Ecological Research Series
MICROBIAL INTERACTIONS WITH PESTICIDES
IN ESTUARINE SURFACE SLICKS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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RESEARCH REPORTING SERIES
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n >c is avf."S'f,10 !he pu5lic throu9h the N^ional Technical Informa-
tion Service, Springfield, Virginia 22161.
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MICROBIAL INTERACTIONS WITH PESTICIDES
IN ESTUARINE SURFACE SLICKS
by
D. G. Ahearn
S. A. Crow
and
W. L. Cook
Department of Biology
Georgia State University
Atlanta, Georgia 30303
Grant No. R-803141
Project Officer
Al W. Bourquin
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publi-
cation. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
The protection of our estuarine and coastal areas from damage caused by
toxic organic pollutants requires that regulations restricting the introduc-
tion of these compounds into the environment be formulated on a sound scien-
tific basis. Accurate information describing dose-response relationships for
organisms and ecosystems under varying conditions is required. The
Environmental Research Laboratory, Gulf Breeze, contributes to this informa-
tion through research programs aimed at determining:
. the effects of toxic organic pollutants on individual species and
communities of organisms;
. the effects of toxic organics on ecosystem processes and components;
. the significance of chemical carcinogens in the estuarine and marine
environments.
Microorganisms are considered ultimate disposers of pollutants, and
therefore microbial considerations are essential in the proper functioning,
response, and recovery of environmental degradation of marine ecosystems.
Information on microbial interactions in specific niches (air-sea interface)
is necessary to assess the effects on both biodegradative potential and on
microbiological activities after a pollutant reaches the estuarine environ-
ment. The air-sea interface is a unique membrane, vitally important to ma-
rine food chains and implicated in concentration and transport of pollutant
chemicals. Studies such as this report can enhance our understanding of this
unique ecotone and contribute significantly to valid environmental assessment
of a pollutant's effect on the marine ecosystem.
Al W. Bourquin
Research Microbiologist
Environmental Research Laboratory
Gulf Breeze, Florida 32561
iii
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ABSTRACT
Estuarine surface films from Escambia Bay, Florida, and adjacent waters
were sampled by using the membrane adsorption technique to enumerate micro-
bial populations. Samples of the upper 10 ym of estuarine surface films
yielded microbial populations up to 108 ml'-1- or 10^ cm"2. These populations
were 10 to 100 times greater than those in underlying waters at a depth of
10 cm. Predominant bacteria in surface films as isolated on Marine Agar
(Difco) were motile, nonpigmented, gram-negative rods. Colony-forming units
of yeasts and molds on Mycological Agar (Difco) prepared with 50 per cent
seawater were found in concentrations to 10^ ml"-'- or 28 cm~^. The predomi-
nant surface film isolates from Marine agar were proteolytic and amylolytic,
but exhibited only weak to negligible hydrocarbonoclastic and lipolytic
activities. A greater proportion of the surface-film bacteria, as compared
to those at 10 cm depth, were capable of growth on freshwater media. With
selective isolation media, amylolytic, and lipolytic bacteria appeared to
comprise a more significant proportion of the total population. Twenty-one
representative bacteria, yeasts, and filamentous fungi from initial sampling
of surface microlayers were tested for the effects of selected pesticides on
utilization of various substrates. No inhibition by heptachlor or methoxy-
chlor was noted with glucose as a carbon source. One bacterium was sensitive
to PCB formulations. In subsequent studies with 53 isolates representative
of more diverse physiological groups, o-chlorophenol, 1-chloronaphthalene,
PCB 1016, and pentachlorophenol were inhibitory to a large portion of the
isolates. Heptachlor inhibited 2 isolates and methoxychlor inhibited only
1 isolate. In contrast, with hydrocarbon as a substrate, microorganisms
were more frequently inhibited by various aromatic or chlorinated hydrocar-
bons. For example, heptachlor, biphenyl, pyrene, and PCB 1016 significantly
reduced hexadecane utilization by representative surface film bacteria. In
a few instances, trace concentrations of pesticides enhanced hydrocarbon
utilization. In studies with yeasts, high concentrations of heptachlor
appeared to have a slight stimulating effect on utilization of hexade'cane
by £. maltosa, but no effect with £. lipolytica. The complex nature of
metabolic responses with varying substrate and pesticide indicates that
multiple assay procedures are required to detect the altering capability of
pesticides within the surface film ecosystem.
This report was submitted in fulfillment of Grant No. R-803141-01-0
by Georgia State University under partial sponsorship of the U.S. Environ-
mental Protection Agency. This report covers the period from May 1, 1974
to September 20, 1976 and was completed as of November 1, 1976.
iv
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CONTENTS
Forward .
Abstract
Tables
ill
iv
.vl
1. Introduction
2. Conclusions
3. Recommendations ....
4. Materials
5. Experimental
Environmental Sampling
Laboratory Studies . .
6. Results and Discussions
, 1
, 3
, 4
5
, 7
, 8
,11
References
,21
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TABLES
Number Page
1 Synthetic Crude Oil Composition ................. 9
2 Compounds Screened for Inhibitory Effects ............ 9
3 Concentration of Microorganisms from Surface Slicks ...... 12
4 Heptachlor Recovery from Hexadecane Growth Medium ....... 13
5 Dry Weight .......................... 13
6 Recovery of 14C Label from Heptachlor Following
Eight Days of Incubation with £. maltosa .......... 14
7 Recovery of 14C Label from Various Fractions of
Yeast Culture After Eight Days Incubation with
14C-Heptachlor
8 Utilization of Synthetic Crude Oil by C,. lipolytica
and C. maltosa .... ~~
9 Utilization of Synthetic Crude Components by
C^. lipolytica and £. maltosa ................ 16
10 Mole Utilization Ratios of Synthetic Crude Oil
Components ............... -i-
11 Effect of Chlorinated and Aromatic Hydrocarbons on
Growth of Selected Microorganisms .............. 17
12 Effect of Aromatic and Chlorinated Hydrocarbons
(10 PPM) on Hexadecane Metabolism by Surface
Slick Bacteria ..................... 19
13 Inhibition of C. lipolytica Isolate by Various Compounds ... 19
14 Effect of Aromatic and Chlorinated Hydrocarbons at 10 PPM
on Hexadecane Metabolism by C. lipolytica Mutants ...... 20
vi
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SECTION 1
INTRODUCTION
The surface microlayer is a unique microbial habitat occurring at the
air-water interface. Accumulations of surface active organic material in
this region leads to production of calm areas in disturbed waters. Chemical
and physical studies of these surface films indicate a wide range of organic
and inorganic components (Baeir 1970, 1972; Ewing 1950, Garrett 1965,
Sutcliffe et al. 1963). Several recent studies propose a significant role
for the surface microlayer in environmental processes (Maclntyre 1973, Parker
and Barsom 1970). Although numerous studies of the physical and chemical
properties of surface films have been conducted, little is known of the
biological activities and interactions occurring within the surface micro-
layer and surface film.
Investigators have reported extensive microbial populations associated
with the surface region (Zobell 1946, Gunkel 1973, Parsons and Takahashi
1973). Coupled with these observations, the rich varied supply of organic
and inorganic nutrients suggest a biologically active region.
Recent studies (Seba and Corcoran 1970, Harvey et al. 1973, Stadler and
Ziebarth 1976) demonstrated the presence of certain chlorinated hydrocarbons
in surface films. Others (Hartung and Klingler 1970, Sayler and Colwell
1976) have suggested that surface' film materials will rapidly concentrate
chlorinated hydrocarbons and aromatic hydrocarbons from aqueous systems.
Accumulation of such compounds within the surface film may alter the physio-
logical activity of microorganisms associated with natural surface films.
Rapid sequestering of chlorinated hydrocarbons in accidentally spilled hydro-
phobic materials may alter response of an ecosystem to this stress, thus
lengthening the residence time of these materials in the environment.
The potential for hydrocarbon pollution of coastal and estuarine waters
is increasing. The role of surface microlayer microorganisms in the dis-
persal of accidental spills and the functions of populations within naturally
occurring surface films are closely associated. An understanding of the bio-
nomics of pollutant molecules is basic to the establishment of tolerable
levels for pesticides, chlorinated hydrocarbons, and aromatic hydrocarbons in
aquatic systems. Determination of baseline microbial populations of natural
surface films, their response to the chemical and physical conditions, as
well as the numerous interactions, are essential to this development.
The basic objectives of this research were: (1) to determine microbial
population numbers and diversity in estuarine surface films, (2) to study the
metabolic activity of surface film isolates on a wide range of substrates,
(3) to determine the interactions of various pollutant molecules with
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microbial metabolism of selected substrates.
To facilitate the presentation of this material, studies will be grouped
into two categories.
Initial Studies: primarily dealing with establishing surface film
microbial populations, with further evaluation of metabolic potential and
pesticide interference on a single culture basis.
Studies of Selected Metabolic Types: evaluation of surface film micro-
bial populations with emphasis on establishing numbers of major metabolic
types (proteolytic, amylolytic, lipolytic, and hydrocanbonoclastic groups)
and laboratory evaluation of pesticide interactions with these types.
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SECTION 2
CONCLUSIONS
It is concluded from this study that estuarine and coastal surface
slicks usually contain dense numbers of aerobic, heterotrophic microorganisms.
No single enrichment medium depicts the true number or metabolic range of the
microorganisms present in any single slick. The growth of representative
surface slick microorganisms may be affected by the presence of certain aro-
matic or chlorinated organic molecules. The effects may stimulate metabolism
of select compounds, (e.g., hexadecane) and concomittantly decrease recovery
of recalcitrant molecules (e.g., naphthalene, biphenyl, heptachlor) from
culture systems. Conversely, the growth or metabolism of certain surface
slick flora may be inhibited by the presence of chlorinated aromatic
compounds. It is suggested that the sequestering of recalcitrant molecules
within surface films affects natural microbial processes.
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SECTION 3
RECOMMENDATIONS
It is recommended that microbiological assay systems be developed for
rapid quantitative measurement of specific toxicity by chlorinated aromatic
compounds. These assays should be functional for a marine system and predict
potential harm from industrial chemicals. Impairment of metabolism, inhibi-
tion of growth, and alteration of the genome should be considered as primary
indices for evaluation.
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SECTION 4
MATERIALS
Areas Sampled
Barataria Bay, Louisiana
Airplane Lake 2 samples
Bayou Per Blanc 2 samples
Gulf Breeze, Florida
Cove adjacent to EPA Laboratory 13 samples
Waste Pond EPA Laboratory 10 samples
Sewage Outfall Pensacola 2 samples
Escambia Bay Mid Bay near Channel 4 samples
Boat Slip EPA Laboratory 2 samples
Range Point 8 samples
Bayou Chico 6 samples
49 samples
Materials and Sources
Polycarbonate membranes, Nuclepore Corporation, Pleasanton, California
Standard Media (prepared with 50 per cent seawater)
Marine Agar 2216
Mycological Agar
Spirit Blue Agar
Tryptic Soy Agar
MOF Medium .
Bushnell Haas Broth
Specialized media (listed below) were prepared according to the methods
of Colwell and Wiebe (1970) and Hankin and Anagnostakis (1975).
Proteolytic Enumeration Media
Amylolytic Enumeration Media
Lipolytic Enumeration Media
Hydrocarbon Enumeration Media (1 per cent Hexadecane in Bushnell-Haas
Broth)
Phosphatase Media
Basal Broth
Yeast: YNB (Difco)
Bacteria: Bushnell-Haas broth
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Chemicals Studied and Sources
l^C-heptachlor, Velsicol Chemical
PCB Formulations, Monsanto Chemical
cyclohexane, Fisher Scientific
methylcyclohexane, Fisher Scientific
ethyl benzene, Eastman Organic Chemicals
naphthalene, Matheson, Coleman and Bell
biphenyl, Eastman Organic Chemicals
tetradecane, Eastman Organic Chemicals
hexadecane", Eastman Organic Chemicals
eicosane, Sigma Chemical Company
mirex, Chem Services
1-chloronaphthalene, Aldrich Chemical
o-chlorophenol, Aldrich Chemical
endrin, Chem Services
methoxychlor, Chem Services
pyrene, Fisher Scientific
pentachlorophenol, Matheson, Coleman, and Bell
anthracene, Matheson, Coleman, and Bell
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SECTION 5
EXPERIMENTAL PROCEDURES
ENVIRONMENTAL SAMPLING
Microbiological
Initial studies lead to the development of a microbiological surface
slick sampling technique (Crow et al. 1973). The polycarbonate membrane
(Nuclepore Corporation) had several suitable characters for surface sampling.
The polycarbonate membranes have a very low density and are thus capable of
floating even when saturated with water. Membranes composed of other mater-
ials (cellulose acetate, etc.) were found to sink rapidly when saturated with
water. As compared to screen samplers, the membranes are easier to manipu-
late and problems with sterilization of sufficient samplers are non-existant.
The membrane (47 mm dia, 0.4 ym pore size) absorbs approximately 50 yl of
surface film permitting collection of a 20 to 40 ym thick sample. The size
of a sample, however, will vary with the nature and thickness of the slick,
a phenomenon common to all surface slick samplers.
The sterile polycarbonate membranes were floated on the water surface.
The membrane and adhering surface film were retrieved with either a sterile
plastic dish which was submerged under the membrane and underlying waters or,
in calm waters, by directly retrieving the membrane with a sterile forceps
from the water surface. The membranes were placed into 100-ml bottles con-
taining sterile seawater or placed directly onto a nutrient agar medium.
Upon return to the laboratory, the bottles were agitated for 3 min on a
wrist-action shaker. Aliquots were serially diluted and 0.1 ml of dilutions
plated onto appropriate media. In initial studies, dominant colonial types
were picked from the enumeration medium, subcultured to assure purity, then
placed on a maintenance medium subsequent to their physiological characteri-
zation.
In more recent studies, selective isolation media were used to detect
major physiological groups. Serial dilutions of the membrane rinse water
were plated onto media designed to detect proteolytic, amylolytic, lipo-
lytic, and hydrocarbonoclastic microorganisms (Colwell and Wiebe 1970;
Hankin and Anagnostakis 1975). Predominant organisms from each physiolo-
gical group were isolated and stored on the appropriate stock culture medium.
Chemical
Large polycarbonate membranes (29 cm d, 0.4 ym pore size) were used to
sample surface slicks. The membranes were retrieved from the water surface
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and placed in 50 ml of petroleum ether. The petroleum ether extracts were
concentrated and analyzed by Gas-Liquid chromatography with electron capture
detector.
LABORATORY STUDIES
Initial Study
The physiological patterns of the predominant aerobic heterotrophs
(41 cultures) from initial isolation medium (Marine Agar 2216) were esta-
blished by using methods described by Colwell and Wiebe 1970; Hankin and
Anagnostakis 1975. Effects of heptachlor, methoxychlor, and PCB formula-
tions (aroclor 1016, 1242) on growth in Marine Agar were determined with a
modified antibiotic sensitivity test. Selected strains were tested for the
effect on ethanol utilization with heptachlor (up to 100 yg/ml) and methoxy-
chlor (up to 200 yg/ml) fortified with 2 per cent ethanol.
Hydrocarbon metabolism of 2 yeasts, Candida maltosa and £. lipolytica,
was studied by using a YNB-broth supplemented with hexadecane. Comparisons
were made between systems containing hexadecane alone, hexadecane and hepta-
chlor, hexadecane and methoxychlor, and hexadecane with the addition of
glucose and glycerol.
To determine if heptachlor was being metabolized by £. maltosa. the
C02 generated during the metabolism of a hexadecane and 14-C-heptachlor sub-
strate was monitored. Cultures were extracted with petroleum ether. The
petroleum ether fraction was separated, and washed with water. Cells were
centrifuged from the culture fluid and PE extracts and all fractions were
assayed for 14-C-heptachlor.
The uptake of constituents of a synthetic crude oil, in the presence of
selected carbohydrates and pesticides, (Table 1) by £. lipolytica and
C. maltosa in a YNB basal broth was evaluated. Substrate combinations tested
included: synthetic crude, synthetic crude and heptachlor, synthetic crude
and methoxychlor, synthetic crude and glucose, synthetic crude and glycerol.
Studies of Selected Metabolic Types
Representative isolates of the major physiological types of micro-
organisms isolated from surface slicks were screened for inhibition by aro-
matic and chlorinated hydrocarbons (Table 2). Organisms were grown in
Marine broth or Mycological broth for 48 hr. A minimal inoculum (.1 ml)
was then added to the appropriate nutrient medium containing 100 ppm, 10 ppm,
or 1 ppm of aromatic or chlorinated hydrocarbon dissolved in acetone. All
cultures were initially screened for sensitivity to acetone. Inhibition
was determined by a reduction of optical density compared to the acetone
control. Hexadecane utilizing bacteria and a series of 5 nitroso-guanidine
induced mutants of £. lipolytica were tested for reduction of hexadecane
utilization in the presence of chlorinated and aromatic hydrocarbons.
Model hydrocarbon (hexadecane) surface slicks were exposed to water
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TABLE 1. SYNTHETIC CRUDE OIL COMPOSITION
" . . , Mole Ratio to
Item Amount Weight (gm) Moles Hexadecane
Cyclohexane 1.2 ml
Methylcyclohexane 2 . 6 ml
Ethyl benzene 3.9 ml
Naphthalene 4.8 gm
Biphenyl 4.8 gm
Tetradecane 8.5 ml
Hexadecane 8.3 ml
Eicosane 4.8 gm
TABLE 2. COMPOUNDS SCREENED
Heptachlor (Kept)
Naphthalene (Nap
Mirex (Mir)
Biphenyl (Bip)
1-Chloronaphthalene (1-Cl-N)
Polychlorinated biphenyl (PCS)
.94 .011
2.00 .020
3.37 .032
4.80 .038
4.80 .031
6.48 .032
6.55 .028
4.80 .017
FOR INHIBITORY EFFECTS
o-Chlorophenol (o-Cl-P)
Endrin (End)
Methoxychlor (Meth)
Pyrene (Pyr)
Pentachlorophenol (PCP)
Anthracene (Anth)
.39
.71
1.14
1.36
1.11
1.14
1.00
.61
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saturated with selected recalcitrant molecules. The slick was maintained in
a closed continuous flow system by slow stirring on a magnetic stirrer.
Liquid from a reservoir of pesticide- or aromatic hydrocarbon-laden water
was directed slowly through the system at a rate to maintain the surface
slick and a constant volume of liquid in the experimental vessel. The sur-
face film was sampled at varying intervals by using 1 cm wide x 6 cm strip
of polycarbonate material (no-hole Nuclepore). These were extracted in
screw cap tubes with 2 ml of petroleum ether. Hexadecane and pollutant
molecules were quantitated by gas chromatographic analysis.
10
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SECTION 6
RESULTS AND DISCUSSION
ENVIRONMENTAL STUDIES
Microbial Populations
The microbial populations in surface films of estuarine, coastal, and
pelagic waters were found to be higher than those previously reported
(Table 3). Selected isolations for varied physiological types, in specific
instances, revealed greater populations than indicated by nonselective
medium for total aerobic heterotrophs. This finding further emphasizes the
failure of a single nutrient medium to detect the total microbiota. The
presence of such dense microbial populations suggests that the surface film
is a site of significant metabolic activity. The varied numbers of physio-
logical types in separate samples may reflect differential composition of
films. None of the samples were collected from weathered hydrocarbon slicks
or chronically oil-polluted areas. This fact may explain the relatively low
number of hydro-carbonoclasts. A few samples from a fresh marine diesel
spill (9-to 12-hr-old) yielded sparse numbers of microorganisms suggesting
possible inhibition or dispersion of the normal film flora. The microbial
populations of surface films may be influenced by the physical and chemical
nature of the slick, the extent of the slick, the prevailing meteorological
conditions, and the age of the slick.
Chemical
Analysis of extracts from large membranes indicated the presence of
numerous electrophilic compounds not found in extraction of membranes without
adsorbed surface film. Such compounds were observed most frequently in
heavy slicks found in regions of intense biological activity or in regions
subject to industrial output or effluents from sewage treatment facilities.
Extracts of selected samples have been provided to the EPA Laboratory, Gulf
Breeze, Florida, for more finite analytical studies.
LABORATORY STUDIES
Initial Studies
Characterization of the aerobic heterotrophs randomly isolated from
Marine Agar 2216 illustrated that the majority of the isolates from coastal
and estuarine films were proteolytic, amylolytic,, and capable of growth on
freshwater medium. None of these isolates utilized hydrocarbons or produced
reactions on spirit blue medium. Studies of interference of growth of 41
isolates as determined by a modified antibiotic sensitivity test with discs
11
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TABLE 3. CONCENTRATION OF MICROORGANISMS FROM SURFACE SLICKS*
Sampling
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Mean
Number of
total
Heterotrophs
1050 highest
60 mean
103 mean
2.5xl05
1x10* 2
1.2x102
S.lxlO2 2
5.3x10* 2
4.8x10* 2
2.3x103
<1
35
17
1.7xl02
57
57
5.8xl03
5.8x103
l.SxlO6
1.15xl06
2.8x107
<6
6.3x103
6.9x10*
1.2x103
1.2x10*
1.6xl03
<10
1.2x102
1.8x102
1.24x106 3.
Number
Hydro-
carbono-
clastic
31 cm2
highest
3xl02
.5xl02
6
.7xl02
.7x103 2.
.7x10* 1.
43 1.
<1
<1
<1
3
4
4.
25 5.
2.
1.
4.
7x.l03 3.
Number
Proteo-
lytic
2 xlO3
1x10
<1
<1
9x103
2x103
2xl02
<1
6
<1
6
<1
<1
6xl02
2xl02
9x103
7 xlO*
6x105
<6
<6
<6
<6
<6
6
6
6
6
2x10*
Number
Amylolytic
23
1.2xl02
3.5x10*
5.2xl02
6
<1
<1
<1
1.7xl02
29
23
1.1x103
3.9x103
3.8x10*
2.9x105
5.8x10?
<6
S.lxlO2
6.9x102
S.OxlO2
9.8xl02
3.5x102
6
6
60
2.8xl06
Number
Lipolytic Reference
Sieburth
1965
Gunkel 1973
Crow et al.
1975
Crow et al.
1976
^^w
__
__
17
6
<1
S.lxlO3
3.8xl05
6.9x10*
1.8x107
<6
5.2xl02
4.6xl02
35
l.lxlO3
35
6.9xl02
<6
^ğ \J
69
1.4xlO^_
Current
data
*Number viable cells/cm2
12
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containing approximately 100 ppm heptachlor and methoxychlor showed no
inhibition. Only one isolate demonstrated sensitivity to PCB formulations.
Similarly, pesticides did not alter phosphatase activity. Concentrations of
up to 100 ppm heptachlor and 200 ppm methoxychlor in basal medium fortified
with 2 per cent ethanol did not alter (growth rates) of ethanol utilizing
strains.
The growth of one yeast isolate, jC_. maltosa, representative of a strain
associated with hydrocarbons, has given increased cell yields on hexadecane
in the presence of heptachlor. Concomitantly, extractable levels of hepta-
chlor in the culture broths have decreased. Results of a representative
experiment are presented in Table 4. Though cell yields were slightly
greater with larger heptachlor concentrations (Table 5) much greater amounts
of heptachlor were unextractable at the higher concentrations. It is not
clear whether this loss of heptachlor is due to metabolic uptake or to
physical binding of heptachlor.
TABLE 4. HEPTACHLOR RECOVERY FROM HEXADECANE GROWTH MEDIUM
Organism Initial Cone.
Heptachlor
(Mg)
Hexadecane
(ml)
Disappearance
Heptachlor
(MS)
Hexadecane
(ml)
Candida maltosa
(R-42, Ahearn)
0.0
956.0
9560.0
0.1
0.1
0.1
0.0
85.1
(91 per cent)
1405.3
(85 per cent)
0.0302
0.0331
0.0308
TABLE 5. DRY WEIGHT
Organism
Heptachlor
Cone.
(yg/ml hexadecane)
Dry Weight
(yg/ml x 10-4)
.Candida maltosa
(R-42, Ahearn)
0.0
956.0
9560.0
7.70
7.52
9.19
Eight days after adding 6.748 x 10^ CPM of labeled heptachlor to the
culture system, 6.734 x 105 CPM, (99.79 per cent) could be recovered from
13
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the various fractions (see Table 6). The amount of ^ CC>2 trapped represented
only 0.385 per cent of the total activity added, while the total recovery
from the system was 100.178 per cent.
TABLE 6. RECOVERY OF 14C LABEL FROM HEPTACHLOR FOLLOWING
EIGHT DAYS OF INCUBATION WITH C. MALTOSA
Item
CMP
Per cent Recovery
Counts
Counts
Counts
Counts
added
recovered
recovered
recovered
from culture
as C02
, total
6.
6.
0.
6,
748
734
026
760
X
X
X
X
105
105
105
105
99.
0.
100.
__
792
385
178
The oil fraction (hexadecane or metabolites of hexadecane) contained the
highest per cent of the label (82 per cent). The supernatant, aqueous
fraction cells, and oil fraction cells contained from 2.6 to 8.5 per cent of
the total label added (see Table 7), while the remaining fractions contained
less than 1.0 per cent of the total l^C added. It is of interest to note
that even though the oil fraction contained the greatest isotope activity,
the cells recovered from the aqueous fraction contained 53 per cent more
radioactivity than those from the oil fraction. This
for entirely in terms of numbers of cells in each fraction, for when cal-
culated on the basis of CPM/ml of cells, the aqueous fraction cells still
contained 9 per cent more activity than the oil fraction cells.
TABLE 7. RECOVERY OF 1*C LABEL FROM VARIOUS FRACTIONS OF
YEAST CULTURE AFTER EIGHT DAYS INCUBATION WITH
14C-HEPTACHLOR
Item
Total counts added
Counts recovered
Oil fraction
Cells
Supernatent
Oil fraction cells
Rinse
Culture flask rinse
Total
CPM
6.748 x 105
5.543 x 105
5.543 x 105
0.576 x 105
0.377 x 105
0.178 x 105
0.018 x 105
0.042 x 105
6.734 x 105
Per cent Recovery
82.143
8.536
5.587
2.638
0.267
0.622
99.793
14
-------
The PE extraction of these cells revealed that only 42.0 per cent of the
label remaining within the cells could be removed with PE. When one compares
, this with the 98.5 per cent recovery of l^C-heptachlor frOm distilled water
using PE, it becomes evident that (1) the heptachlor is not free to be ex-
tracted due to some form of binding, or (2) that the labeled compound is no
longer heptachlor but one of its less soluble metabolites.
The data indicate that the majority of the l^C-heptachlor remained
solubilized in the hexadecane. However, some wa* found in all of the fractions
that resulted from centrifuging the yeast containing media. Moreover, the
loss of label due to metabolism to 14cc>2 appeared to be negligible; as did
loss due to glass binding. This does not mean,however, that certain frag-
ments of the molecule were not being utilized for energy production by the
organisms, only that the portion of the molecule containing the label was
not being utilized for C02 production. In fact, the extraction of the cells,
with various solvents has given preliminary evidence that the heptachlor is
being broken down, leaving a metabolite of metabolites which are not as
soluble in PE as the heptachlor. The radioactivity remaining in the cellular
fractions could have been due to: (a) hexadecane containing l^C-heptachlor
adhering to the outside of the cell, (b) the hexadecane containing l^C-hepta-
chlor located within -the cells in vacuoles, (c) the l^C-heptachlor being
degraded and the 14c incorporated into cellular structure, or (d) metabolites
of hexadecane bound to or within the cell. The radioactivity remaining
within the cells may be due to a combination of all these factors; however,
the importance of each factor has not yet been determined.
In studies of the utilization of synthetic crude by £. lipolytica and
£. maltosa, heptachlor consistently increased the utilization of synthetic
crude over systems with synthetic crude alone (Table 8). With the addition
of glucose and glycerol this effect was not evident. The recoverable amounts
of naphthalene and biphenyl from the synthetic crude were consistently
reduced after growth of both yeasts (Table 9). Neither of these compounds
supported the growth of the yeasts as sole sources of carbon. Examination of
molar utilization ratios of various components of synthetic crude indicate
that the per cent reduction in the recovery of naphthalene and biphenyl is
always greater in the presence of heptachlor (Table 10). This suggests that
heptachlor is preferentially affecting the uptake of naphthalene and bi-
phenyl in these systems.
Studies of Selected Metabolic Types
Fifty-three selected isolates from the four major physiological groups
of bacteria from surface slicks showed variable responses to chlorinated and
aromatic hydrocarbons (Table 11). PCP, 1-chloro-naphthalene, and o-chloro-
phenyl were the most inhibitory compounds, whereas only a few strains were
inhibited by heptachlor, methoxychlor, endrin, and mirex. The hydrocarbono-
clastic group proved to be suprisingly sensitive to naphthalene, biphenyl,
PCB 1016, and 1-chloro-naphthalene when grown in a peptone-based media,
whereas the aerobic heterotrophs were less affected. The inhibition or
stimulation of growth by various compounds appeared dependent upon the
medium. The proceeding results (Table 11) were determined with a convention-
al nutrient medium. In a basal salt medium with hexadecane as the carbon
15
-------
TABLE 8. UTILIZATION OF SYNTHETIC CRUDE OIL BY
C. LIPOLYTICA AND C. MALTOSA
Item
Synthetic crude
+ Glycerol
+ Glucose
+ Methoxychlor
+ Heptachlor
C. maltosa
35.65*
25.20
26.77
30.85
47.10
C. lipolytica
. - _.
26.27
29.20
23.85
30.70
47.20
*Per cent utilization based on uninoculated control
Each value is the result of average of duplicate flasks for two separate
experiments
TABLE 9. UTILIZATION OF SYNTHETIC CRUDE COMPONENTS
BY C. LIPOLYTICA AND C. MALTOSA
Synthetic Crude
+ Glycerol
+ Glucose
+ Methoxychlor
+ Heptachlor
Ethyl-
benzene
29.3*
17.5
26.7
22.5
49.6
C. maltosa
Naphtha-
lene Biphenyl
23.5 17.3
17.6
20.3
23.6
50.4
21.8
23.1
29.9
52.6
Tetra-
decane
43.1
31.3
26.2
36.3
52.9
Hexa-
decane
44.1
30.7
28.2
36.8
52.6
Eico-
sane
43.8
30.2
27.6
37.2
50.3
C. lipolytica
Synthetic Crude
+ Glycerol
+ Glucose
+ Methoxychlor
+ Heptachlor
29.4
27.8
46.7
14.4
49.6
14.6
20.0
22.0
26.4
44.8
34.6
19.6
21.8
29.2
47.5
32.9
36.8
23.6
32.7
49.0
32.7
36.1
22.7
35.5
47.9
35.3
29.8
23.8
26.6
41.0
*Per cent utilization compared to an uninoculated control
16
-------
TABLE 10. MOLE UTILIZATION RATIOS OF SYNTHETIC CRUDE OIL COMPONENTS
Synthetic crude
Synthetic crude
+ Heptachlor
Nap/
Hex
.76
1.38
C
. maltosa
Nap/ Nap/
Hex + Tetra Hex + Tetra + Eic
.36
.65
.28
.51
Bip/
Hex
Bip/ Bip/
Hex + Tetra Hex + Tetra + Eic
.81
1.10
.39
.51
.30
.41
C. lipolytica
Nap/
Hex
Nap/ Nap/
Hex + Tetra Hex + Tetra
Synthetic crude .56
ta Synthetic crude
+ Heptachlor 1.31
.25
1.00
.20
.49
+ Eic
Bip/
Hex
1.20
1.15
TABLE 11 EFFECT OF CHLORINATED AND AROMATIC HYDROCARBONS
Physiological
Type
Aerobic
Heterotroph
Lipolytic
Hydrocarbono-
clastic
Amylolytic
Total
Number
Isolate
Tested
25
9
11 & 15
4
53.49
ON GROWTH
Bip/ Bip/
Hex + Tetra Hex + Tetra + Eic
.55
.88
.44
.42
OF SELECTED MICROORGANISMS
COMPOUNDS (100 yg)
0-C1-P
12/25
5/9
0/15
3/4
20/53
I-C1-N
18/25
7/9
12/15
4/4
41/53
1016
PCB
6/25
0/9
10/15
1/4
17/53
Nap Hept
2/25 1/25
4/9 0/9
7/11 0/11
3/4 1/4
16/49 2/49
Meth
1/25
0/9
0/15
0/4
1/53
Bip
1/25
4/9
6/11
2/4
13/49
End
2/25
0/9
0/11
4/4
2/49
PCP Mir
25/25 1/25
9/9 0/9
15/15 0/15
4/4 0/4
53/531/53
Pyr
0/25
0/9
0/15
0/4
0/53
Total
Inhibitory
Responses _
69/275
29/99
50/149
16/44
^M
-------
source, the effect of various compounds appeared organism-related (Table 12)
In general, bacteria which showed the greater utilization of hexadecane were
inhibited by a wider range of compounds. This phenomenon may be related to
transport and concentration of the pesticide within the cell. The eleven
cultures of c. lipolytica grown on a carbohydrate medium were more resistant
to most compounds than the hydrocarbonoclastic bacteria (Table 13). in con-
trast to the bacteria, the yeasts were resistant to PCB 1016, naphthalene
and blphenyl. Both bacteria and yeasts were sensitive to 1-chloronaphthalene.
In a hexadecane-based medium the yeast appeared to be less affected by addi-
tion of aromatic and chlorinated compounds than by bacteria (Table 14) ln
model surface slick systems, hexadecane films rapidly accumulated both'naph-
thalene and PGP from subsurface water. PGP reached concentrations of .02 to
.03 per cent in hexadecane after 2 days of continuous exposure to water
containing less than 80 ppm pentachlorophenol. Concentrations of .08 per
cent were measured after an additional 24 hr. Recalcitrant molecules in
experimental accumulation studies rapidly reached concentrations several
orders greater than the magnitude that inhibited representative surface slick
isolates. Accumulation or sequestering of recalcitrant molecules within a
surface film may alter essential metabolic processes with this system,
18
-------
TABLE 12. EFFECT OF AROMATIC AND CHLORINATED HYDROCARBONS (10 PPM) ON HEXADECANE
METABOLISM BY SURFACE SLICK BACTERIA AAunuAWt.
Hexa-
Culture decane
No. control 0-Cl-P 1-C1-N PCB 1016
19-2 34* 0 +++ 0
18-3 59 -H- o ++
9-1 50 0 + o
H-17 7 +++ o l l I
H-16 84 0 - 0
H-1 q 52 0 0 III
H-14 72 0 in
H-28 35+0
Total 3/8 2/8 4/8
^er cent recovery hexadecane
b+ 10-20 per cent inhibition
-H- 21-30 per cent inhibition
-H+ greater than 31 per cent inhibition
- enhanced utilization
0 no effect
TABLE 13. INHIBITION OF
No. Tested 0-Cl-P 1-C1-N PCB 1016
11 4/11 11/11 3/11
Total
Inhibitory
Nap Kept Meth Bip End PCP Mir Pyr Responses
0 Ob 0 +++00++++++ 4/11
0 + - +++++++00 6/11
0 +++ - 0 0 +++-+++ 4/11
+++ +++ +++ +++ +++ +++ +++ +++ 10/11
+ 0 0 - 0 1/11
0 - +0+++-0 3/11
- +++ 0 - 2/11
+++ - - +++ + +++ 5/11
1/8 4/8 2/8 4/8 2/8 6/8 3/8 4/8
C. LIPOLYTICA ISOLATE BY VARTnTTS rnMpOTTNPS
-LUU tig Tofcal Inhibl_
jap_h Kept Meth Bip End PCP Mir Pyr tory Responses
0/11 0/11 0/11 0/11 0/11 11/112/11 3/11 34/121
-------
TABLE 14. EFFECT OF AROMATIC AND CHLORINATED HYDROCARBONS AT 10 PPM ON
HEXADECANE METABOLISM BY C. LIPOLYTICA MUTANTS
Mutant
No.
37-1
5-2
12-1
5-1
8-2
4-2
25-2
Total
Hexa-
decane
control
67a
96
28
27
28
41
83
0-C1-P
-
0
+++
+
0
0
0
2/7
1-C1-N
0
0
0
0
0
0
0
0/7
PCB 1016 Nap
0
0 0
+-H- +-H-
+ 4-H-
0 +
0 0
0 0
2/7 3/7
Kept
Ob
0
-H-
0
0
0
0
1/7
Meth
0
0
+
0
++
0
0
2/7
Bip
0
0
+++
++
-H-
-
0
3/7
End
4-H-
0
0
0
+
0
0
2/7
PCP Mir
0 0
0 0
-H-+ 0
+++ 0
-H- 0
+ 0
0
4/7 0/7
Pyr
0
0
0
0
0
+
0
1/7
Total
Inhibitory
Responses
1/11
0/11
7/11
5/11
5/11
2/11
0/11
aPer cent recovery hexadecane
b+ 10-20 per cent inhibition
-H- 20-30 per cent inhibition
+++ greater than 30 per cent inhibition
- enhanced utilization
0 no effect
-------
REFERENCES
Baler, R. E. 1970. Surface Quality Assessment of Natural Bodies of Water.
Proc. Great Lakes Res. 13th, 114-127.
Baler, R. E. 1972. Organic Films on Natural Waters: Their Retrieval, Iden-
tification, and Modes of Elimination. J. Geophys. Res., 77:5062-5075.
Colwell, R. R., and W. T. Wiebe. 1970. "Core" Characteristics for Use in
Classifying Aerobic, Heterotrophic Bacteria by Numerical Taxonomy.
Bull. Ga. Acad. Sci., 28:165-185.
Crow, S. A., D. G. Ahearn, W. L. Cook, and A. W. Bourquin. 1975. Densities
of Bacteria and Fungi in Coastal Surface Films as Determined by a
Membrane Adsorption Procedure. Limnol. Oceanogr., 20:644-646.
Crow, S. A., W. L. Cook, D. G. Ahearn, and A. W. Bourquin. 1976. Microbial
Populations in Coastal Surface Slicks. Proc. Third International Bio-
degradation Symposium. J. M. Sharpleyad, A. M. Kaplan (eds.). Applied
Science, 93-98.
Ewing, G. 1950. Slicks, Surface Films and Internal Waves. J. Mar. Res.,
9:161-187.
Garrett, W. D. 1965. Collection of Slick Forming Materials from the Sea
Surface. Limnol. Oceanogr., 10:602-605.
Gunkel, W. 1973. Distribution and Abundance of Oil-Oxidizing Bacteria in
the North Sea. In: The Microbial Degradation of Oil Pollutants,
(D. G. Ahearn anTs. P. Meyer, eds.), Center for Wetland Resources,
Louisiana State University, Publication LSU-SG-73-01, 127-139.
Hankin, L., and S. L. Anagnostakis. 1975. The Use of Solid Media for De-
tection of Enzyme Production by Fungi. Mycologia, 67:597-607.
Hartung R., and G. W. Klinger. 1970. Concentration of pp DDT by Sedimented
Polluting Oils. Environ. Sci. Technol., 4:407-409.
Harvey, G. R., W. G. Steinhauer, and H. P. Miklas. 1974 Decline of PCB
Concentrations in North Atlantic Surface Water. Nature, 252:387-388.
Maylntyre, F. 1974. The Top Millimeter of the Ocean. Sci. Am., 230:62-77.
21
-------
Parker, B., and G. Barsom. 1970. Biological and Chemical Significance of
Surface Microlayers in Aquatic Ecosystems. Bio. Sci., 20:87-94.
Parsons, T. R., and M. Takahashi. 1973. Biological Oceanographic Processes.
Pergamon Press. 230 pp.
Sayler, G. S., and R. R. Colwell. Partitioning of Mercury and Polychlori-
nated Biphenyl by Oil, Water, and Suspended Sediment. Env. Sci. Tech.,
10:1142-1145.
Seba, D. B., and E. F. Corcoran. 1969. Surface Slicks as Concentrations of
Pesticides in the Marine Environment. Pesticides Monitoring Journal,
3:190-193.
Sieburth, J. McN. 1965. Bacteriological Samplers for Air-Water and Water-
Sediment Interfaces. In; Trans. Joint Conf. Ocean Sci. Ocean Eng.,
MTS-ASLO, Washington, D. C., 1064-1068.
Stadtler, D., and U. Ziebarth. 1976. p,p-DDT, Dieldrin, and Polychlorierte
Biphenyle (PCB) in Oberflachenwasser der Westlichen Ostsee 1974.
Deut. Hydrograph. Zeits., 29:25-31.
Sutcliffe, W. H., Jr., E. R. Baylor, D. W. Menzel. 1963. Sea Surface
Chemistry and Langmuir Circulation. Deep-Sea Res., 10:233-243.
ZoBell, C. E. 1946. "Marine Microbiology." Chronica Botanica, Waltham,
Mass.
22
-------
. REPORT NO.
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
_ ;r^r.
3. RECIPIENT'S ACCESSION>NO.
TITLE AND SUBTITLE
MICROBIAL INTERACTIONS WITH PESTICIDES
IN ESTUARINE SURFACE SLICKS
5. REPORT DATE
March. 1977
AUTHOR(S)
D.G. Ahearn, S.A. Crow and W.L. Cook
6. PERFORMING ORGANIZATION CODE
, Georgia State University
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Georgia State University
Biology Department
33 Gilmer St. N.E.
Atlanta, Georgia 30303
2. SPONSORING AGENCY NAME AND ADDRESS
Gulf Breeze Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze. Florida 32561^
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
EPA Grant R-803141
13. TYPE OF REPORT AND PERIOD COVERED
May 1974 - Sept. 1976
14. SPONSORING AGENCY CODE
EPA-ORD
B. SUPPLEMENTARY NOTES
"is. ABSTRACT Estuarine surface films trom Escambia Hay, ^^^^^^l^*
lations. Samples of the upper 10 pm o_f ^""^^i^ions^ere ^to^OO
unts of
uiij.i.a oi
and molds on Mycological Agar prepared with 50 per cent seawater were
ii , , 2 The predominant surface film isolates
rĞe "ytS .J^lS^ L 5*iblt- only Ğ* to negligible
and lipolytic activities. A greater proportion of the surface-
sudies with 53 isolates representative of more diverse physiological groups o-chloro-
naphthaline PCB 1016, and pentachlorophenol were inhibitory to a large portion of the
Solates and heptachlor, biphenyl, pyrene, and PCB 1016 significantly reduced
hexadecane utilization.
17.
DESCRIPTORS
Mlcrobial Interactions
Microbial Degradation
Air-Sea Interface
Hydrocarbon Degradation
Pesticide-Hydrocarbon Interaction
Surface Microlayers
KEY WORDS AND DOCUMENT ANALYSIS
b.IDENTIFIERS/OPEN ENDED TERMS
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