DOC
EPA
United States
Department of
Commerce
National Oceanic and
Atmospheric
Administration
United States
Environmental Protection
Agency
Office of Environmental Engineering
and Technology
Washington DC 20460
EPA-600 7-80-133
June 1980
Research and Development
Petroleum
Biodegradation
Potential of Northern
Puget Sound and
Strait of Juan de Fuca
Interagency
Energy/Environment
R&D Program
Report
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide range of energy-related environ-
mental issues.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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PETROLEUM BIODEGRADATION POTENTIAL OF
NORTHERN PUGET SOUND AND
STRAIT OF JUAN DE FUCA ENVIRONMENTS
by
Dr. D.W.S. Westlake
Department of Microbiology
University of Alberta
Edmonton, Alberta
Canada T6G 2E9
Dr. F.D. Cook
Department of Soil Science and Microbiology
University of Alberta
Edmonton, Alberta
Canada T6G 2E9
Prepared for the MESA (Marine Ecosystems Analysis) Puget Sound
Project, Seattle, Washington in partial fulfillment of
EPA Interagency Agreement No. D6-E693-EN
Program Element No. EHE625-A
EPA Project Officer: Clinton W. Hall (EPA/Washington, D.C.)
NOAA Project Officer: Howard S. Harris (NOAA/Seattle, WA)
This study was conducted
as part of the Federal
Interagency Energy/Environment
Research and Development Program
Prepared for
OFFICE OF ENERGY, MINERALS, AND INDUSTRY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
MARCH 1980
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Completion Report Submitted to
PUGET SOUND ENERGY-RELATED RESEARCH PROJECT
MARINE ECOSYSTEMS ANALYSIS PROGRAM
ENVIRONMENTAL RESEARCH LABORATORIES
by
UNIVERSITY OF ALBERTA
EDMONTON, ALBERTA
CANADA T6G 2E9
This work is the result of research sponsored by the Environmental
Protection Agency and administered by the Environmental Research
Laboratories of the National Oceanic and Atmospheric Administration.
The Environmental Research Laboratories do not approve, recommend,
or endorse any proprietary product or proprietary material mentioned in
this publication. No reference shall be made to the Environmental
Research Laboratories or to this publication furnished by the Environmental
Research Laboratories in any advertising or sales promotion which would
indicate or imply that the Environmental Research Laboratories approve,
recommend, or endorse any proprietary product or proprietary material
mentioned herein, or which has as its purpose an intent to cause directly
or indirectly the advertised product to be used or purchased because of
this Environmental Research Laboratories publication.
n
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FOREWORD
Substantially increased petroleum tanker traffic, pipeline transport
and refining operations are anticipated in the region of Northern Puget
Sound and Strait of Juan de Fuca with the Alaskan pipeline in operation.
To assess the potential future environmental impact arising from these
activities, the Puget Sound Energy-Related Project contracted the Univer-
sity of Alberta to undertake a study on the "Microbial Degradation of
Petroleum Hydrocarbons" in Northern Puget Sound and Strait of Juan de Fuca.
This study was supported by U.S. Environmental Protection Agency "pass-
through" funds administered by the NOAA Marine Ecosystem Analysis Program.
This final report presents the results of two years of research.
m
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.ABSTRACT
The oil-degrading activity of the microbial flora present in marine
samples from three sites in the northern Puget Sound - Samish Bay,
E. Fidalgo and Pt. Partridge and several sites in the Pt. Angeles area
were investigated in this study. Activity was measured in terms of changes
in the n-alkane and isoprenoid gas chromatographic profile of the saturate
fraction and reported in terms of a "Degradative Capacity Index". Oil-
degrading activity was greatest in areas adjacent to oil refineries and
areas of relatively high levels of commercial activity. The levels of
nitrogen and phosphorus were the primary environmental factors controlling
the activity of the oil-degrading microbial flora. The fact that oil-
degrading bacterial populations were readily isolated under enrichment con-
ditions similar to those existing in the natural environment whereas fungi
and yeast were only obtained under selective enrichment conditions (i.e.
low pH) suggests that bacteria would be the most active group in removing
oil spilled from this environment. Oil-degrading populations consisted
predominantly of Flavobacterium and Pseudomonas genera with occasional
populations containing a predominance of members of Acinetobacter and
Alcaligenes genera.
The highest rates of removal (i.e. weight loss) of Prudhoe Bay oil
by the microbial flora present in water column materials were obtained in
areas where maximum oil-degrading activity was observed (i.e. near refiner-
ies and commercially active sites). This loss in weight was due to the
removal of compounds in the saturate and aromatic fractions of Prudhoe Bay
oil. Components in the saturate fraction are slowly used (long lag phase)
followed by an extended period of a high rate of removal whereas aromatics
are continuously used at a low rate followed by a short period where a
rapid loss of weight in this fraction occurs. Studies determining the rate
of release of l^C02 from ^C-labelled hydrocarbon-"spiked" Prudhoe Bay oil
by the microbial flora present in water column and sub-tidal sediments
showed that [9-11+C]-phenanthrene yielded the highest rates of lltC02 release
followed by [l-11+C]-naphthalene, [l-C^j-hexadecane and [9-ll*C]-anthracene.
However lt*C02 was released very quickly (i.e. with a very short lag) from
[l-^C^j-naphthalene-spiked 0-ns indicating that the natural indigenous
microbial flora very quickly adapted to this substrate. The shortest lag
times for llfC02 evolution from all labelled substrates was observed with
samples near oil refineries and commercially active areas. The highest
yields of ltfC02 (greater than 50%) were obtained with [9-14C]-phenanthrene.
Studies with glass capillary gas chromatographic analysis of the aromatic
fraction indicate that substituted benzenes, naphthalene, and 1-methyl- and
2-methyl-naphthalenes were used before changes in the n-alkanes and iso-
prenoids were detected. It was also observed using this gas chromatographic
technique that changes take place in the components of the aromatic -and
saturate fraction without nitrogeji and phosphorus supplementation of marine
samples although at a lower rate than when such nutrients were added.
IV
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CONTENTS
Foreword i i i
Abstract i v
Figures vi
Tables ix
Acknowledgement xi i i
1. Introduction 1
2. Conclusions 5
3. Recommendations 7
4. Materials and Methods 9
5. Results 27
6. Discussion 102
References 113
Appendix 117
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FIGURES
Number Page
1 Map showing location of sample sites used in 1977 -
1978 study 10
2 Maps showing location of sample sites used in 1978 - 1979
study 11
3 Typical G.C. profiles of saturate fraction of Prudhoe Bay
oil before and after growth of enrichment populations
(N = no degradation; S = selective metabolism, n-alkanes
Ci2 to C19; P = partial removal, n-alkane peaks still
discernible; I = only isoprenoids remaining; C = complete
utilization of n-alkanes and isoprenoids) 15
4 Typical G.C. profiles of saturate fraction of Minas oil
before and after growth of enrichment populations
(- = no degradation; ± = selective degradation;
+ = degraded profi1e) 16
5 Typical G.C. profiles of saturate fraction of Murban oil
before and after growth of enrichment populations
(- = no degradation; ± = selective degradation;
+ = degraded profile) 17
6 Typical G.C. profiles of saturate fraction of Seria oil
before and after growth of enrichment populations
(- = no degradation; ± = selective degradation;
+ = degraded profile) 18
7 Type of Erlenmeyer flask used in radiometric studies 22
8 Weight percent loss of Prudhoe Bay oil as a result of
incubation (8°C) with water column samples from
E. Fidalgo and Pt. Partridge (April, 1979) 40
9 Changes in the components of Prudhoe Bay oil as a result
of incubation with water column samples from northern
Puget Sound (April, 1979) 42
10 Weight percent loss of Prudhoe Bay oil as a result of
incubation (8°C) with water column samples from the
Pt. Angeles area (June, 1979) 43
vi
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Number Page
11 Changes in the components of Prudhoe Bay oil as a
result of incubation (8°C) with water column samples
from the Pt. Angeles area (June, 1979) 44
12 14C02 evolution patterns for Prudhoe Bay oil "spiked"
with one of n-[l-14C]-hexadecane, [l-1IfC]-naphthalene,
[9-14C]-phenanthrene or [9-ll*C]-anthracene for water
column samples from E. Fidalgo and Pt. Partridge
(April, 1979) 50
13 14C02 evolution patterns for Prudhoe Bay oil "spiked"
with one of n-El-^Cl-hexadecane, [l-lltC]-naphthalene,
[9-14C]-phenanthrene or [9-14C]-anthracene for water
column samples from Ediz Hook, Peabody Creek and
Dungeness Spit #2 (June, 1979) 52
14 14C02 evolution patterns for Prudhoe Bay oil "spiked"
with one of n-[l-14C]-hexadecane, [l-^Cl-naphthalene,
[9-ll*C]-phenanthrene or [9-11+C]-anthracene for sub-tidal
sediments from Ediz Hook, Peabody Creek and Dungeness
Spit #2 (June, 1979) 55
15 Typical G.C. profile (glass capillary column) of aromatic
fraction of Prudhoe Bay oil (hmb = hexamethylbenzene) ... 63
16 Changes in the G.C. profiles (glass capillary column) of
the aromatic fraction of Prudhoe Bay oil after 6, 10 and
14 days incubation with water column samples from
E. Fidalgo supplemented with nitrogen and phosphorus
(+N,P) (hmb = hexamethylbenzene) 64
17 Changes in the G.C. profiles (glass capillary column) of
the aromatic fraction of Prudhoe Bay oil recovered from
control incubations (i.e. sterile artificial sea water)
after 6, 10 and 14 days incubation (hmb = hexamethyl-
benzene) 65
18 Changes in the G.C. profiles (glass capillary column)
of the aromatic fraction of Prudhoe Bay oil recovered
after 27 days incubation; control = sterile artificial
sea water and E. Fidalgo water column sample without
(-N,P) and with (+N.P) addition of nitrogen and phos-
phorus (hmb = hexamethyl benzene) 67
vii
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Number
19 G.C. profiles (glass capillary column) of aromatic
fraction of Prudhoe Bay oil recovered after 27 days
incubation with water from one of Peabody Creek,
Pt. Partridge or E. Fidalgo supplemented with
nitrogen and phosphorus (+N,P) (hmb = hexamethyl-
benzene) 68
20 G.C. profiles (glass capillary column) of aromatic
fraction of Prudhoe Bay oil recovered after 27 days
incubation from a control (i.e. sterile artificial
sea water) and water from one of Peabody Creek,
Pt. Partridge or E. Fidalgo without nutrient supple-
mentation (-N,P) (hmb = hexamethylbenzene) 69
21 G.C. profiles (glass capillary column) of saturate
fraction of Prudhoe Bay oil recovered after 27 days
incubation from a control (sterile artificial sea water)
and water from one of Peabody Creek, Pt. Partridge or
E. Fidalgo supplemented with nitrogen and phosphorus
(+N,P) (hmb = hexamethylbenzene, Pr = pristane,
Ph = phytane) 70
22 G.C. profiles (glass capillary column) of saturate
fraction of Prudhoe Bay oil recovered after 27 days
incubation with water from one of Peabody Creek,
Pt. Partridge or E. Fidalgo without nutrient supple-
mentation (-N,P) (hmb = hexamethylbenzene,
Pr = pristane, Ph = phytane) 71
viii
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TABLES
Number
1 Sampling locations ................................... ^2
2 Characteristics used in classifying bacterial
isolates ............................................. 24
3 G.C, profile of pentane extract of recovered Prudhoe
Bay oil after 28 days incubation at 8°C with water
column samples supplemented with nitrogen and
phosphorus ........................................... 28
4 G.C. profile of pentane extract of recovered Prudhoe
Bay oil after 28 days incubation at 8°C with beach
samples supplemented with nitrogen and phosphorus ---- 29
5 Summary of oil -degrading activity at sites sampled
from 1977 - 1979 ..................................... 30
6 Degradative capacity index for sites sampled
1978 - 1979a ......................................... 31
7 Degradative capacity index for Pt. Angeles samples
sites9 (1978 - 1979). 32
8 Oil -degrading capability of inter- tidal core samples. 34
9 Utilization of oil by bacteria attached to seaweeds
(collected August 1978) .............................. 36
10 Utilization of oil by "washed" and "unwashed" seaweed
(collected August and October 1979) .................. 37
11 Utilization of Prudhoe Bay oil by bacteria attached
to cobbl es ..... . ..................................... 38
12 Gravimetric and G.C. changes in Prudhoe Bay oil
brought about by incubation with water column samples
from E. Fidalgo ....................................... 4'
13 Gravimetric and G.C. changes in Prudhoe Bay oil
brought about by incubation with water column samples
from Pt, Angeles harbor (Ediz Hook, June, 1979) .......
14 Gravimetric and G.C. changes in Prudhoe Bay oil
brought about by incubation with water column samples
from Pt. Angeles harbor (Peabody Creek, June, 1979)... 46
ix
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Number Page
15 Gravimetric and G.C. changes in Prudhoe Bay oil
brought about by incubation with water column
samples from Dungeness Spit #2 (June 1979) 47
16 Rate of 11+C02 evolution from n-[l-li+C]-hexadecane,
[l-1£tC]-naphthalene, [9-14C]-anthracene and [9-1£tCJ-
phenanthrene by water column samples from the
northern Puget Sound area 49
17 Rate of 1(+C02 evolution from n-[l-ll+C]-hexadecane,
[l-^Cj-naphthalene, [9-1IfC]-anthracene and [9-lt+CJ-
phenanthrene by water column samples from the
Pt. Angeles area 51
18 Rate of 1(tC02 evolution from n-H-^Cj-hexadecane,
[l-14C]-naphthalene, [9-lltC]-anthracene and [9-11+C]-
phenanthrene by sub-tidal sediment samples from the
Pt. Angel es area 54
19 Rate of lkC02 evolution from n-H-^Cj-hexadecane,
[l-ll+C]-naphthalene by beach samples from the
Pt. Angeles area 56
20 Chemical composition of oils 57
21 Utilization of Minas (Sumatra) Murban (Jabal Dhanna)
and Seria (Malaysia) by oil-degrading enrichments
from northern Puget Sound 58
22 Utilization of Minas, Murban and Seria oils by water
and beach samples - northern Puget Sound and
Pt. Angeles area (June, 1979) 59
23 Effect of incubation time at (8°C) on the utilization
of Prudhoe Bay oil by water column samples from
northern Puget Sound (April 2-3, 1979) and
Pt. Angeles area (June 18-20, 1979) 61
24 Oil biodegradative activity (20°C) of fungi isolated
by plating "in situ" water column samples - northern
Puget Sound (August 20-21, 1978) 73
25 Oil biodegradative activity (20°C) of fungi isolated
from laboratory plating*of water and beach material-
northern Puget Sound (August 20-21, 1978) 74
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Number Page
26 Oil biodegradative activity (8°C) of fungi isolated
by enrichment procedure from water and beach material -
northern Puget Sound (August 20-21, 1978) 75
27 Oil biodegradative activity (20°C) of fungi isolated
from water and beach material-northern Puget Sound
(November 21-22, 1978) 76
28 Oil biodegradative activity (8°C) of fungi isolated
by enrichment procedure from water and beach material -
northern Puget Sound (November 21-22, 1978) 77
29 Oil biodegradative activity (20°C) of fungi isolated
from water and beach material - Pt. Angeles area
(October 1-3, 1978) 78
30 Oil biodegradative activity (20°C) of fungi isolated
from water and beach samples - Pt. Angeles area
(January 14-16, 1979) 80
31 Effect of culture technique on the utilization of the
n-alkanes in Prudhoe Bay oil 83
32 Bacterial, yeast and fungal colony forming units -
water and beach samples - northern Puget Sound
sites (August 20-21, 1978) 84
33 Bacterial, yeast and fungal colony forming units -
water and beach samples - northern Puget Sound sites
(November 21-22, 1978) 86
34 Bacterial, yeast and fungal colony forming units -
water and beach samples - northern Puget Sound
sites (April 2-3, 1979) 87
35 Generic composition of bacterial populations in
E. Fidalgo and Pt. Partridge before and after enrich-
ments in the presence of added nitrogen and phosphorus
(8°C) (April 2-3, 1979) 88
36 Generic composition of bacterial populations in water,
beach and cobble samples from E. Fidalgo and
Pt. Partridge (April 2-3, 1979) after enrichment
with Prudhoe Bay and added nitrogen and phosphorus ... 89
37 Bacterial, yeast and fungal colony forming units -
water, beach and sub-tidal samples - Pt. Angeles
area (October 1-3, 1978) 90
XI
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Number Page
38 Generic composition of bacterial populations in
sub-tidal sediments from the Pt. Angeles area
(October 1-3, 1978) 92
39 Bacterial, yeast and fungal colony forming units -
water and beach samples - Pt, Angeles area
(January 14-16, 1979) 93
40 Bacterial, yeast and fungal colony forming units -
water, beach and sub-tidal samples - Pt. Angeles
area (June 18-20, 1979) 95
41 Generic composition of bacterial populations in water,
beach and sub-tidal samples from the Pt. Angeles area
(June 18-20, 1979) before enrichment at 8°C with
Prudhoe Bay oil and nitrogen and phosphorus 96
42 Generic composition of bacterial populations in water,
beach and sub-tidal samples from the Pt. Angeles area
(June 18-20, 1979) after enrichment at 8°C with
Prudhoe Bay oil and nitrogen and phosphorus 97
43 Total heterotrophic count on basal marine agar, marine
agar (Difco-2216) and TCBS (Difco) agar in water,
beach and sub-tidal samples from Pt. Angeles area
(June 18-20, 1979) 98
44 Bacterial, yeast and fungal colony forming units -
water and sediment samples - Duwamish River
(August 20-21, 1979) 99
45 G.C. profile of pentane extract of recovered Prudhoe
Bay oil after 28 days incubation at 8°C with Duwamish
River water or sediment samples with and without
nitrogen and phosphorus supplementation and the
chemical analysis of samples - (August 20-21, 1979) .. 100
xi i
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ACKNOWLEDGEMENTS
We wish to thank Mr. D. Horsfield and Miss K. Semple for technical
assistance. The effort of Miss J. Foght in the development and application
of the radiometric technique and Mr. P. Fedorak in the development of the
glass capillary gas chromatographic technique and the handling of the data
management aspects of this research program are greatly appreciated. The
cooperation of Mr. L.S. Ramos, Ms. P.G. Prohaska'and Dr. W.D. McLeod, Jr.
of the NOAA National Analytical Facility at Seattle, Washington in the
development of our glass capillary gas chromatography capability is
gratefully acknowledged. We wish to thank the Managers of the Shell Oil
Company and Texaco refineries at Anacortes, Washington for supplying
samples of Minas, Murban and Seria oils used in part of these studies.
xm
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SECTION 1
INTRODUCTION
The Strait of Juan de Fuca and northern Puget Sound serve as the
'main transportation corridor for marine traffic proceeding to U.S. ports
in the Puget Sound area (e.g. Seattle), and to Canadian ports (e.g.
Vancouver). Until recently the incidence of oil-tanker traffic in this
area was light, as the oil needs of the Pacific Northwest were being
primarily supplied by pipeline from western Canada (1). However, as a
result of the re-evaluation of Canada's oil needs, the Canadian government
in the early 1970's re-allocated Canadian oil supplies so that western
Canadian crude oil was no longer available to U.S. refineries in the Pacific
Northwest area. The major refineries i.e. at Anacortes, Wash. (Shell Oil
Co. and Texaco, Inc.) and at Cherry Point, Wash. (Mobil Oil Corp. and
Atlantic Richfield Co.) now receive their crude oil exclusively via oil-
tankers. A limited amount of oil is also delivered to refineries in the
Vancouver area by oil-tankers. These developments have resulted in a tre-
mendous increase in the volume of oil being transported by water in this
area. When this is considered in conjunction with the general expansion of
commercial marine traffic resulting from the increased uses of the Ports
of Seattle and Vancouver together with the possibility of Pt. Angeles
serving as a crude oil transhipment center there is an increased risk of
an oil spill taking place.
Oil entering the marine environment can be removed from the water
column by evaporation, or sedimentation either into inter-tidal or sub-
tidal materials, or by biodegradation, or physical-mechanical clean-up.
Only the components lost by evaporation (i.e. the low molecular weight
volatile compounds) and physical-mechanical removal are not available for
biodegradation. The rate of disappearance of oil from the marine environ-
ment depends on the physical and chemical properties of the oil spilled,
and on chemical, mechanical, thermal and biological energies available in
the system (2). It is the biological (i.e. microbiological) component of
marine environmental systems on the southern shores of the Strait of Juan
de Fuca and the northern Puget Sound area of the State of Washington which
is the subject of this study.
Bacteria, yeasts and fungi (i.e. microorganisms) are unique biological
species in that many of them have the genetic capability of using hydro-
carbons either singly or in a mixture (as found in crude oil) as sources
of food and energy. This capability has only occasionally been found in
other forms of life (3). The microbial ability to degrade hydrocarbons is
not a genetically stable characteristic; it is readily lost in Gram negative
bacteria but is more stable in Gram positive bacteria, yeasts and fungi.
If substrates (i.e. microbial food) like oil or hydrocarbons are continually
introduced into a marine or terrestrial environment at low levels there will
be a greater incidence of oil-utilizing microorganisms and a faster rate
of removal of freshly added hydrocarbo/is than in areas where such additions
are not taking place. The ubiquitous distribution of microorganisms in
aquatic and terrestrial environments provides a biological system which
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contributes to the natural removal of oil spilled in the marine environment.
In addition to the presence of microorganisms with oil-degrading capability,
the oil spilled must be a biodegradable one (i.e. an oil which will sustain
microbial growth) and environmental conditions have to be suitable for
microbial growth. Parameters like temperature, pH, salinity, oxygen con-
centration, and nutritional factors such as nitrogen and phosphorus have
to be in ranges which will support the growth of oil-degrading microorgan-
isms. Any one of these parameters can control the rate of removal of oil
by microbial activity. Information on the interaction of these factors
affecting the activities of oil-degrading microorganisms in marine environ-
ments has been the subject of recent reviews (4, 5, 6) and a recent sympo-
sium (7). However, since no data are available on the Strait of Juan de Fuca
and northern Puget sound areas of the Pacific Northwest, a study was designed
to investigate factors affecting the activities and distribution of oil-
degrading microorganisms under the environmental conditions prevalent in
this area. The initial study (8) was designed to provide information on the
effect of geographic and seasonal variation and proximity to oil sources on
the activity, distribution and types of oil-degrading microbial flora found
in waters, beaches and inter-tidal sediments of this area. The object of
this study is to examine the microbiological oil-degradation process in
greater detail in northern Puget Sound and Pt. Angeles area of the Strait
of Juan de Fuca.
Investigations have been concerned with the enumeration of hydro-
carbon utilizing microorganisms and the determination of their incidence as
a fraction of the normal heterotrophic population. The problems encountered
in such enumeration studies have been investigated by Walker and Colwell (9).
Substrates used for enumeration techniques include pure hydrocarbons, syn-
thetic oil mixtures (11), whole crude oils and whole crude oils spiked with
^C-labelled hydrocarbons (10). Such experimentation has shown that oil-
or hydrocarbon-polluted areas will have a greater incidence of hydrocarbon-
utilizing microorganisms than adjacent unpolluted areas. Data obtained
using pure hydrocarbons are difficult to evaluate regarding their value in
assessing oil-degradation since it has been shown (11) that many fungi (e.g.
Cladosporium resinae) which can grow on a pure hydrocarbon cannot grow on
a whole oil containing that compound. Techniques based on the measurement
of the increase in mass or number (e.g. plate count or Most Probable Number)
of oil-degrading heterotrophs grown on a complex substance such as oil do
not yield information regarding which compound or compounds are supporting
growth. The use of 1/tC-hydrocarbon-"spiked" oil yields the most valuable
data but is limited by the availability of 14C-labelled hydrocarbons. Growth
also can be monitored indirectly by following changes in the concentrations
of the compound(s) present (12). Of the four major components of crude oil
i.e. asphaltenes, saturates, aromatics and the polar N (nitrogen)-, S (sul-
fur)-, 0 (oxygen)-containing molecules, only the n-alkanes (and to a lesser ex-
tent the isoprenoids) of the saturate fraction (13) and the mono-, di- and
tri-ring compounds of the aromatic fractions (14, 15) are readily degraded
by microorganisms. Changes in the content of n-alkanes and the isoprenoids
can be readily monitored by gas chromatography (G.C.), while the resolution
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of aromatic compounds by G.C. requires more rigorous pre-treatment of the
oil than is required for monitoring changes in the n-alkanes and iso-
prenoids. The approach used to assess distribution and the factors affect-
ing the activity of oil-degrading microorganisms was based on an enrichment
technique using crude oil (e.g. Prudhoe Bay) as sole carbon and energy
source. Changes in the n-alkane and isoprenoid profile of recovered oil
were determined by G.C. and used as an indicator of oil-degrading microbial
activity. Such changes were used in the establishment of a "Degradative
Capacity Index" for comparing the oil-degrading activities of microorganisms
found in this marine environment.
The majority of techniques used for the enumeration of microorganisms
employ conditions which favor bacterial growth but limit the development of
yeast and fungi. Yet representative species of both these organisms are
known hydrocarbon degraders and some grow on crude oil (11). The incidence
and role of such organisms in the biodegradation process were investigated
using selective enrichment and plating conditions followed by the examination
of isolates regarding their oil-degrading capability.
The study of oil-degrading microorganisms has been shown to be influ-
enced by temperature (16, 17) and by the nutrient status, especially the
nitrogen and phosphorus content (18, 19, 20, 21) of the environment. The
effects of both these parameters on the degradation of oil were studied in
the initial investigation (8) and the effect of nutritional conditions was
determined on all samples examined in this investigation.
The chemical composition of oil has been reported to have an influence
on the biodegradation process (16, 22). In addition to oil from the north
slope of Alaska, i.e. Prudhoe Bay, oil is received from many other areas
(e.g. Malaysia and the Middle East) of the world (1). The possible effects
of the differences in the physical-chemical composition of such oils on the
biodegradation process were also investigated.
The rate of removal of oil from the marine environment will depend on
the types of microorganisms which are present and the status of the physical-
chemical parameters at the time the oil is introduced to the system. Some
of the problems involved in the generation and the evaluation of oil-biode-
gradation rates were discussed in the early seventies (23). Two approaches,
gravimetric and radiochemical (using Prudhoe Bay oil "spiked" with different
l£tC-labelled hydrocarbons), were used in this study to obtain data on the
rate of removal of oil and specific components of oil from water column
material from pristine sites and sites which would be subject to hydrocarbon
exposure. Data were also obtained with the radiochemical technique on the
hydrocarbon-degrading activity of sub-tidal sediments and beach materials.
The initial studies (8) provided information on the distribution and
factors affecting the activities of oil-degrading microorganisms in the
marine environment of northern Puget Sound and the southern shore of the
Strait of Juan de Fuca. This initial data base was expanded in these studies.
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The oil-degrading capabilities of microorganisms in the water column, beach,
inter-tidal and sub-tidal sediments from these areas were evaluated and the
biodegradation process investigated. Particular emphasis was placed on
obtaining data on the Pt. Angeles area, since there is a possibility of its
development as a deep water oil tanker port. As our investigations were
carried out in the laboratory the effect of oil on the microbial population
under field conditions was studied in cooperation with the Battelle
Institute at Sequim, Wash. A mini-study of the oil-degrading capability
of water and sediment samples from the Duwamish River, which flows through
the southern part of Seattle and empties into Elliot Bay, was also under-
taken.
-------
SECTION 2
CONCLUSIONS
Oil-degrading microorganisms are ubiquitous in the waters, beaches,
inter-tidal and sub-tidal sediments of northern Puget Sound and the south
shore of the Strait of Juan de Fuca. Their level of activity (as deter-
mined by loss of n-alkanes and isoprenoids) is quite variable, being great-
est in areas adjacent to oil-refineries (e.g. Anacortes) or where relatively
high levels of commercial and recreational marine activities take place
(e.g. south shore of Pt. Angeles harbor). There was no relationship however
between the total number of bacteria or yeast and fungi detected and the
level of oil-degrading activity observed. The temperatures in this area
are relatively constant and result in the presence of psychrotrophic (cojd
tolerant) and psychrophilic (cold-requiring) microbial flora. The levels
of nitrogen and phosphorus are the primary environmental parameters which
control the rate of activity of oil-degrading microorganisms in this marine
system. Seasonal shifts in the observed levels of activity probably reflect
in part natural nutrient cycling processes, e.g. phytoplankton blooms which
could remove temporarily the available nitrogen and phosphorus. The oil-
degrading microorganisms present in these environments consist of bacteria,
fungi and to a lesser extent yeasts. Since oil-degrading bacterial popu-
lations (predominantly members of Flavobacterium and Pseudomonas genera
with occasional populations containing a predominance of members of the
Acinetobacter and Alcaligenes genera) are readily recovered under enrichment
conditions similar to those which exist in the natural environment, it is
projected that the bacteria would be the group most active in removing oil
spilled in this environment. Oil-degrading fungi, and to a lesser extent
yeasts, are also present and could function under conditions which retard
or prevent the growth of bacteria.
Microbial activity (i.e. mineralization) can remove approximately 1/3
the weight of Prudhoe Bay oil; another 1/3 can be lost by weathering leaving
a residue of 1/3 the original weight of oil. The highest rates of weight
loss of Prudhoe Bay oil were obtained in areas where maximum oil-degrading
activity was observed (i.e. near refineries and commercially active sites).
Studies on the changes in the 4 major components of this oil (i.e. the
asphaltenes, saturates, aromatics and polar N,S,0-containing compounds) in-
dicate that loss in weight was a result of utilization of aromatics and
saturate components. The pattern of aromatic utilization suggests that com-
ponents of this fraction are continuously used at a low rate with only short
periods during which a rapid loss of weight of this fraction is observed.
In contrast, components of the saturate fraction are initially slowly used
(long lag phase) followed by an extended period when a high rate of weight
loss is observed. There is a gradual increase in the proportion of polar
N,S,0-containing compounds as a result of the microbiological degradation
process.
This pattern of utilization of components of Prudhoe Bay oil was con-
firmed using carbon-14 labelled hydrocarbon-"spiked" oil and glass capillary
-------
gas chromatographic examination of recovered oil. The release of 1"*C02
from specifically labelled hydrocarbons added to Prudhoe Bay oil indicates
that the maximum rate of removal of the hydrocarbons studied by microbes
present in water and sub-tidal sediments was observed with labelled phenan-
threne, followed by naphthalene, hexadecane and anthracene. This, coupled
with the fact the l*CQ2 was released from [l-^Cj-nephthalene by microbes
present in water column samples with a very short lag, supports the obser-
vation that certain aromatics are rapidly used, prior to the period of
rapid n-alkane utilization, by the indigenous microbial flora. The short-
est lag times were observed with samples taken near oil refineries and from
commercially active harbor areas. The glass capillary gas chromatographic
data indicate that the substituted benzenes, naphthalene, and 1-methyl- and
2-methyl-naphthalenes were being utilized prior to the catabolism of ri-
al kanes. Analysis of changes in the aromatic and n-alkane components with
and without nutrient supplementation by this G.C. technique indicates that
changes take place in these fractions without nutrient supplementation of
marine samples, although at a lower rate than when exogenous nitrogen and
phosphorus were present.
Pt. Angeles harbor area (Ediz Hook Pilot Station to Dungeness Spit)
is a heterogeneous area regarding oil-degrading activity, with maximum
activity observed in the harbor area (i.e. west of Morse Creek to Ediz Hook,
Pilot Station). Water column samples from Peabody Creek were more active
(i.e. shorter lag time) in releasing ll*CQ2 from Prudhoe Bay oil "spiked"
with specifically carbon-14 labelled hydrocarbons than water samples from
Ediz Hook (Pilot Station) or Dungeness Spit. In contrast sub-tidal sedi-
ments from Ediz Hook (Pilot Station) were as active or more active than
those from Peabody Creek. The activity of water column and sub-tidal
samples from Dungeness Spit was generally lower than that observed with
the other two sites studied.
The Duwamish River is an example of an aquatic system which has a
very high level of oil-degrading activity present. Hydrocarbons were
readily visible at some sites as a sheen on the surface of water as well
as being extruded from sediments when samples were being taken.
The data presented in this and the previous report (8) are inter-
preted as showing the ubiquitous presence of oil-degrading microorganisms
in the marine system investigated. Changes in an actual oil spill at the
northern end of Vancouver Island are cited as revealing the potential of
marine environments in this Pacific Northwest area to recover from oil
pollution.
-------
SECTION 3
RECOMMENDATIONS
One of the main difficulties in defining the oil-degrading capability
of the marine environment is that it is a diverse, multiphasic, open system
(i.e. consisting of water, inter-tidal and sub-tidal sediments and beach
materials). Oil entering this environment may spread or partition in any
or all of the components of this system. Each phase has different physical -
chemical parameters like pH, dissolved oxygen level, temperature, nitrogen
and phosphorus levels etc. which can influence the activity of oil-degrading
microorganisms. As an open system these parameters are continuously chang-
ing, although at different rates for the phases under consideration, so
observations made are relevant to the type of sample and the time and condi-
tions existing when they are taken. In particular, the water column and
inter-tidal sediments represent phases where the physical-chemical parameters
are continuously changing whereas beach (i.e. at and above the high tide
level) and sub-tidal materials represent more stable environments. The fate
of oil incorporated into sub-tidal sediments is of particular interest as
the environmental parameters existing there (e.g. low oxygen level) would
result in the slow biodegradation of such oil. Under these conditions par-
tially oxidized aromatics could be generated, resulting in a greater chance
of generation of mutagenic compounds.
The main natural factors involved in the recovery of an oiled temper-
ate marine environment are physical-mechanical ones (vigorous wave and
tidal action) and microbial degradation. Physical-mechanical processes
will be most operative in high energy areas like those found on the west
side of Dungeness Spit and Pt. Partridge, but will have less effect in low
energy areas like E. Fidalgo or depositional zones like Skagit Bay. It is
in these latter stable, low energy areas that microbial degradation of oil,
although a slow process under natural conditions, would assume more impor-
tance in removing oil spilled in the marine environment. If there is a
possibility that such areas could be exposed to oil spilled from an existing
or planned pipeline then the physical-chemical and biological parameters
existing in such systems should be investigated. Such knowledge would be of
use in accelerating the rate of recovery of such environments from the
effects of an oil spill.
Information obtained to date on the marine environment as found in
the northern Puget Sound and the south shore of the Strait of Juan de Fuca
area indicate a ubiquitous distribution of oil-degrading microorganisms,
although their activity varies markedly depending on the geographic location
of the site being investigated. While a few low energy areas were .studied
in these investigations more information is required on the effect of micro-
organisms on oil in inter-tidal and sub-tidal sediments from such areas.
Data on fuel oil spills in Buzzard's Bay, Mass. (42) indicate that aromatic
compounds can persist in sediments for relatively long periods of time. In
particular, the possible production of mutagens and the production and fate
of aromatics and compounds which are recovered as N,S,0's should be investi-
-------
gated. The fate of asphaltenes, the most recalcitrant fraction of oil,
should also be monitored, if a meaningful assay for determining changes
in this fraction can be developed. Investigations should include studies
on the effect of varying parameters (e.g. oxygen level and nutrient level -
nitrogen and phosphorus) on the rate of removal of oil from such inter-
and sub-tidal environments. Studies should be carried out under conditions
as closely related to those found in the natural environment as possible
(e.g. as per the Battelle-Sequim experiment) and under laboratory conditions.
The latter studies are particularly important as the effect of physical -
chemical parameters can be more closely monitored under laboratory conditions
than under the "in vivo" conditions of the Battelle-Sequim experiments.
Such investigations should follow the qualitative and quantitative changes
taking place in the oil and microbial population present. The oil recovered
from both field and laboratory studies should also be monitored for the
production of mutagens.
Since the chemical composition of oil can have a marked influence on
the effect of oil on an environment, studies on the fate and effect of
other oils being brought into this area should be investigated. In partic-
ular, as the present studies show that changes can be brought about in
Prudhoe Bay oil by the microflora in marine samples without nutrient supple-
mentation, long term studies should be carried out on the effect and fate
of different oils in the various environments found in this area, e.g.
Pt. Partridge (pristine area), Pt. Angeles harbor (a commercially contami-
nated area), Dungeness Spit (a high energy area) and Skagit Bay (a low
energy area).
The information resulting from the studies outlined would provide
data complementary to those already obtained and would improve the ability
to predict how an oil spill would affect and be affected by the microbial
flora present in this marine environment.
8
-------
SECTION 4
MATERIALS AND METHODS
Sample Sites and Sampling Procedures
The locations of the sample sites used in the initial study (8) are
shown in Figure 1 and those used in this investigation in Figure 2. The
latitudes and longitudes of these sites are presented in Table 1. The initial
sample sites were chosen to represent the diverse environments found in the
northern Puget Sound and Strait of Juan de Fuca areas of the State of
Washington and on the basis of their accessibility. The sites used in this
study from the northern Puget Sound area were selected on the basis of the
initial results (8) and represent the range of environmental types found in
this area. The sites selected in the industrialized Pt. Angeles harbor are
contrasted with sites chosen in the adjoining more "pristine" environments
found in the Dungeness and Freshwater Bay areas. The environmental codes
characterizing each site are available in NOAA's computerized data bank.
Whenever tide and terrain permitted both a water column and a beach sample
were obtained at each site.
Sub-tidal sediment samples were collected by a scuba diver. These
were taken from Freshwater Bay and Dungeness Spit at a depth of approximately
6 m and from Peabody Creek and Ediz Hook Pilot Station at a depth of 13 m.
Cores of inter-tidal sediments were obtained with sterile plastic (PVC) tubing
(4.5 cm dia. x 35 cm) with one end beveled to facilitate coring. Tubes were
carefully pushed vertically into sediment, capped, withdrawn, and transported
to the laboratory. Such cores were sectioned into samples (lOg) and assayed
for oil-degrading activity and bacterial numbers.
Water samples were obtained at approximately 30 to 45 cm depth using
a wide mouth 4L plastic bottle. Microbial and physical examinations were
carried out on a sample which was obtained after rinsing the bottle 3 times
with the water to be examined. Large volumes of water for laboratory use
were taken and transported in sterile 4L bottles.
In this survey beach samples refer to sand obtained above the detritis
(upper inter-tidal) left by spring tidal action. Such samples were a composite
of 200 to 500 g of surface beach materials from approximately a 1 cm depth
and 0.5 square meter area and were transferred using sterile tongue depressors
to sterile 250 mL centrifuge bottles. Beach, inter-tidal cores and sub-tidal
sediment samples were stored at -20°C unless otherwise indicated.
All materials were transported in cooler chests containing sufficient
ice packs to keep the temperature at 10°C ± 5°C. Samples were obtained from
sites in August, October and November 1978 and January, April, June and August
-------
CANADA
UNITED STATES
2iCherry Point
Vancouver
Island
Victoria,
Strait
of Juan de Fuca
Angeles
45
30
15'
Figure 1. Map showing location of sample sites used in 1977 - 1978 study.
-------
Anacortes
Point
Partridge
JuandeFuca
SEATTLE
Strait of Juan de Fuca
Freshwater
Bay
34
Dungeness
bert
Creek
Figure 2. Maps showing location of sample sites used in 1978 - 1979 study.
11
-------
TABLE 1. SAMPLING LOCATIONS
Site No.a
1
2
3
4
5
Description
Birch Bay State Park
Between Atlantic Richfield and
Mobil Docks (refinery site)
Beach Access of Sucia Road
Samish Island Public Beach
2 miles east Jet. 20W and East
Latitude (N)
485405
485108
485124
483450
482930
Longitude (W)
1224610
1224500
1224349
1223235
1223320
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
March Point Road
Mud Flats 2 miles from Jet.
East March Point Road and
East Fidalgo Road
Rosario Beach
Point Partridge Park
Entrance Dungeness State Park
Clallam Bay
Pillar Point
Salt Creek Recreation Campground
Port Angeles Southside #3
(Below Red Lion Inn)
Fort Worden State Park
Jamestown
Mean Bay
Lopez Island - Shoal Bight
San Juan Island - South Beach
Orcas Island - Crescent Beach
Deception Pass State Park-
West Beach
Orcas Island - Terrill Beach
San Juan Island - False Bay
Freshwater Bay (Western Edge)
Pt. Angeles-Ediz Hook #1
(Pilot Station)
482930
1223550
482510
481330
480935
481540
481251
481000
480710
480845
480831
482337
482737
482723
484141
482408
484244
482915
480829
480829
1223935
1224605
1230900
1241720
1240603
1234217
1232525
1224525
1230623
1243821
1224906
1230013
1225351
1223946
1225252
1230352
1233755
1232529
12
-------
Table 1. continued ....
Site No.
25
26
27
28
29
30
31
32
Description
Pt. Angeles-Ediz Hook #2
(Between Piles)
Pt. Angeles Southside #1
(Public Marina)
Pt. Angeles Southside #2
(Peabody Creek)
West of Morse Creek
West of Green Point
(Near Cavern)
Green Point #1 (East of
Siebert Creek)
Green Point #2 (West of Crevice)
Green Point #3 (West of
Latitude (N)
480823
480742
480714
480650
480650
480702
480709
480811
Longitude (W)
1232653
1232718
1232542
1232232
1231917
1231634
1231404
1231207
33
34
35
36
37
38
39
40
41
Dungeness Spit)
Dungeness Spit #1 (Opposite First 480926
Navigational Marker)
Dungeness Spit #2 (Opposite Second 481037
Navigational Marker)
Dungeness Lagoon (Opposite Second 481037
Navigational Marker)
Christiansen Rd. and Strandner 472715
Blvd. (Christiansen Green Belt
Pk., Tukwila)
119 St. S. East Side of Foot 472950
Bridge over Duwamish River.
Monroe St. and 10th Ave. 473153
(Duwamish Waterway Pk.)
S.W. Dakota and Duwamish River 473402
(Westside of Marina)
Klikitat Ave. Opposite Fisher 473424
Mills (Longshoremans Parking Lot)
Pacific N.W. Bell Cable Crossing 473510
(Southside)
1231026
1230834
1230834
1221430
1221652
1221903
1222057
1222116
1222029
a sites 1 to 22 used in 1977/1978 study; sites 4, 6, 8 and 13 and 23-41
inclusive were used in the 1978/1979 study.
13
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Protocol for Measuring Oil-Degrading Activity
Water Column, Beach and Sediment Samples
The chemical compositions of all microbiological media used are re-
reported in Appendix A. An enrichment technique using a mineral medium with a
neutral pH and Prudhoe Bay oil as sole carbon source was used to screen samples
for the presence of oil-degrading microorganisms. The effect of nutrition (i.e.
nitrogen and phosphorus levels) on oil-degrading activities of such populations
was investigated using this technique. A similar procedure was used in assess-
ing the biodegradability of Minas, Murban and Seria oils.
The oil-degrading activity of water column material was determined by
setting up "in situ" duplicate enrichments consisting of 200 ml of water and
0.2 ml of Prudhoe Bay, Murban, Seria or 0.15 g of Minas oils in 500 ml screw-
capped Erlenmeyer flasks. One of the duplicate flasks received 2.0 ml of a
nitrogen and phosphorus supplement. To permit aeration the screw-caps were
replaced in the laboratory with sterile foam plugs prior to incubation on a
rotary shaker. In a laminar flow hood 10 g beach, inter-tidal cores or sub-
tidal sediment material were removed aseptically from the field samples and
added to 500 mL Erlenmeyer flasks each containing 200 mL of sterile artificial
sea water medium (Appendix A) and oil. Duplicate flasks were prepared for
each sample, one of which received a nutrient supplement of nitrogen and phos-
phorus, and all flasks were incubated on a rotary shaker. Unless otherwise
specified all rotary shakers were run at 250 r.p.m. with an eccentricity of
3.8 cm and at a temperature of 8°C. After a suitable incubation period the
oil was recovered by pentane flotation technique and the pentane-soluble frac-
tion subjected to gas chromatographic (G.C.) analysis.
Oil-degrading activity was determined by comparing the G.C. profile of
the saturate fraction of recovered oils with that of undegraded Prudhoe Bay
oil (i.e. sterile controls) subjected to the same incubation and recovery
procedures. Typical G.C. profiles for Prudhoe Bay oil at various stages of
degradation are shown in Figure 3. To aid in evaluating bacterial oil-degra-
ding activity each profile was given a numerical value as follows: a com-
pletely degraded n-saturate fraction (i.e. C) equal to 4; presence of residual
isoprenoids" (i.e. I) equal to 3; partial reduction of n-alkanes profile (i.e.
P) equal to 2; selective removal of n-alkanes, C^ to C^ in carbon content
(i.e. S) equal to 1; no degradation of n-alkanes (i.e. N) equal to 0. Thus,
for each sample type from each sample site, a "Degradative Capacity Index"
was calculated by summing the numerical values and dividing by the number of
samples. Not enough data are available to set up a similar system for Minas,
Murban and Seria oils. Utilization patterns with these oils can only be sub-
jective and are reported on a plus or minus basis for utilization (Figures 4,
5 and 6).
Fungi and Yeast
The oil-degrading capability of pure yeast and fungal cultures was
initially tested by the technique used by Davies and Westlake (11) with the
14
-------
cm
Figure 3. Typical G.C. profiles of saturate fraction of Prudhoe Bay oil
before and after growth of enrichment populations (N = no
degradation; S = selective metabolism, n-alkanes C12 to C19;
P = partial removal, n-alkane peaks still discernible; I =
only isoprenoids remaining; C = complete utilization of n-
alkanes and isoprenoids).
15
-------
Ml MAS -
MINAS ±
Figure 4. Typical 6.C. profiles of saturate fraction of Minas oil before
and after growth of enrichment populations (- = no degradation;
± = selective degradation; + = degraded profile).
16
-------
MURBAN
MURBAN-
MURBAN ±
Figure 5. Typical G.C. profiles of saturate fraction of Murban oil
before and after growth of enrichment populations (- = no
degradation; ± = selective degradation; + = degraded profile)
17
-------
Figure 6. Typical G.C. profiles of saturate fraction of Seria oil
before and after growth of enrichment populations (- =
no degradation; ± = selective degradation; + = degraded
profile).
18
-------
exception that a saline basal agar was used for the growth of these marine
isolates. One set was incubated at 20°C for 4 weeks and another set at 8°C
for 8 weeks. The degree of oil degradation was evaluated by comparing tne
G.C. profile of the saturate fraction of recovered oil with that of undegraded
(i.e. sterile control) Prudhoe Bay oil. The ability of selected fungi and
yeasts to degrade Prudhoe Bay oil was also evaluated under highly aerobic
conditions of the shake culture technique. All cultures were incubated at
20°C for 4 weeks before recovering the oil by pentane extraction and examining
the status of the n-alkane and isoprenoid profile by gas chromatography.
Seaweeds and Cobbles
Samples of seaweeds were collected from various sites at different
times throughout the study period using sterile scissors and forceps. Dupli-
cate samples of approximately equal surface area of a given variety were
collected. One sample was placed directly into a 500 ml, screw-capped
Erlenmeyer flask containing 200 ml of sterile solution A supplemented with
2.0 ml of nitrogen and phosphorus solution and 0.2 ml of Prudhoe Bay oil. The
other seaweed sample was placed in a plastic bottle containing 100 ml of ster-
ile solution A and shaken "vigorously" 25 times. The seaweed was transferred
to another bottle and the procedure repeated. The "washed" seaweed was then
placed in a screw-capped Erlenmeyer flask containing a nitrogen and phosphorus
supplement and Prudhoe Bay oil. The screw caps were replaced with sterile
foam plugs prior to incubation under shaking conditions for 4 weeks at 8 C.
The presence of oil-degrading microorganisms on cobbles was determined
by aseptically picking up cobbles (free of sand and weeds) on the beach and
placing them in sterile 4L wide-mouth plastic jars containing 200 nt of sterile
solution A. The jars and cobbles were shaken for 30 min after which the
liquid was decanted into screw-capped 500 ml Erlenmeyer flasks and 2 ml of
Prudhoe Bay oil were added to each flask. All flasks were incubated at 8°C
with shaking for 4 weeks.
The residual oil was recovered from seaweed and cobble cultures using
a pentane flotation technique and the status of the n-alkane and isoprenoid
components of the saturate fraction determined by gas chromatography.
Gravimetric Studies
The rate of oil loss in water column samples by weathering and mineral-
ization was determined using a gravimetric procedure. The loss of oil as a
result of weathering (i.e. physical-chemical processes) was determined by
monitoring as a function of incubation time the recovery of oil from 500 ml
Erlenmeyer flasks containing 200 ml of sterile solution A and 0.4 g of
Prudhoe Bay oil. The loss as a result of weathering and mineralization (i.e.
physical-chemical and microbial action) was monitored in a similar manner
except that the flasks contained 200 ml of water column material, 0.4 g of
oil and 2.0 ml of a nitrogen and phosphorus solution. The difference in
weight loss between the two series of flasks was considered to be a result of
mineralization (i.e. microbial action).
19
-------
The residual oil was recovered using chloroform which was subsequently
evaporated at room temperature. The residue was extracted with benzene, trans-
ferred to a tared beaker, the benzene evaporated at room temperature and the
residue weighed and reported as recovered oil. The chemical composition of
the recovered oil was determined by extracting the residue from the benzene
procedure with pentane. An aliquot of the pentane-soluble components was
analyzed for residual n-alkanes and isoprenoids by gas chromatography. The
remainder of the pentane soluble material was "topped" for 18 hr at 31°C in
preparation for determining its chemical composition by a liquid chromato-
graphic technique (13).
Gas Chromatography
Oil was recovered from cultures for gas chromatography by a pentane
flotation technique. Cultures were cooled in an ice-water bath, and acidified
with 50% HC1 to pH < 1 to minimize emulsion formation. Five mL of n-pentane
were added and the culture flasks were stoppered with rubber bungs previously
washed with pentane and benzene. The inner surfaces of the flasks were gently
rinsed with the pentane layer and then the contents mixed for 25 min on a
rotary shaker at 200 r.p.m. The oil/pentane mixture was recovered by replacing
the solid stopper with one containing a short fine bore glass tube (3 mm
internal diameter) and a larger bore glass tube (5 mm internal diameter)
reaching the bottom of the flask. Tap water was slowly added via the larger
tube so that the pentane/oil mixture slowly floated to the top of the Erlen-
meyer flask and was recovered via the fine-bore tube using a micro!itre
hypodermic syringe. Samples which could not be analyzed immediately were
acidified and stored at 4°C.
The n-alkane and isoprenoid content of the pentane-soluble fraction of
recovered oil was determined by gas-liquid chromatography using a Varian
(Model 1740) gas chromatograph equipped with flame ionization detectors and
containing stainless steel columns (6.1 m x 0.32 cm) packed with 3% SE 30
Ultraphase on Chromosorb W (AW-DMCS), 80/100 mesh. Nitrogen was used as the
carrier gas with a flow rate of 15 mL/min. The oven temperature was programmed
from 100°C to 300°C at a rate of 10°/min and the upper temperature was held
for 9 min. The injection ports and detectors were maintained at 325°C and
350°C, respectively.
Selected samples of aromatic fractions collected from the gravimetric
analysis were analyzed by glass capillary gas chromatography. A Hewlett-
Packard (Model 5710) gas chromatograph was modified to accept a capillary
column as outlined (24,25). A 30 m x 0.25 mm WCOT SE54 (J & W Scientific Inc.)
column was operated with helium as a carrier gas at a flow rate of 2 mL/min
(split ratio 10:1). The initial column temperature of 100°C was held for 2
min and then programmed at a rate of 4°C/min to 270°C and held for 16 min.
The injection port temperature was 300°C and the detector was 350°C.
The degradation of aromatic compounds also was studied by adding 200 ul
Prudhoe Bay crude to replicate flasks containing 200 ml of sea water samples
(some of which were supplemented with 2 ml nitrogen and phosphorus solution).
20
-------
Sterile controls containing 200 ml artificial sea water plus oil were incubated
on rotary shakers at 8°C. At various times, flasks were removed and acidified
with 2 ml concentrated HC1. Prior to extraction, 100 yL of chrysene solution
(2.08 mg/mL in methylene chloride) were added to each flask as a "recovery
standard" and the flask was extracted with one 15 ml and two 10 mL portions of
methylene chloride. TJie extracts were filtered through anhydrous Na2SOif,
pooled, and methylene chloride added to a final volume of 40 mL. A 10 ml
aliquot of this solution was concentrated to a volume of approximately 2 mL
using a Kontes tube heater, appropriate concentrator tubes and reflux columns
(Kontes Glass Co.). Two mL of hexane were added through the condenser and the
sample was further concentrated to a final volume of 0.7 - 1.0 mL. This con-
centrate was fractionated (25) on a silica gel column prepared with n-pentane
being substituted for petroleum ether. The sample was eluted with 5 mL n-pen-
tane followed by 5 mL 20% methylene chloride in n-pentane and 17 mL methylene
chloride. The first 17 mL of column effluent contained the saturate fraction
and the final 10 mL contained the aromatic fraction. The eluant solvents were
displaced, using a Kontes tube heater, with hexane and the saturates were con-
centrated to 0.7 to 0.9 mL and the aromatics concentrated to 0.6 to 0.75 mL.
Fifty yL of a hexamethylbenzene standard were added to each aromatic sample.
The saturates were made up with hexane to 1 mL and the aromatics to 0,8 mL
total volume. These samples were resolved using the glass capillary column
and conditions given above. However the temperature program was altered to
80°C for 4 min followed by an increase of 4°/min to 270°C which was held for
32 min.
Radiometric Procedures
The rate and extent of liberation of 14C02 from aliquots of Prudhoe Bay
oil "spiked" with one of n-H-^Cl-hexadecane, [l-^C]-naphthalene, [9-llfC]-
anthracene or [9-lt+C]-phenanthrene were compared for representative beach, sub-
tidal and water column samples. Twenty yL of "spiked" oil were added to 25 mL
of water column samples or 1:3 dilutions of beach and sub-tidal samples in
sterile solution A. The Erlenmeyer flasks had been modified (Figure 7) and a
pleated filter paper was suspended in the plastic cup. Blanks were established
by treating reaction mixtures at the initiation of the experiments with HgCl2
at a rate of 100 yg/mL of liquid for beach and water column samples. Sub-tidal
sediments (1:3 dilution) were heat sterilized. All flasks were incubated at
8°C with shaking. The amount of ltfC02 liberated was determined by injecting a
flask with 0.5 mL of 4N, H2S04 and adding 0.2 mL of C02-trapping agent (Carbo-
Sorb II, Packard) to the filter paper suspended in the plastic cup and incuba-
ting with shaking for 60 min. The cup-filter paper assembly system was placed
in scintillation vials containing 10 mL of Monophase-40 (Packard) and incubated
for 24 hr at 6°C in the dark. The DPM's present were determined using a Mark
III-6881-C-Analytical Liquid Scintillation Counter (Searle). Rates of hydro-
carbon utilization were determined by periodically removing replicate flasks,
counting the L1*C02, and plotting the cumulative values. "Lag time" was defined
as the time required for test values to exceed blank values by 5000 DPM. Rates
of total specific hydrocarbons .released as ^CO^ per day per unit of inoculum
were calculated after correcting for blanks and for changes in specific activi-
ties due to dilution by cold substrate present in Prudhoe Bay oil. Values
21
-------
Figure 7. Type of Erlenmeyer flask used in radiometric studies.
22
-------
(w/w%) of 0.45% for hexadecane and 0.1% for anthracene (based on laboratory
data) and 2.0% for naphthalene and 2.0% for phenanthrene (26) in Prudhoe Bay
oil were used in calculating dilutions of added radioactive compounds.
Microbial Numbers
Bacteria
The spread plate technique using a basal marine agar was used in all
studies for determining total viable count. All dilution blanks contained
artificial seawater salt solution at pH 7.3. Plates were dried before using
by incubating for 2-3 days at 22°C and were stored at 4°C in sealed plastic
bags. Plates used in the field were transported, inverted, in sealed plastic
bags. Five replicate plates were prepared of each dilution and viable counts
are reported as the average ± one standard deviation. Microbial counts from
water column samples are reported per ml and beach and sediment data per g
of dry material with the exception of the inter-tidal cores which were re-
ported per g of wet weight.
Isolates for taxonomic studies were obtained by grouping colonies
appearing on the 5 plates of the dilution used for enumeration as to colonial
color and morphology. Colonies representative of each type were checked for
purity by streaking on basal agar. Isolates were examined for cellular
morphology, Gram reaction, presence of catalase and oxidase, and the utiliz-
ation of glucose and lactose under aerobic and anaerobic conditions. The
presence or absence of flagella (motility) of these isolates was determined
either by examining negatively-stained specimens (1% phosphotungstic acid) by
electron microscopy or by examining wet mounts with a light microscope.
Bacteria were placed in genera based on the reactions to the above tests as
outlined in Table 2. Type cultures of isolates were kept by storing at 4°C
on basal marine agar on sealed agar slants. Some oil-degrading populations
were maintained by monthly transfer in the enrichment medium containing
Prudhoe Bay oil.
Fungi and Yeast
Fungal and yeast colony-forming units were enumerated using Sabouraud's
agar plus antibiotics prepared in an artificial seawater solution (saline
Sabouraud's agar). Three or 5 replicates of each dilution in artificial sea-
water solution were plated using the spread plate technique. Counts of colony
forming units are reported as the average ± one standard deviation.
Fungal and yeast isolates were purified by repeated platings on saline
Sabouraud's agar and pure cultures were stored on this agar at 4°C. Jungi
were identified by the structure and color of colonies and their mycelium and
spore-bearing hyphae when grown on malt agar. Fungi which could not be iden-
tified by these procedures were examined by the slide culture technique (see
Appendix). Yeasts were tentatively classified on the basis of the colonial
color and morphology when grown on saline Sabouraud's agar, their response to
the API-20C (Analytical Products) sugar tests on their cellular morphology
23
-------
TABLE 2. CHARACTERISTICS USED IN CLASSIFYING BACTERIAL ISOLATES
ro
Test3
«o
Genus °°
E
to
CD
Acinetobacter sp. . -
Aeromonas sp.
Alcaligenes sp.
Bacillus sp. +
Chromobacterium sp.
corynebacteria +
Cytophaga sp.
Flavobacerium sp.
Pseudomonas sp.
Vibrio sp.
Acid from
Rod(R) Motile Spores Catalase Oxidase Growth Glucose Lactose
°r +00b -00 +00 -09 +09 -09
Coccus 2 2 22 22
(C)
R - - + _ +.+/--+/--
R/C+ - + + +...__
R + - + + ++/_ + +/__-
R - - + - + + + + + +
R + +/- + + +/- +/- +/- +/- +/-
R +/- + + + +/- +/- +/- +/- +/~
R + - + + + + + ++/- +/-
Characteristics used for primary computer classification. Some isolates required the use of
additional information and Bergey's Manual of Determinative Bacteriology, 8th Ed.
-KL = aerobic; -(L = anaerobic
-------
on malt agar and presence of spores as determined by light microscopy.
Sub-tidal Samples (Battelle Institute, Environmental
Protection Agency - National Oceanic and Atmospheric
Administration)
Samples were supplied by the Battelle Institute (Sequim) frozen at
-20°C and they were maintained at this temperature until analyzed.
Samples were thawed overnight at 4°C and 10-fold dilutions of cores
were prepared in sterile solution A. These dilutions were used to inoculate
test tubes containing one of three different media. To enumerate oil-degrad-
ing heterotrophs, diluted samples were inoculated into test tubes containing
5 ml of solution A, 0.05 ml of nitrogen and phosphorus solution and 0.05 ml
of Prudhoe Bay oil. Total heterotrophs were estimated using 5 mL of a medium
enriched with amino acids, and the number of autotrophs determined using a
medium containing 5 ml of solution A. All dilutions of samples were set up
in quintuplicate and incubated at 20-22°C for 4 weeks. Growth was scored
as plus or minus and statistical tables were used to calculate the Most
Probable Number (MPN) of bacteria present. Data are reported as MPN/g wet
weight.
Physical and Chemical Measurements
Physical Analyses
All instruments were calibrated before and after each sample trip in
addition to the usual adjustments made prior to each measurement.
The pH of water column samples was determined using a portable Radiom-
eter pH meter (Model #29) which was standardized using a Radiometer (51001)
buffer system (pH 7.0). Salinity and water column temperature were measured
in the field using a Yellow Springs SCT meter (Model #33). The salinity
function of this instrument was calibrated according to the manufacturer's
specifications (i.e. with a standard solution of KC1 in distilled water).
Beach temperatures were obtained using a standard laboratory thermometer
(0-50°C scale). Dissolved oxygen content of water column samples was deter-
mined on site using a Yellow Springs Oxygen meter (Model #52) which was
calibrated against a dropping mercury electrode system at the City of
Edmonton's Goldbar Waste Water Treatment Plant analytical control laboratory.
Visual observations of habitat parameters were recorded and submitted
as part of the computer data bank but are not included in this report.
The dry weights of beach and sediment samples were determined Jjy
accurately weighing, in triplicate, 1 to 2 g samples and drying for 72 h
at 100°C. The average difference between the wet and dry weights was used
to calculate a correction factor for reporting results on a dry weight basis.
The weight of suspended material in water column samples was obtained by
filtering aliquots through glass fibre filters (Reeve Angel) and weighing
after drying to constant weight at 104°C.
25
-------
Chemical Analyses
Water samples taken for grease and oil analysis were collected in wide-
mouth, one-quart glass jars fitted with teflon-lined caps. Each container
was rinsed with carbon tetrachloride, and allowed to dry prior to sampling.
The samples were preserved by adding 50% ^SOt, (to pH<2) and stored at 4°C
until analyzed. Standard Methods (27), Method 502B, was used for the analysis
with carbon tetrachloride rather than using freon for extraction.
In all cases, sand samples were collected in wide-mouth plastic bottles
and were stored frozen (-20°C) until they were analyzed. Grease and oil
analyses of beach samples were done using the Soxhlet extraction method with
freon [Standard Methods (27), Method 502D]. Grease and oil concentrations
were measured spectrophotometrically using a Perkin-Elmer 297 IR spectropho-
tometer with 50 mm quartz cells.
A 10 ml aliquot of the preserved water sample for grease and oil analysis
was used for total organic carbon analysis. Inorganic carbon was removed from
the acidified sample by sparging with C02-free N2 as outlined in Standard
Methods (27), Method 505. Carbon analyses were performed using a Beckman 915A
Total Organic Carbon Analyzer.
Water samples for ammonia-nitrogen and nitrate-nitrogen analyses were
collected in plastic bottles and acidified to pH<2 with 50% H2SOU. Ammonia-
nitrogen was determined by the distillation method using a 50 ml sample in a
micro-Kjeldahl apparatus, followed by Nesslerization [Standard Methods (27).
Method 418B]. The brucine method [Standard Methods (27), Method 419D] was
used for nitrate-nitrogen analysis of water samples.
The amount of ammonia-nitrogen present in beach samples was determined
by placing an accurately known amount of beach material (approximately 2 g
wet weight) into a micro-Kjeldahl flask, then adding 10 ml 2f[ KC1 and 0.1 g
MgO (28). The ammonia was steam distilled, trapped and determined by
Nesslerization. Nitrate-nitrogen was extracted from accurately known weights
of sand (approximately 10 g wet weight) with 50 ml 0.015 M CaS04 and vigorous
shaking for 10 min (28). The extract was filtered througF Whatman 42 paper
and nitrate-nitrogen determined by the brucine method given above.
Water samples were analyzed for total orthophosphate using the ascorbic
acid method [Standard Methods (27), Method 425F]. These samples were collected
in plastic bottles and kept on ice during transport and stored frozen in the
laboratory until analyzed. Available orthophosphate was extracted from 15 g
of sand dried at 105°C for 24 h, with 75 ml 0.03 H_ NH4F in 0.03 N^ H2S04.
After 2 min shaking, the extract was filtered through Whatman 30 filter paper
and a 50 mL aliquot was analyzed for orthophosphate using the ascorbic acid
method.
Data Management
All the data collected or obtained by the physical-chemical and micro-
biological methods used in this study together with the characteristics of
sample sites and types are available from the N.O.A.A., Washington, D.C.,
National Oceanographic Data Center.
26
-------
SECTION 5
RESULTS
Survey for Oil-Degrading Activity
Water Column and Beach Samples
The state of biodegradation of recovered Prudhoe Bay oil, as shown by
the G.C. profiles of the saturate fraction, after 28 days incubation at 8°C
with water column and beach samples supplemented with nitrogen and phosphorus,
is presented in Tables 3 and 4. The extent of n-alkane and isoprenoid utilir
zation varied both with sample site and sampling time. The samples taken in
the fall showed the greatest oil-degrading capability. No degradation of
these Prudhoe Bay oil fractions was observed in a series of similar experiments
which did not receive a supplement of nitrogen and phosphorus.
Table 5 is a summary of the oil-degrading capability of sites which
were sampled over a 2 year period. Greater seasonal variation in oil-degrad-
ing ability as determined by "Degradative Capacity Index" is noted in water
column samples, and in particular in those samples from "pristine" areas (e.g.
Pt. Partridge). In contrast, beach samples from areas more likely to have
contact with hydrocarbons (e.g. E. Fidalgo) show a much more consistent oil-
degrading capability as a function of season. Samples taken between April and
September show a slightly higher degradative capability {Table 5) for both
beach and water column samples than those taken between October and March
(ratios of 2.2:1.6 for beach and 2.0:1.4 for water column).
The "Degradative Capacity Indices" for all sites sampled during the
current study period (fall of 1978-1979) are presented in Table 6. As pre-
viously noted, samples taken from areas more likely to be subjected to oil or
hydrocarbon exposure (e.g. Pt. Angeles harbor area) show a greater ability to
degrade Prudhoe Bay oil than do samples taken from a "pristine" area (e.g.
Dungeness spit area). This is clearly shown by the data presented in Table 7
where the "Degradative Capacity Index" for water and beach samples from the
Pt. Angeles area is summarized. The area west of Morse Creek to the Ediz Hook
Pilot Station has a much greater capability to degrade Prudhoe Bay oil than the
area east of Morse Creek to the outer Dungeness Spit navigational markers.
Inter-tidal Samples
The oil-degrading ability of inter-tidal core samples is presented in
Table 8. Only the surface cm of the core taken in Pt. Angeles harbor at
Peabody Creek was able to completely utilize the n-alkanes and isoprefioids
present in Prudhoe Bay oil. The microorganisms present in the rest of-the
material in this core were relatively ineffective in bringing about chanqes
in the n-alkane portion of this oil. In contrast, the oil-degrading-activity
27
-------
TABLE 3. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL AFTER 28 DAYS INCUBATION
AT 8°CWITH WATER COLUMN SAMPLES SUPPLEMENTED WITH NITROGEN AND PHOSPHORUS.
ro
oo
Site
Samish Island
E. Fidalgo
Pt. Partridge
Freshwater Bay
Pt. Angeles - Ediz Hook #1
Pt. Angeles - Ediz Hook #2
Pt. Angeles - Southside #1
Pt. Angeles - Southside #2
Pt. Angeles - Southside #3
West of Morse Creek
West of Green Pt. (near cavern)
Green Pt. #1
Green Pt. #2
Green Pt. #3
Dungeness #1
Dungeness #2
Dungeness Lagoon
1978
Aug. 22 Oct. 1
S
P
I
-b S
S
C
C
C
C
S
N
S
S
S
N
N
-
G.C. Profile3
1979
Nov. 21 Jan. 14 Apr. 2
N - S
P - S
S - S
I
S
S
S
C
S
S
_
_
_
N
S
S
_
June 18
-
-
-
-
P
S
S
I
N
S
S
S
-
S
N
S
N
" M - nrt s1asiv>a/4af -inn« C = enl a/-M Via n-f i 1 i -»at inn f\f n_a! Isanoc P, ~ tn f., « ; P = Partial rpmrwal -
n-alkane peaks still discernible; I = only isoprenoid peaks remaining; C = complete utilization
of n-alkanes and isoprenoids.
not sampled
-------
TABLE 4. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL AFTER 28 DAYS
INCUBATION AT 8°C WITH BEACH SAMPLES SUPPLEMENTED WITH NITROGEN AND PHOSPHORUS.
ro
vo
Site
Samish Island
E. Fidalgo
Pt. Partridge
Freshwater Bay (Western edge)
Pt. Angeles - Ediz Hook #1
Pt. Angeles - Ediz Hook #2
Pt. Angeles - Southside #1
Pt. Angeles - Southside #2
Pt. Angeles - Southside #3
West of Morse Creek
West of Green Pt. (near cavern)
Green Pt. #1
Green Pt. #2
Green Pt. #3
Dungeness #1
Dimgeness #2
Dungeness Lagoon
1978
Aug. 22 Oct. 1
C
I
N
-b I
I
I
I
I
C
I
I
N
S
N
S
P
-
G.C. Profile3
1979
Nov. 21 Jan. 14 Apr. 2
S - S
I - P
N - S
I
C
I
- -
I
I
S
_
_
- -
P
S
N
- - -
June 18
--
-
-
S
I
S
S
P
P
-
S
-
-
S
N
N
S
a M - nn ,4anv<3/4a-t--;nn> c = c ai of + 1 »/ Q iiti i i-rati nn t\f n.al kanp<; P., rv t.n P.,«i P = nartial removal.
|^ i |^ Vl^y I UV1U \* I Wl I J *S **^IV»N»VIF^ «W«IPfc**VI**<« V • »• *« • i »»- i • w—• ± £_ L J * ' — -_- — - __.__. ^
n-alkane peaks still discernible; I = only isoprenoid peaks remaining; C = complete utilization
of n-alkanes and isoprenoids.
not sampled
-------
TABLE 5. SUMMARY OF OIL-DEGRADING ACTIVITY3 AT SITES SAMPLED
FROM 1977 - 1979.
Water
Column
Degradative
Index
Beach
Degradative
Index
Date
1977 April 16
Sept. 19
1978 Jan. 8
Aug. 22
Oct. 1
Nov. 21
1979 Jan. 14
April 2
June 18
Capacity
1977 April 16
Sept. 19
1978 Jan. 8
Aug. 22
Oct. 1
Nov. 21
1979 Jan. 14
April 2
June 18
Capacity
Samish
Island
I
I
N
S
_b
N
-
S
-
1.3
S
P
S
C
-
S
-
S
-
1.7
East
Fidalgo
C
I
S
P
-
P
-
S
-
2.2
C
I
C
I
-
I
-
P
-
3.2
Pt.
Partridge
S
I
S
I
-
S
-
S
-
1.7
I
P
S
N
-
N
-
S
-
1.2
Pt. Angeles
Southsi de #3
P
P
I
_
C
-
S
-
N
2.0
I
I
I
-
C
-
I
-
P
3.0
a N = no degradation; S = selective utilization, C12 to C19;
P = partial removal, n-alkane peaks still discernible;
I = only isoprenoid peaks remaining; C = complete utilization of n-alkanes
and isoprenoids
= not sampled
30
-------
TABLE 6. DEGRADATIVE CAPACITY INDEX FOR SITES SAMPLED 1978 - 1979a.
Site
Samish Island
E. Fidalgo
Pt. Partridge
Freshwater Bay
Pt. Angeles-Ediz Hook #1
Pt. Angeles-Ediz Hook #2
Pt. Angeles-Southside #1
Pt. Angeles-Southside #2
Pt. Angeles-Southside #3
West of Morse Creek
West of Green Point
(near cavern)
Green Point #1
Green Point #2
Green Point #3
Dungeness #1
Dungeness #2
Dungeness Lagoon
Degradative
Capacity Index
Beach
2.0
2.7
0.3
2.3
3.3
2.3
2.0
2.7
3.0
2.0
2.0
0
1.0
1.0
0.7
0.7
1.0
Water
Column
0.7
1.7
1.7
2.0
1.3
2.0
2.0
3.7
1.7
1.0
0.5
1.0
1.0
0.7
0.3
0.7
0
Combined
1.3
2.2
1.0
2.2
2.3
2.2
2.0
3.3
2.3
1.5
1.3
0.5
1.0
0.8
0.5
0.7
0.5
28 days incubation at 8°C supplemented with nitrogen and phosphorus
average value of beach plus water column indices
31
-------
TABLE 7. DEGRADATIVE CAPACITY INDEX FOR PT. ANGELES SAMPLES SITESa (1978 - 1979)
CO
ro
Sample
Degradative
Phosphorus
Supplementation Oct 1/1978
Capacity Index
1979
Jan 14 June 18
All Sample Sites
Water
Beach
Area West of Morse
Water
Beach
Area East of Morse
Water
Beach .
+ 1.7
0
+ 2.2
0
Creek to Ediz Hook Pilot Station
+ 2.7
0
+ 3.2
0
1.4 1.0
0 0
2.2 1.2
0 0
1.5 1.7
0 0
2.8 1.8
0 0
Creek to Outer Dungeness Navigational Marker
+ 0.5
0
+ 1.2
0
0.7 0.8
0 0
1.0 0.5
0 0
a 28 days incubation at 8°C
-------
TABLE 8. OIL-DEGRADING CAPABILITY*1 OF INTER-TIDAL CORE SAMPLES.
co
CO
Bacterial Counts
initial5 final0
Site
Pt. Angeles
(Southside,
site 27,
Peabody Creek;
June 1979)
E. Fidalgo
(site 6,
October 1979)
Depth
(cm)
0-1
2-4
4-6
6-8
8-10
10-12
12-14
1-2
16-18
32-34
Description
coarse sand
coarse sand &
small gravel
small gravel
small gravel &
decaying plant
material
small gravel &
decaying plant
material
decaying plant
material
clay & muck
reddish brown
medium size
sand particles
reddish brown
medium size
sand particles
red sand with
G.C. number
Profile per gm
C 2.0(±0.4)xl07
S -d
S
S
S
S
S 4.2(±0.5)xl05
P/I 4.2(±1.3)xl06
P/I
P/I 3.7(±0.4)xlO't
% number %
pigmented per ml pigmented
40 1.8(±0.2)xl08 55
_
_
_ _ _
_
-
7 6.8(±0.7)xl07 14
45 6.9(±0.6)xl07 12
3.5(±0.8)xl08 35
12 1.2(±0.1)xl06 5
clay particles
-------
TABLE 8. continued
00
Bacterial Counts
initialb final0
C-iY-o
o 1 Lc
Dungeness Spit
(site 33,
October 1979)
Depth
(cm)
1-2
10-12
20-22
Description
grey; coarse
sand and
pebbl es
grey; coarse
sand and
pebbles
grey; coarse
sand and
pebbl es
6.C. number % number
Profile per gm pigmented per ml
S < 101* - 5.3(±0.5)xl07
N/S - - 7.8(±1.2)xl07
f
N/S 4.9(±0.9)xlOIt 30 5.3(±0.6)xl07
%
pigmented
50
30
5
a incubated with nitrogen and phosphorus at 8°C for 28 days
bacterial colony forming units in original samples
bacterial colony forming units in enrichment cultures
samples not analyzed
-------
present in the E. Fidalgo core, while only bringing about the partial modi-
fication of the n-alkanes, is uniformly distributed throughout the core.
The two-fold reduction in bacterial colony forming units between the surface
and bottom of this'core has not affected the oil-degrading activity present.
Very little oil-degrading activity is demonstrated in the material recovered
in the Dungeness core although the bacterial numbers detected are similar
to those found in the bottom of the E. Fidalgo core. The difference noted
in the proportion of pigmented colonies in the samples indicates the pres-
ence of different bacterial types in the populations. However such differ-
ences are not related to the oil-degrading capability of such populations.
Seaweeds
The results in Table 9 show that very little change was brought about
by microorganisms associated with seaweeds during the initial
16 day incubation period. However the presence of oil-degrading micro-
organisms is indicated as some of the transfers after 28 days incubation
did bring about major changes in the chemical composition of Prudhoe Bay
oil. The data in this Table also indicate that increasing the amount of
nitrogen and phosphorus added was detrimental to oil degradation.
The oil-degrading capability of "washed" and "unwashed" seaweed samples
is compared in the data presented in Table 10. If an oi1-degrading microbial
flora is present on seaweed the "washing" technique did not remove it. In
fact the data suggest that a greater degree of oil degradation occurs with
"washed" than with "unwashed" seaweed.
Cobbles
The oil-degrading activity of microorganisms released from cobbles by
"washing" is presented in Table 11. Samples taken from cobbles originating
from areas more likely to have been exposed to hydrocarbon or oil spillage
("e.g. Pt. Angeles harbor (Ediz Hook #2) and E. Fidalgo) result in a greater
change in the chemical composition of Prudhoe Bay oil than samples from more
"pristine" environments (e.g. Pt. Partridge). Transfer from initial enrich-
ments, as for seaweed samples, showed better degradation than was observed
in the initial enrichments.
35
-------
CO
TABLE 9. UTILIZATION OF OIL BY BACTERIA ATTACHED TO SEAWEEDS
(COLLECTED AUGUST 1978).
G.C.
Initial Enrichment
Kelp
Bleached Kelp
Eel Grass
"Cedar-like" leaves
"Lettuce"
Ic
N
N/S
N
N/S
N
IId
N
N
N
N
N
Profile
Transfer*5
I
N
I
I
I
I
II
N
N
I
I
N
a 16 days incubation at 8°C
28 days incubation at 8°C; 5% inoculum
c regular concentration of nitrogen and phosphorus
twice the concentration used in "c"
-------
TABLE 10. UTILIZATION OF OIL BY "WASHED" AND "UNWASHED" SEAWEED (COLLECTED AUGUST AND OCTOBER 1979),
U)
••J
(A) AUGUST 1979
G.C. Profile3
Site
Pt. Partridge
Type "Surface Area
(cm2)
green -brown 10
seaweed
green-brown 50
seaweed
green "lettuce" 16
green "lettuce" 16
Washed /Unwashed Enrichment
W
U
W
U
W
U
W
U
N
N
N
N
N
N
P
N
Transfer
N
N
N
N
S
N
_b
-
(B) OCTOBER 1979
Pea body
Pt. Partridge
E. Fidalgo
green-brown 40
seaweed
Kelp (dead) 30
Eel grass 40
W
U
W
U
W
U
S/P
S
S/P
S
I
S
-
-
-
-
-
28 days "incubation at 8°C supplemented with nitrogen and phosphorus
not done
-------
TABLE 11. UTILIZATION OF PRUDHOE BAY OIL BY BACTERIA ATTACHED TO COBBLES.
CO
00
(A) JANUARY 1979
Sample
Site
Condition
Tide Level
Surface Are
(on*)
G.C. Profile
a
Enrichment3
Transfer^
Enrichment Bacterial
Count/ml
Pt. Angeles
Ediz Hook #2
Pt. Partridge
wet
damp
dry
wet
damp
dry
low
high
low
high
51.8
60.4
50.3
106.4
93.0
72.4
S
P
S
N
N
N
I
I
I
I
I
N
1.2(± 0.2)x 108
1.1(± 0.2)x 108
4.5(± 1.3)x 107
1.8(± 0.1 )x 107
7.4(± 1.4)x 107
2.2(± 0.4)x 106
(B) APRIL 1979
E. Fidalgo
Pt. Partridge
wet
damp
dry
wet
damp
dry
low
high
low
high
44.5
48.0
47.2
35.6
35.8
55.1
S
S
P
N
P
N
I
P
P
N
N
N
1.3(±0.1)x 108
2.8(±0.6)x 108
1.7(±0.1)x 108
1.4(±0.1)x 107
8.0(±0.1)x 107
l.l(±0.1)x 106
4 weeks incubation at 8°C supplemented with nitrogen and phosphorus
inoculum 5% v/v of original enrichment; 4 weeks incubation at 8°C
-------
Rate of Oil Utilization
Gravimetric Studies
The rate of weight loss of Prudhoe Bay oil was determined after
incubation with water column samples (April, 1979) from E. Fidalgo and
Pt. Partridge supplemented with nitrogen and phosphorus. Changes in the
weight of recoverable oil were observed only with the E. Fidalgo water
samples and these are presented in Figure 8 and Table 12. The maximum
rate of mineralization of oil observed with the E. Fidalgo water column
samples was 50.5 mg/L/day and occurred during the 28th to 38th day of incu-
bation. The changes in the weight percentage recoveries of the asphaltene,
saturate, aromatic and N,S,0 components of the oil recovered from the
E. Fidalgo experiments are presented in Table 12 and Figure 9. The changes
resulting from the incubations of oil with Pt. Partridge water and a
control series are also presented in Figure 9. Very little change was
observed between the recovery patterns of the components of Prudhoe Bay
oil between the control and the Pt. Partridge sample. However the saturate
and aromatic components in the E. Fidalgo series start to decrease between
the 20th and 25th day of incubation. This decrease terminated between the
35th and 40th day of incubation. The slightly increased level of these
components observed after the 40th day of incubation is a result of recovery
problems incurred by the formation of very stable oil/water emulsions by
cultures during the latter part of the incubation period. Data on the G.C.
profile of the saturate fraction of oil recovered from the E. Fidalgo series
of cultures (Table 12) indicate a rapid utilization of the n-alkanes and
isoprenoids during the 20th to the 35th days of incubation. The status of
the G.C. profile of recovered oil is also presented in Table 12.
The results of similar studies using water column samples from the
Pt. Angeles area (June, 1979; Ediz Hook, Peabody Creek and Dungeness Spit #2)
are presented in Figures 10 and 11. The data on weight loss (Figure 10 and
Tables 13, 14, and 15) indicate a more rapid removal of oil by microorganisms
present in the water column material from Pt. Angeles harbor (Peabody Creek
site). The longest lag, 40 days, (before oil was degraded) occurred with
water samples from Ediz Hook. Water samples from Peabody Creek and Dungeness
area initiated oil degradation after approximately 22 and 28 days incubation
respectively. Greater variation between samples also is observed with water
samples from the more "pristine" areas(e.g. Ediz Hook and Dungeness Spit) than
from more "polluted" areas(e.g. Peabody Creek). The maximum rates of removal
of oil were calculated to be 37.7, 14.6 and 13.5 mg/L/day for water column
samples from Peabody Creek, Dungeness and Ediz Hook respectively. Changes
in the four major components of Prudhoe Bay oil are presented in Figure 11,
and indicate (as have other studies) that the saturate and aromatic-fractions
are readily metabolized with lags similar to those shown in Figure 9 for
weight loss of oil. The status of the G.C. profile of recovered oil is also
presented in Tables 13, 14, and 15 for Ediz Hook, Peabody Creek and Dungeness
incubations respectively.
39
-------
30
Weight
20
percent
10
Loss
0
WATER COLUMN
•
East Fidalgo/ .
\
•s
•
o
Pt. Partridge a-.0., c
---- -9
I I
I
I
I
10
20 30 40
Incubation Time (days)
50
60
Figure 8. Weight percent loss of Prudhoe Bay oil as a result of incubation (8°C) with water
column samples from E. Fidalgo and Pt. Partridge (April, 1979).
-------
TABLE 12. GRAVIMETRIC AND G.C. CHANGES IN PRUDHOE BAY OIL BROUGHT ABOUT
BY INCUBATION WITH WATER COLUMN SAMPLES FROM E. FIDALGO.
% Composition of
Incubation
Time (Days)
0
8
13
15
17
20
22
24
27
29
31
34
36
38
41
43
45
48
50
52
55
57
59
G.C.a
Profile
N
N
N
N
N
N
S
S
I
P
I
C
c
c
c
c
c
c
c
c
c
c
c
% Weight
Loss of
Oil
28.9
25.3
27.9
26.9
28.5
27.9
27.9
27.8
34.4
31.1
46.7
46.2
53.5
57.8
53.2
50.1
45.1
48.4
47.4
48.4
45.3
45.3
48.6
Corrected
% Weight
Loss
-1.29
-1.47
0.23
-0.12
3.14
2.22
1.87
1.67
10.28
6.66
24.03
19.84
29.42
34.81
33.61
29.37
22.11
25.62
26.90
26.36
21.85
21.69
25.36
to
O)
S-
ra
CO
25.9
25.3
25.1
25.9
25.3
25.3
25.1
24.8
20.8
23.4
16.0
14.9
12.1
10.7
11.0
12.2
13.6
12.6
13.5
12.7
14.1
13.1
12.3
to
o
•r-
(O
E
i_
)^£
25.2
23.9
23.4
24.5
22.9
22.5
23.3
23.0
21.9
22.7
17.3
18.9
16.6
15.1
16.6
17.4
18.9
17.9
19.3
17.9
19.0
18.4
16.7
Residual Oilc
to
O)
c:
O)
£
i-
Q.
to
10.0
11.3
10.6
10.9
11.0
11.6
11.9
11.9
11.4
7.1
11.3
10.7
9.5
8.8
10.3
11.0
12.7
12.8
12.4
12.9
12.8
12.3
12.0
00
0
CO
10.4
11.8
12.8
11.4
11.6
12.1
11.9
12.3
12.2
15.9
10.4
10.6
10.1
10.0
9.9
10.4
11.0
10.6
9.9
10.6
10.8
13.4
12.6
N = no degradation; S = selective utilization of n-alkanes C12 to C19;
P = partial removal, n-alkane peaks still discernible; I = only
isoprenoid peaks remaining; C = complete utilization of n-alkanes
and isoprenoids.
Corrected by subtracting respective uninoculated control values
The %'s of each oil fraction were calculated based on the initial wt.
of oil present in each flask and not as a percentage of the residual
weight.
41
-------
30
Weight
20
Percent
10
0
30
Weight
20
Percent
10
0
CONTROL
• saturates ,*.v-f.. - «-> — f-»--
I • • * * •
aromatics " *
NSO's p ^*-*_» "»«,
«.-8— ^_v__r_zi2:-^'t^ — *-F ' o — ^o—
•«•-"""" 8 v asphaltenes *
- 1 . i.i.i .1.1.1
0 10 20 30 40 50 60
Incubation Time (days)
_ WATER COLUMN - PL Partridge
saturate^ r—~
aromatics T *
-i . I , 1 _, 1 , 1 1 1 1 1_
10 20 30 40
Incubation Time (days)
30|_ WATER COLUMN - East Fidalgo
Weight
20-
Percent
10
0-,
- ..... .T
• ""
NSOs
^« saturates
aromatics
asphaltenes
a
10 20 30 40
Incubation Time (days)
50
60
Figure 9. Changes in the components of Prudhoe Bay oil as a result of
incubation with water column samples from northern Puget Sound
(April, 1979).
42
-------
20
10
WATER COLUMN SAMPLES
Ediz Hook (pilot station)
20
0)
••-.-*--:"
Peabody Creek
O
UJ
20-
10
0
Figure 10.
Dungeness Spit No.2.
•
10
40
50
INCUBATION TIME (days)
60
Weight percent loss of Prudhoe Bay oil as a result of
incubation (8°C) with water column samples from the
Pt. Angeles area (June, 1979).
43
-------
30
Weight
20
Percent
10
0
WATER COLUMN -Edis Hook (pitot station)
--T: — ^ J^~" o ^ \saturates
aromatics •*•"----•»— *..\ B %
X^^
o
•
?.rh?M.n.c „ £_.* . JL vqf^-^~""ISlir * *
===»j~-"rr -
0 10 20 30 40 50
Incubation Time (days)
30
Weight
20
Percent
10
WATER COLUMN - Peabody Creek
' \Varomatics
\ "*• • * -,._^ »»..» »
saturates\ ~ *"
N»— »-•-• — *-.___-a-,-- ^-t—r-
NSOs 0 a— -o <; n
° ,*••**"'& v
asphaltenes
10 20 30
Incubation Time (days)
34 WATER COLUMN - Dungeness Spit Na2
Weiflht ...^.—.^^.. \saturates
20
Percent
10|- „ v'c
NSO's
0 L_l_
10 20 30
Incubation Time days
40
50
Figure 11. Changes in the components of Prudhoe Bay oil as a result
of incubation (8°C) with water column samples from the
Pt, Angeles area (June, 1979).
44
-------
TABLE 13. GRAVIMETRIC AND G.C. CHANGES IN PRUDHOE BAY OIL BROUGHT ABOUT
BY INCUBATION WITH WATER COLUMN SAMPLES FROM PT. ANGELES
HARBOR (EDIZ HOOK, JUNE, 1979).
% Composition of Residual Oil
Incubation G.C.a
Time(Days)
7
14
20
22
25
27
32
34
36
39
41
43
47
48
50
53
55
57
60
Profile
N
N
N
S
N
N
N
S
N
S
S
P
P/I
P/I
I
P
I
I
N
a N = no degradation;
P =
% Weight
Loss of
Oil
28.46
28.00
25.23
27.37
25.99
22.81
25.02
28.54
23.67
25.08
24.66
32.02
34.39
37.34
44.44
26.68
33.31
31.92
24.92
Corrected
% Weight
Loss
-1.93
2.47
-2.25
-0.81
-1.70
7.51
-0.14
2.59
-2.10
-0.06
1.29
5.76
10.14
12.32
19,70
-0.05
10.05
6.66
-0.91
CO
OJ
"£
3
to
in
27.10
27.22
27.31
26.34
26.59
26.26
26.87
25.55
26.43
28.20
22.06
22.62
19.87
18.14
15.17
22.42
20.37
20.64
27.00
CO
0
•1 —
4J
gr\
fU
1
0
tf)
w t
•zz
9.08
11.13
9.99
9.98
11.68
13.39
13.97
12.11
12.00
10.27
13.66
14.15
13.02
13.18
12.80
16.48
14.28
12.83
11.34
Ci Q ;
I = only"
isoprenoid peaks remaining.
Corrected by subtracting respective uninoculated control values.
The %'s of each oil fraction were calculated based on the initial weight
of oil present in each flask and not as a percentage of the residual
weight.
45
-------
TABLE 14. GRAVIMETRIC AND G.C. CHANGES IN PRUDHOE BAY OIL BROUGHT ABOUT
BY INCUBATION WITH WATER COLUMN SAMPLES FROM PT. ANGELES HARBOR
(PEABODY CREEK, JUNE, 1979).
Incubation
Time( Days)
14
20
22
25
29
32
34
36
39
41
43
47
48
50
53
55
57
60
62
G.C.a
Profile
N
P
S
P
C
c
C
c
c
c
c
c
c
c
c
c
c
c
c
a N = no degradation;
P = parti;
il removal
% Weight
Loss of
Oil
26.73
29.87
26.93
34.65
41.35
41.86
43.59
39.12
39.46
39.95
43.94
41.67
41.04
43.01
43.17
43.52
46.06
45.97
44.64
I
Corrected
% Weight
Loss
1.20
2.39
0.37
6.95
13.80
16.69
17.64
15.24
14.33
16.58
17.69
17.42
16.01
18.28
16.44
20.26
20.81
20.80
16.07
; Composition of Residual Oilc
O>
O) O 0)
-M ••" C -
(O 4-> 0) O
i- O fO
ia i- -c
OO
-------
TABLE 15. GRAVIMETRIC AND G.C. CHANGES IN PRUDHOE BAY OIL BROUGHT ABOUT
BY INCUBATION WITH WATER COLUMN SAMPLES FROM DUNGENESS SPIT #2
(JUNE 1979).
% Composition of
Incubation
Time( Days)
14
20
22
25
27
29
32
34
36
39
41
43
47
48
50
53
55
57
60
62
G.C.a
Profile
N
S
S
N
N
P
C
C
I
C
P
C
c
c
c
c
c
c
c
S
% Weight
Loss of
Oil
29.41
26.42
26.40
25.81
25.23
31.50
48.91
39.57
48.44
48.31
32.22
41.76
38.89
40.54
37.47
53.67
39.56
45.11
43.18
27.26
Corrected
% Weight
Loss
3.88
-1.07
-0.16
-1.88
9.93
3.93
23.75
13.62
24.55
23.19
8.84
15.51
15.04
15.51
17.73
26.95
16.30
19.86
18.01
-1.35
a;
[ %
£
3
to
co
28.15
27.84
28.64
26.70
26.60
23.19
13.19
14.96
14.68
11.86
19.77
14.44
13.84
14.33
14.45
11.06
13.52
13.13
13.65
24.84
to
o
(O
i.
ef.
24.89
24.97
24.91
23.92
23.77
23.44
17.44
20.88
17.19
16.26
21.66
19.44
19.08
19.10
19.36
15.14
19.40
17.54
18.65
22.46
Residual Oilc
OJ
0)
+J
f-
Q.
to
10.97
10.85
11.68
11.19
12.86
11.41
8.85
10.98
9.55
19.36
13.51
11.74
13.08
12.70
12.93
11.63
13.69
10.52
12.58
13.53
•/>
o
CO
9.14
10.70
9.31
13.20
11.93
11.84
12.88
14.40
10.76
12.71
14.01
14.51
15.08
15.40
16.72
9.79
15.07
12.91
12.15
10.46
N = no degradation; S = selective utilization of n-alkanes C16 to C19;
P = partial removal, n-alkane peaks still discernible; I = only iso-
prenoid peaks remaining; C = complete utilization of n-alkanes and iso-
prenoids.
corrected by subtracting respective uninoculated control values.
c The %'s of each oil fraction were calculated based on the initial weight
of oil present in each flask and not as a percentage of the residual
weight.
47
-------
Radiometric Studies
The rates of release of ll4C02 and length of lags from Prudhoe Bay oil
"spiked" with one of n-[l-14C]-hexadecane, [l-11+C]-naphthalene, [9-11+C]-
anthracene or [g-^C]-phenanthrene as a result of incubation of water column
samples from northern Puget Sound, supplemented with nitrogen and phosphorus,
are presented in Table 16. Data showing the length of lags and yields of
IL*CQ2 from the April 1979 sample series are illustrated in Figure 12. With
one exception (August 1978 sample from Samish Island) the lag period before
5000 DPM of 11}C02 is released is shorter for [l-lttC]-napthalene-"spiked" oil
than for n-[l-ll+C]-hexadecane-"spiked" oil, and the rate of release of 14C02
is always faster from [l-1£tC]-naphthalene than from n-[l-ll4C]-hexadecane.
A comparison of the data obtained for the April 1979 samples (Table 16 and
Figure 12), when [9-ll*C]-anthracene and [9-*4C]-phenanthrene were included
for comparative purposes, indicates that naphthalene is metabolized first,
but on the basis of the evolution of lt+C02 relatively incompletely. This is
in contrast to the catabolism of [9-ll*C]-phenanthrene, which requires a
longer lag before 14C02 is detected, but yields very much higher levels of
1'tC02. The rate of utilization of these two aromatics is much faster than
the metabolism of n-[l-11+C]-hexadecane or [l-ll*C]-anthracene (Table 16).
This latter aromatic is degraded to a greater extent by water column samples
from E. Fidalgo than those from the "pristine" Pt..Partridge area. A com-
parison of the G.C. profiles of recovered oils (data not reported) indicates
that extensive release of 14C02 has occurred from oil samples "spiked" with
[1-i^C]-naphthalene and [9-ll+C]-phenanthrene before changes are noted in
the n-alkane and isoprenoid components of recovered oils. With the exception
of [l-^C]-naphthalene where there was very little difference in the pattern
of ^C02 from water column samples from E. Fidalgo and Pt. Partridge shortest
lags (i.e. time before ll+C02 was rapidly released) were always observed for
E. Fidalgo water column samples.
The rates of release of ll*C02 and length of lags from a similar series
of experiments involving water column samples from the Pt. Angeles area are
presented in Table 17 and the length of lags and yields of 14C02 are presented
in Figure 13. As previously noted with water column samples, ^C02 was re-
leased with the shortest lag time from [l-ll4C]-naphthalene and, with the
exception of [9-llfC]-phenanthrene, the fastest rates of release (Table 17)
were obtained with naphthalene. The metabolism of phenanthrene by the micro-
flora present in water from oil sites again yielded the greatest amount of
ll*C02. Water column samples from Peabody Creek site were the most active in
releasing llfC02 from all substrates tested with least difference being shown
on [l-llfC]-naphthalene. Samples from this site also yielded higher levels
of 5l*C02 from labelled hexadecane, anthracene and naphthalene "spiked" oil
than water samples from Dungeness Spit and Ediz Hook. A comparison of the
G.C. profiles of recovered oils again indicates that extensive metabolism of
naphthalene and phenanthrene occurs before significant changes in the G.C.
profiles of the saturate fraction are detectable. Yields of 20,000 DPM of
^C02 from the hexadecane experiments were noted to occur in this experiment
without any major changes (e.g. N/S or S/P category) in the n-alkane profile
48
-------
TABLE 16. RATE3 of 14C02 EVOLUTION FROM n-[l-lltC]-HEXADECANE, [1-14C]-NAPHTHALENE, O^C]-ANTHRACENE
AND [g-^OPHENANTHRENE BY WATER COLUMN SAMPLES FROM THE NORTHERN PUGET SOUND AREA.
Date
Aug. 1978
Nov. 1978
April 1979
Site
Samish Island
East Fidalgo
Pt. Partridge
Samish Island
East Fidalgo
Pt. Partridge
East Fidalgo
Pt. Partridge
n-Cl-^Cl-Hexadecane
lag (days)
11.0
6.0
8.0
12.5
9.0
13.5
6.0
9.0
yg/L/day
65.7
184
18.7
45.0
97.0
22.3
97.3
65.7
[l-lltC]-Naphthalene [Q-^C] -Anthracene [9-14C]-Phenanthrene
lag (days)
12.0
4.0
2.0
8.5
3.5
9.8
3.0
2.5
ug/L/day lag (days) yg/L/day lag (days)
731 _b _
990 - -
164
233
240
140
451 11.0 61.6 8.0
1112 14.5 14.9 10.5
yg/L/day
-
-
-
-
-
-
1083
1083
a based on the rate of release of 14COo from n-H-^Cl-hexadecane, [l-lltC]-naphthalene, [g-^Cj-anthracene
and [g-^Ci-phenanthrene from "spiked" Prudhoe Bay oil.
samples not examined
-------
40
30
*w
20
C02
10
released
0
60
released
Pt. Partridge .A o in
..-•"
a -
/
_g • B "Tr-
•,,,.71.111111
04 8 12 16 20
TIME -days
9-14C-phenanthrene ,, . M
WATER X "~^5<£
COLUMN / q.-*~
/ °
/ / 2°
// Pt. Partridge ':O2
10
/
released
Q _n 4 "°
"> • 1 1 1 I 1 1 I 1 1 I O
04 8 12 16 20
TIME -days
1-14C-naphthalene
WATER
COLUMN . . ° ^
•~~y^ * /' *
s'~ /
°''' / %> /'
° / / Pt. Partridge
* f
t f
!•' /E. Fidalgo
D '' / *
• /
»J/
"i i i i i i i . i , i
04 8 12 16 20
TIME -days
1-14C-hexadecane ^*»
WATER / *
a f
COLUMN /
• / '°
/
I /
E. Fidalgo/
./ D D /*-. Partridge
/
•^ " *'"'
^-^t-**^
11 I i 1 " i I i i i
0 4 8 12 16 20
TIME -days
Figure 12. ltfCOo evolution patterns for Prudhoe Bay oil "spiked" with one of n-[l-14C]-hexadecane,
[l-^Cl-naphthalene, [g-^Cj-phenanthrene or [Q-^Cl-anthracene for water column samples
from E. Fidalgo and Pt. Partridge (April, 1979).
-------
TABLE 17. RATE3 OF 14C02 EVOLUTION FROM n-[l-11+C]-HEXADECANE, [1-14C]-NAPHTHALENE, [9-14C]-ANTHRACENE
AND [g-^Cj-PHENANTHRENE BY WATER COLUMN SAMPLES FROM THE PT. ANGELES AREA.
1, -••
Date
Oct.
Jan.
June
Site
1978 Freshwater Bay
Pt. Angeles-
Ed iz Hook #1
Pt. Angeles-
Souths ide
(Peabody Creek)
Dungeness Spit
#2
1979 Freshwater Bay
Pt. Angeles-
Souths ide
(Peabody Creek)
Dungeness Spit
#2
1979 Pt. Angeles-
Ed iz Hook #1
Pt. Angeles-
Souths ide
(Peabody Creek)
Dungeness Spit
#2
n-[l-ll+C]-Hexadecane
lag (days)
8.0
10.0
6.0
7.0
12.0
8.5
12.5
8.5
6.0
10.0
yg/L/oay
65.9
138
39.6
78.3
113
139
67
34
133
42.4
[l-lltC] -Naphthalene [9-14C]-Anthracene [9-14C-Phenanthrene
lag (days)
6.0
6.0
4.0
7.0
4.0
4.0
4.0
6.0
2.0
4.0
ug/L/day
598
472
428
236
545
723
535
464
654
308
lag(days) yg/L/day lag (days) yg/L/day
-b
-
— — _ _
- _ _
-
.
- _
14.0 21.8 11.5 2750
7.5 102 5.5 1640
10.5 47.0 9.5 2090
a based on the rate of release of ll*CQ2 from n-[l-ll*C]-hexadecane, [l-llfC]-naphthalene, [9-14C]-anthracene
and [9-ll*C]-phenanthrene from "spiked" Prudhoe Bay oil.
samples not examined
-------
Ol
6O
40
released
9 -^c-phenanthrene
VOTER
COLUMN
40
14,
'CO,
24
16-
released
1-14C- hexadecane
WATER
COLUMN
Peabody , _
' / Dungeness o
Creek/ S
Hook
I . i
4 a
TIME-days
12
14co2
40
32
24
16
released
1-14C-naphthalene
WATER
COLUMN
Dungeness Spit
"5-^.
0 -
4 8
TIME-days
12
°to
40
30
20
"co,
10
released
9-14C-anthraeene
WATER
COLUMN
.'Ediz Hook
8 12
TIME-days
16
Figure 13. 11+C02 evolution patterns for Prudhoe Bay oil "spiked" with one of n-[l-14C]-hexadecane,
[1-i^C]-naphthalene, [9-!ltC]-phenanthrene or [9-llfC]-anthracene for water column samples
from Ediz Hook, Peabody Creek and Dungeness Spit #2 (June, 1979).
-------
being detectable by the G.C. procedure used.
Data on the rate of release of 14C02 and length of lags from a series
of experiments using sub-tidal sediments are presented in Table 18 and the
June 1979 data showing length of lags and yields of 14C02 are presented in
Figure 14. On the basis of length of lag time,[1-14C]-naphthalene again is
the first hydrocarbon of those studied to yield significant levels of 14C02.
The fastest rates of 14C02 evolution occur with [l-ll+C]-naphthalene- and
[9-ll*C]-phenanthrene-"spiked" oils (Table 18). Sub-tidal samples from Ediz
Hook and Peabody Creek were more active, i.e. shorter lag before rapid rate
of 1£tC02 evolution is observed, than Dungeness Spit sediment in releasing
14C02 from [l-11+C]-hexadecane, [g-^Cj-phenanthrene and [9-14C]-anthracene.
In contrast the patterns of 14C02 evolution from [l-lltC]-naphthalene are
similar for all three sub-tidal sediment samples. Since very little change
was detectable in the n-alkane and isoprenoid profiles of the saturate
fractions as determined by G.C., the metabolism of these aromatics is taking
place before n-alkane utilization. The release of 11+C02 from [9-1I+C]-
phenanthrene (Figure 14) is in the form of a biphasic curve for the sub-tidal
sediment samples from Pt. Angeles harbor. The sub-tidal sediment samples
from Ediz Hook (Pt. Angeles harbor) also yielded a biphasic curve for [9-14C]-
anthracene catabolism.
Data on the production of 14C02 in a similar series of experiments
using beach samples as sources of inoculum are presented in Table 19. The
results are similar to those previously reported regarding the preferential
utilization of naphthalene before hexadecane.
Utilization of Other Oils Imported into the Puget Sound Area
In 1978 the oil refineries operating at Anacortes, Washington were
importing oil from Sumatra (Minas), the Middle East - Jabal Dhanna (Murban)
and Malaysia (Seria). Minas is physically a different oil as it is a waxy
crude with a pour point of approximately 95°F (i.e. it is a solid at room
temperature) whereas Murban and Seria are liquids at room temperature. The
chemical compositions of these oils, as well as Prudhoe Bay for comparative
purposes, are presented in Table 20 and the G.C. profile of the saturate
fractions in Figures 3, 4, 5 and 6. The results of an experiment to deter-
mine the biodegradability of these new oils using oil-degrading marine cul-
tures enriched on Prudhoe Bay oil are presented in Table 21. Murban and Seria,
like Prudhoe Bay, were readily used by these cultures at both 8°C and 20°C.
Very little activity, as judged by changes in the G.C. profile of the saturate
fraction, was observed when Minas, the "solid" oil from Sumatra, was used as
sole carbon source even when supplemented with nitrogen and phosphorus.
Data on the utilization of these new oils by microorganisms present
in water column and beach samples from the northern Puget Sound and Pt. Angeles
area are presented in Table 22. Changes in the G.C. profiles of the saturate
fraction of Murban and Seria were readily brought about by samples from areas
likely to have been contaminated with petroleum and/or its products (i.e.
53
-------
01
TABLE 18. RATE3 OF 14C02 EVOLUTION FROM n-[l-lltC]-HEXADECANE, [I-1 "^-NAPHTHALENE, [9-14C]-ANTHRACENE
AND [9-11+C]-PHENANTHRENE BY SUB-TIDAL SEDIMENT SAMPLES FROM THE PT. ANGELES AREA.
n-D-^Cl-Hexadecane [l-ll*C]-Naphthalene [Q-^Cl-Anthracene [Q-^Cl-Phenanthrene
Date
Oct.
June
Site
1979C Freshwater Bay
Pt. Angel es-
Ediz Hook #1
Pt. Angeles-
Southside
(Peabody Creek)
1979e Pt. Angel es-
Ediz Hook #1
Pt. Angeles-
Souths ide
(Peabody Creek)
Dungeness Spit
#2
lag
(days)
14.0
16.0
13.0
6.5
8.5
10.5
yg/Kg /day
1175
575
416
688
473
214
lag
(days)
9.0
6.0
9.0
5.0
6.0
5.0
b lag b lag
yg/Kg /day (days) yg/Kg /day (days)
1121 -d
1799
3057
1756 6.5 235 6.5
1660 9.0 1036 6.5
1215 13.0 291 8.5
yg/Kg /day
-
-
™
17,930
13,170
7,999
a based on the rate of release of 14C02 from n-D-^Cl-hexadecane, [l-^Cl-naphthalene, [g
anthracene and [9-luC]-phenanthrene from "spiked" Prudhoe Bay oil.
b Kg (dry weight)
c degradative capability after 28 days incubation at 8°C = S, C and C for Freshwater Bay, Pt. Angeles,
Ediz Hook #1 and Southside (Peabody Creek) respectively
samples not examined
e degradative capability after 28 days incubation at 8°C =C, C and S for Pt. Angeles, Ediz Hook #1, Southside
(Peabody Creek) and Dungeness Spit #2 respectively.
-------
60 •
Ul
"to
released
30
20
14,
10
released
1 - C-hexadecane
SUB TIDAL
SEDIMENT
Ediz/A
Hook/
/ Peabodv/
Dungeness
Spit
8
TIME - days
12
TIME - days
30
20
14C02
10
released
1-14C-naphthalene
SUB-TIDAL
SEDIMENT
Peabody
Creek
4 8
TIME-days
12
Figure 14 14C02 evolution patterns for Prudhoe Bay oil "spiked" with one of n-[l-14C]-hexadecane,
[1-^C]-naphthalene, [9-14C]-phenanthrene or [9-llfC]-anthracene for sub-tidal sediments
from Ediz Hook, Peabody Creek and Dungeness Spit #2 (June, 1979).
-------
tn
TABLE 19. RATE3 of lltC02 EVOLUTION FROM n-[l-11+C]-HEXADECANE, [1-11+C]-NAPHTHALENE BY BEACH SAMPLES
FROM THE PT. ANGELES AREA.
Date
Jan. 1979
n -[1 -1 4C] -Hexadecane
Slte lag (days) Pg/kgb/day
Freshwater Bay 10.8 131
Pt. Angel es-Southside 6.8 249
(Peabody Creek)
Dungeness Spit #2 18 22
[l-^C] -Naphthalene
lag (days) yg/kg /day
2 1737
5.2 1032
8.6 1009
a based on the rate of release of lt4C02 from n-[l-14C]-hexadecane and [1-11*C]-naphthalene from
"spiked" Prudhoe Bay oil.
Kg (dry weight)
-------
TABLE 20. CHEMICAL COMPOSITION OF OILS.
Crude Oil
Fraction
Asphaltenes
Saturates
Aromatics
NSO's
Minas
18.8(±1.2)
60.3(±4.5)
15.6(±0.1)
7.9(±1.9)
% Composition
Murban
5.2(±0.5)
54.0(±1.0)
34.8(±6.6)
8.6(±3.7)
of Oil3
Prudhoe
Bay
7.3(±0.7)
38.7(±0.7)
36.4(±0.5)
18.7(±2.3)
Seria
2.1(±0.3)
60.4(±0.4)
33.8(±1.3)
9.5(±1.4)
average weight percent composition (±1 standard deviation) of three
replicate samples after "topping" for 18 hrs at 30°C under forced
draft conditions.
57
-------
en
oo
TABLE 21. UTILIZATION3 OF MINAS (SUMATRA) MURBAN (JABAL DHANNA) AND SERIA (MALAYSIA) BY
OIL-DEGRADING ENRICHMENTS FROM NORTHERN PUGET SOUND.
on
Minas
Murban
Seria
Incubation
Time
(days)
10
15
20
30
10
15
20
30
10
15
20
30
G.C. Status
Water Column
(March Pt. Rd)
8°C
NS
-
NS
-
NS
+/-
NS
+
NS
+/-
NS
+
20°C
-
NS
-
NS
+
NS
+
NS
+/-
NS
+
NS
of Saturate Fraction
Intertidal Sediment
West Beach-Deception St. Park
8°C
NS
-
NS
-
NS
+
NS
+
NS
+
NS
NS
20°C
-
NS
+/-
NS
+
NS
+
NS
+
NS
+
NS
a G.C. pattern of the saturate profile, see Figures 4, 5 and 6 (- = no degradation;
+/- = selective degradation; + = degraded profile).
cultures (28 days at 8°C with added nitrogen and phosphorus).
c NS - no sample
-------
TABLE 22. UTILIZATION OF MINAS, MURBAN AND SERIA OILS BY WATER AND BEACH
SAMPLES - NORTHERN PUGET SOUND AND PT. ANGELES AREA (JUNE, 1979).
5artlpie G.C. Status of Saturate Fraction
Minas Murban Seria
Northern Puget Sound - + +
East Fidalgo
(water)
Pt. Partridge -
(water)
Pt. Angeles Area SL - +
Ediz Hook
(water)
(beach) - - +/-
Pt. Angeles Harbor - + +
Peabody Creek
(water)
(beach) + + SL
Dungeness Spit - SL +
#2
(water)
(beach) + SL
a G.C. status of saturate profile, see Figures 4, 5 and 6; (- = no degradation;
+/- = selective degradation; + = degraded profile) after 28 days incuba-
tion at 8°C with added nitrogen and phosphorus.
sample lost
59
-------
E. Fidalgo and Pt. Angeles harbor-Peabody Creek). More water column samples
contained microorganisms which were able to bring about chemical changes in
Seria than Murban oil. Minas, the oil which is a solid at room temperature,
was not susceptible to alteration by incubation with water column samples.
However the n-alkanes present in this oil were readily metabolized by
microorganisms present in beach samples. Sub-tidal samples from Pt. Angeles
harbor-Ediz Hook and Peabody Creek as well as from Dungeness Spit (#2) also
contained microbes which readily metabolized the n-alkanes in Minas oil.
Very little biodegradative activity was detectable with samples that
did not receive a nutrient supplement of nitrogen and phosphorus.
Studies on the Biodegradation Process
Incubation Time
Data on the effect of extended incubation time at 8°C on the loss in
weight and changes in the G.C. status of Prudhoe Bay oil recovered from in-
cubations with water column samples are presented in Table 23. Of the two
samples taken from northern Puget Sound, water from E. Fidalgo brought about
changes in oil more quickly and completely if nitrogen and phosphorus were
present than water from Pt. Partridge. In the absence of added nutrients no
significant changes in the oil were detected over the 3 month incubation
period.
The results from a similar study (Table 23) using water samples from
the Pt. Angeles area indicate that, in the presence of added nitrogen and
phosphorus, water from Peabody Creek was the most active in bringing about
changes in Prudhoe Bay oil. Water samples from Dungeness spit also brought
about major changes in oil but required a longer incubation time to initiate
and complete the changes. The water samples from Ediz Hook produced variable
changes in the parameters measured. In the absence of added nitrogen and
phosphorus the weight of oil lost varied markedly whereas the status of the
G.C. profiles was consistent and indicated that the n-alkanes and isoprenoids
had not been utilized.
Utilization of Aromatics and Saturates
A qualitative glass capillary G.C. analysis of the aromatic fraction
of Prudhoe Bay oil is shown in Figure 15. Peaks have been identified on the
basis of retention time comparisons with authentic standards, or by compari-
son with similar chromatograms (25). Chrysene was added as a recovery stan-
dard and hexamethylbenzene (hmb) as an internal standard. The data in
Figure 16 represent the changes as resolved by this technique in the aromatic
components of Prudhoe Bay oil after 6, 10 and 14 days incubation with water
column samples from E. Fidalgo supplemented with nitrogen and phosphorus.
Changes brought about under similar cultural conditions with control (sterile)
water samples are presented in Figure 17. The recovery of the aromatic
components after 27 days incubation for a control, and for water column
samples with and without a nutrient supplement of nitrogen and phosphorus are
60
-------
TABLE 23. EFFECT OF INCUBATION TIME AT (8°C) ON THE UTILIZATION OF PRUDHOE BAY OIL BY WATER COLUMN
SAMPLES FROM NORTHERN PUGET SOUND (APRIL 2-3, 1979) AND PT. ANGELES AREA (JUNE 18-20, 1979),
Nitrogen Incubation
Site Phosphorus Period-8°C
Addition (Months)
Northern Puget Sound
East Fidalgo - 1
2
3
+ 1
+ 2
+ 3
Pt. Partridge - 1
2
3
+ 1
+ 2
+ 3
5
Total
27.53
27.08
26.98
29.16
43.97
44.29
28.45
26.86
_c
27.54
26.21
48.98
3 Weight Loss
Biodegradation3
-0.45
0
-0.10
2.08
16.89
17.21
1.37
-0.24
-
0.46
-0.87
21.90
G.C. Profileb
N
N
N
S
C
C
N
N
-
N
N
I
-------
TABLE 23. continued
Nitrogen Incubation
PhosDhoru*; Ppriorl-ft°r
riivso|jiiwiuo r c i i wvj \j \s
Addition (Months)
Port Angeles Area
Ediz Hook - 1
(Pilot Station) _ 2
3
+ 1
+ 2
+ 3
Peabody Creek - 1
2
3
+ 1
+ 2
+ 3
Dungeness Spit #2 - 1
2
3
+ 1
+ 2
+ 3
%
Total
26.52
24.10
22.81
31.92
23.71
36.89
26.87
20.15
41.39
45.97
-
25.65
28.23
25.02
31.50
43.18
43.69
Weight Loss
Biodegradation
-0.96
-2.98
-4.97
4.84
-3.37
9.81
-0.21
-6.93
14.31
18.89
-
-1.43
1.15
-2.06
4.42
16.10
16.61
G.C. Profileb
N
N
N
P
N
N
N
N
C
C
-
N
N
N
P
C
C
? value of 27.08 (weight
° see Figure 3
c flask broken
used a loss due to physical-chemical processes
-------
2-CH3- naphthalene
1- CH naphthalene
naphthalene
CHUphenanthrenes
O y
hmb
phenanthrene ^.
i T
li!1 dibenzothiophene
ii i ^ ••
-naphthalenes
substituted
benzenes
naphthalenes
chrysene
Figure 15. Typical G.C. profile (glass capillary column) of aromatic fraction of Prudhoe Bay oil
(hmb = hexamethylbenzene).
-------
+ N,P
hmb DAY 6
phenanthrene.
chrysene
Figure 16. Changes in the G.C. profiles (glass capillary column) of the
aromatic fraction of Prudhoe Bay oil after 6, 10 and 14 days
incubation with water column samples from E. Fidalgo supple-
mented with nitrogen and phosphorus (+N,P) (hmb = hexamethyl-
benzene).
64
-------
hmb CONTROLS
DAY 6
chrysene
~ -v^_MAAsft*Jlll
Figure 17. Changes in the G.C. profiles (glass capillary column) of the
aromatic fraction of Prudhoe Bay oil recovered from control
incubations (i.e. sterile artificial sea water) after 6, 10
and 14 days incubation (hmb = hexamethylbenzene).
65
-------
presented in Figure 18.
The chromatograms from 3 day incubations (not included in this report)
showed only a slight loss in naphthalene as a result of microbial activity
when nitrogen and phosphorus were present. The G.C. (aromatic) profile of
the 3 day control culture was similar to that of the 6 day sample which is
presented in Figure 17. A comparison of the 6 day G.C. profiles (Figures
16 and 17) indicates that the more volatile components, e.g. substituted
benzenes, naphthalene and 1-methyl-and 2-methyl-naphthalenes were being
utilized during this period provided nitrogen and phosphorus had been added
to the water. After 10 days incubation these compounds had been mostly used
and the degradation of the dimethyl naphthalenes, dibenzothiophene and
phenanthrene components of this oil had been initiated. The G.C. profile
after 14 days incubation when the water column sample had been supplemented
with nitrogen and phosphorus showed extensive utilization of aromatics up
to and including the methylphenanthrenes. The chromatogram from 18 day in-
cubations (not included in this report) differed little from those presented
in Figures 16 and 17 for the 14 days sampling.
The samples recovered after 27 days incubation (Figure 18) indicate
the extensive utilization of all of the aromatics resolved by this capillary
technique. Considerable changes are also evident in the oil recovered from
the culture which did not receive an addition of nitrogen and phosphorus.
The lower molecular weight components, e.g. substituted benzenes, naphthalene,
mono-methyl and dimethyl-benzenes, naphthalene derivatives, dibenzothiophene
and phenanthrene show varying degrees of utilization as reflected by this
G.C. technique.
Changes in the aromatic and saturate fractions of Prudhoe Bay oil
after incubation for 27 days with water column samples from Peabody Creek,
Pt. Partridge and E. Fidalgo with and without supplementation with nitrogen
and phosphorus, are presented in Figures 19, 20, 21 and 22. Extensive util-
ization of the aromatic components occurred when water samples were supple-
mented with nitrogen and phosphorus (Figure 19). The microorganisms present
in the Pt. Partridge water samples did not bring about as great a change in
the aromatic G.C. profile as did those from E. Fidalgo and Peabody Creek.
The "control" aromatic profile for a 27 day incubation for comparative pur-
poses is presented in Figure 20. Marked changes in these aromatic components
also were observed even when nitrogen and phosphorus were not added to in-
cubation mixtures (Figure 20). However these changes were not as great as
when the nutrients had been added. The microbes present in the water samples
from Peabody Creek and Pt. Partridge brought about greater changes in the
aromatic fraction than those present in the E. Fidalgo water sample in the
absence of added nutrients.
The changes in the saturate content of recovered oil, under nitrogen
and phosphorus-enriched conditions and in the absence of added nutrient
supplements, are presented in Figures 21 and 22 respectively. The n-alkanes
and isoprenoids were completely used when nitrogen and phosphorus had been
66
-------
DAY 27
Figure 18. Changes in the G.C. profiles (glass capillary column) of the
aromatic fraction of Prudhoe Bay oil recovered after 27 days
incubation; control = sterile artificial sea water and
E. Fidalgo water column sample without (-N,P) and with (+N,P)
addition of nitrogen and phosphorus (hmb = hexamethylbenzene)
67
-------
hm
PEABODY CREEK
+ N,P
chrysene
PT. PARTRIDGE
+ N.P
E. FIDALGO
+ N.P
Figure 19.
G.C. profiles (glass capillary column) of aromatic fraction of
Prudhoe Bay oil recovered after 27 days incubation with water
from one of Peabody Creek, Pt. Partridge or E. Fidalgo supple-
mented with nitrogen and phosphorus (+N,P) (hmb = hexamethyl-
benzene).
68
-------
hmb
naphthalene
CONTROL
chrysene
phenanthrene
p-
anthracene
"a"
PEABODY CREEK
-N,P
PT. PARTRIDGE
-N,P
a E.FIDALGO
-N,P
Figure 20. G.C. profiles (glass capillary column) of aromatic fraction of
Prudhoe Bay oil recovered after 27 days incubation from a con-
trol (i.e. sterile artificial sea water) and water from one of
Peabody Creek, Pt. Partridge or E. Fidalgo without nutrient
supplementation (-N,P) (hmb = hexamethylbenzene).
69
-------
nC18
Ph
CONTROL
hm
PEABODY CREEK
+ N,P
PT. PARTRIDGE
+ N.P
hmb
E.FIDALGO
+ N,P
Figure 21. G.C. profiles (glass capillary column) of saturate fraction of
Prudhoe Bay oil recovered after 27 days incubation from a con-
trol (sterile artificial sea water) and water from one of
Peabody Creek, Pt. Partridge or E. Fidalgo supplenented with
nitrogen and phosphorus (+N,P) (hmb = hexamethylbenzene,
Pr = pristane, Ph = phytane).
70
-------
,mb
PEABODY CREEK
-N,P
hmb
Ww
Pr
'
Ph
PT. PARTRIDGE
-N,P
hm
J
i.
•
ui
.
Pr
Jri
Ph
E. FIDALGO
-N,P
Figure 22. G.C. profiles (glass capillary column) of saturate fraction of
Prudhoe Bay oil recovered after 27 days incubation with water
from one of Peabody Creek, Pt. Partridge or E. Fidalgo without
nutrient supplementation (-N,P) (hmb = hexamethylbenzene,
Pr = pristane, Ph = phytane).
71
-------
added to water column samples from Peabody Creek and E. Fidalgo. Residual
peaks (possibly isoprenoids) are still clearly visible in the G.C. profile
of the saturates in the oil recovered from the Pt. Partridge incubation.
In the absence of added nitrogen and phosphorus (Figure 22) little change
in the saturate profile from the control profile (Figure 21) was noted in
the oil recovered from the Peabody Creek and Pt. Partridge incubations.
Changes were clearly apparent in the saturate fraction of the oil recovered
from the E. Fidalgo incubation.
The results of this latter study show that changes take place in the
aromatic fraction, but not the saturate fraction, in the absence of added
nitrogen and phosphorus as a result of microbial activity in the Peabody
Creek and Pt. Partridge water samples. In contrast, the microbes present
in the E. Fidalgo water column samples brought about greater changes in the
saturate than in the aromatic fraction of Prudhoe Bay oil without the addi-
tion of nitrogen and phosphorus. A comparison of the capillary G.C. data
in Figures 19, 20, 21 and 22 indicates that the degradation of the biode-
gradable components of the aromatic fraction is less dependent on nitrogen
and phosphorus supplementation than is the degradation of the saturate
fraction of Prudhoe Bay oil.
Role of Fungi and Yeasts
Data on the number of colony forming units, their genera, and oil-
degrading capability as measured by changes in the G.C. profile of the n-
alkane and isoprenoid profile of recovered oil after 30 days incubation at
20°C are presented in Tables 24, 25, 26, 27 and 28 for sites studied in
northern Puget Sound and in Tables 29 and 30 for the Pt. Angeles area.
The data presented in Table 24 were obtained by "in situ" plating of
water column material and those in Table 25 by plating the water column and
beach samples in the laboratory. Since water plated "in situ" yielded fungi
(from samples taken at Samish Island and Pt. Partridge) whereas the labor-
atory platings did not, all subsequent water samples were plated "in situ".
Because of logistic problems a similar comparison was not carried out using
beach or sub-tidal samples. Therefore these samples were always plated in
the laboratory. The data also show that more fungal colony forming units
were isolated from E. Fidalgo samples and that isolates showing maximum oil-
degrading activity were obtained from these samples.
A comparison of the growth (numbers and types) of fungal colony form-
ing units on a basal marine agar containing streptomycin (100 yg/ml) and
penicillin G (30 units/ml), and Sabouraud's agar (plus marine salts) was
carried out on these samples. The types of fungal colony forming units
growing on both media were similar; however the basal marine agar supported
the growth of more bacteria than did Sabouraud's. Therefore Sabouraud's
agar was used for all attempts at the isolation of fungal colony forming
units. In order to minimize the growth of bacteria on subsequent platings,
antibiotics were included in the saline Sabouraud's agar (Appendix).
72
-------
TABLE 24. OIL BIODEGRADATIVE ACTIVITY3 (20°C) OF FUNGI ISOLATED13 BY PLATING "IN SITU"
WATER COLUMN SAMPLES - NORTHERN PUGET SOUND (AUGUST 20-21, 1978).
Site
01
-------
TABLE 25. OIL BIODEGRADATIVE ACTIVITY3 (20°C) OF FUNGI ISOLATED5 FROM LABORATORY PLATING OF WATER
AND BEACH MATERIAL-NORTHERN PUGET SOUND (AUGUST 20-21, 1978).
Site
CD
Q.
E
ra
to
Irt
d)
r— 4J
03 to
•M r—
O O
I— 10
Status of G.C. Profile of Recovered Oil
Samish Island
E. Fidalgo
Pt. Partridge
water
beach
water
beach
water
beach
14 Aureobasidium(l)
Cladosporium(l)
Penicillium(l)
Unidentified(lO)
7 Cladosporium(l)
TrichodermaO)
Unidentified(4)
19 Gliocladium(l)
Penicillium(l)
Stachybotrys(l)
TrichodermaO)
Verticillium(l)
Unidentified(B)
Unidentified(l)
Penicillium(l)
Penicillium(l)
Unidentified(1)
Penicillium(l)
Penicinium(6)
based on n-alkane profile of recovered oil
colony forming units growing on Sabouraud's agar
c see Figure 3
no colony forming units isolated under experimental conditions used
e Genus (number of isolates tested)
-------
Ol
TABLE 26. OIL BIODEGRADATIVE ACTIVITY3 (8°C) OF FUNGI ISOLATED6 BY ENRICHMENT PROCEDURE FROM WATER
AND BEACH MATERIAL-NORTHERN PUGET SOUND (AUGUST 20-21, 1978).
Site
Samish Island
E. Fidalgo
Pt. Partridge
a based on n-al
r>fi1f\n\r 'fnvm'iin
O>
(~1
E
ro
i— fO
(O r—
•M O
O
I— HH
6
5
2
4
oe
7
kane profile of
rt 1 1 irt T ^ o
n v* rtiii "i n n
Status of 6.C. Profile of Recovered Oilc
N S P I
Cladosporium(l)
Unidentified(B)
Unidentified(2) Penicillium(2)
Cladosporium(l)
Unidentified(l)
Unidentified(2) Paecilomyces(l)
Penicillium(l )
AcremoniumO ) Beauveria(l)
Cladosporium(l } Penicillium(l )
Unidentified(S)
recovered oil
f\n CaKni ii^a *
see Figure 3
Genus (number of isolates tested)
e no colony forming units isolated under experimental conditions used
-------
TABLE 27. OIL BIODEGRADATIVE ACTIVITY3 (20°C) OF FUNGI ISOLATED6 FROM WATER AND BEACH MATERIAL-
NORTHERN PUGET SOUND (NOVEMBER 21-22, 1978).
Site
Samish Island
E. Fidalgo
Pt. Partridge
0)
r—
ra
water
beach
water
beach
water
beach
Total
Isolates
4
1
3
12
1
1
Status of G.C. Profile of Recovered Oil0
N S
Unidentified(3)d
Unidentified(l)
Unidentified (2)
Unidentified(G) Unidentified (1 )
Penicillium(l )
Unidentified (1)
P
Unidentified(l)
Unidentified(l)
I
Penicill ium(l )
Penicillium(4)
a based on n-alkane profile of recovered oil
b colony forming units (water samples plated "in situ"; beach samples plated in laboratory) growing
on Sabouraud's agar
c see Figure 3
Genus (number of isolates tested)
-------
TABLE 28. OIL BIODEGRADATIVE ACTIVITY9 (8°C) OF FUNGI ISOLATED5 BY ENRICHMENT PROCEDURE FROM WATER
AND BEACH MATERIAL-NORTHERN PUGET SOUND (NOVEMBER 21-22, 1978).
Site
Samish Island
E. Fidalgo
Pt. Partridge
O)
ID.
(O
water
beach
water
beach
water
beach
Total
Isolates
1
1
2
4
Oe
1
Status of G.C.
N S
Unidentified(l)d
Unidentified(l)
Verticil lium(l)
Fusarium(l )
Unidentified(l)
Profile of Recovered Oil
P I
Penicillium(l )
Unidentified(l ) Unidentif ied(l )
based on n-alkane profile of recovered oil
colony forming units growing on Sabouraud's agar
see Figure 3
Genus (number of isolates tested)
no colony forming units isolated under experimental conditions used
-------
TABLE 29. OIL BIODEGRADATIVE ACTIVITY3 (ZO°C) OF FUNGI ISOLATED5 FROM WATER AND BEACH
MATERIAL - PT. ANGELES AREA (OCTOBER 1-3, 1978).
CO
Site
Freshwater
Bay
Pt. Angeles-
Ediz Hook
(Pilot
Station)
Pt. Angeles-
Southside
(marina)
i/i
o>
O> -M
r- r- ro
Q- fO r—
E +-> 0
-------
TABLE 29. continued
vo
Site
Pt. Angel es-
Southside
(Pea body
Creek)
Pt. Angeles-
Southside
(Red Lion Inn)
Green Point
(west of
cavern)
Green Point
(west of
crevice)
Dungeness Spit
(2nd naviga-
tion marker)
i— (O
-------
00
o
TABLE 30. OIL BIODEGRADATIVE ACTIVITY3 (20°C) OF FUNGI ISOLATED6 FROM WATER AND BEACH SAMPLES -
PT. ANGELES AREA (JANUARY 14-16, 1979).
Site
Freshwater
Bay
Pt. Angeles
Southside
(Pea body
Creek)
Dungeness
Spit (2nd
navigation
marker)
•»-»
i— to
to i—
+-> 0
o to
1— t-t
3
1
10
6
Oe
2
Status of G.C. Profile of Recovered Oilc
N S P I
Unidentified(3)d
Unidentified(l)
Penicillium(l)
Unidentified(9)
Unidentif ied(5) Unidentif ied(l )
Penicillium(2)
based on n-alkane profile of recovered oil
colony forming units (water samples plated "in situ"; beach samples in laboratory) growing on
Sabouraud's agar
c see Figure 3
Genus (number of isolates tested)
e no colony forming units isolated under experimental conditions used
-------
The water and beach samples which were plated directly onto
Sabouraud's agar were also subjected to our enrichment procedure. Data
on the fungal colony forming units isolated by this procedure are presented
in Table 26. This technique resulted in the recovery of fungal colony
forming units from all beach samples from Samish Island and E. Fidalgo
water column samples, but not from the Pt. Partridge-water. All enrichments,
even though the pH was in the range of 4 to 5, contained readily detectable
levels of bacterial contaminants which complicated the purification of the
fungal colony forming units present.
None of the 93 fungi isolated from the August, 1979 samples taken in
northern Puget Sound were able to use the isoprenoids present in Prudhoe Bay
oil. Twelve fungi, Aspergillus (1), Beauveria (1), Paecilomyces (1) and
Penicillium (9) were able to completely utilize the n-alkanes; 4 fungi, 3
Penici11iu¥ and 1 unidentified species, were able to partially modify the
n-alkanes; 1 PeniciIlium and 2 unidentified fungi were able to selectively
utilize the n-alkanes present in Prudhoe Bay oil under the experimental
conditions used.
The fungal colony forming units isolated from the November, 1978
samples obtained from northern Puget Sound and their oil-degrading capability
are presented in Tables 27 and 28. The results are similar to those obtained
in the August samples. Thirty-one fungal colony forming units were isolated
by the plating and enrichment techniques (Tables 27 and 28). Seven of these
isolates degraded all the n-alkanes, 3 partially removed them and 1 showed a
selective utilization pattern. As noted for the August samples, members of
the Penicillium genus predominate among those fungi capable of utilizing the
n-alkanes present in Prudhoe Bay oil.
Data on the isolation of fungal colony forming units from the
Pt. Angeles area are presented in Table 29 and Table 30. The results from
the October sampling (Table 29) include data on isolations from water column,
beach and sub-tidal sediment samples. Ninety-five isolates were obtained, 15
of which utilized all the n-alkanes, 3 partially removed them and 7 showed a
selective utilization profile. As previously observed, isolates from the
genus Penicillium predominate among those fungi capable of utilizing the
n-alkanes present in Prudhoe Bay oil.
The addition of antibiotics (streptomycin 100 yg/ml and penicillin G
(60,000 units/L) to enrichment cultures set up from Pt. Angeles samples taken
in January, 1979 was not effective in controlling the growth of bacteria in
enrichments. As the types, numbers and oil-degrading activities obtained
from these enrichments were similar to the data already presented, tbey are
not included in this report.
A total of 237 fungi were isolated by direct plating and enrichment
techniques from water, beach and sub-tidal sediments of northern Puget Sound
and the Pt. Angeles area of the Strait of Juan de Fuca. The majority of the
isolates (75.5%) were not able to utilize the n-alkanes present in Prudhoe Bay
81
-------
oil for growth. Approximately one-quarter of them were able to bring about
changes in the G.C. profile of the saturate fraction of this oil: 4.6%
brought about selective changes; 4.2% partially degraded the n-alkanes
present; 15.6% completely utilized the n-alkanes. None however were able
to utilize the isoprenoids present in Prudhoe Bay oil.
The data previously reported on oil degradation were obtained by in-
cubation at 20°C for 30 days. A similar series of studies was carried out
with incubation at 8°C for 60 days. The majority of the 237 isolates (83%)
did not utilize the n-alkanes in Prudhoe Bay oil for growth at this tem-
perature. Of the 17% which did modify the n-alkane profile only 3.4% were
able to completely utilize these components, 7.2% partially removed them
and 6.4% selectively utilized them. Only one isolate was able to bring
about greater changes in the n-alkane components at 8°C than at 20°C. The
ability of these fungi to grow on Prudhoe Bay oil as indicated by changes
in the n-alkane and isoprenoid profiles, is much less at 8°C (in spite of a
longer incubation time), than at 20°C. The incidence of n-alkane-utilizing
fungi isolated (with one exception - the August "in situ" water platings from
northern Puget Sound) is in the range of 17±5% for all sites studied.
Yeasts were always present in samples from all sites, particularly in
those platings of enrichment cultures. Seventy-four yeasts were screened for
their ability to grow on the n-alkanes present in Prudhoe Bay oil and only 3
completely utilized the n-alkanes. These were tentatively identified (via
the Analytab Products-API20 Clinical Yeast System for in Vitro Diagnostic
Use; and by_morphology studies) as being strains of Candida tropical is,
Candida marina and Leucosporidium antarctica. None of the other yeasts were
capable of bringing about any changes in the n-alkane profile of Prudhoe Bay
oil. As the basal medium used for screening of oil-degrading capability does
not contain vitamins, 54 of the yeast isolates, including the 3 degraders,
were grown on a medium containing Bacto Yeast Nitrogen Base with Prudhoe Bay
oil as the sole carbon source. The G.C. profile results were identical to
those obtained using the basal agar, thus the presence of vitamins does not
improve the oil-degrading capability of these marine yeast isolates.
The results presented in Table 31 indicate that the high aeration and
cultural conditions obtained by liquid shake culture do not enhance oil
degradation by the fungi and yeasts tested.
Microbial Numbers and Bacterial Taxonomy
The numbers of bacterial and yeast-fungal colony forming units found
in water and beach samples from northern Puget Sound sites obtained in August
1978 are presented in Table 32. There was a slight increase in the number
of bacterial colony forming units obtained in water samples that had been
transported to the laboratory before plating compared with the numbers ob-
tained for "in situ" plating. Such differences were not noted with data
obtained for the number of yeast-fungal colony forming units. In order to
minimize changes in the number of colony forming units all water samples
82
-------
TABLE 31. EFFECT OF CULTURE TECHNIQUE ON THE UTILIZATION OF THE
N-ALKANES IN PRUDHOE BAY OIL.
Isolate
Asperqillus sp.
Aspergillus sp.
Beauveria sp.
Gliomastix sp.
Paecilomyces sp.
Penicillium sp.
Penicillium sp.
Penicillium sp.
Penici Ilium sp.
Penicillium sp.
Verticillium sp.
Candida marina
Candida tropical is
Leucosporidium antarctica
Rhodotorula sp.
Status of G.C. Profile
Static Culture
I
I
I
S
I
P
I
P
I
S
I
S
I
I
N
of Recovered Oil3
Shake Culture0
I
S
I
S
P
P
P
S
S
S
P
S
S
P
N
see Figure 3
slant culture technique
rotary shaker @ 240 RPM for 30 days at 20°C
83
-------
00
TABLE 32. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER AND BEACH
SAMPLES - NORTHERN PUGET SOUND SITES (AUGUST 20-21, 1978).
Yeast and Fungi3
"in situ"
Site
r—
Q.
oo
Bacteria3
"in situ" in laboratory
Sabouraud's
medium
,-§
(O '1-
"o
(S E
in laboratory
Sabouraud's
medium
E
"re •!-
m E
Samish
Island
E.
Fidalgo
Pt.
Partridge
water
beach
water
beach
water
beach
i.yyuo.igjxio4 s.yuo.ejxio1*
b 6.3(±l.l)xl06
7.1(±1.2)xlOtt 1.61(±0.25)xl05
8.9(±1.2)xl05
6(±2)xl01 4(±2)xl01
l(±l)xlC
J1 2(±2)xl01
1.28(±0.08)xl0lt 1.05(±0.11)xl05 4(±5)xlO° 2(±3)xl01
4.8(±0.6)xlO't
4(±5)xlO°
2.8(±0.5)xl03
3(±l)xl01
2.5xl03
2(±4)xlO°
< 101
2.0(±0.3)xl03
6(±3)xl01
1.9(±0.5)xl03
2(±4)xlO°
< 101
a colony forming units (± 1 standard deviation) per mL of water or g (dry weight) of
beach material from 5 replicate plates
not plated under these conditions
-------
were plated "in situ". No significant differences were noted in the yeast-
fungal counts obtained on Sabouraud's agar (salinity adjusted) and those
obtained on the basal marine agar. However fewer bacterial colonies appeared
on Sabouraud's medium, therefore it was routinely used for the enumeration
of yeast-fungal colony forming units. Low levels of the antibiotics strep-
tomycin and penicillin (see Appendix for composition) to minimize bacterial
growth were also incorporated into Sabouraud's agar.
The data on colony forming units for the November, 1978 and April,
1979 samplings of the northern Puget Sound sites are presented in Tables 33
and 34 respectively. The numbers of bacteria and yeast-fungal colony form-
ing units were similar for the three sampling times investigated. However,
the numbers of bacteria found were 2 to 3 orders of magnitude greater than
those obtained for yeast-fungi.
The generic composition of the bacterial populations present in
E. Fidalgo and Pt. Partridge water and beach samples obtained in April, 1979
before and after enrichment with Prudhoe Bay oil and added nitrogen and
phosphorus, are presented in Table 35. The water column samples have a
more diverse flora than do the beach samples before enrichment. Members of
the Flavobacterium and Pseudomonas genera predominate in enriched, oil-
degrading populations.
Table 36 presents the generic composition of enriched, oil-degrading
populations obtained from cobble stones from E. Fidalgo and Pt. Partridge.
Comparing these results with data obtained from water and beach samples from
the respective sites, the composition of the cobble populations varies con-
siderably although members of the Flavobacterium and Pseudomonas genera
predominate in 3 of the 6 populations studied.
The numbers of bacterial and yeast-fungal colony forming units found
in water, beach and sub-tidal samples from the Pt. Angeles area are pre-
sented in Table 37. The numbers of bacterial colony forming units are 2 to
3 orders of magnitude greater than those obtained for yeast-fungal colony
forming units. Very little difference occurs between numbers of bacterial
or yeast-fungal colony forming units from pristine areas (e.g. as in the
Dungeness Spit area) and counts obtained from Pt. Angeles harbor sites
(e.g. Peabody Creek). The generic compositions (Table 38) of the bacterial
populations found in the sub-tidal sediments show a relatively high percen-
tage of colonies which did not grow on transfers.
The numbers of bacterial and yeast-fungal colony forming units found
in the January 1979 samples from Freshwater Bay, Pt. Angeles - Peabody Creek
and Dungeness Spit #2 are presented in Table 39. The counts are similar to
those obtained from the October samples.
Data on the number of colony forming units in water, beach and sub-
tidal sediment samples (June, 1979) taken from Pt. Angeles harbor (Ediz
Hook-pilot station and Peabody Creek) and Dungeness Spit are presented in
85
-------
00
TABLE 33. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER AND BEACH
SAMPLES - NORTHERN PUGET SOUND SITES (NOVEMBER 21-22, 1978).
Site
Samish Island
E. Fidalgo
Pt. Partridge
O)
"a.
1
CO
water
beach
water
beach
water
beach
Bacteria3
"in situ" in laboratory
3.28(±0.30)xl03 1.36(±0.24)xlOlt
c 1.79(±0.45)xl05
i.sgcto.ogjxio4 3.82(±o.so)xiolf
2.90(±0.31)xl05
2.17(±0.35)xl03 2.22(±0.26)xl03
5.3(±0.6)xl04
Yeast and
"in situ"
8(±8)xlO°
2(±2)xl01
2(±5)xlO°
Fungi3 'b
in laboratory
2.1(±4.6)xl01
5.8(±2.5)xl02
4.1(±5.6)xl01
a colony forming units(± 1 standard deviation) per mL of water or g
(dry weight) of beach material from 5 replicate plates
Sabouraud's medium
not plated under these conditions
-------
TABLE 34. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER AND
BEACH SAMPLES - NORTHERN PUGET SOUND SITES (APRIL 2-3, 1979).
CO
Site
Samish Island
E. Fidalgo
Pt. Partridge
CD
"a.
to
VI
Bacteria3
"in situ"
in laboratory
Yeast and Fungi3'
"in situ" in laboratory
water 3.3(±0.7)xl03
beach
water 1 .SUO.GjxlO1*
beach
water 2.2(±0.6)xl03
beach
5.0(±0.2)xlO"
4.8(±].0)xlO"
5.3(±0.6)xl05
3.6(±0.6)xl05
2.2(±0.5)xlOlt
3(±2)xl01
2(±1.3)xl01
I(±0.2)xl03
a colony forming units (± 1 standard deviation) per mL of water or g
(dry weight) of beach material from 5 replicate plates
Sabouraud's medium
c not plated under these conditions
-------
TABLE 35. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN E. FIDALGO AND PT. PARTRIDGE BEFORE AND
AFTER ENRICHMENT IN THE PRESENCE OF ADDED NITROGEN AND PHOSPHORUS (8°C) (APRIL 2-3, 1979).
00
c»
% Generic Composition
Genus
Acinetobacter sp.
Aeromonas sp.
Alcaligenes sp.
Coryneforms
Cytophaga sp.
Flavobacterium sp.
Pseudomonas sp.
Vibrio sp.
Unidentified
Non-Transferabl ee
Colonies
E. Fidalgo
Water Beach
Before3
_c
21.4
25.8
28.1
3.4
14.6
6.7
After Before
13.4 31.4
0.8
54.2 50.2
8.1 8.0
24.5 9.2
0.4
After
5.0
0.2
2.2
3.0
72.5
2.3
14.9
Pt. Partridge
Water Beach
Before
2.9
20.9
13.4
3.0
17.9
4.5
14.9
16.4
6.0
After Before
0.2 23.4
3.7
0.4 61.7
98.6 0.9
0.6 10.3
0.2
After
6.0
15.2
12.0
65.0
1.8
a before enrichment (i.e. original samples)
after enrichment with nitrogen, phosphorus and Prudhoe Bay oil present
c no colonies present on dilution plate used to assess population composition
not classifiable on the basis of the characteristics used
e colonies which did not grow on transfer
-------
00
vo
TABLE 36. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN WATER, BEACH AND COBBLE SAMPLES FROM
E. FIDALGO AND PT. PARTRIDGE (APRIL 2-3, 1979) AFTER ENRICHMENT WITH PRUDHOE BAY AND
ADDED NITROGEN AND PHOSPHORUS.
% Generic Composition
Genus
Acinetobacter sp.
Aeromonas sp.
Alcaligenes sp.
Bacillus sp.
Coryneforms
Cytophaga sp.
Flavobacterium sp.
Pseudomonas sp.
Vibrio sp.
Unidentified6
Non-Transf erabl e
Colonies
E. Fidalgo
Weta
Water Cobble
_d
2.8
13.4 25.8
0.9
5.6
1.8
54.2 19.5
8.1 13.6
24.5 19.2
10.8
Damp
Cobble
1.8
73.0
9.9
14.5
0.9
Dryc
Cobble
63.0
22.6
3.8
0.3
2.6
7.3
0.3
0.3
Beach
5.0
0.2
2.2
3.0
72.5
2.3
14.9
Water
0.2
0.4
98.6
0.6
0.2
Pt. Partridge
Wet
Cobble
3.2
24.2
3.2
1.3
0.9
66.9
0.3
Damp
Cobble
54.9
0.5
17.5
19.5
7.7
Dry .
Cobble
0.4
13.2
29.1
0.6
46.5
10.4
Beach
6.0
15.2
12.0
65.0
1.8
closest to tide
mid-way between tide and high tide line
dry; just below high tide line
no colonies present on dilution plate used to assess population composition
not classifiable on the basis of the characteristics used
colonies which did not grow on transfer
-------
TABLE 37. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER, BEACH
AND SUB-TIDAL SAMPLES - PT. ANGELES AREA (OCTOBER 1-3, 1978).
Site
Fresh water
Bay
Pt. Angeles-
Ed iz Hook
(Pilot Station)
Pt. Angeles-
Southside
(Marina)
Pt. Angeles-
Souths id e
(Pea body Creek)
Pt. Angel es-
cu
Q.
to
1/5
water
beach
sediment
water
beach
sediment
water
water
beach
sediment
water
Bacteria3
"in situ" in laboratory
5.5(±2.9)xlOIt 4.5(±0.9)xlO't
c 4.3(±0.9)xl05
1.0(±0.2)xl06
8.1(±l.l)xl03 3.9(±0.6)xlOlf
4.7(±l.l)xl06
7.1(±2.3)xl05
4.0(±1.2)xl0lt
4.6(±0.7)xlO't 3.83(±0.13)xlOlt
1.22(±0.28)xl06
5.9(±1.3)xl05
2.08(±0.13)xl01*
Fungi and
"in situ"
l.l(±0.4)x!02
3(±l)xl01
2(±1)xl01
6(±2)xl01
2(±l)xl01
Yeasts3 'b
in laboratory
7(±3)xl01
4(±2)xl02
4(±3)xl02
6(±5)xlO°
1.2(±0.4)xl03
3(±l)xl02
3(±2)xl01
5(±2)xl02
1.5(±0.3)xl03
Southside (Red
Lion Inn)
-------
Site
Green Point
(west by
cavern)
Green Point
(west of
crevice)
Dungeness (by
2nd navigation
marker)
O)
"a.
1
t/3
water
beach
water
water
beach
Bacteria Fungi an
"in situ" in laboratory "in situ"
1.38(±0.11)xlOlt 4.5(±0.8)xlO't 6(±3)xl01
1.54(±0.41)xl07
3.1(±1.4)xlO" JftDxlO1
7.8(±1.6)xl03 2.9(±0.7)xlOI> 3(±l)xl01
2.34(±0.84)xl06
d Yeasts '
in laboratory
lUDxlO1
7.6(±0.6)xl03
SttSjxlO1
1.4(±0.2)xl03
a colony forming units (± 1 standard deviation) per ml of water or g (dry weight) of
beach or sub-tidal sediment material from 5 replicate plates
b Sabouraud's medium
c not plated under these conditions
sub-tidal sediment
-------
TABLE 38. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN SUB-TIDAL
SEDIMENTS FROM THE PT. ANGELES AREA (OCTOBER 1-3, 1978).
Genus
Acinetobacter sp.
Aeromonas sp.
Al call genes sp.
Bacillus sp.
Coryneforms
Cytophaga sp.
Flavobacterium sp.
Pseudomonas sp.
Vibrio sp.
Unidentified
[Ion -Transferable0
Colonies
Freshwater
Bay
2.0
5.9
3.1
0.6
0.3
0.3
9.0
9.8
2.0
30.2
37.2
% Generic Composition
Pt. Angeles
Ediz Hook Peabody Creek
(Pilot Station)
12.0
_a
21.8
0.4
5.6
16.7
30.8
0.4
12.4
7.4
1.7
5.7
2.3
0.6
1.7
17.6
3.4
2.8
56.8
no colonies present on dilution plate used to assess population composition
not classifiable on the basis of the characteristics used
c colonies which did not grow on transfer
92
-------
vo
co
TABLE 39. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER AND BEACH
SAMPLES - PT. ANGELES AREA (JANUARY 14-16, 1979).
Site
O)
"a.
CO
Bacteria0
Fungi and Yeasts3'
"in situ"
in laboratory "in situ"
in laboratory
Freshwater
Bay
Pt. Angeles-
Southside
(Peabody Creek)
Dungeness (by
2nd navigation
marker)
water
beach
water
beach
water
beach
2.3(±0.16)xlO't 1 .12(±0.10)xl05 6(±1.3)xlO° _
_ c 4.6(±0.6)xl05 _ 2.1(±4.7)xl01
1 .55(±0. 17)xl05 3.19(±0.31 )x!05 2.6(±1 .1
_ 3.09(±0.26)xl05 _ 4.0(±3.2)xl02
1 .04(±0.14)xl05 1 .72(±0.25)xl05 2(±4.5)xlO° _
_ 6.6(±1 .3)xlOl+ 1 .4(±1 .4)xl02
a colony forming units (+ 1 standard deviation) per mL of water or g (dry weight)
of beach material from 5 replicate plates.
Sabouraud's medium
c not plated under these conditions
-------
Table 40. The generic composition of the bacterial populations before and
after enrichment from these samples are presented in Table 41 and 42 respec-
tively.
The numbers of colony forming units found in the June 1979 samples
are similar to those reported for January, 1979 and October, 1978 samples.
Members of the Cytophaga and Flavobacterium genera predominate in unenriched
populations (Table 41). The composition of the enriched populations varies
markedly although members of the Flavobacterium genus predominate (i.e.
greater than 30% of the population) in 6 of the 9 populations studied.
Data in Table 43 show total heterotrophic counts on the basal marine
agar used in these studies, a commercial marine agar (Difco 2216) and a
commercial medium (Difco TCBS) which is selective for disease-causing
vibrios. Similar levels of bacterial colony forming units were obtained on
both the basal marine agar and the commercial product (Difco 2216). Very
few members of the Vibrio genus (common marine bacteria) were recovered on
either of these marine agars. The incidence of vibrios in the samples
analyzed is reflected in the numbers of colony forming units growing on TCBS
agar. Typical vibrios produce yellow colored colonies when grown on this
medium. Such colored colonies formed from 25% to 88% (average 52%) of the
colonial counts reported on TCBS agar for water, beach and sediment samples
analyzed (Table 43). Thus the actual vibrio count would be approximately
one-half of the total number of colonies growing on TCBS agar and could
represent up to 5% of the total bacterial colony forming units capable of
growth under the conditions used in this study. Transfers of these
"presumptive" vibrio isolates readily grew on both marine agars used for
determining the number of heterotrophic colony forming units. Therefore
the failure to detect these typical marine bacteria in enriched, oil-degrad-
ing populations is not due to their inability to grow on basal marine agar
but rather to their lack of direct participation in the oil-degrading process.
Oil-Degradation by Duwamish River Samples
The numbers of bacterial and yeast-fungal colony forming units de-
tected in water and inter-tidal sediments from the Duwamish River are pre-
sented in Table 44. The numbers of bacteria obtained from water column
samples using saline basal marine agar and non-saline plate count agar are
similar and are of the same order of magnitude as reported for water column
samples in northern Puget Sound and the Pt. Angeles area. The number of
yeast-fungal colony forming units in water column samples are also similar
to those reported for northern Washington waters. The numbers of micro-
organisms found in the Duwamish inter-tidal sediments are similar to those
found in such materials from the other sites investigated in this study.
The oil-degrading capability of these microbial populations with and
without nitrogen and phosphorus supplementation, and data on the chemical
analyses of samples are presented in Table 45. Very high levels of
94
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UD
tn
TABLE 40. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER, BEACH
AND SUB-TIDAL SAMPLES - PT. ANGELES AREA (JUNE. 18-20, 1979).
Site
101
> 101
> 101
3 colony forming units (± 1 standard deviation) per mi_ of water or g (dry weight)
of beach or sub-tidal sediment material from 5 replicate plates.
Sabouraud's medium
not plated under these conditions
sub-tidal sediment
-------
vo
en
TABLE 41. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN WATER, BEACH AND SUB-TIDAL SAMPLES FROM
THE PT. ANGELES AREA (JUNE 18-20, 1979) BEFORE ENRICHMENT AT 8°C WITH PRUDHOE BAY OIL
AND NITROGEN AND PHOSPHORUS.
% Generic Composition
Genus
Pt. Angeles Area
Ediz Hook (Pilot Station)
Water Beach Sediment
(sub-tidal)
Acinetobacter sp.
Aeromonas sp.
Al cali genes sp.
Bacillus sp.
Coryneforms
Cytophaga sp.
Flavobacterium sp.
Pseudomonas sp.
Unidentified15
Non-Transferable
Coloniesc
4.0 21.8
0.5
1.5 2.7
5.0 38.2
72.2 19.1
4.6
1.0 13.6
15.9
0.9
0.3
70.5
3.0
3.1
11.4
3.3
0.4
7.1
Peabody
Creek
Water Beach Sediment
(sub-tidal'
-a 22.2
0.6
4.8 1.4
46.8 17.4
40.7 14.4
7.7 0.7
43.3
6.9
47.4
1.7
16.6
10.3
1.7
9.7
5.7
Dungeness Spit #2
Water Beach Sediment
1 (sub-tidal)
1.3
11.5
61.8 41.4 8.9
31.8 51.9 70.7
6.5 6.7 5.7
1.9
no colonies present on dilution plate used to assess population composition
not classifiable on the basis of the characteristics used
c colonies which did not grow on transfer
-------
10
TABLE 42. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN WATER, BEACH AND SUB-TIDAL SAMPLES FROM
THE PT. ANGELES AREA (JUNE 18-20, 1979) AFTER ENRICHMENT AT 8°C WITH PRUDHOE BAY OIL
AND NITROGEN AND PHOSPHORUS.
% Generic Composition
Genus
Aeromonas sp.
Alcaligenes sp.
Coryneforms
Cytophagas sp.
Flavobacterium sp.
Pseudomonas sp.
Unidentified5
Non-Transferable0
Colonies0
Pt. Angeles Area
Ediz Hook (Pilot Station)
Water
_a
61.0
0.9
37.3
0.8
Beach
12.7
3.5
0.1
83.1
0.4
0.3
Sediment
(sub-tidal)
7.5
19.1
61.0
10.2
2.3
Water
45.6
0.8
4.7
10.2
18.9
19.7
Peabody Creek
Beach Sediment
(sub-tidal)
7.1
5.3
18.6
36.3
1.8
31.0
Dungeness Spit #2
Water
21.1
8.1
23.2
6.0
41.6
Beach Sediment
(sub-tidal)
27.5 6.6
23.1
1.7
11.1
61.4 32.2
26.5
9.9
a no colonies present on dilution plates used to assess population composition
b not classifiable on the basis of the characteristics used
0 colonies which did not grow on transfer
-------
00
TABLE 43. TOTAL HETEROTROPHIC COUNT ON BASAL MARINE AGAR, MARINE AGAR (DIFCO-2216) AND TCBS (DIFCO)
AGAR IN WATER, BEACH AND SUB-TIDAL SAMPLES FROM PT. ANGELES AREA (JUNE 18-20, 1979).
Site
Pt. Angel es-
(Ediz Hook,
Pilot Station)
Pt. Angeles-
(Peabody Creek)
Dungeness
Spit #2
Sample
water
beach
sediment
water
beach
sediment
water
beach
sediment
Bacterial
Basal Marine3
Agar
7.4(±1.2)xlO't
1.5(±0.2)xl05
1.9(±0.1)xl05
8.3(±1.5)xlOIt
1.3(±0.2)xl07
5.1(±0.5)xl05
2.9(±0.4)xlO't
2.0(±0.5)xlOt*
4.3(±0.7)xl05
Colony Forming Units
Marine Agar
2216 (DIFCO)
5.Q(±Q.7)xlQk
2.3(±0.5)xl05
3.3(±0.2)xl05
1.5(±0.2)xl05
3.9(±0.4)xl07
7.5(±1.7)xl05
3A(±0.5)x1Ql*
2.2(±0.6)xlOlt
4.2(±1.0)xl05
TCBSC
Agar
2.5(±0.7)xl02
4.0(±0.6)xl03
7.0(±3.2)xl03
1.6(±0.8)xl03
4.7(±1.2)xl05
6.7(±0.5)xl04
1.8(±0.4)xl03
7.0(±3.8)xl02
1.4(±2.7)xl04
see Appendix for composition
Bacto Marine Agar (Difco) for culture and enumeration of heterotrophic marine bacteria
Thiosulfate Citrate Bile Salts Agar (selective medium for isolation of pathogenic vibrios
sub-tidal sediment
-------
TABLE 44. BACTERIAL, YEAST AND FUNGAL COLONY FORMING UNITS - WATER AND SEDIMENT SAMPLES-
DUWAMISH RIVER (AUGUST 20-21, 1979).
Bacteria
a,b
Site
Sample
Marine Agar (Basal)
in situ
Nate
Count Agar
in laboratory in laboratory
Yeast
a,b
Fungi
,a,b
<£>
UD
Tukwila water 7.6(±1.7)xl03 -d
sediment6 - 1.7(±0.17)xl06
Monroe &
10th Ave.
water 1.2(±Q.29)x'\Olt
sediment
Klikitat Ave. water
(Fisher Mills)
Pacific N.W.
Bell Cabel
Crossing
(Southside)
8.3(±2.0)xl03
water 8.8(±1.6)xl03
1.2(±0.64)xlOlt 5(±2)xl01 1.5(±0.'41 )x!02
1.7(±0.31)xl06 1.9(±0.5)xl03 6.2(±0.63)xl03
8(±l)xl03 2(±0.7)xl01 5(±3)xl01
3.0(±0.26)xl05 9.8(±6.5)xl05 1,8(±0.5)xl03 2.0(±0.8)xl03
6(±3)xl03 HiO.SjxlO1 3(±2)xl01
3(±0.6)xl03 0.4(±0.6)xlO° 2(±l)xl01
a colony forming units (± 1 standard deviation) per mL of liquid or per g (wet weight) of sediment
b water samples plated "in situ" and sediments plated in the laboratory
c standard plate count agar (i.e. non-saline)
not plated under these conditions
e inter-tidal sediment
-------
o
o
TABLE 45. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL AFTER 28 DAYS INCUBATION AT
8°C WITH DUWAMISH RIVER WATER OR SEDIMENT SAMPLES WITH AND WITHOUT NITROGEN AND PHOSPHORUS
SUPPLEMENTATION AND THE CHEMICAL ANALYSIS OF SAMPLES - (AUGUST 20-21,1979).
Site
Tukwila
119 St. &
Duwamish Rv.
Monroe &
10th Ave.
S.W. Dakota
&
Duwamish Rv.
Klikitat Ave.
(Fisher Mills)
Pacific N.W.
Bell Cable-
Crossing
CD
'a.
1000
16
670
6
570
8.0
550
200
Total
Total Organic Greases
rthophosphate Carbon and oils
(yg)a (mg/L) (yg)a
38
77
440
280
220
120
150
39
170
88
8 200
-e 0.58
10 310
1.2
9 300
0.2
8 190
0.51
310
9 290
Salinity
(o/oo)
NDC
-
ND
-
12.0
-
17.8
—
18.8
22.8
. yg per L water or mgs per g (wet weight) sediment
see Figure 3
d not detectable by analytical methods used
inter-tidal sediment
not analyzed
-------
oil-degrading activity occurred in those samples which received a nitrogen-
phosphorus supplement. Without nutrient supplementation 5 of the 10 samples
examined were able to bring about readily detectable changes in the n-alkane
profile of Prudhoe Bay oil within the 28 day incubation period at 8°C.
101
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SECTION 6
DISCUSSION
Three northern Puget Sound sites were selected to represent the
diverse marine environmental conditions found in these waters and were
investigated in this study: Samish Island - an area rich in organic matter;
E. Fidalgo - a bay area rich in organic matter which is likely to have been
contaminated with hydrocarbons (i.e. close proximity to oil refineries);
and Pt. Partridge - a pristine area low in organic matter. The sites used
in the Pt. Angeles area were selected to provide detailed information on
the waters, beaches and sediments within the harbor and were contrasted with
more pristine sites found in Freshwater Bay and in the area from Green Point
to Dungeness Spit.
The temperature, often a rate-limiting parameter in microbial
activities such as oil-degradation (17), of samples taken in this inves-
tigation ranged from 4°C to 22°C (Appendix) with beach samples being a few
degrees warmer than water column samples. The pH of water column samples
ranged from 6.9 to 8.3 (8), while the dissolved oxygen varied from 3.6 to
14.4 ppm and the salinity from 18.2 to 28.3 ppt. All of these physical
characteristics are in the range which would support the growth of marine
psychrotrophic heterotrophic microbial species.
As long as the physical parameters of an environment are in ranges
which support microbial growth, the removal of exogenous carbon sources
like oil will be dependent on the nitrogen and phosphorus levels in that
environment (4, 5, 29). The phosphorus level, in particular, has been
cited (21) as the key nutrient in controlling the rate of oil removal in
open oceans whereas other studies (8, 29) indicate that the removal of oil
from marine waters is reported to be more sensitive to low levels of nitrogen
than phosphorus. The data obtained in this current study confirm the impor-
tance of added nutrients (i.e. nitrogen and phosphorus) in accelerating the
mineralization of Prudhoe Bay oil by the microbial flora present in these
marine environments.
Oil-degrading capability has been reported in terms of a "Degradative
Capacity Index". This reflects the microbial flora's ability to remove the
n-alkanes and isoprenoids present in the saturate fraction of oils (e.g.
Prudhoe Bay). That is, activity is based on the demonstration of changes
in the chemical composition of the oil brought about by a biological response
(8, 12).
Differences in oil-degrading activity were observed in the psychrotro-
phic oil-degrading capability of water column and beach samples obtained at
each site as well as between samples from different sites (Tables 3 and 4).
Higher levels of activity were found in beach material than in the corres-
ponding water column sample and the activity tended to persist on a seasonal
basis for a longer period of time in beach samples (Tables 3, 4, 5). The
102
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viable counts of beach samples were 1 to 2 and occasionally 3 orders of
magnitude greater than those found in the corresponding water column samples
(Tables 32, 33, 34,-37, 38, 39, 40). Neither this observation nor the
differences noted in generic composition of the component bacterial
population (Tables 35, 41, 42) could be correlated with differences in
oil-degrading activity. Such oil-degrading activity, as previously reported
(8), was more closely associated with proximity to oil refineries (e.g.
E. Fidalgo) and to heavy commercial and recreational use (e.g. Pt. Angeles
harbor). This latter point is demonstrated (Tables 6, 7) in that samples
taken from within the Pt. Angeles harbor area (i.e. Ediz Hook to the site
west of Morse Creek) had higher levels of oil-degrading activity than those
obtained from the sites east of Morse Creek to Dungeness Spit.
Oil spilled in the marine environment may be incorporated into inter-
tidal and sub-tidal sediments. Examination of oil-degrading ability of
sections of inter-tidal cores indicates that a marked variation in activity
exists between sites (Table 8). For example, oil-degrading activity was
found only in the surface centimeter of a core taken at Peabody Creek
(Pt. Angeles harbor), whereas activity was uniformly distributed throughout
the E. Fidalgo core and was hardly detectable in a core taken on Dungeness
Spit. The variation in oil-degrading activity does not correlate with
initial viable count of bacteria present in samples nor with the total viable
count reached after enrichment with Prudhoe Bay oil and an exogenous source
of nitrogen and phosphorus. It is probably related to the types of bacteria
present which not only vary from site to site but also from section to
section of a sample. The changing incidence of pigmented colony forming
units both within and between cores, although not related to oil-degrading
capability, illustrates the heterogeneity of the bacterial populations
present in these cores.
If oil is spilled in a marine environment the seaweed and cobbles
present are likely to be contaminated with oil. Such materials will in all
likelihood have an attached (i.e. periphytic) microbial flora (30). The
presence of such bacteria with the capability of bringing about chemical
changes in Prudhoe Bay oil was demonstrated when enrichments from seaweeds,
cobbles and cobble washes in the presence of added nitrogen and phosphorus
brought about changes in the composition of Prudhoe Bay oil (Tables 9, 10,
11). The observation that transfers of the initial enrichments were more
active than the original enrichments indicates that oil-degrading bacteria
were present in very low numbers and required a longer incubation time to
bring about the changes observed in the chemical composition of this oil.
It would also appear that "washing" (i.e. rinsing) the seaweed has a stim-
ulatory effect on the oil-degrading capability of the microbial flora present.
Not enough samples of seaweed were examined to see if there was any* relation-"
ship between the variety of seaweed, or its geographic location or a*je and
the degradation of oil. Similarly, not enough cobbles were examined to
determine whether there was any correlation between geographic location, type
of cobble or its position relative to tide and the degradation of oil. The
surface area examined, at least between 10 cm2 and 40 cm2 for seaweed and
between 35 cm2 and 106 cm2 for cobbles, does not appear to be correlated to
103
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the ability to utilize Prudhoe Bay oil.
While the generic composition of the original marine population
(Tables 35, 38, 41) can range from low levels of many different genera to
ones with only one or two genera predominating, the composition of oil-
degrading populations consists of only one or two prominent generic types.
Most oil-degrading populations of this type consist of members of the
Flavobacterium genus with occasional populations showing high levels of
members of the Pseudomonas. Acinetobacter or Alcaligenes genera. The
absence of vibrios (common marine bacteria) as a significant proportion of
oil-degrading cultures is due to their inability to grow on oil, as the
medium used in this study readily supported the growth of members of the
genus Vibrio (Table 43).
The concensus in the literature is that crude oil degradation in
marine and terrestrial environments results primarily from bacterial activ-
ity. The literature on the role of fungi and yeasts in oil-degradation
has been recently reviewed (4,6, 36) and it was concluded that they could
play a role in the removal of oil spilled in the environment. Fungi and
yeasts (237 cultures) isolated by direct plating and enrichment techniques
from northern Puget Sound and the Pt. Angeles area were screened by a
stationary culture technique (11) for their ability to grow (i.e. utilize
n-alkanes and isoprenoids) on Prudhoe Bay oil at 8°C or 20°C. The major-
ity of these isolates, 76% at 20°C and 83% at 8°C, would not grow on
Prudhoe Bay oil under the experimental conditions used in this study.
Those which did grow were primarily members of the Aspergillus, Penicillium
and VerticiIlium genera and in most cases they completely utilized the n-—
alkanesTOnly three yeasts of the 74 isolated were capable of utilizing
the n-alkanes present in Prudhoe Bay oil. None of the fungal or yeast
cultures isolated were able to utilize the isoprenoids present in this oil.
Twenty cultures identified as Cladosporium resinae, which has been associ-"
ated with oiled marine environments (37), were isolated in this study.
However, as previously reported (11) for terrestrial isolates and cultural
collection stains, none of the marine C_. resinae isolates were able to
grow on Prudhoe Bay oil. Not enough fungi were obtained by the enrichment
technique to compare its efficiency in isolating oil-degrading fungi and
yeasts with the direct plating technique. Considering the selective
pressures, e.g. low pH and low aeration required to enhance the recovery of
fungi rather than bacteria from environmental samples, it is doubtful
whether oil-degrading fungi and yeasts are competitive with bacteria in
removing oil from aquatic or terrestrial environment under non-stressed
environmental conditions. However there is a relatively high percentage
of the fungal isolates (apparently restricted to a few genera) with the
ability to utilize n-alkanes such that under conditions where bacterial
growth is restricted these organisms could initiate the degradation of oil.
Studies on the rate of mineralization of Prudhoe Bay oil under
non-nutrient-limiting conditions, using a gravimetric procedure (Figures
8, 10), showed maximum rates of 50.5, 13.5, 37.7 and 14.6 mg of oil
104
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removed per litre of water per day at 8°C for water column samples from
E. Fidalgo, Ediz Hook (Pilot Station), Peabody Creek, and Dungeness Spit
(No. 2) respectively. No degradation of this oil was observed under the
conditions of this experiment with the water column sample taken from
Pt. Partridge (April, 1979). Samples from E. Fidalgo (near Anacortes oil
refineries) and Peabody Creek (southside of Pt. Angeles harbor), showed
the highest rates of mineralization of oil and the shortest lag times
(i.e. period before rapid oil utilization takes place). This is not un-
expected as both these sites are probably being exposed to chronic low
levels of hydrocarbon contamination. Sediments from March Pt. (near
the Anacortes refineries) have been found to contain relatively high
levels of saturate and aromatic hydrocarbons (40); summary in Appendix.
This would result in a selective pressure being brought to play on the
"normal" marine microbial flora and result in natural enrichment of hydro-
carbon-degrading bacteria. As reported in the literature (13, 14), loss
of weight of oil was brought about by the utilization of components of the
saturate and aromatic fractions (Figures 9, 11). Little change is noted
in the proportion of asphaltenes whereas the recovery of polar (i.e.
N,S,0's) compounds increases with time. This latter observation suggests
a conversion of non-polar hydrocarbons found in the saturate and aromatic
fractions to polar compounds, probably via partial oxidation or co-oxida-
tion (31). The data (Figures 9, 11) on the rates of change of these
fractions suggest a gradual utilization of the aromatic compounds (e.g.
Ediz Hook and E. Fidalgo water) whereas a definite lag phase followed by
a period of very rapid utilization occurs with compounds present in the
saturate fraction (e.g. n-alkanes) suggesting that there is an inherent
ability present in the microbial flora in some water samples to utilize
components present in the aromatic fraction. However an induction period
is required before the rapid utilization of components in the saturate
fraction occurs. The extensive utilization of Prudhoe Bay oil which occurs
with prolonged incubation- that is, longer than the 28 days used in the
activity survey (Figure 11) - indicates that the survey data has to be
interpreted carefully. The results observed, therefore, are related to the
length of the incubation period and/or the sample size used as source of
inoculum. Increasing either or both variables would with some samples
increase the degree of chemical change observed in recovered oil.
Oil-utilization is a biochemical characteristic which is wide-
spread, although at markedly different levels, in the microbial flora
found in this marine environment. This is reflected in the varied re-
sponse, even with nutrient supplementation, of the microbial flora in
different samples to bring about changes in the chemical composition
of Prudhoe Bay oil.
Greater variation in gravimetric recovery studies was observe^
with samples from pristine areas, e.g. Ediz Hook (Pilot Station) and
Dungeness Spit No. 2 than from areas likely to be subject to hydrocarbon
contamination. This is probably a result of a heterogeneous distribution
of microorganisms in the sample size used in pristine areas and could be
105
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overcome by using a larger sample size. Difficulties were also experienced
with emulsion formation on long term incubation of oil-water mixtures which
interfered with the recovery of the oil. Attempts to carry out gravimetric
studies on sediment samples also were unsuccessful because of the formation
of stable emulsions making quantitative recovery of oil impossible.
Investigations on the rate of mineralization using Prudhoe Bay oil
"spiked" with one of n-[l-llfC]-hexadecane, [I-1 ^-naphthalene, [9-^C]-
phenanthrene or [9-ll4C]-anthracene produced data on the fate of individual
hydrocarbons in oil, whereas the gravimetric procedure determined total
changes in the components of oil. The radiometric technique relies on the
release of ltfC02 from compounds specifically labelled with carbon-14. Thus
the position of the label will be a factor in the time required and the
amount of metabolism required before 14C02 is released. The dilution of
added 1LtC-hydrocarbons by the corresponding component in oil also has to be
considered in the determination of the rates of removal of individual hydro-
carbons. The contents of these hydrocarbons in Prudhoe Bay oil used for
calculating rates of removal of individual hydrocarbons are taken from the
current literature available to us.
The metabolism of n-alkanes can proceed via a, a> or internal oxida-
tion (32). The first step in the a and u terminal oxidation processes is
the formation of a primary alcohol which is subsequently oxidized to an acid.
Further pathways of catabolism of such acids, although not firmly established,
probably proceed via 3-oxidation to yield acetate units whose metabolism
would yield 1'tC02. Regardless of the site of initial oxidation, many enzy-
matic steps, and thus time, are required to yield 14C02 from n-p-^C]-
hexadecane. Pathways (33) involved in the catabolism of the aromatic com-
pounds naphthalene, anthracene and phenanthrene by pseudomonads are summa-
rized in Figure 23 (pseudomonads and related organisms predominate in oil-
degrading enrichments). Many enzymatic steps are required before 14C02 will
be released from the catabolism of these aromatic compounds. All of them,
and naphthalene in particular, are labelled in symmetrical sites. Therefore
the number of steps involved before the release of ll*CQ2» and "thus the time
elapsed, will also depend on the site where the initial oxidation takes place
relative to the ^-labelled carbon atom. But in all cases, cleavage and
extensive catabolism of the aromatic ring(s) are required before 14C02 will
be released. The shape of the 14C02 release curves for a given compound
reflects the time required (i.e. lag) before the microbial flora adapts to
the respective substrates and maximum rates of 14C02 evolution are observed.
As these compounds are added in oil it is also possible that some differences
observed in lags, rates of l^CQ2 released and recovery are a result of the
response of the microbial flora to other compounds present in oil.
The rapid release of 14C02 from ll*C-labelled naphthalene by the
microbial flora present in all water column samples tested (Figures 1?, 13)
indicates that such populations quickly adapt to the addition of this sub-
strate. It is well established that, in the control and regulation of
procaryotic (i.e. bacterial) metabolism, the substrate which supports
the fastest growth rate will be used first and its use will inhibit the
106
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phenanthrene
C09H
•, z
salicylic acid
co
FURTHER METABOLISM
(pyruvate; salicylic acid)
Figure 23. Pathways of catabolism of anthracene, naphthalene and phenanthrene by pseudomonads
(34): ( C14 used in pathway; asymmetric sites, number of reactions between
intermediates).
-------
synthesis of the enzymes required to metabolize other more slowly utilized
substrates. The microbial flora present in waters from areas likely to have
been exposed, or being exposed, to chronic low level hydrocarbon contamina-
tion, i.e. E. Fidalgo and Peabody Creek, much more quickly adapt (as deter-
mined by length of incubation i.e. days before 1HC02 is rapidly released) to
the catabolism of hexadecane, anthracene and phenanthrene. In contrast,
longer incubation times are required before the microbial flora present in
other waters release ltfC02 from these compounds. The differences in yields
of 14C02 relative to the amount of 14C-labelled substrate added reflects in
part the differences in the catabolic pathways used and/or the degree of
assimilation (i.e. incorporation of carbon into cell material) which occurs
with the different substrates and populations. For example the high degree
of recovery of carbon-14 from [Q-^Cl-phenanthrene as llfC02 indicates that
this carbon is readily released as l!iC02 whereas the lower recoveries from
[l-^C] -hexadecane could reflect a higher degree of assimilation taking
place. In addition, since there is an increase in the polar components of
crude oil as a result of microbial catabolism of oil, some of the 11+C-
labelled substrates could be converted by partial oxidation or co-oxidation
to such compounds. Therefore the total ability of the microbial flora
present to remove specific hydrocarbons in oil will not be known until the
distribution of carbon-14 in other potential products of metabolism is
investigated.
The 14C02 release patterns from the microbial flora present in the
sub-tidal sediments (Figure 14) show a rapid release of 14C02 from [l-14Cl-
naphthalene. In contrast to the 1!*C02 release patterns from the water column
samples taken at the same time, where Peabody Creek was the most active,
these results show that the sub-tidal sediment from Ediz HooK was more active
!U a?J™ en? and nexadeca|ie. Very little difference exists however between
the 11+C02 release patterns brought about by Peabody Creek and Ediz Hook on
phenanthrene.
The highest rates (not related to lag) of removal of these compounds
from Prudhoe Bay oil by both water column and sub-tidal sediments were ob-
served for phenanthrene (Tables 16, 17, 18). This was followed by naphthalene
with hexadecane generally being removed at a slightly higher rate than anthra-
cene. The length of lag observed (i.e. time before a rapid rate of
release is achieved) (Tables 16, 17, 18) confirms the observation from
Figures 12, 13, 14 that naphthalene is the most readily utilizable hydrocarbon
tested. The lengths of lags observed with the other substrates varies to
some degree with the type of sample being studied (i.e. water column versus
sub-tidal sediment). The ability of sub-tidal flora to catabolize polycyclic
aromatic hydrocarbons, even under highly aerobic conditions, is important as
it was recently shown (34) that 14C-anthracene added to two 80-litre pond
microcosms rapidly accumulated in the sediment. Therefore the presence of a
sub-tidal microbial flora capable of catabolizing such compounds is very
important if such pollutants are going to be removed or maintained at low
levels in this area of the marine environment. The release of !1|C02 from
spiked" oils by microbes found in beach samples (Table 19) indicates that
they are capable of catabolizing hexadecane arid naphthalene (samples were
108
-------
not tested on [9-1J*C]-anthracene or [9-14C]-phenanthrene).
Oil-degrading activity of microbial flora in this study is based on
changes occurring in the n-alkane and isoprenoid components of the saturate
fraction of oils. It has been shown however (35, 15) that aromatic compo-
nents, in particular mon-, di- and tri-cyclic ring systems, are readily re-
moved from oil by microbial action. This was confirmed by some of the
gravimetric data obtained and in the studies using carbon-14 "spiked"
Prudhoe Bay oil. However the gravimetric study reflects gross changes in
oil as a result of microbial catabolism and the radiometric technique pro-
vides information on the fate of an individual hydrocarbon (albeit in the
presence of other components of crude oil). Therefore an investigation of
the utilization patterns of saturates and aromatics of Prudhoe Bay oil was
undertaken using glass capillary gas chromatography which requires extensive
pre-fractionation of oil but provides detailed information on other changes
taking place in these fractions of Prudhoe Bay oil.
The rates of utilization of saturate and aromatic components as a
function of time at 8°C in the presence of exogenous nutrients was investi-
gated using water column samples from E. Fidalgo. No changes were noted in
either the n-alkane or aromatic fractions of recovered oil (data not re-
ported) after 3 days incubation. Very few changes were detected in the
saturate fraction (data not reported) after 6 days incubation whereas the
more volatile components like substituted benzenes, 1-methyl- and 2-methyl-
naphthalenes were being utilized at this time (Figures 16, 17). However,
between day 6 and 10 the n-alkanes and isoprenoids were completely used
whereas only the substituted benzenes and methyl naphthalenes had been used
in the aromatic fraction and the degradation of dimethyl naphthalenes, di-
benzothiophene and phenanthrene had been initiated. Extensive utilization
of these aromatic components had occurred after 14 days incubation although
the phenanthrene and anthracene peaks were still discernible. After 27
days incubation in the presence of exogenous nutrients the glass capillary
G.C. profile (Figure 18) shows that extensive metabolism of aromatic com-
ponents has taken place as a result of microbial activity. A comparison of
the G.C. profiles of uninoculated controls (Figures 17 and 18) shows that
extensive loss of these components also occurs via volatilization. These
results confirm the previous data which indicated aromatic catabolism pro-
ceeds prior to the extensive, spectacular catabolism of n-alkanes and iso-
prenoids, and continues after the saturate components have been removed from
the oil.
The effect of the presence or absence of exogenous nutrients on the
changes in the chemical composition of the saturate and aromatic components
of Prudhoe Bay oil was studied using water column samples from Peabody Creek
(Pt. Angeles harbor), E. Fidalgo (near Anacortes refineries), and Pt. Partridge,
The glass capillary profiles of these fractions (Figure 27) show that in the
absence of added nitrogen and phosphorus the microbial flora present in water
column samples from Peabody Creek and Pt. Partridge brought about little
change in the n-alkane and isoprenoid profile (Figure 21). In contrast,
water from E. Fidalgo brought about quite readily detectable changes in the
109
-------
n-alkanes present in Prudhoe Bay oil. The profiles of the corresponding
aromatic fraction (Figure 20) show more changes in the aromatic fraction
of the oil recovered from the Peabody Creek and Pt. Partridge water samples
than from E. Fidalgo. In the presence of exogenous nutrients the microbial
flora present in water samples from Peabody Creek and E. Fidalgo completely
utilized the n-alkanes and isoprenoids whereas water from Pt. Partridge
only partially removed the n-alkanes and did not utilize the isoprenoids
present in Prudhoe Bay oil (Figure 21). Similarly, greater changes were
brought about in the aromatic components by the microbial flora present in
water from Peabody Creek and E. Fidalgo than by water from Pt. Partridge
(Figure 19) where the anthracene peak is still discernible.
These results indicate that catabolism of saturates and aromatics
do take place in the absence of added nutrients although at a very much
slower rate. The degree of utilization of these fractions also varies with
source of the sample. Water from E. Fidalgo, which is adjacent to oil
refineries, brought about changes in the saturate fraction but was less
active on the aromatic components. In contrast, water from the pristine
Pt. Partridge area and Peabody Creek (a heavily used commercial, industrial
area) brought about greater changes in the aromatic components than in the
saturate fraction.
The chemical composition of oil has been shown to affect its biode-
gradability (16, 22). Since refineries in the Puget Sound area, particularly
those near Anacortes, receive oil from areas of the world other than the
north slope of Alaska an investigation of the biodegradability of represent-
ative oils was undertaken. The data (Table 21) indicate that the n-alkanes
in Murban and Seria oils were readily catabolized by stock, laboratory oil-
degrading cultures from the Puget Sound area. These populations were not
able to bring about similar changes in Minas oil which is a solid at 8°C.
The n-alkanes in Seria oil were readily catabolized by the microbial flora
present in waters and beach samples from the northern Puget Sound and
Port Angeles area. However Murban oil was not as readily metabolized and
Minas oil was only utilized by the microbial flora present in beach samples
from Peabody Creek (Pt. Angeles harbor) and Dungeness Spit No. 2. These
results indicate that attention must be paid to the type of oil being shipped,
since biodegradability differs considerably. A spill of Minas oil, even
though it is not readily degraded, would not be a problem as at temperatures
prevalent in the air and waters of this area it would be a solid and thus
physically removable. However it has been noticed that under the laboratory
conditions used this "oil" occasionally formed "tar-balls" which sank in the
artificial seawater solution used in these experiments.
During the course of the sub-tidal oil-degrading experiment conducted
by the Battelle laboratories at Sequim Bay the incidence of oil-dearading
heterotrophs increased in oil treated materials until they become a signifi-
cant proportion (up to 10%) of the heterotrophic population (Table A8). How-
ever the fact that a similar shift, at a slightly lower level, occurred in
the non-oiled plots suggests that oil cross-contamination of plots occurred.
no
-------
The incidence of oil-degrading heterotrophs initially was low and similar
values were found for the fine and coarse material examined in the prelim-
inary study. This incidence changed quite rapidly in the coarse material,
whereas a considerably longer period of time was required to bring about
a similar change in the fine material. This is a very sound approach to
studying the effect of oil on sub-tidal biological activities. However
from a microbial aspect more samples are required to follow the changing
trends in the activities of the oil-degrading flora present as well as the
use of more sensitive techniques. For example the incorporation of carbon-
14 labelled hydrocarbons in the oil-degrading enumeration procedure (10)
and the use of the radiometric technique employed in this investigation
would be useful. These sensitive procedures coupled with G.C. glass capil-
larly analysis of the aromatic and saturate fractions of the oil recovered
periodically should provide the data required to interpret changes in the
microbial flora.
The oil-degrading capability of the microbial flora in the Duwamish
River flowing through south Seattle and entering Puget Sound at Elliott Bay
was investigated. The numbers of bacterial, yeast and fungal colony forming
units (Table 44) are similar to those found in other areas of these coastal
waters studied. All samples (both water column and sediments) readily uti-
lized the n-alkanes and isoprenoids present in Prudhoe Bay oil (Table 45)
under these experimental conditions. Significant changes also were noted
in enrichments which did not receive a nitrogen and phosphorus supplement.
There is a relatively high level of oil-degrading activity amongst the
microbial flora present (even though total viable numbers are similar) in
this water system as compared to that in other sites investigated. This
undoubtedly is a result of the presence of relatively high levels of hydro-
carbons and greases in this environment (hydrocarbon sheens were readily
visible on the surface of the water at many of the sites sampled and hydro-
carbon sheens were visible in the sediment samples taken from this area).
Their presence would result in a very active oil-degrading component being
present and functional in the micro-flora of this water system.
The information presented in this and the previous report (8) in-
dicates a viable but ubiquitous distribution of oil-degrading microorganisms
in the water, beaches, inter-tidal and sub-tidal sediments of northern Puget
Sound and the southern shores of the Strait of Juan de Fuca. Samples
from areas which have been or are subject to chronic, low-level contamination
by hydrocarbons(e.g. oil refineries or commerical traffic) have a higher rate
of activity than those from other areas. As noted in other studies the oil-
degrading activity of these microorganisms is limited by the nitrogen and/or
phosphorus content of the various marine environments found in these areas.
It is doubtful whether "fertilizing" marine oil spills even with oleophilic
fertilizers containing nitrogen and phosphorus (37) can be effectively and
efficiently used on open water spills. However such fertilizers could be of
use in treating oil spills reaching inter-tidal and beach areas. The ability
of the marine environment in this area to recover from an oil spill is illus-
trated by the fate of an oil, classed as a No. 5 fuel oil, in the Alert Bay
111
-------
area of northern Vancouver Island (38, 39). Studies (39) over a 4-year
period followed the fate of the oil in a bay area (Reserved Bay, Pearse
Island group) which was not subjected to clean up procedures. The n-
alkanes were removed during the first year following the spill whereas
pristane and phytane were not completely utilized until the fourth year.
The distribution pattern of residual hydrocarbons (tentatively identified
as oil pentacyclic terpenes) recovered in the fourth year had a distribu-
tion pattern similar to that of the original oil. The inter-tidal biota
which was initially adversely affected by the spill was well on its road
to recovery within a year of the spill. These observations suggest that
normal nutrient cycling of nitrogen in particular, and the level of phos-
phorus, are sufficient to support the oil-degrading activities of the
indigenous microbial population and that over an extended period of time
significant microbial degradation of oil will take place. This action
together with the normal physical-chemical processes (e.g. photolysis,
evaporation, dissolution, etc.) involved in removing oil in the marine
environment were able to remove most of the oil spilled within a 4-year
period.
112
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24. MacLeod, W.D. Jr., D.W. Brown, R.G. Jenkins, L.S. Ramos and V.D. Henry.
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34. Giddings, J.M., B.T. Walton, G.K. Eddlemon and K.G. Olson. Transport
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A. Tonolo. Microbial Degradation of Oil Spills Enhanced by a Shaw-
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of a Heavy Fuel Oil in a Spill Contaminated B.C. Coastal Bay.
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116
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APPENDIX
Composition of Microbiological Media
(i) Artificial Seawater Solution
NaCl 23.40 g
KC1 0.75 g
MgSOv7H20 7.00 gm
Distilled water 1000 ml
pH 7.3
(ii) Basal Marine Agar (for viable cell counts;
storage of bacterial cultures and for
oxidase and catalase tests).
proteose peptone #3 (Difco) 1 g
yeast extract (Difco) 1 g
agar (Difco purified) 20 g
artificial seawater 1000 ml
pH adjusted to 7.5 before autoclaving
final pH 7.3
(iii) O.F. Medium for Sugar Utilization Tests
Solution A - NaCl 23.40 g
KC1 0.75 g
MgSO^M 7.00 g
yeast extract (Difco) 1.00 g
Bromthymol blue 0.03 g
agar (Difco purified) 15.0 g
distilled water 750 mL
Solution B - glucose or lactose 10.0 g
distilled water 200 ml
Solution C - NHitH2POit 0.50 g
KzHPOit 0.50 g
distilled water 50 ml
The pH of all solutions was adjusted to 7.15 before autoclaving.
After cooling to 50°C solutions A and B were mixed, solution C
added and mixed and then the medium was dispensed into Petri plates,
(iv) Saline Sabouraud's agar - 65 g of Sabouraud dextrose agar (Difco)
added to 1000 ml of artificial seawater solution; after minimum
sterilization the medium was cooled to 50°C and 100 mg of strepto-
mycin and 60,000 units of penicillin-G (both filter sterilized)
added to 1 L of medium.
117
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(v) Malt agar - prepared as per Difco's instructions.
(vi) TCBS agar - 89 g added per 1 L of artificial seawater solution.
(vii) Marine agar 2216 - 37.4 g of purified Difco agar added per 1 L
of distilled water.
(viii) Anino acid medium for Most Probable Number determinations.
Thirty g of vitamin-free cosamino acids added per 1 L of
artificial seawater solution.
(ix) Modified But!in's for sulfide-generating bacteria.
Solution A - NHj+Cl 1.00 g
Na2SOit 2.00 g
MgSOv7H20 0.02 g
Sodium lactate 1.5 ml (60% syrup)
yeast extract (Difco) 1.0 g
K2HPOij 0.5 g
artificial seawater 1000 ml
Ten mis dispensed per 18x150 mm test tube and 2 iron finishing
nails added per tube for poising the medium before sterilization.
Solution B - 3 drops of freshly prepared 10% Na2S03 solution
(sterilized by filtration) added to each tube
of medium A prior to inoculation.
(x) Nitrogen-and phosphorus-containing solution used for nutrient
supplementation.
10% K2HPOi, 420 ml
10% KH2P04 180 ml
NHi,N03 60 g
pH adjusted to 7.3 with ION NaOH prior to autoclaving; used at
the rate of 1 ml/100 ml of natural or artificial seawater medium.
Biochemical Tests for Bacterial Identification
(a) Oxidase reagent - 1% solution of N,N,N',N'-tetramethyl-p-phenylene-
diamine-dihydrochloride; 1 to 3 drops added to
colonies growing on basal marine agar.
(b) Catalase reagent - 1 to 3 drops of freshly prepared 3% H202 added
to colonies growing on basal marine agar.
118
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TABLE Al. DISSOLVED OXYGEN, SALINITY AND TEMPERATURES OF SAMPLES FROM
NORTHERN PUGET SOUND.
Water Column
Site
Samish Island
E. Fidalgo
Pt. Partridge
Date
Aug. 14/78
Nov. 21/78
April 2/79
Aug. 14/78
Nov. 21/78
April 2/79
Aug. 14/78
Nov. 21/78
April 2/79
Aug. 21/79
Dissolved
Oxygen
(ppm)
8.1
11.2
9.8
3.6
11.8
9.4
8.5
9.5
7.8
9.1
Salinity
(o/oo)
21.5
22.0
21.8
22.6
22.5
24.3
24.8
24.0
24.9
28.3
Temperature
(°c)
beach
15
3
13
15
8
17
14
2
25
_a
water
14
7
10
15
5
12
13
8
12
14
sample not taken
119
-------
TABLE A2. DISSOLVED OXYGEN, SALINITY AND TEMPERATURES OF SAMPLES FROM PT. ANGELES AREA.
PO
o
Site
Freshwater
Bay
Pt. Angeles-
Ed iz Hook #1
Pt. Angel es-
Ediz Hook #2
Pt. Angel es-
Southside
(Marina)
Pt. Angel es-
Southside-
(Peabody Creek)
Date
Oct. 2/78
Jan. 15/79
June 19/79
Oct. 2/79
Jan. 15/79
June 19/79
Oct. 2/78
Jan. 15/79
June 19/79
Oct. 2/78
Jan. 15/79
June 19/79
Oct. 2/78
Jan. 15/79
June 19/79
Water Column
Dissolved Oxygen Salinity
(ppm) (o/oo)
top
10.6
10.6
9.1
9.9
9.9
9.3
7.8
12.6
14.0
8.5
11.2
11.8
8.0
11.1
13.6
bottom top bottom
8.4a 19.0 20. 5a
27.8
21.9
7.9C 21.4 21. 0C
27.1
21 .0
26.0
27.6
22.8
20.1
27.4
23.3
7.3d 24.6 21.3d
16.2
18.3
Temperature
(°C)
beach
_b
5
14
-
2
19
13
6
18
13
19
11
4
18
water
column
12
-
-
14
6
11
14
6
11
16
6
11
13
5
12
bottom
12a
-
-
14C
-
-
-
-
-
-
-
15d
-
-------
TABLE A2. continued
Site
Pt. Angeles-
Souths ide
(Red Lion
Inn)
West of
Morse Creek
West of
Green Point
Green Pt. #1
Green Pt. #2
Green Pt. #3
Date
Oct. 2/78
Jan. 15/79
June 19/79
Oct. 2/78
Jan. 15/79
June 20/79
Oct. 2/78
Oct. 2/78
Oct. 2/78
Oct. 2/78
Jan. 16/79
June 19/79
Water Co
Dissolved Oxygen
(ppm)
top bottom
7.8
12.2
11.7
8.2
14.4
12.6
11.2
8.3
9.1
8.8
10.8
10.0
lumn
Salinity
(o/oo)
top bottom
26.1
27.3
18.2
24.7
27.1
20.5
25.0
24.9
25.1
24.3
27.0
23.0
beach
12
5
16
11
4
-
11
11
14
21
6
18
Temperature
(°C)
watpr
column
13
6
12
17
7
15
17
16
16
17
7
13
bottom
-
-
-
-
-
-
-
-
-
-------
ro
ro
Site
Dungeness
Spit #1
Dungeness
Spit #2
Dungeness
Lagoon
Date
Oct.
Jan.
June
Oct.
Jan.
June
June
Water Column
Dissolved Oxygen
(DDIH)
2/78
16/79
19/79
2/78
16/79
19/79
19/79
top
8.4
11.2
10.9
10.4
10.1
9.9
10.6
bottom top
24.
26.
23.
21.
27.
23.
22.
Salinity
(o/oo)
bottom
9
9
3
5
1
0
8
Temperature
beach
-
5
18
21
4
19
23
water
col umn
17
6
13
17
6
14
15
bottom
-
-
-
-
-
-
-
a bottom water - 7.62 meters depth
no sample taken
c bottom water - 9.14 meters depth
d bottom water ^ 15.24 meters depth
-------
TABLE A3- CHEMICAL ANALYSIS OF WATER SAMPLES FROM NORTHERN PUGET SOUND SITES.
ro
oo
Date
August 13-14,
1978
April 2-3,
1979
June 18-20,
1979
Site
Samish Island
E. Fidalgo
Pt. Partridge
Samish Island
E. Fidalgo
Pt. Partridge
Samish Island
E. Fidalgo
Pt. Partridge
Nitrogen
(yg/L)
NO ~ NH.+
3 4
70 750
70 770
230 540
150 90
78 130
120
_
-
-
Total
orthophosphate
(yg/L)
80
100
70
76
1740
46
-
-
Total
organic carbon
(mg/L)
15
16
13
9
13
2
-
-
Greases Suspended
and oils solids
(yg/L) (mg/L)
_a
-
-
-
-
170 13.4
500 13.8
260 19.3
not analyzed
-------
TABLE A4. CHEMICAL ANALYSIS OF BEACH SAMPLES FROM NORTHERN PUGET SOUND SITES.
ro
Nitrogen
(yg/g)
Date
August 13-14, 1978
April 2-3, 1978
Site
Samish Island
E. Fidalgo
Pt. Partridge
Samish Island
E. Fidalgo
Pt. Partridge
N03-
3.7
NDa
0.16
3.3
3.0
2.5
NH/
0.02
0.003
0.007
2.3
1.3
1.2
Total
orthophosphate
(yg/g)
1.1
0.7
0.02
2.4
360
0.11
Greases
and solids
(mg/gj
0.16
0.06
0.07
_b
-
~
3 not detected by method used
not analyzed
-------
TABLE A5. CHEMICAL ANALYSIS OF WATER SAMPLES FROM PT. ANGELES AREA SITES.
ro
01
Date
October 1-3,
1978
Site
Freshwater Bay
Pt. Angeles-
Ed iz Hook #1
Pt. Angel es-
Ediz Hook #2
Pt. Angeles-
Sou thside
Marina
Pt. Angeles-
Pea body Creek
Pt. Angeles-
Red Lion Inn
West of Morse
Creek
West of Green
Point
Green Point #1
Green Point #2
Green Point #3
Dunqeness Spit
Nitrogen
(yq/L)
N03-
120
170
230
260
270
270
310
290
400
410
490
380
NH/
130
ND"
350
ND
100
100
180
250
ND
240
110
Total
orthophosphate
(yg/L)
51
34
31
37
40
44
41
41
44
50
46
43
Total
organic carbon
(mg/L )
6.2
5.3
4.5
3.6
3.6
4.3.
3.3
3.3
5.2
3.7
4.2
3.5
Greases Suspended
and oils solids
(yg/L) (mg/L)
_a
_ _
™ •• •
-
_ _
-
-
-
-
-
- -
#1
Dungeness Spit
#2
400
220
44
3.7
-------
ro
en
1 f 1LS ^k- f tw • V
Dafp
Nitrogen Tr»4-=*i
/ / 1 \ 1 O td *
CT ^ p ... v M SJ/ *- / . _._ .„_ AV*+" hnnhrtc nh^ to n
N03" NH4 (yg/L)
Total
v*1"hnnhnQnh3l"P
(mg/L)
Greases Suspended
anH nil<; ^nliH^
(yg/L) (mg/L)
June 18-20, Pt. Angeles- 110 260 51 4 260 8.2
1979 Ediz Hook #1
Pt. Angeles- ND 210 43 15 360 18.6
Peabody Creek
Dungeness Spit ND 580 65 7 300 115
#2
a not analyzed
not detected by method used
-------
TABLE A6. CHEMICAL ANALYSIS OF BEACH AND SUB-TIDAL SAMPLES FROM PT. ANGELES AREA SITES.
ro
Date Sample Site
October 1-3, 1978 Beach Freshwater Bay
Pt. Angel es-
Ediz Hook #1
Pt. Angeles-
Ed iz Hook #2
Pt. Angeles-
Southside
Marina
Pt. Angel es-
Peabody Creek
Pt. Angel es-
Red Lion Inn
West of Morse
Creek
West of Green
Point
Green Point #1
Green Point #2
Green Point #3
Dungeness Spit #1
Dungeness Spit #2
Nitrogen
(yg/g )
N03-
0.29
1.5
0.85
0.7
0.7
0.45
1.8
2.7
0.2
0.03
1.1
0.1
1.0
NH/
9.0
2.9
4.6
5.9
4.1
3.5
5.2
7.7
4.0
4.2
14
NO
12
Total
rthophosphate
(yg/g)
9.9
0.93
1.2
5.1
9.8
12
NDa
0.07
ND
0.10
4.4
1.1
0.36
Greases
and oils
(mg/g)
0.034
0.094
0.160
0.052
0.091
0.046
0.009
0.010
0.010
0.110
0.007
0.005
0.025
-------
TABLE A6. continued
ro
oo
Date Sample
October 1-3, 1978 Sub-tidal
June 18-20, 1979 Beach
Sub-tidal
Site
Freshwater
Bay
Pt. Angeles-
Ediz Hook #1
Pt. Anqeles-
Peabody Creek
Pt. Angel es-
Ediz Hook #1
Pt. Angel es-
Peabody Creek
Dungeness Spit
#1
Pt. Angeles-
Ediz Hook #1
Pt. Angeles-
Peabody Creek
Dungeness Spit
#1
Nitrogen
(yg/g)
N03- NH/
0.29
0.038
0.13
1.9
2.5
1.4
6.3
8.4
2.9
14
34
13
ND
4.8
ND
13
ND
ND
Total
orthophosphate
(yg/g)
34
21
47
3.5
5.0
0.79
150
250
5.0
Greases
and oils
(mg/g)
0.16
0.59
0.37
0.06
0.33
0.01
0.42
0.50
0.01
not detected by methods used
-------
TABLE A7. MOST PROBABLE NUMBERS (±1 STANDARD DEVIATION) OF HETEROTROPHS, OIL-DEGRADING HETEROTROPHS
AND AUTOTROPHS - BATTELLE EXPERIMENTS (SEQUIM BAY - LAGOON II, 1978-1979)
ro
Most Probable Numbers3
Particle
Size
Fine
Treated
Untreated
Treated
Untreated
Treated
Heterotrophs
0.5-5.6xl04
1.5-16.2xl05
1.0-10.9xl05
1.5-16.2xl06
0.4-4.3xl05
0.3-3.6xl07
1.5-16.2xl05
0.7-7. 6x1 O6
0.7-7.9xl08
1.5-16.2xl06
2.4-26.1xl06
0.4-4.3xl06
2.4-26.1xl03
0.3-3. 6x10^
Average
±1 S.D.c
2.79(±2.4)xl05
5.34(±5.4)xl06
8.09(±13.7)xl07
4.70(±3.3)xl06
Oil-Degrading Average
Heterotrophs6 ±1 S.D.C
0.7-7.6X101
0.7-7.6xl02 1.10(±l.l)xl02
2.4-26.8X101
0.7-7.6X101
1.54(±l.l)xl01
0.2-2.6X101
0.7-7.3xl03
0.5-5.6X101* 2.27(±2.4)xlOt>
1.5-16.2X1014
0.7-7.6xl03
0.5-5. 6x10^ 6.67(±9.0)xl03
2.0-23.1xl02
2.1-23.1X101
2.1-23.1xl03
Autotrophs
^Qie
<1 0^
<1 0^
<101
0.3-3.6x10^
2.4-26.1xl03
1.5-16.2xl03
7.83(±5.5)xl03
1.5-16.2xl02
0.4-4.3xl03
1.5-16.2xl02
1.0-10.9xl02
3.99(±6.4)xl02
-------
TABLE A7 continued
Most Probable Numbers3
Particle
Size
Fine
•M
S
Summer
1979
0)
IS.
to
oo
Untreated
. Average
Heterotrophs0 ±1 S.D.C
0.7-7.3x10^
0.4-4.3xl04
Oil -Degrading Average
Heterotrophs" ±1 S.D.C
1.0xl0.9xl02
0.7-6.9xl02
Autotrophs
00
o
Coarse
Sunnier
1978
(preliminary)
Summer
1978
Treated
Untreated
Treated
Untreated
1.5-16.2xl03
1.5-16.2x1O3
0.7-7. BxlO1*
1.4-15.2xl03
0.7-7.6xl06
2.5-27.7x1O7
0.4-4.3xl06
1.5-16.2xl07
0.4-4.3xl06
0.4-4.3xl08
2.4-26.1xl05
1.4-15.2xl05
0.4-4.3xl05
2.4-26.1xl05
0.4-4.3xlOG
8.65(±7.2)xl03
2.92(±4.7)xl07
6.01(±6.5)xl07
4.60(±3.3)xl05
1.05(±0.4)xl06
1.4-15.2X101
1.4-15.2X101
0.7-7.6x1O2
0.5-5.GxlO1
0.7-7.6X101
0.7-7.6X101
1.0-10.9X101
0.4-4.3x1O2
0.7-7.6X101
0.4-4.3x1O2
0.4-4.6xl05
1.5-16.2xl02
2.4-26.1xl02
0.7-7.6X104
2.4-26.U101
7.06(±5.1)xl02
1.94(±1.6)xl02
4.71(±8.0)xlOt+
1.19(±1.6)xl04
-------
Most Probable Numbers9
OJ
"o
•i —
-M (LI
ro -i-
-------
TABLE A8. RATIOS OF OIL-DEGRADING HETEROTROPHS/HETEROTROPHS FOR BATTELLE EXPERIMENT (SEQUIM BAY-
LAGOON II, 1978-1979).
CO
INJ
Average MPN's <,/
Particle
Size
Fine
Coarse
Sample
Preliminary Treated
Untreated
Summer- 1978 Treated
Untreated
Sumner- 1979 Treated
Untreated
Preliminary Treated
Untreated
Summer - 1978 Treated
Untreated
Summer - 1979 Treated
Untreated
Heterotrophs
3x1 05
5x1 06
8x1 07
5x1 06
8xl03
9xl03
3x1 07
6x1 07
5xl05
IxlO6
IxlO1*
IxlO1*
Oil -Degrading
Heterotrophs
IxlO2
ZxlO1
2x1 01*
7xl03
4x1 02
7x1 02
2x1 02
9x1 01
5x1 04
IxlO1*
5x1 02
IxlO2
Oil -Degrading
Heterotrophs
0.033
0.0004
0.025
0.14
5.0
7.8
0.0007
0.0002
10.0
1.0
5.0
1.0
-------
TABLE A9. SUMMARY OF HYDROCARBON ANALYSES (40) OF SELECTED NORTHERN
PUGET SOUND AND PT. ANGELES HARBOR AREA SEDIMENTS.
ng/g of sediment
saturates
(aromatics)
Site
Dungeness
Spit
Dungeness
Three Crabs
Ediz
Hook
Whidby
Island
March
Point
Spring
24a
(2.6)a
(860)b
(61)b
390a
(33)a
7.7a
(4.2)a
480C
(160)c
Summer
83
(4.6)
(340)a
(26)a
301
(17)
128
(6.2)
280a
(220)a
Fall
16
(0)
620
(0)
228
(0)
23
(2.0)
282
(60)
Winter
56
(0)
787
(32)
262
(21)
27
9
207
(84)
a average of two values
average of 3 values
c average of 4 values
133
------- |