R. CORNER

Department of Microbiology and Public Health, Michigan State University, East Lansing. Michigan 48823

Received for publication 23 August 1972

NO detrimental effects of l, 1,1-trichloro-2,2-bis(p-chlorophenyl)ethane
(DDT) were observed when cells Of Bacillus megaterium were grown from small
inocula in nutrient media containing up to 100 pg of DDT/ml. However, when
the ratio Of DDT to biomass of resting cells was held constant, levels of
DDT as low as 1 rig/ml (0.5 rig/mg of cell dry weight) enhanced the rate of death
in the population. The lethal action of DDT was both time- and dose-dependent
so that higher doses required less time to effect the same killing than did lower
doses. Intact cells bound 3 maximum Of about 1.7 pg of DDT/mg of cell dry
weight, of which about 75% was localized in the protoplast membrane. Much of
the bound DDT was subsequently lost to the suspending medium and the
aqueous stability of the returned DDT was enhanced, possibly by association
with solubilized cell materials. A small quantity Of bound DDT was converted to
1, l-dichlorO-2,2-bis(p-chlorophenyl)ethane, which was released from cells
somewhat faster than DDT. Apparently the lethal action Of DDT was related to
its binding in the membrane, but respiration was not inhibited. The atypical
macroscopic appearance of membranes isolated from treated cells suggested

that cell death may result from altered membrane chemistry.

 

1, 1,1 - Trichloro - 2, 2 - bis(p - chlorophenyl)—
ethane (DDT) continues to be a heavily used
organochlorine insecticide worldwide (21),
though recently its use in the United States has
been restricted. Its buildup and persistence in
the biosphere has aroused considerable inter-
est in the effects of this pesticide on soil micro-
organisms. When spread on agar containing
surface films of various organochlorine insecti-
cides, gram-positive bacteria are generally
more susceptible to growth inhibition than are
gram-negative bacteria (16). Other effects re-
ported in microorganisms range from increased
growth of some species to complete inhibition
of others (3, 9, 18). In addition to these gross
effects, more detailed mechanisms of action
have been suggested. Winely and San Clemente
(18) demonstrated inhibition of respiratory ac-
tivity in Nitrobacter agilis by 1, 1-dichloro—2, 2-
bis(p-chlorophenyl)ethane (DDD; a common
degradation product of DDT) and other pesti-
cides. DDT also may inhibit certain specific
enzymes (7).

DDT is known to accumulate in the lipid-
containing tissues of many higher organisms,

particularly those high in food chains (6), but
soil microorganisms also accumulate it.
Chacko and Lockwood (2) reported that bacte-
ria centrifuged from an aqueous suspension
containing 1 ug of DDT/ml retained between
38% (Bacillus subtilis) and 100% (Agrobacter-
ium tume/aciens) of the DDT to which they
were exposed. Selected chlorinated hydrocar-
bons were taken up from both water and soil by
fungal and actinomycete myelia (2, 8). Cells of
Euglena gracilis also have been reported to
take up DDT from their environment (4).
These studies were concerned with uptake by
intact cells, and little is known of the distribu-
tion of DDT within the cells.

In this paper, we report on experiments
designed to relate the gross lethal effects of
DDT to the site Of localization within cells of B.
megaterium, a representative gram-positive
soil bacillus. Even with high ambient concen-
trations of DDT, no effect could be elicited
when the ratio of DDT to biomass was allowed
to vary. When the ratio of DDT to biomass was
held constant, cells of B. megaterium were
killed by concentrations of DDT as low as 0.5

381

 

382

pg/mg of cell dry weight. Cells bound a consid-
erable amount of the DDT to which they were
exposed, and most of the bound pesticide was
located in the limiting membrane. However,
inhibition of membrane-bound or other re-
spiratory enzymes was not detected.

MATERIALS AND METHODS

Organism and growth conditions. Cells of the
asporogenous KM strain of B. megaterium were
grown in aerated 2% (wt/vol) Oxoid peptone (Flow
Laboratories, Rockville, Md.) broth (pl-I 7.0) at 30 :i:
1 C. Growth was monitored by following absorbance
at 700 nm in a Spectronic-‘ZO spectrophotometer
(Bausch & Lomb, Inc.) in 1.1-cm light path cuvettes
with distilled water as a blank. Optical densities were
correlated with viable cells per milliliter and with cell
dry weight per milliliter. Viable counts were made by
serially diluting the cultures in 1.2% (wt/vol) BactO-
peptone (Difco) and spreading in triplicate on Tryp-
ticase soy agar (BBL). Colonies were counted after
approximately 30 h of incubation at room tempera-
ture.

Exposure of cells to DDT in growth experi-
ments. Approximately 101 cells were spread on
Trypticase soy agar containing 1, 10, or 100 pg of
DDT/ml. The appropriate amount. of pesticide in 0.1
ml of acetone was added to 100 ml of the warm agar
prior to pouring plates. In other experiments. paper
disks were wet with acetone solutions of DDT con-
taining 1, 10, or 100 pg of DDT/ml. allowed to dry,
and placed on Trypticase soy agar plates previously
spread with about 101 bacteria. Bacterial growth was
determined visually.

About 3 x 10‘ cells were also inoculated into each
of several flasks Of Oxoid peptone broth which
contained, variously, 0.1, l, 10, 50, or 100 pg of
DDT/ml, acetone alone, or broth alone. DDT solu-
tions were added by means of a pipette rather than a
syringe. The final concentration Of acetone was
0.0136 M, or 0.1%. Optical densities at 700 nm were
recorded before the addition of cells and at various
times through the culture cycle until exponential
growth was completed. Viable counts and micro-
scopic observation were also used to assess possible
pesticide effects.

Exposure of cells to DDT in survival experi-
ments. Cells were harvested from peptone broth by
centrifugation when the Optical density at 700 nm
reached from 2.20 to 2.40, or about 2.2 :i: 0.1 mg of
cell dry weight/ml, and were washed twice by resus-
pension in sterile 0.1 M potassium phosphate buffer,
pH 7.00 :t 0.05 (hereafter called buffer). These and
all succeeding centrifugations were carried out at 4 C.
Cells at this optical density were in the late exponen-
tial phase of growth. They were then introduced to 99
ml of buffer-DDT-acetone mixtures to bring the final
concentrations to 2.0 mg of cell dry weight/ml and
the final volume to 100 ml. Controls consisted of cells
in buffer alone and cells in buffer plus 0.0136 M
acetone (initial concentration). The flasks containing
these mixtures were agitated for 24 h. Periodically,
1.0-ml samples were taken, and the number of

HICKS AND CORNER

APPL. MICROBIOL.

survivors was determined by the viable counting
procedure. Similarly, the effects of 0.136 or 0.00136
M acetone on these cells were determined.

Respiratory measurements. Oxygen uptake at 30
C was measured by standard Warburg techniques
(17). Cells were suspended to a concentration of 2.0
mg of cell dry weight/ml in buffer containing 1, 10.
50, or 100 pg of DDT/ml. Oxygen uptake was moni-
tored for various time periods. When desired, 0.1 ml
of 2% (wt/wt) glucose was added from the side arm.
For oxygen measurements after long-term exposure
to DDT, 100 ml of a mixture of cells and DDT in
buffer was incubated with aeration at 30 C prior to
being placed in the Warburg vessels.

DDT extraction and gas chromatography. Each
sample to be analyzed for pesticide content was
extracted initially with a mixture of n-hexane and
isopropanol (3:1) followed by two more extractions
with hexane alone. The hexane fractions were pooled
and washed three times with equal volumes of
distilled water. If a sample contained a high concen-
tration of sucrose, three volumes of distilled water
were added to the sample before the extraction
procedure was begun. Any necessary evaporations of
the pesticide-hexane solutions were performed at
room temperature and were Of limited duration. In
any case, only a small percentage of the total volume
was evaporated. Under these conditions, losses of
DDT due to evaporation were negligible. All imple—
ments and chemicals were extracted with the hex—
ane-isopropanol solution to test for interfering sub-
stances. Glassware was washed with acid and with
hexane prior to use. Polypropylene or cellulose ni-
trate materials were washed with hexane alone.

A Beckman gas chromatograph, model GC-5,
equipped with an electron capture detector and a
1.83-m glass column (3-mm inner diameter) was used
for pesticide analysis. Three different packings were
used on 60 to 80 mesh Gas Chrom Q: (i) 2% DC 11.
(ii) 1.5% 0V-17/1.95% QF-l, or (iii) 4% SE 30/6959.
QF-l. Column temperatures were 190, 205, and 205
C, respectively. Inlet temperatures in all cases were
220 C. The carrier gas (helium) had a flow rate of 25
ml/min. Stable base lines were Observed in all
experiments reported.

Separation of suspended DDT and cells. Dis-
continuous gradients comprised of six layers, succes-
sively 4 ml each of 70, 60, 50, 40, 30, and 20% (wt/wt)
sucrose solutions, were employed to separate cells
from sedimentible DDT. Cells. suspended at 2.0 mg
of cell dry weight/ml in 50 ml Of buffer containing 10
pg of DDT/ml, were centrifuged (10,400 x g, 10 min),
washed with 100 ml of buffer, and resedimented. This
procedure eliminated virtually all acetone and non-
sedimentible pesticide. The pellet was thoroughly
suspended in 0.8 t 0.1 ml of buffer and layered on
the gradient. Buffer was added to bring the total
volume of the layer to 1.0 ml. The gradient was then
centrifuged (4,470 x g, 4 min) in a swinging-bucket
rotor (Sorvall. PIS-4), which was allowed to coast to a
stop. The cells formed a band above the 50% sucrose
solution, whereas most DDT was forced into the 70%
layer (see Fig. 2). The cell band and other fractions
were separated by use of a Cornwall syringe, and each
fraction was quantitatively assayed for pesticide.

 

 

VOL. 25, 1973

Other experiments were undertaken to test the
efficiency of this separation procedure. An identical
preparation of cells, not exposed to DDT, was treated
in the same fashion, and the resultant gradient
fractions were analyzed for protein by the method of
Lowry et al. (11). To facilitate a reasonably accurate
protein analysis, each fraction was washed twice
(16,300 x g, 10 min) with 100 ml of buffer to reduce
the sucrose content (5). Similarly, a 1.0-ml prepara-
tion containing approximately 500 pg of DDT and
virtually no acetone was sedimented through an
identical gradient. Acetone was removed by heating
the suspension at 80 C for 2 h. The gradient was then
fractionated and analyzed for DDT.

DDT uptake by whole cells. A 500-ml sample of
buffer containing 10 pg of DDT/ml was inoculated
with 1 g (dry weight) of cells harvested from the
exponential phase of the culture cycle when the
culture optical density at 700 nm was between 2.4
and 2.6. These optical densities are equivalent to
from 2.3 to 2.4 mg of cell dry weight per ml. At
periodic intervals over 30 h, 50-ml samples of this cell
suspension were prepared for gradient centrifugation
and were centrifuged (4,470 x g, 4 min). On removal
of the cells from the gradient, 50 ml of buffer was
added to dilute the sucrose. The cells were sedi-
mented (10,400 x g, 10 min), resuspended in 10 ml of
hexane-isopropanol (3: 1), and stored in the cold
until the pesticide extraction procedure could be
completed. Larger relative centrifugal forces (a speed
of 55,000 rpm in an A-321 rotor, representing from
100,000 to 280,000 x g, 1 h) were employed, with the
use of preparative ultracentrifuge (IEC model 860)
in an attempt to sediment additional material from
the supernatant fluids.

DDT uptake by limiting membranes. The proce-
dure was identical to the one for whole cells up to the
addition of the hexane-isopropanol mixture, except
that the 50-ml samples were prepared and layered on
each of two gradients. The cells from the gradients
were pooled before sedimentation from the diluted
sucrose solution. The cells were then subjected to
lysozyme treatment (0.5 mg of lysozyme/20 mg of cell
dry weight) for about 2 h at 30 C. After lysis was
completed, about 5 mg of deoxyribonuclease was
added to the mixture to reduce viscosity. After 1 h of
incubation with deoxyribonuclease, the mixture was
centrifuged (3,300 x g, 6 min) to remove whole cells,
cell debris, and most poly-fl-hydroxybutyrate gran-
ules. The supernatant fluid was centrifuged (19,600

x g. 20 min) to sediment the membranes. The
membrane pellet was washed in 100 ml of buffer and
resedimented under identical conditions. The pellet
was then suspended in 20.0 ml of buffer and divided
into two 10.0-ml portions. One was mixed with the
hexane-isopropanol solution and the other was used
for dry weight analysis. The supernatant fluids of
several successive buffer washes from a membrane
preparation not exposed to DDT had approximately
equal 260 to 280 nm absorbance ratios. Hence. one
wash was deemed adequate to remove most soluble
material from the membrane preparation. A Beck-
man spectrophotometer, model DB-G, was used for
these determinations. Observations of KMnO.-
stained membrane preparations made with a Philips

EFFECT OF DDT ON 8. MEGATERIUM

383

300 electron microscope evidenced little, if any,
contaminating material.

Chemicals. The composition of 99 + % p,p’-DDT
(City Chemical Corp., New York, NY.) was verified
by gas-liquid chromatography. Nanograde n-hexane
and N anograde isopropanol were Obtained from Mal-
linckrodt Chemical CO. (St. Louis, Mo), lysozyme
and deoxyribonuclease from the Sigma Chemical Co.
(St. Louis MO.), and column packing materials from
Applied Science Laboratories (State College, Pa.).

RESULTS

Action of DDT on resting cells. Resting
cells of B. megaterium were killed by exposure
to all concentrations of DDT tested (Fig. 1).
After 15 h Of exposure, the number of viable
organisms in the flask containing 10 pg Of
DDT/m1 was reduced about 10-fold as com-
pared with the number of viable cells in the
control flasks. Initially, the cell count per
milliliter in the mixtures without DDT, with
acetone alone, or with 1 pg Of DDT/ml ap-
peared to increase. Microscopic examination of
the preparations suggested that these rises in
count were due to fragmentation of chains of
organisms. Such fragmentation may have re-
sulted from the agitation which the cells suf-

 

lO’I I I r I I I I I I I

I0'

VIABLE CELLS] ml
3
fl

 

 

 

 

'05 I I I I I I I I I I I I
0 2 4 6 8 0 l2 l4 l6 IO 20 22
HOURS

FIG. 1. Effect of DDT on the viability of B.
megaterium. Cells (2.0 mg of dry weight/ml) were
agitated in buffer containing 1 pg of DDT/ml (O). 10
pg of DDT/ml (A), 50 pg of DDT/ml (El), 100 pg of
DDT/ml (V), acetone alone (A), or no addition (O).
Values are representative of six experiments.

384

fered during the exposure period. Alternatively,
endogenous energy and carbon sources may
have been able to provide for a limited number
of cell divisions. Microscopic examinations dis-
counted the possibility that plate counts were
reduced by the aggregation of cells around fine
crystals of DDT or other substances. In other
experiments, acetone at initial concentrations
up to 0.136 M was found to have a negligible
effect on cell viability.

Action of DDT on growing cells. Cells
grown in peptone broth culture containing
DDT were not measurably affected by concen-
trations up to 100 pg of DDT/ml. No significant
differences in culture optical densities or viable
counts were found among the controls and
pesticide-containing cultures. Optical densities
at 700 nm of the broth media were not affected
by the addition of DDT or acetone or both.
Moreover, growth was not inhibited on plates
containing up to 100 pg of DDT/ml of agar nor
on agar surfaces with filter-paper disks satu-
rated with DDT solutions of 1, 10. or 100 pg/ml.

Respiratory measurements. No significant
differences in the rates of oxygen utilization
(measured with and without the addition of
glucose) were observed among the various con-
centrations of pesticide and controls (Table l).
The rates of oxygen uptake dropped evenly
over a 24-h period.

Separation of suspended DDT from
cells. It soon became apparent that routine
centrifugation procedures could not provide
adequate separation of cells and tine particles
of DDT. Consequently, the separation proce-
dure based on density gradient centrifugation
in sucrose solution was developed. In a typical
experiment (Fig. 2), the cell pellet from a
mixture exposed to 10 pg of DDT/ml (or 5 pg of
DDT/mg of cell dry weight) for 4 h was layered

HICKS AND CORNER

Arm. MICROBIOL.

on the gradient and centrifuged. The bulk of
the DDT went to the 70% sucrose layer,
whereas the DDT in the 40% layer was as-
sociated with the cells that banded there (Fig.
2B). The distributions of cells alone as assessed
by protein determination (Fig. 2A) or DDT
alone (Fig. 2C) confirmed this observation.
Most DDT settled into the 70% sucrose,
whereas cell protein remained mostly in the
40% layer. When cells not previously exposed to
DDT were mixed with 0.5 mg of crystalline
DDT just prior to centrifugation, results virtu-
ally identical to those shown in Fig. 2C were
obtained.

DDT uptake by whole cells or isolated
membranes. The amount of pesticide detected
in the cells initially increased and then de—
creased as time progressed (Fig. 3). Decreases
were accompanied by corresponding increases
in the amounts of pesticide in cell supernatant
fluids and cell washes. Thus, the total recov-
ered pesticide, including both DDT and the
only detectable degradation product (DDD; see
below), remained essentially stable over 24 h.
About 48 and 50% of the pesticide could be
sedimented from the exposure medium super-
natant fluids by ultracentrifugation at 0 and
1.5 h, respectively. compared with 34 and 2697:.
at 8 and 24 h, respectively. The initial amount
of DDT in each 50-ml sample was approxi-
mately 500 pg. Hence, the maximal uptake of
the pesticide by 100 mg (dry weight) of whole
cells was about 175 pg or about 35% by weight.
of the DDT.

Exposure to DDT did not markedly interfere
with cell wall dissolution by lysozyme or with
subsequent. protoplast lysis. However, mem-
branes isolated from treated cells were of a
whitish color, whereas those from untreated
cells were a pale yellow-brown. Membranes

TABLE 1. Effect of exposure to DDT on oxygen uptake by intact cells of B. megaterium“

 

 

 

 

 

 

Q”, (pliters of 0, per hr per mg of cell dry wt)
Exposure time Endogenous Exogenous
(h) .

Control Acetone DDT (”g/ml) Control Acetone DDT (pg/ml)
l 10 50 100 l 10 50 100
0 38 35 38 39 36 110 118 105 107 105

4 l5 17 15 I4 18 16 117 112 110 112 100
8 11 11 1‘2 9 1'2 90 79 77 81 81 82

16 7 5 6 6 5 6 80 80 73 80 80
24 6 6 3 7 3 55 62 60 59 59 55

 

 

 

 

 

 

 

 

 

 

 

 

 

“Cells were treated with 0.0136 M acetone, 0.0136 M acetone containing DDT, or untreated for the
indicated number of hours. Then samples (2.8 ml) were placed in the main compartments of the Warburg
flasks. After 20 min of equilibration at 30 C, 0.1 ml of 2% (wt/wt) glucose solution or 0.1 M phosphate buffer
was tipped in to start the determination. Center wells contained 0.1 ml of 20% (wt/wt.) KOH.

 

 

 

VOL. 25, 1973

 

T I T T

80"A

50 i PROTEIN ‘

 

 

so~B ~

    

5° CELLS + our
40

20

PERCENT OF TOTAL

 

mo

80

60

4O

 

20

 

 

 

 

FRACTION

FIG. 2. Relative amounts of DDT and protein
found in each of six fractions of the sucrose gradients.
Percentages are based on the total pesticide recov-
ered, which varied from 89 to 96% of the DDT origi-
nally added. Figures 2A and ZC show the distribu-
tion of protein or DDT, respectively, when cells
only or DDT only were subjected to gradient centri-
fugation. Figure 28 illustrates the typical DDT dis-
tribution obtained from preparations containing cells
treated for 4 h with DDT.

isolated from cells treated with 0.5 pg of
DDT/mg of cell dry weight showed an uptake
pattern qualitatively similar to that of whole
cells (Fig. 3). For the first 1.5 h of exposure, the
pesticide content of the membranes increased
to a maximum of about 120 pg/IOO mg of cell
dry weight, and then decreased at a rate similar
to the decrease seen with whole cells.

The reported values for the pesticide content
of the membranes were calculated on the
assumption that the membrane comprised
6.5% of the cell dry weight as given by Yudkin
(22). Membrane yields from untreated cells
were actually about 6.1%, but those from
exposed cells were only about 2.5 to 3.3% of the
initial dry weight. The maximal amount of

EFFECT OF DDT ON B. MEGA TERIUM

385

pesticide in the membranes isolated under
these conditions represented about 4% (wt/wt)
of the recovered membrane dry weight.

The recorded times in Fig. 3 do not reflect
the preparation time (approximately 40 min)
between the removal of the sample from the
exposure medium and the gradient separation
of the suspended pesticide and cells. Factors
such as lowering of temperature (through cen-
trifugation) and sequential reduction of ace-
tone concentration (through the washing of
cells) may have affected the rate of uptake
during these intervals.

Conversion of DDT to DDD. DDD was
produced almost immediately after the addi-
tion of the cells to the buffer containing DDT
(Fig. 4). However, after about 2 h of exposure,
DDD was more easily eluted from the cells than
was DDT. Analysis of pesticide in suspension
in buffer or in sucrose solution or mixed with
heat-killed cells in buffer indicated that DDD
production took place only in the presence of
viable cells. To exclude the possibility that
DDD was being produced after the addition of
hexane-isopropanol to the cell or membrane
suspensions, DDT was added to unexposed
cells previously layered with hexane-iso-
propanol solution. Under these conditions, no
DDD was detected. The solvent also eliminated
any viable organisms as determined by plate
counting. Periodic checks of known DDT solu-
tions suggested that photoconversion to DDD
did not take place.

DISCUSSION

The discrepancy between the accelerated
reduction in viability observed with resting
cells (Fig. 1) and the apparent lack of effect of
DDT on growing cells may be due to a number

 

 

 

 

z I I T ‘T r j I T
§Iso~ 0
5(60’ ° 5 ‘I
s o “
i140” 4

:0»
E 2 fl 5 l
BnOh ‘ o -(
\ .0> . 4
3 so- a
Ill 0
Ecol

)- -«
g 20
l 1 l A l J l A A

§ 0 o I 2 3 s s s r a Van

HOURS

FIG. 3. Uptake of DDT by resting cells of B.
megaterium (open symbols) or by membranes iso-
lated from similar cells (closed symbols) after expo-
sure to 10 pg of DDT/ml. Amounts of 2 mg dry weight
of cells/ml of buffer (containing 10 pg of DDT/ml)
were exposed for 24 hr. The circles and triangles
denote two separate experiments for each curve.

 

 

 

 

 

 

r I T I M
8- -(
3
o s- A ..
I.“ O
.t 5- -
3
3 4L1} -1
E 3- -
8 2- ~
0
at I- -
o I I I In]
0 2 4 6 8 24
HOURS

FIG. 4. DDD content of the total pesticide recov-
ered from whole cells of B. megaterium. Different
symbols denote different experiments.

of factors. The most prominent of these con-
cerns the rather drastic change in the ratio of
DDT to biomass in a growing culture. This
ratio is halved after each doubling of the
population. Under the described growth condi-
tions, the generation time of B. megaterium is
approximately 80 min. At all DDT levels tested,
significant numbers of cells survived after
only 80 min of exposure. Hence, a small initial
reduction of living cells in broth cultures con-
taining DDT would have little measurable
effect on the culture cycle. Differences between
the environment of the growing cells and that
of the resting cells (peptone-water as opposed
to buffer) may have altered the osmotic re-
sponses or metabolic pathways of the cells. The
extent of uptake of DDT may be influenced by
environmental conditions which alter the solu-
bility of DDT or by interactions of DDT with
various medium components. The lack of inhi-
bition observed on the agar plates containing
DDT might be accounted for in a similar way.
Recent results (16) indicated that B.
megaterium is totally inhibited on agar con-
taining a surface film of 0.5 mg of DDT/cm”, a
much higher concentration than any used here.

The conditions under which the resting cells
were exposed would probably more closely
approximate the physiological state of bacteria
in soils than would bacteria grown in rich
media. Much of the time, undomesticated
bacteria are in a condition of relative starvation
(1). However, the temptation to draw direct
parallels to a soil ecosystem should be avoided.

HICKS AND CORNER

Arm. Micaosrm.

Soils vary greatly in biological and chemical
complexity, and fluctuating parameters, such
as water content, affect the degree of adsorp-
tion of DDT to soil particles (14). Thus, pesti-
cides may be less readily available to microor-
ganisms in soil than they were in the experi-
ments here.

The solubility of DDT in water, as defined by
gravitational stability, has been reported to be
about 5 ng/ml (14). The precise solubility of
DDT in buffer was neither investigated nor
defined, but can be considered to be less than 1
pg/ ml as evidenced by the fine suspensions that
resulted when DDT in acetone was introduced
into buffer. As the DDT suspensions were
shaken over a period of hours, slightly larger
aggregates of DDT or DDT complexes ap-
peared. These paracrystalline aggregates prob-
ably resulted from a reduction in acetone
concentration by evaporation. When distilled
water was substituted for the buffer, virtually
identical results were obtained.

Clearly, the cells took up DDT rather slowly
(Fig. 3), but ultimately a maximal concentra-
tion of 1.7 pg/mg of cell dry weight was at-
tained. This represented about a 190-fold in-
crease over the environmental concentration of
DDT. Thus, the cells act as a DDT sink. The
rate at which the cells accumulate DDT apo
pears to be a function of the low water solubil-
ity of DDT and seemingly is governed by the
rate at which DDT can be pumped from the
microcrystal reservoirs through the aqueous
medium into the cell sink. A certain amount of
the observed uptake may have resulted from
direct interactions between cells and solid-
phase DDT. Thus, the rate of uptake would
have been influenced by the rate at which such
transfer occurred. However, it seems that most
of the DDT taken up must first pass through a
solubilization process because, as previously
pointed out, when cell pellets were mixed with
crystalline DDT in short-term experiments,
there was no detectable uptake. In this context,
it has been suggested that various aromatic
hydrocarbons are taken up by cells from aque-
ous solution rather than from the solid phase
(19, 20).

Our findings that the cells accumulated a
maximum of about 35% of the DDT after 1.5 h
of exposure, which dropped to 9% at 24 h, are in
apparent quantitative disagreement with pre-
vious results. Chacko and Lockwood (2) re-
ported that various soil microorganisms took
up between 38 and 100% of the DDT to which
they were exposed. However, differences in
organism and pesticide concentration, different

 

VOL. 25, 1973

techniques for separating suspended pesticide
from cells, and possible differences in cell
concentration and viability all preclude direct
comparison.

The progressive loss of DDT from the mem-
brane fraction (Fig. 3) was reflected in a
concomitant loss by intact cells. Thus, the
decrease in DDT in the membrane did not
result from partitioning into other subcellular
fractions such as the cytoplasm. Rather the
DDT returned to the suspending medium.
That this DDT may have repartitioned into
solid-phase DDT is possible but unlikely, since
less DDT could be sedimented by ultracentrif-
ugation of the exposure medium after long
exposure periods than after short ones. More-
over, the pesticide did not crystallize as it left
the cells because pesticide crystals would have
been detected by the gradient technique. The
increased aqueous stability of the released
DDT may have been caused by the association
of the DDT with cell materials, perhaps mem-
brane lipids, lost to the medium as the cells
gradually deteriorated.

Many of the enzymes of terminal oxidative
respiration are bound to the protoplast mem-
brane in B. megaterium (15). Thus, it would
not have been suprising had respiratory inhibi-
tion resulted from the extensive accumulation
of DDT detected in the membrane fraction
(Fig. 3). However, there are a number of other
important processes that are intimately as-
sociated with the limiting membrane, includ-
ing those involved in cell division, chromosome
replication and segregation, and protein syn-
thesis (12). Of particular significance in this
regard is the qualitative finding that mem-
branes from treated cells apparently have a
slightly different color and probably a different
chemistry than those from untreated cells.
Several other compounds are known to inhibit
selectively the synthesis of certain membrane
components. e.g., diphenylamine (13) or to
solubilize certain membrane components se-
lectively, e.g., alkylamine HCl (10). Thus, this
observation may represent a clue to the way
DDT kills bacterial cells.

ACKNOWLEDGMENTS

This work was supported by grant GB-31663 from the
National Science Foundation.

We thank Jerol Hatinger for technical assistance and A.
R. Wolcott, Y.-O. Shin and A. Filonow for the use of their gas
chromatograph and for advice on gas chromatography proce-
dures. This article was assigned journal article no. 5984 of
the Michigan Agricultural Experiment Station.

 

EFFECT OF DDT ON B. MEGA TERIUM

l

2.

14.

15.

16.

19.

‘21.

22.

387

LITERATURE CITED

Alexander, M. 1961. Introduction to soil microbiology.
John Wiley & Sons, New York.

Chacko, C. 1., and J. L. Lockwood. 1967. Accumulation
of DDT and dieldrin by microorganisms. Can. J.
Microbiol. I3:1123~1126.

. Dalton. S. A., M. Hodkinson. and K. A. Smith. 1970.

Interactions between DDT and river fungi. I. Effects of
p,p'-DDT on the growth of aquatic hyphomycetes.
Appl. Microbiol. 20:662—666.

. de Koning, H. W., and D. C. Martinez. 1971. DDT

uptake and growth of Euglena gracilis. Bull. Environ.
Contam. Toxicol. 6:244—248.

. Gerhardt, B., and H. Beevers. 1968. Influence of sucrose

on protein determination by the Lowry procedure.
Anal. Biochem. 24:337—338.

. Harrison. H. L., O. L. Loucks, J. W. Mitchell. D. F.

Parkhurst, C. R. Tracy, D. G. Watts, and V. J.
Yannacone. Jr. 1970. Systems studies of DDT trans-
port. Science 170:503—508.

. Janicki, R. H., and W. B. Kinter. 1971. DDT inhibits

Na*,K‘,Mg"-ATPase in the intestinal mucosae and
gills of marine teleosts. Nature N. Biol. 233:148-149.

. Ko. W. H., and J. L. Lockwood. 1968. Accumulation and

concentration of chlorinated hydrocarbon pesticides
by microorganisms in soil. Can. J. Microbial.
14:1075-1078.

. K0, W. H., and J. L. Lockwood. 1968. Conversion of

DDT to DDD in soil and the effects of these com-
pounds on soil microorganisms. Can. J. Microbial.
14:1069-1073.

. Kondo, T., and M. Tomizawa. 1969. Hemolytic actions

of short-chain alkylamine hydrochlorides. J. Pharm.
Sci. 58:255-256.

. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J.

Randall. 1951. Protein measurement with the Folin
phenol reagent. J. Biol. Chem. 193:265—275.

. Salton, M. R. J. 1967. Structure and function of bacte-

rial cell membranes. Annu. Rev. Microbiol.

21:417-422.

. Salton. M. R. J., and M. D. Schmitt. 1967. Effects of

diphenylamine on carotenoids and menaquinanes in
bacterial membranes. Biochim. Biophys. Acta
l35:196~207.

Shin. Y. 0., J. J. Chodan, and A. R. Wolcott. 1970.
Adsorption of DDT by soils. soil fractions. and biologi-
cal materials. J. Agr. Food Chem. 18:1129—1133.

Starck, R., and J. T. Wachsman. 1957. Enzyme localiza-
tion in Bacillus megaterium. J. Bacteriol. 73:784—790.

Trudgill, P. W., R. Widdus, and J. S. Rees. 1971. Effects
of organochlorine insecticides on bacterial growth.
respiration and viability. J. Gen. Microbiol. 69:1-13.

. Umbreit, W. W., R. J. Burris, and J. F. Stauffer. 1964.

Manometric techniques, 4th ed. Burgess Publishing
Co., Minneapolis.

. Winely, C. L., and C. L. San Clemente. 1970. Effects of

pesticides on nitrite oxidation by Nitrobacter agilis.
Appl. Microbiol. 19:214—219.

Wodzinski, R. S., and D. Bertolini. 1972. Physical state
in which naphthalene and bibenzyl are utilized by
bacteria. Appl. Microbial. 23:1077—1081.

. Wodzinski, R. S., and M. J. Johnson. 1968. Yields of

bacterial cells from hydracarbons. Appl. Microbiol.
16:1886—1891.

Woodwell, G. M., P. P. Craig. and H. A. Johnson. 1971.
DDT in the biosphere: where does it go? Science
174:.1101-1107.

Yudkin, M. D. 1966. Isolation and analysis of the
protoplast membrane of Bacillus megaterium. Bio-
chem. J. 98:923—928.

 

 

SECTION 3 (ARTICLE 2)

DISTRIBUTION AND EFFECTS OF 2,4,S-T IN

CELLS OF BACILLUS MEGATERIUM

BY
G. F. Hicks, Jr. and T. R. Corner

67

68

Distribution and Effects of 2,4,S—T in
Cells of Bacillus megateriuml

*
G. F. Hicks, Jr.2 and T. R. Corner

Department of Microbiology and Public Health
Michigan State University

East Lansing, Michigan 48824

Running title: 2,4,S-T interactions

(Manuscript prepared for Applied Microbiology)

Journal article no. 7134 from Michigan Agricultural
Experiment Station.

Present address: Department of Biology, Valparaiso
University, Valparaiso, Indiana 46383

69

ABSTRACT

Cell death in a resting population of Bacillus mega-
terium was accelerated by ambient concentrations of
2,4,S-trichlorophenoxyacetic acid (2,4,5-T) equal to or
greater than 10 ug/ml, or 5 ug/mg of cells (dry weight),
but only after prolonged exposure. Conversely, popula-
tions of growing cells were not markedly influenced even
at 100 ug/ml. Effects on cell respiration were not mani-
fest until the ambient concentration reached 1000 ug of
2,4,5-T/m1, or 500 ug/mg. Cells of B. megaterium did,
however, accumulate 2,4,5-T passively to a level approxi-
i mately Z-fold above the ambient concentration. Most of
the accumulated compound was easily washed from the cells
but, of the firmly bound herbicide, about 0.5 ug/mg of
cells (dry weight), nearly 60% by weight, was localized
in the protoplast membrane. The foregoing results,
obtained with a purified preparation of 2,4,S-T, were
also elicited by 2,4,S-T analytical standards. The
extracted contaminants did not produce the results alone,
nor did they influence the results when present in combi-

nation with 2,4,5-T.

70
(INTRODUCTION)

The herbicide 2,4,S-trichlorophenoxyacetic acid
(2,4,S-T) has been used extensively to control broadleaf
woody vegetation throughout the world. Some 2,4,5-T
preparations contain various amounts of chlorodibenzo-
pfdioxins (CDDs) and chlorodibenzofurans, principally the
extremely toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin that
has been an inherent contaminant in the manufacturing
process (Kimbrough, 1972). The occupational and environ-
mental hazards of 2,4,5-T preparations of various degrees
of purity have been investigated (see Lee and Falk, 1973).
But, apart from degradation, the interactions of 2,4,5-T
with bacterial cells are largely unknown (loos, 1969).

Daugherty et al. (1971) reported that 2,4,S-T
inhibits growth of Bacillus thuringiensis. The minimum
‘inhibitory concentration depended on the solvent system
used to deliver the agent but was not less than 0.01 M
(2.55 mg/ml). By means of a soil perfusion system, Loos
(1969b) attempted to select for microflora that would
decompose 2,4,S-T. A perfusing solution containing 200
ug 2,4,S-T per m1 caused the soil to lose biological
activity.

Although 2,4,5-T has been reported to persist in the
environment for up to 270 days (Loos, 1969), it is never-
theless subject to slow biodegradation. The pathway may

be similar to that by which the much less recalcitrant

71
2,4,-dichlorophenoxyacetic acid is degraded (Horvath,
1971, 1972).

The subcellular distribution of 2,4,S-T apparently
has not been examined. Since the molecule contains both
hydrophilic and hydrophobic regions, it is amphipathic
and might well lodge in membranes. Such accumulations
could be important in the physiology of cells and might
influence the way in which the herbicide moves through
the biosphere.

In this paper, we show that binding of 2,4,S-T to
cells of B. megaterium is easily reversible. However,
small quantities of 2,4,S-T are firmly sequestered by

cell membranes.
MATERIALS AND METHODS

Organism andgrowth conditions. Cells of B. mega-

 

terium, strain KM, were grown and harvested as described

previously (Hicks and Corner, 1973).

Stock 2,4,S-T preparation and extraction of con-

 

taminants. A preparation of 2,4,S-T (K and K Laboratories,

 

Plainview, N.Y.) was cleaned by recrystallization three

times from benzene, washing with n—hexane in a separatory
funnel, and employing the methods of Woolson et al. (1972)
to remove possible CDD contaminants. The resulting solu-

tion was dried under nitrogen to provide a pesticide-free

72

extract of contaminants for determination of interference
or synergistic effects in the reported experiments.

Stock solutions of 2,4,S-T (1 mg/ml) were made by
dissolving KZHPO4 (6.80 g) and NaOH (0.02 g) in about 900
m1 of distilled water to which solution 2,4,S-T (l g) was
added. Following agitation for approximately 3 h, or

until all the 2,4,S-T was dissolved, KH PO4 (8.71 g) and

2
distilled water to one liter were added to yield 1 g
herbicide per liter of 0.1 M potassium phosphate buffer,
pH 7.0. For long term storage, the stock solution was
filtered and sterilized by repetitive steaming. This

process did not affect the herbicide as determined by

comparison with an identical but unheated solution.

Growth experiments. The medium used in the growth

 

experiments consisted of 20 g Oxoid peptone (Flow Labs,
Rockville, Md.), 900 ml distilled H20 and 100 m1 potassium
phosphate buffer. Sterilized buffer alone or buffer con-
taining herbicide was added to the previously autoclaved
peptone solution. Final concentrations of herbicide in
the media were 1, 10, 50 or 100 pg per m1. Media were
inoculated with about 108 cells taken from the late expo-
nential phase of growth. The cultures were incubated at
30 C with aeration by means of shaking. Growth of bac-
teria in media containing herbicide was monitored both
by following changes in optical density at 700 nm and by

microscopic observation over a 30 h period.

73

Survival measurements. Cells (2 mg dry weight/ml)

 

were suspended in buffer containing 1, 10, $0 or 100 ug
of 2,4,S-T per ml. Flasks containing these mixtures were
agitated at 30 C for periods up to 24 h. Periodically,
measurements of colony-forming units were made by appro-
priately diluting culture samples in 1.2% (weight/vol)
peptone (Difco) and spreading the diluted samples on 3

Trypticase soy agar (BBL) plates.

Respiratory measurements. Respiration of cells was

 

measured using conventional Warburg respirometry (Umbreit
et al., 1964). Cells were exposed to 10, 50, 100, 500

or 1000 pg of 2,4,5-T per ml of cell suspension which
contained 2 mg of cells (dry weight)/ml in phosphate
buffer.

Analysis of 2,4,S-T retention by intact cells.
Intact cells were placed in a 2-1iter flask containing
500 ml of buffer (pH 7.0) and 10 ug of 2,4,S-T per ml.
The final biomass concentration in the flask was 2 mg of
cells (dry weight) per m1. Hence the effective concentra-
tion of the herbicide was 5 ug per mg of cells. At
selected intervals, 50 m1 samples of cell suspension were
withdrawn from the flask, sedimented in the cold (10,400
x g; 10 min) and washed once by centrifugation with approxi-
mately 100 ml of buffer and twice with 100 ml of distilled

H20. The 2,4,5-T was then extracted from the cells in

74
preparation for analysis by gas chromatography. A control
flask without added herbicide was treated identically to
check for substances that might have interfered with the

herbicide assay.

Analysis of 2,4,5-T retention by membranes. Cells

 

were treated as described above, but membranes were iso-
lated from cells exposed to 2,4,5-T prior to extraction
of the herbicide. Cells were washed twice with buffer
(10,400 x g; 10 min) and resuspended in buffer. Lysozyme
(mucopeptide-N-acetylmuramylhydrolase; EC 3.2.1.17) was
added at 0.5 ug per mg of cells (dry weight) and the
mixture was allowed to incubate for 2 h at 30 C. Five
milligrams of deoxyribonucleate oligonucleotidohydrolase
(EC 3.1.4.5) were then added to reduce the viscosity of
the mixture. The preparation was centrifuged once at

low speed (3,300 x g; 6 min) to remove debris and most
poly-B-hydroxybutyrate granules. The membranes were
sedimented (19,000 x g; 30 min) and washed once with
buffer and twice with distilled H20; one-half was assayed
for herbicide content and the other half was used for a
dry weight determination. The latter was necessary

because membrane yield tended to decrease in older cells.

Determination of absorptive capacity of the cells

 

to 2,4,5-T. The basic methods of Scherrer and Gerhardt

 

(1971), the so-called space or thick suspension technique,

75
were followed except that equilibration periods ranged
from 0.5 to 10 h. In addition, the amounts of pesticide
in the cell pellets as well as those in the supernatant
solutions were determined. Known wet weights of pre-
viously washed cells were therefore exposed to 10 pg of
2,4,5-T per m1 (initial concentration) and, at selected
intervals, the cells were sedimented by centrifugation.
The pesticide contents of both the cell pellets and the
supernatants were determined by gas chromatography.
Little or no herbicide absorbed to the walls of the cen-
trifuge tube and temperature-induced precipitation did

not occur .

Calculation of amounts of 2,4,5-T taken up by cells.
The interstitial space (the aqueous volume between the
packed B. megaterium cells in the pellet) is 21.6% of
the pellet volume (Scherrer and Gerhardt, 1971). The
void volume within the cell pellet, which includes the
space within the peptidoglycan cell wall and the inter-
stitial space, is between 43.4% (pellet space infiltrated
by stachyose) and 50.5% (pellet space infiltrated by
sucrose); neither sucrose nor stachyose penetrate the
protoplast membrane in significant levels (Corner and
Marquis, 1969). These data are in general agreement with
those of Marquis (1968), who reported that about 36% of
the total cell volume of a nonplasmolyzed cell consisted

of "sucrose space" of the cell wall. The sucrose space

76
calculated from the data of Scherrer and Gerhardt (1971),
is 28.9%. The 36% wall space value and the 21.6% inter-
stitial space value were used in our calculations in order
to minimize the possibility of overestimating the magni-
tude of 2,4,S-T absorption. The ambient pesticide con-
centration in the supernatant after equilibration was
considered to approximate the concentration of pesticide
in the void volume. This quantity was subtracted from
the total pesticide found in the entire cell pellet; the
remaining pesticide in the pellet was associated with
the membrane, cytoplasm or cell wall materials, exclusive

of peptidoglycan void space.

Extraction and analysis of 2,4,5-T. Samples con-

 

taining herbicide were suspended in 10 ml of distilled
H20, acidified to pH 2.0 with 2 N HCl and extracted three
times with several volumes of diethyl ether in a separa-
tory funnel. Samples containing cells or membranes
frequently formed emulsions that would not break spon-
taneously. When this occurred, about 1 gm of anhydrous
NaZSO4 (previously washed with diethyl ether) was added
to the emulsion and the mixture was allowed to stand for
24 h. This procedure resulted in good separation of the,
ether and H20 phases. The spent NaZSO4 was then extracted
three times with diethyl ether and all ethereal extracts
were pooled and appropriately reduced in volume by

evaporation at room temperature under N2. The NaZSO4

77
did not affect the extraction efficiency or introduce
measurable contaminants.

The herbicide extracted from each sample was esteri-
fied by dropwise addition of diethyl ether solution of
diazomethane. Diazomethane was freshly prepared from
Diazold (Aldrich Chemical Co., Inc., Milwaukee, Wisc.)
by distilling an ethereal Diazold solution in the presence
of 95% ethanol and KOH. Analytical standards of 2,4,5-T
and standards made from our cleaned preparation were
esterified as above. Actual quantitative analysis of
the 2,4,5-T ester was made by use of a Beckman GC-4 gas
chromatograph equipped with an electron capture detector
and a column containing 10% DC 200 on Gas Chrom Q, 60/80
mesh. Helium was the carrier gas and the column tempera-

ture was 180 C.
RESULTS

Purity of herbicide and effects of contaminants.

The cleaned herbicide preparation produced no extraneous
peaks when analyzed by electron capture gas chromatography.
However, several relatively small unidentified peaks were
detected in the analysis for CDD contaminants in the
uncleaned preparation. The contaminants extracted from
the uncleaned preparation did not affect the growth rate,
the survival of resting cells, cell respiration or cause

fluctuations in uptake values when used alone or in

78

combination with the cleaned preparation. Furthermore,
when respiration or survival of resting cells was
determined, 2,4,S-T analytical standards of high but
unknown purity yielded results identical to those pro-
duced by the cleaned preparation. No evidence of 2,4,5-T
degradation or cometabolism was found during the course
of the reported experiments. Therefore, effects reported
here are attributable solely to 2,4,5-T rather than to

degradation products or contaminants.

Action of 2,4,5-T on resting cells. Exposure of

 

cells, aerated in phosphate buffer, to various concen-
trations of 2,4,5-T revealed that levels as low as 10
ug/ml (S ug/mg of cells, dry weight) did accelerate death
in the population (Figure 1). However, the acceleration
was only slight at 10 ug/ml but became greater when the
2,4,5-T concentration was raised to 50 or 100 ug/ml.
Moreover, the major increases in death rate occurred

only after about 24 h of exposure.

Action of 2,4,5-T ongrowing cells. Although

 

exposure to 10 ug/mg was at least marginally lethal to
resting cells and higher concentrations did accelerate
ldeath, growing cells were not affected by exposure to
concentrations less than 100 ug/ml (Figure 2). Even at
this particular conCentration, the inhibition was

expressed as a slight increase in the length of the lag

79

Figure 1. Survival of cells of B. megaterium (2
mg of cells, dry weight, per ml) in buffer containing
10 ug/ml 03), 50 ug/ml (A), 100 ug/ml (I) or no
addition (0). Cells were incubated at 30 C with shak-
ing. Data averaged from 5 experiments.

80

 

 

,IA

'/‘

 

_

 

I09

b
8 7
o m

A _E\m._._za @2335... *20400vmm0>_>m3m no mmmZDZ

Io“ '

20 25 30 35 40

IS
HOURS

IO

Figure 1

81

Figure 2. Effects of 2,4,5-T on cells of B. mega-
terium grown in buffered peptone. Optical densities at
700 nm were determined during the growth cycles. The
upper curve represents cell growth at 2,4,S—T concentra-
tions of l ug/ml (O), 10 ug/ml (D), 50 ug/ml (A) and no
herbicide (O); the lower curve illustrates cell growth
at 100 ug 2,4,S-T/ml (A). Data averaged from 6
experiments.

82

 

00.

mm

0N

N ouswflm

9501
n.

0_

l

l

_ 00.0

000.0

0.0.0 1.

000.0

00.0

 

 

83

period and a reduced total cell yield. The exponential
growth rate was not markedly affected by the presence of

100 ug of 2,4,5-T/ml.

Effect of 2,4,5-T on cell respiration. The rates

 

of endogenous or exogenous (glucose) metabolism were not
significantly different among cells exposed to up to 500
Hg 2,4,5-T/m1 (250 ug/mg of cells, dry weight) and unex-
posed cells (Figure 3). The rate of endogenous metabolism
was markedly reduced in cells exposed to 1000 ug/ml and
the rate at which glucose was oxidized was somewhat less

than that exhibited by unexposed cells.

Uptake characteristics. The 2,4,5-T found in the

 

cell pellet under static conditions, exclusive of the
interstitial space and the peptidoglycan, was about 2-
fold higher than the theoretical ambient concentration
that would have existed in cells freely permeable to the
herbicide (Table 1). However, after washing, equivalent
cells retained only about 0.49 ug of the herbicide (Table
2) so that much of the bound compound was easily removed
from the cells. Although this herbicide retention was
well below the theoretical ambient concentration of

about 1 ug/100 mg cells (dry weight), the amount retained
by membranes, about 0.3 ug, was greater than the theoreti-
cal ambient level of 0.065 ug/mg of membranes (dry weight).

The amount of herbicide bound to membranes of exposed

84

Figure 3. Oxygen uptake at 30 C by cells of B.

megaterium (2 mg of cells, dry weight, per ml), exposed
to 10 pg 2,4,S-T/ml (A), 50 ug/ml GB), 100 ug/ml (O),

500 ug/ml (V), 1000 ug/ml (I) or no addition (0). Arrow
indicates addition of glucose.

85

 

Anv-

 

220

Lav

— p _ b _ p _ _
nu nu nu mw .U nu nu nv
m M W. I 8 6 4 2

200'
I80“

. E., :3 .38 95335.5 zmo>xo

40 60 80 I00 I20 I40 I60

20

 

0

MINUTES

Figure 3

86

Table l. Uptake of 2,4,5-T by cells of Bacillus

 

 

megaterium
Equilibration Predicteda Experimentalb Fold
time (b) value (ug) value (ug) increase
0.5 2.1 4.3 2.0
l 2 7 5.2 l 9
3 Z 2 4.6 2 1
10 2.2 5.1 2.3

 

aTheoretical ambient concentration of 2,4,5—T in
500 mg (wet weight) cells.

bUptake as determined by the thick suspension
technique (Scherrer). Data averaged from four
experiments.

87

Table 2. Retention of 2,4,S-T by 100 mg of cells (dry
weight) of Bacillus megaterium or by membranes

 

 

 

Exposure period Residual 2,4,5-T (pg) .
(h) In cellsa In membranesD
0-3 0.49 0.30
4-9 0.48 0.29
10-13 - 0.34
23:24 0.49 0.29

 

aResidual herbicide in 100 mg cells (dry weight)
after exposure to 10 pg 2,4,5-T/m1 (5 pg/mg cells).
Data averaged from three experiments.

bResidual herbicide in membranes isolated from
100 mg exposed cells. Membranes were considered to
comprise 6.5% of cell dry weight. Data averaged from
four experiments.

88

cells represented about 60% of the total sequestered

pesticide.

DISCUSSION

The experimental conditions used here were not
designed to simulate conditions in the natural environ-
ments of organisms in soil or water but rather to permit
elucidation of the potential responses of a model bac-
terium to 2,4,S-T. Moreover, the pesticide concentra—
tions were not typical of 2,4,5-T concentrations normally
found in contaminated soils, waters or sediments.

The methods used to remove possible contaminants
from the crude 2,4,5-T preparation were adequate to mini-
mize possible influence of the contaminants on the experi-
mental results. However, the reported extreme toxicity
of some CDD isomers (Kimbrough, 1972) necessitates
exhaustive analytical and toxicological studies on each
individual 2,4,5-T preparation before making judgments
about absolute purity or toxicity to higher organisms.

Low concentrations of 2,4,5-T accelerated the death
of resting cells, but similar concentrations did not kill
cells in growing cultures wherein the biomass was increas-
ing. In accord with observations on the lethal effects
of other organochlorine biocides such as DDT (Hicks and
Corner, 1973) and hexachlorophene (Silvernale et al.,
1971), this finding supports the contention that the

detrimental effects of a pesticide depend largely on the

89
ratio of pesticide to biomass rather than on the ambient
concentrations in the immediate microbial environment
(Wurster, 1968).

K0 and Lockwood (1968) and Chacko and Lockwood
(1968), among others, have demonstrated that water-
insoluble compounds such as DDT and dieldrin may become
highly concentrated within the soil microflora many-fold
above ambient concentrations. Previously Hicks and
Corner (1973) reported that DDT is taken up by B. mega-
terium, with most of the accumulated DDT sequestered in
the limiting membranes of the cells. Similarly, hexa-
chlorophene was found to be concentrated in membranes of
B. megaterium (Silvernale et al., 1971). Thus, the uptake
of 2,4,S-T by membranes is not a singular phenomenon.
However, the quantities taken up were much smaller com-
pared to the amounts of DDT or hexachlorophene. The
residual content of the cells may have gone unnoticed
or thought insignificant had not herbicide assays been
performed on membranes isolated from exposed whole cells.
The trace amounts of 2,4,5-T in the intact cells suggest
that the herbicide does not partition into biological
systems to the extent necessary for biological magnifi-
cation within food chains. Indeed, to our knowledge
there have been no reports of 2,4,5-T accumulation in
higher predators.

In addition to the actual sequestering of 2,4,5-T

by B. megaterium membranes, a more subtle interaction may

90
have occurred. The 2,4,5-T molecule possesses hydrophobic
(the substituted benzene ring) and hydrophilic (the
carboxyl group) regions. Thus, it may act analogously
to detergents and selectively associate with those parts
of the cell that are composed of adjacent hydrophobic
and hydrophilic areas. This interaction may partially
account for the relatively large quantities of herbicide
that become reversibly associated with the cells under
static conditions. Since the active concentrating mechan-
isms of a mesophile operate slightly, if at all, at the
low temperature (DC) at which uptake was studied, the
association is probably entirely passive. Moreover,
this organism has not undergone the selective pressure
needed to develop transport mechanisms specific for this
man-made compound, though the 2,4,5-T molecule could
conceivably act as an analog of a biomolecule of similar
structure, such as phenylalanine or tyrosine.

However, much of the herbicide may not, in fact,
have penetrated the cell membranes, as membranes are
relatively impermeable to dissociated acids (Bell et al.,
1959). Consequently, the difference between the residual
herbicide in the whole cells and residual herbicide in
membranes isolated from exposed intact cells may repre-
sent 2,4,5-T bound to the cell wall. The affinity of
2,4,S-T for cells of B. megaterium, at least, may be

primarily restricted to membranes and cell walls.

91

Lockwood and his colleagues (l968a,b) have suggested
that the concentration of certain chlorinated pesticides
in biological material may facilitate the persistence of
these compounds in soil. The degree of 2,4,5-T accumu-
lation exhibited by the cells studied here reduces the
likelihood of this mechanism as a major contributor to
2,4,5-T persistence. Nevertheless, that amount of 2,4,S-T
sequestered in the cells would be less available to

degradative organisms.

ACKNOWLEDGEMENTS
We thank A. Filonow for technical advice and A.
Wolcott for use of his gas chromatograph. This work was
supported by Grant No. BMS 71-01493 from the National

Science Foundation.

LITERATURE CITED

1. Bell, T. A., J. L. Etchells, and A. F. Borg. 1959.
Influence of sorbic acid on the growth of certain

species of bacteria, yeasts, and filamentous fungi.
J. Bacteriol. 77:573-580.

2. Chacko, C. 1., and J. L. Lockwood. 1967. Accumu-
lation of DDT and dieldrin by microorganisms.
Can. J. Microbiol. 13:1123—1126.

3. Corner, T. R., and R. E. Marquis. 1969. Why do
bacterial protoplasts burst in hypotonic solu-
tions? Biochim. Biophys. Acta 183:544-558.

4. Dougherty, E. M., C. F. Reichelderfer, and R. M.
Faust. 1971. Sensitivity of Bacillus thuringiensis
var. thuringiensis to various insecticides and
herbicides. J. Invert. Pathol. 17:292-293.

10.

11.

12.

13.

14.

15.

16.

92

Hicks, G. F., and T. R. Corner. 1973. Location and
consequences of l,l,l-trichloro-2,2-bis(p-chloro-
phenyl)ethane uptake by Bacillus megaterium.

Appl. Microbiol. 25:381-387.

Horvath, R. S. 1971. Microbial co-metabolism of
2,4,S-trichlorophenoxyacetic acid. Bull. Environ.
Contam. Toxicol. 5:537-541.

Horvath, R. S. 1972. Microbial co-metabolism and
the degradation of organic compounds in nature.
Bacteriol. Rev. 36:146-155.

Kimbrough, R. D. 1972. Toxicity of chlorinated
hydrocarbons and related compounds. Arch. Pathol.
94:125-131.

Ko, W. H., and J. L. Lockwood. 1968. Accumulation
and concentration of chlorinated hydrocarbon pes—
ticides by microorganisms in soil. Can. J.
Microbiol. 14:1075-1078.

Lee, D. H., and H. L. Falk (ed.). 1973. Environ-
mental Health Perspectives, Issue No. S. U.S.
Department of Health, Education, and Welfare.

Loos, M. A. 1969. Degradation of phenoxyalkanoic
acid herbicides by soil microorganisms. J.
Suid-Afrikaanse Chem. Inst. 22:871-878.

Loos, M. A. 1969. Phenoxyalkanoic acids, p. 1-49.
In P. C. Kearney and D. D. Kaufman (ed.), Degra-
dation of Herbicides. Marcel Dekker, Inc., New
York.

Marquis, R. E. 1968. Salt-induced contraction of
bacterial cell walls. J. Bacteriol. 19:775-781.

Scherrer, R., and P. Gerhardt. 1971. Molecular
sieving by the Bacillus megaterium cell wall and
protoplast. J. Bacteriol. 107:718-735.

Silvernale, J. N., H. L. Joswick, T. R. Corner,
and P. Gerhardt. 1971. Antimicrobial actions
of hexachlorophene: cytological manifestations.
J. Bacteriol. 108:482-491.

Umbreit, W. W., R. J. Burris, and J. F. Stauffer.
1964. Manometric.Techniques, 4th ed. Burgess
Publishing Co., Minneapolis.

 

93

17. Woolson, E. A., R. F. Thomas, and P. D. J. Ensor.
1972. Survey of polychlorodibenzo-p-dioxin
content in selected pesticides. J.—Agr. Food
Chem. 20:351-354.

18. Wurster, C. F. 1968. IDDT reduces photosynthesis
by marine phytoplankton. Science 15921474-1475.

19. Yudkin, M. D. 1966. Isolation and analysis of the
protoplast membrane of Bacillus megaterium.
Biochem. J. 98:923-928.

SECTION 4. MEMBRANE MODIFICATION
INDUCED BY DDT

Introduction

 

DDT and 2,4,5-T accumulate in the limiting membranes
of Bacillus megaterium (Hicks and Corner, 1973; Hicks and
Corner, manuscript in preparation). Therefore, the pes-
ticide effects on cells may be exerted at the level of
the membrane. Others have suggested that DDT may exert
toxic effects on organisms by inhibiting processes asso-
ciated with membranes (Hilton and O'Brien, 1970; Janicki
and Kinter, 1971; Matsumura and Patil, 1969; Rogers et
al., 1971; Tinsley et al., 1971).

Both DDT and 2,4,5-T inhibit cell division in B.
megaterium at concentrations of 5 pg pesticide/mg of
cells (dry weight), but no well defined biochemical or
physiological event could be implicated in the gradual
killing effect of these compounds, even at concentrations
as high as 50 pg pesticide/mg of cells (dry weight)
(Hicks and Corner, 1973; Hicks and Corner, manuscript).
In addition, membranes isolated from cells exposed to DDT

had atypical macroscopic appearances, which suggested that

94

95
the membranes were chemically modified (Hicks and Corner,
1973).
The suggestion that DDT affects the gross chemical
composition of membranes is confirmed by the results
reported below. However, membranes from cells exposed

to 2,4,5-T were identical to control membranes.

Materials and Methods

 

Membrane Isolation

 

Membranes were isolated from the cells of B. mega-
terium by the methods of Hicks and Corner (1973). Prepa-
rations of membranes were obtained direclty from cells
taken from (i) the late exponential phase of growth
(control) and from the following mixtures after 1.5 h
exposure in phosphate buffer, pH 7.0; (ii) buffer alone;
(iii) buffer amended initially with 0.1% (vol/vol)
acetone; (iv) buffer containing 10 pg 2,4,5-T/ml; and
(v) buffer containing 0.1% (vol/vol) acetone and 10 pg
DDT/ml. A sixth preparation was made from cells subjected
to a sucrose density gradient procedure (Hicks and Corner,
1973) after exposure in buffer containing 0.1% acetone.
After isolation, the various membrane preparations were
frozen at about —35 C and then lyophilized. Yields

varied from 40 to 110 mg membrane material.

96

Lipid Extraction

 

Dried membrane materials (5, 10 or 20 mg) were
extracted with about 10 ml chloroformzmethanol (2:1) in
the‘cold with agitation under a partial vacuum for periods
that ranged from 8 h to 40 h. The membrane residue was
then washed by filtration and the chloroform:methanol
extracts were dried and weighed. As a reference for
insoluble material that may have adhered to or passed
through the filter, the following alternate method was
used. Insoluble material in the chloroformzmethanol
solvent was allowed to settle to the bottom of the extrac-
tion vessel and the supernatant was removed with a pipette.
The insoluble residue was washed several times with

chloroformzmethanol and all supernatants were pooled.

Fatty Acid Analysis of Lipid Extracts

 

The DDT incorporated in the membranes severely inter-
fered with resolution of conventional thin layer chroma—
tographic phospholipid assays, whereas DDT did not markedly
distort the flame ionization characterization of component
fatty acids. Hence, fatty acids were used as indicators
of phospholipid and thus approximated relative amounts
of gross lipid in the membranes. One-half milliliter
of 15% BFS (vol/vol) in methanol (Supelco or Applied
Sciences) was added to the dried chloroformzmethanol
residue and boiled for about 3 min. The esterified fatty

acids in the boiled mixture were partitioned into 30 ml

97
petroleum ether in a separatory funnel containing the
petroleum ether and 20 ml H20. The aqueous bottom layer
was discarded and the ether was evaporated to dryness.
Subsequently the residue was dissolved in petroleum ether
and analyzed by flame ionization gas chromatography.

Fatty acid standards were obtained from Applied Sciences.

Protein Determinations

Protein was measured using the procedure of Lowry
et al. (1951) with bovine serum albumin as a standard.
Protein determinations were performed on the intact
membranes and on the membrane residues left after lipid
extraction. This latter procedure was necessary to
equate the effects that DDT or 2,4,5-T may have had on
the protein determinations of the intact membranes. The
remaining protein fractions and lipid extracts were
assayed for DDT by use of procedures previously described
(Hicks and Corner, 1973). This served to confirm the
DDT content of the membranes and to determine how much
DDT, if any, would be available to interfere with the

Lowry procedure.

Poly-B-hydroxybutyric Acid (PHB) Analysis
A routine PHB analysis was performed on all membranes.

The methods.of Law and Slepecky (1961) were used in_addi-

tion to the PHB extraction technique of Bishop et al.

(1967). Subjective microscopic measurements of the final

memb‘

COHI

mem
mar

THEY

C0

EX

r1

98

membrane preparations were also used to estimate PHB

content .

Results

Of the six membrane preparations studied, only
membranes prepared from cells exposed to DDT showed any
marked difference in gross composition. The DDT in the
membranes or in the exposure medium did not measurably
affect the protein content (Table 1). The fatty acid
content of the Chloroform:methanol extracts of the
exposed membranes was markedly lower than that of all
other samples (Table 2). Hence, the DDT but not 2,4,5-T
replaced or eluted some lipid material from the membranes.

DDT interfered with conventional thin layer chromato-
graphic analysis of phosopholipids in both the membrane
preparation and similar test'preparations with an equiva-
lent amount of DDT added. However, DDT at concentrations
found in the membranes had no detectable effect on gas
chromatographic resolution of fatty acids. Hence, the
fatty acid content of these membranes was used as an
indicator of relative lipid content exclusive of PHB.
No qualitative differences among the fatty acids were
evident in the membrane preparations.

The Lowry procedure performed on intact membranes
produced erratic and irreproducible results when DDT
was present; similar results were observed when DDT was

added to bovine serum albumin. Chloroform:methanol

99

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.momonpcmsmm
:H co>wm mosfim> mo omens “flammflv .Ne pm xuzog mo mwogpoe map an confiEpopoom

 

 

 

mm~-wav I
mm o HN H+H flmn-moU on Hopscoo
mmNIOHV I ucofivmsm omoHUSm
mm o ma H+H mwu-eoV we monouoom .umwwsm
mum-mfiv I
mm o NN H+H men-hov HA H-m.e.~
I flmH-MHv I
mm o+N ms H+H Aqn-moV NA caucus as mucosae .eaa
AVN-wHV I
om o HN H+H “NA-VOU mo commas as o:ouou<
mm~-oflv I .
mm 0 ON H+H 545-009 HA sane powmam
w owfiUwumom w pogwo mmm mflmpou mo w :fiououm moonsom ocmunEoEv
HmHOH owmmwa w mHHou mo ucoecouw>cm

 

Hmfihmpms ofinmuomhuxo 3002\vqou w

 

:OflufimoQEoo onmunsoa mmoum mo thassm .H oanmh

100

Table 2. Relative percent fatty acid and pesticide in
Chloroform:methanol extracts of purified
membranes

 

Membranes isolated from

 

cells exposed to Fatty acid %a Pesticide %
Buffer only 91 0
Acetone in buffer 91 0
DDT, acetone in buffer 77 9
2,4,5-T 105 0
Sucrose gradient 95 0
Control 100 0

 

aControl = 100%.

EXII'II

which

101
extraction removed the DDT and 2,4,S-T from the membranes

which were then measured for total protein content.

Discussion

 

Direct protein determination with the Lowry method,
and membrane phospholipid analysis with thin layer chroma-
tography, were clearly unsuitable for studies with membranes
isolated from DDT-exposed cells. The alternate methods
devised to determine protein and lipid content of membranes
were illustrative and adequate for relative comparison of
membrane chemical composition. These methods may not,
however, precisely measure absolute quantities of any one
component.

DDT enters the membrane slowly, attaining a maximal
concentration of about 2% after 1.5 h exposure (Hicks and
Corner, 1973). Of the six membrane preparations only
the one containing DDT appeared to have a somewhat dif-
ferent texture and color in the hydrated state. The
apparent solubility of DDT, as determined by sedimenta-
tion characteristics, increased with increased time of
exposure to cells. Thus, presumptive evidence suggested
that DDT became associated with extracellular biological
material. Since DDT in its crystalline form is soluble
in solvents with hydrophobic character, membrane lipid
or other hydrophobic cell components could well play a

role in changing the physical state of DDT.

102
This is the first report of DDT-induced physical
modification of a cell. Though 10 pg DDT/m1 is consider-
ably higher than environmental concentrations, it is
clear from these results that DDT does have the physical
potential to disrupt the cell membrane without the out-
ward display of dramatic effects such as immediate cell

lysis or' instantaneous death of cells.

Literature Cited

 

Bishop, D. G., L. Rutenberg, and B. Samuelson. 1967.
The chemical composition of the cytoplasmic
membrane of Bacillus subtilis. Euro. J. Biochem.
2:448-453.

Hicks, G. F., and T. R. Corner. 1973. Location and con-
sequences of l,l,l-trichloro-Z,2-bis(p-chloro-
phenyl)ethane uptake by Bacillus megaterium.

Appl. Microbiol. 25:381-387.

Hilton, H. D., and R. D. O'Brien. 1970. Antagonism by
DDT of the effects of valinomycin on a synthetic
membrane. Science 168:841-842.

Janicki, R. H., and W. B. Kinter. 1971. DDT inhibits
NaI, K+, Mg2+-ATPase in the intestinal mucosae

and gills of marine teleosts. Nature New Biol.
233:148-149.

Law, J. H., and R. A. Slepecky. 1961. Assay of poly-
B-hydroxybutyric acid. J. Bacteriol. 82:33-36.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J.
Randall. 1951. Protein measurement with the
Folin phenol reagent. J. Biol. Chem. 193:265-
275.

Matsumura, F., and K. C. Patil. 1969. Adenosine tri-
phosphatase sensitive to DDT in synapses of rat
brain. Science 166:121-122.

Tinsley, I. J., R. Haque, and D. Schmedding. 1971. Bind—
ing of DDT to lecithin. Science 174:145-147.

Yudkin, M. D. 1966. Isolation and analysis of the pro-
toplast membrane of Bacillus megaterium. Biochem.
J. 98:923-928.

DISCUSSION

The trend expressed in the scientific literature
was to characterize DDT degradation and movement hypo-
thetically in accord with fundamental experimental obser-
vations: some DDT from the insoluble pesticide pool in
the biosphere slowly partitions into microorganisms,
further magnifies with predation and, after much cycling,.
enters anaerobic environments where a mixed microbial
community could presumably degrade the compound.

The present results, in combination with past
observations, allow refinement of the above hypothesis.
Many microorganisms, including B. megaterium, are not
eliminated from a viable population by low environmental
DDT concentrations. This would enable DDT to become
sequestered, delaying degradation and providing direct
entrance through predation to other lower organisms.
However, long exposure to the insecticide could result
in high or threshold concentrations within autonomous
cells and the DDT could elute in a more soluble form,
thus increasing the mobility of the compound in aqueous
environments. This provides a hypothetical mechanism

whereby DDT or DDD could partition more extensively from

103

104
sediments and associated microbial populations into
ambient waters, thereby reducing the quantities accessible
for anaerobic biodegradation while concurrently increas-
ing concentrations in surface waters where UV-mediated
photodecomposition can occur. Obviously, a thorough
study to resolve the nature of DDT degradation in situ
is critical to ascertain the validity of this hypothesis.

The precedents and accuracy of the DDT analytical
procedures led to the finding that the membrane of B.
megaterium is the primary site of very low but detectable
2,4,5-T accumulation. However, the water soluble herbi-
cide is not eluted in the fashion of DDT even as viability
reduction occurs. Although this physical entrapment may
contribute somewhat to the recalcitrance of the compound
in nature, analogously to DDT, it is evident from these
and previous studies that 2,4,S-T will not partition into
biological systems over the observed trace amounts.

The development of gradient centrifugation tech-
niques and the application of the space technique to the
study of pesticide interactions with microflora repre-
sent an advance in analysis of pesticide residues at the
microbial or subcellular level. These procedures should
aid investigations of pesticide uptake in situ and
resolve some questions about the movement and fate of
recalcitrant compounds in the biosphere. Similarly,

chemical modification of membranes apparently is the first

105
evidence presented to date about the precise physical effects
that DDT may have at the subcellular level. Applicability
of this observation specifically or generally to the mode of

action of DDT on target organisms remains to be seen.

MICHIGAN STATE UNIV. LIaRnRIEs
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