Pergamon

0025-326X(94)00110-3

Marine Pollution Bulletin, Vol. 30, No. 2, pp. 125-132, 1995 Copyright 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/95 $9.50+0.00

Distribution of Hydrocarbon-Degrading Microorganisms in Sediments from Prince William Sound, Alaska, following the Exxon Valdez O!! Spill
JOAN F. BRADDOCK*t, JON E. LINDSTROM* and EDWARD J. BROWN:~ *Institute of Arctic Biology, P.O. Box 757000, University of Alaska, Fairbanks, A K 99775, USA ~Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614, USA
tTo whom correspondence should be addressed.

Biodegradation by naturally occurring populations of microorganisms is a major mechanism for the removal of petroleum from the environment. Therefore, measurements of microbial populations are an important component of contaminated site assessment studies. Over a 3 year period following the T/V Exxon Vaidez oil spill in Prince William Sound, Alaska, we counted numbers of hydrocarbon-degrading microorganisms in intertidal and subtidal sediments affected by the spill. We found significantly higher numbers of hydrocarbon-degrading microorganisms at sites within the path of the oil slick than at reference sites, indicating rapid acclimation of the resident microbial populations. In offshore surface sediments, we saw a temporal increase in numbers of hydrocarbondegrading microorganisms. Our data suggest that microbial measurements are good indicators of exposure of sediments in Prince William Sound to hydrocarbons and of mobilization of oil to surface sediments offshore over time.

The grounding of the tanker vessel Exxon Valdez on Bligh Reef on 24 March 1989 released about 35 500 t of crude oil into the waters of Prince William Sound (PWS). The oil spread south-west by coastal circulation and winds to the shorelines of many islands within PWS and eventually into the Gulf of Alaska (Royer et al., 1990). Shortly after the grounding, the National Oceanic and Atmospheric Administration (NOAA) organized a multi-investigator survey cruise to document the extent of oil contamination of coastal habitats in Alaska. This first survey cruise was followed by five seasonal cruises over the next 2 years organized as a joint effort of NOAA and the Alaska Department of Environmental Conservation (ADEC). The purpose of these cruises was to document oil concentration distributions and assess the relative effects of the spill on various communities in intertidal and subtidal areas. Assessment of microbial populations was an important

component of the surveys since a major fate of petroleum contaminants in marine environments depends on the ability of microorganisms to use hydrocarbons as a source of carbon and energy (Leahy & Colwell, 1990). Several studies have been conducted following marine petroleum spills to document microbial populations and/or activity and have provided valuable insight on the responses of microorganisms in marine waters and sediments to acute petroleum contamination. Elevated heterotrophic bacterial populations were observed in oil-contaminated beach sands 2 years after the Metula spill in 1974 in the Straits of Magellan (Colwell et al., 1978) and an enrichment of the numbers of hydrocarbon-utilizing bacteria relative to total bacteria was observed in sediments collected 1 year after the Amoco Cadiz spill off the coast of France in 1978 (Ward et al., 1980). Microbial populations in sediment samples contaminated with crude oil by the Amoco Cadiz spill were able to degrade a wide variety of hydrocarbon fractions to different extents, while generally low degradation potentials were seen for hexadecane in samples collected from the Bahia Paraiso spill of jet and diesel fuel in 1989 off the coast of Antarctica (Karl, 1992). One study of an intentional spill (Baffin Island Spill Project) tracked the response and activity of heterotrophic microbial populations following both a crude oil spill and a chemically dispersed crude oil spill (Bunch, 1987). With the exception of the Baffin Island Spill Project these studies rely on data from a single set of samples collected from 6 weeks to 2 years following a petroleum spill. The legal requirements of the Natural Resource Damage Assessment process after the Exxon Valdez oil spill required collection of a large amount of information, providing an unprecedented quantity of data following an oil spill. We followed the response of microbial populations in sediments from many sites in PWS from about 3 months after the spill and then periodically over the course of the next 2 years. We believe this study is unique by virtue of the extensive 125

Marine Pollution Bulletin

data set that was generated from frequent sampling at a large number of sites after a major spill. Such data provide insights into the extent of both temporal and spatial microbial responses to an environmental perturbation. We sampled 38 sites within PWS over a 3 year period following the oil spill. In these samples we measured numbers of hydrocarbon-degrading microorganisms in shoreline sediments and in subtidal surface sediments offshore at depths of up to 100 m. We found statistically significant differences in the populations of hydrocarbon degraders in shoreline sediments collected from bays within the path of the oil slick relative to populations at reference sites which were not oiled during the Exxon Valdez spill. We also saw differences in populations of hydrocarbon degraders with time following the spill. These differences were observed despite mounting evidence that substantial quantities of older weathered oil may exist in Prince William Sound from other sources such as natural seeps (Page et al., 1993) or from spills (Kvenvolden et al., 1993) likely related to the 1964 earthquake (Kvenvolden as cited in the New York Times, p. C1, 1 December 1993). Measurements of numbers of hydrocarbon-degrading microorganisms provide evidence of the presence of less weathered oil that can be readily used by microorganisms and, therefore, may be useful in determining the extent, movement and persistence of labile hydrocarbon contamination following an oil spill. All of the data generated during this study are contained in a public report to the Exxon Valdez Oil Spill Trustee Council (Braddock et al., 1993) available from the Oil Spill Information Center librarian in Anchorage, Alaska.

TABLE 1 Locations of sites sampled. Date of cruise 01/07 to 22/08/1989 (summer 1989) 07/11 to 08/12/1989 (winter 1989) 31/05 to 10/06/1990 (spring 1990) 27/06 to 05/08/1990 (summer 1990) 05/09 to 15/09/1990 (fall 1990) 15/06 to 25/06/1991 (summer 1991) Site No. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) R(12) (13) (14) (15) (16) (17) (18) R(19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) R(30) R(31) (32) (33) R(34) (35) (36) (37) (38) (39) R(40) R = Reference site. Sites sampled 1,2, 3,4,5,6, 8,9, 10, 11, R12, 13, 14, 15, 16, 17, 18, R19, 39, R40 6, 8, 10, 13, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, r~30, a31, 32 1, 7, 8, 9, 10, 13, 14, 15, 17, 18, R19, 20, 21, 25, R30, 33, R34, 35 1,6,8, 10, 13, 14, 15, 17, 18, R19,20, 21, 25, R30, 33, R34, 35, 36, 37, 38 1, 4, 7, 8, 9, 13, 14, 15, 17, rtl9, 20, 21, 22, 25, R30, 33, 35, 38 6, 13, 14, 15, 17, 18, R19, 20, 22, 25, 33, R34, 36, 37, 38 Latitude 59058.43'N 6000.17'N 6006.52'N 6006.00'N 60 17.26'N 60 14.38'N 6015.77'N 60 16.30'N 6023.03'N 6031.79'N 6039.35'N 6059.00'N 6033.05'N 6029.93'N 6025.90'N 6026.82'N 6004.14'N 6020.28'N 6045.13'N 6031.82'N 6026.35'N 6022.62'N 6020.92"N 6009.80'N 6019.82'N 6026.55'N 6029.33'N 6031.68'N 6037.67'N 6050.28'N 6043.98'N 6041.80'N 5952.80'N 60 16.10'N 6050.70"N 60 12.50'N 6024.37'N 6019.20'N 6032.63'N 5956.20'N Longitude 14810.52'W 14758.90'W 14757.73'W 14759.84'W 14754.28'W 14743.1 I'W 14745.92'W 14726.29'W 14744.90'W 14720.8 I'W 14726.20'W 14701.38'W 14734.62'W 14739.51'W 14747.15'W 14758.48'W 14750.58'W 14708.15"W 14611.50"W 14736.44"W 14737.74'W 14742.45'W 14738.45'W 14745.35'W 14800.40'W 14744.00'W 14743.12'W 14740.03'W 14808.32'W 146 16.50'W 14634.40'W 14744.81 'W 14745.70'W 14705.92'W 14646.00'W 147 18.04'W 14747.82'W 14744.03'W 14543.60'W 149 19.00'W

Methods
Sampling Surface sediment (top 0-3 cm) samples were collected for microbial analysis on six cruises (see Table 1): R / V Fairweather (summer 1989), M/V Nautilus (winter 1989), R / V Cobb (spring 1990), R / V Davidson (summer 1990), R / V Cobb (fall 1990) and F/V Big Valley (summer 1991). The season designators for the cruises used here are meant to reflect the weather in Prince William Sound during the time of the cruise even though the dates of the cruise do not strictly follow traditional season definitions. During the three summer cruises surface sediments were collected in the intertidal zone (referred to as shoreline or 0 m) and at 3, 6, 20, 40 and 100 m depths offshore at mean low tide. On the other cruises surface sediment samples were collected only in the intertidal zone and at the shallower water depths offshore (winter 1989, 0 and 3 m; spring and fall 1990, 0, 3, 6 and 20 m), owing either to the restricted capabilities of the support vessel or the abbreviated cruise schedules for those sampling trips. Surface sediments at the 40 or 100 m depths were collected using either a Van Veen grab or a SmithMclntyre grab. Composite samples from three grabs were obtained by subsampling surface sediment (top 0 3 cm) into sterile Whirl-Pak bags. Samples at the 3, 6 126

and 20 m depths were collected by SCUBA divers while shoreline samples were collected by either SCUBA divers or a shore party in the low intertidal zone as close to low tide as was feasible. The intertidal (shoreline), 3, 6 and 20 m samples were composites of eight subsamples collected at random intervals along a 30 m transect parallel to the shoreline. Only one bag of sediment was collected for each site at each depth on the summer 1989 cruise while three replicate bags were collected at each depth for all subsequent cruises so that statistical procedures could be used to compare the

Volume 3 0 / N u m b e r 2/February 1995

study sites to reference sites. All sediment and water samples were placed in coolers at the time of collection for transport to the support vessel. Processing for microbiological analyses was performed on the support vessel within 3 h of collection. Owing to the prevalence of rocks and coarse-grained sediments at several locations and depths in PWS, we assayed samples from which all rocks greater than 1 cm in diameter were removed.

Direct counts of microorganisms The direct count method was used to determine the total population of microorganisms in sediments (summer 1989 cruise only). Ten grams (wet weight) of sediment were mixed with 90 ml of filtered (0.2 ~tm filter) saline solution. Duplicate aliquots were preserved in 3.7% formaldehyde, providing two replicates to be counted for each sediment sample (Montagna, 1982). The formaldehyde-fixed replicates were processed by sufficiently diluting with filtered water to provide between 30 and 100 microorganisms per field on a filter (Kirchman et al., 1982). They were then incubated for approximately 10 min with 5 ~tg m1-1 of the fluorochrome, 4'-6-diamidino-2-phenylindole dihydrochloride (DAPI) at 4C in the dark (Porter & Feig, 1980). The replicates were filtered onto pre-stained (Irgalan black) polycarbonate filters (0.2 ~tm, 25 mm diameter, Poretics Corporation, Livemore, CA) and counted at a magnification of 1250 with a Zeiss Standard microscope equipped with an epifluorescent light source. Ten fields per filter were counted (for a minimum of 300 cells) and the counts from replicates combined to provide 20 fields counted per sediment sample. A mean was determined for the 20 fields, corrected for background and multiplied by the appropriate dilution factors. Background microbial numbers were determined by running 10-ml blanks of stained filtered water with the samples. The average of all blanks run was 2 lO 4 cells m1-1. Most probable number of hydrocarbon-degrading microorganisms The number of hydrocarbon-degrading microorganisms in each sediment sample was estimated using the Sheen Screen most probable number technique (MPN; Brown & Braddock, 1990). While no technique to enumerate specific metabolic types of microorganisms in marine systems is absolute, the MPN technique can give consistent results that are appropriate for relative comparisons among stations and depths. Hydrocarbon-degrading microorganisms were defined as those microbes capable of dispersing a sterile Prudhoe Bay oil sheen layered on Bushnell-Haas marine mineral salts (Difco Laboratories, Detroit, MI) broth. On the summer 1989 cruise, duplicate sets of cell well plates were prepared for each subtidal depth at each site. For all other cruises one set of cell well plates was prepared from each replicate sediment sample at a given site and depth to yield triplicate values. The Sheen Screen plates were incubated at approximately 15C for 3 weeks before being scored for disruption of the oil sheen.

Data analysis The MPN data for hydrocarbon degraders at study sites were compared to reference site MPN data using non-parametric statistical analysis. The Mann-Whitney two-sample U Test (Zar, 1984) was used to determine whether the replicate sample data from each study site and depth were significantly greater (ct=0.05) than those from reference sites. We used this technique to examine the relative ranks of all the replicate data from the two classes (study vs reference) of samples compared. Three replicates from the study site/depth were compared with all replicates from the same depth at all reference sites visited during that cruise. This distribution-free method allows comparison of data which may not be normally distributed, but requires a minimum sample size of three for each class of samples compared. Thus, this analysis was not performed on the data from the summer 1989 cruise, as too few replicates were collected.

Results
The distribution of the surface slick of oil after the grounding on Bligh Reef was dependent on winds and currents and generally resulted in a spread of the oil to the south-west (Royer et al., 1990; Galt et al., 1991). By about 6 days after the spill the surface slick had heavily coated many shorelines around Knight Island and other smaller islands in that area (Fig. 1). The study sites sampled followed, in general, the path of the oil slick. Total numbers of microorganisms in surface sediments collected on the summer 1989 cruise varied slightly in samples collected from various depths, but did not differ between study sites and the designated reference sites (see Table 2). We observed that particle sizes generally decreased with distance offshore which may account for the slight differences in total numbers

Fig. 1 Distribution of E x x o n Valdez oil in Prince William Sound approximately 6 days after the spill which began when the tanker ran aground on Bligh Reef at midnight 24 March 1989. The figure is redrawn from output from the National Oceanic and Atmospheric Administration On-Scene Spill Model described in Gait et al. (1991 ).

127

Marine Pollution Bulletin

TABLE 2

Log median numbers of microorganisms in surface sediments in Prince William Sound as measured by direct count microscopy for the Summer 1989 cruise. Study sites Log median cells g-1 dry wt sediment 7.9 7.9 8.2 8.4 8.4 8.9 Log (HC degraders/ total cells) -3.7 -5.8 --6.2 < -7.1 < -7.1 < -7.6 Log median cells g-1 dry wt sediment 8.4 8.3 8.1 8.3 8.4 8.2 Reference sites Log (HC degraders/ total cells) --6.8 -7.0 --6.5 --6.3 < -7.1 < -6.9

Depth (m) Shore 3 6 20 40 100

Range 6.9-8.8 7.4-8.9 7.6-8.7 7.1-9.0 7.4-9.2 8.0-9.5

n 18 17 17 17 17 17

Range 7.0-8.8 7.6-8.9 7.3-8.6 8.2-8.6 8.2-8.8 8.2-9.2

n 3 3 3 3 3 3

Ranges of values at n n u m b e r of sites visited are also shown. In 1989 the ratio of median hydrocarbon degraders (see also Table 3) to median total numbers of microorganisms decreased with depth at the study sites.

of microorganisms enumerated. In contrast, numbers of hydrocarbon degraders did vary with depth and location. Thus, the ratio of hydrocarbon degraders to total number of cells also changed with depth and site. In summer 1989, a higher proportion of the total microbial population was able to degrade hydrocarbons in shoreline sediments than in surface samples collected at other depths (Table 2). The geographical distribution of hydrocarbondegrading microorganisms in shoreline sediments and 40 m subtidal surface sediments collected during the three summer cruises (1989-1991) exhibits a pattern with time and with respect to the path of the slick (Fig. 2(A)-(C); Fig. 3(A)-(C)). Numbers of hydrocarbondegrading microorganisms in shoreline sediments were greater than 10 000 cells g-i dry wt sediment at many sites within the path of the slick in the summer of 1989 (Fig. 2(A)). Reference sites for this cruise included Columbia Bay (site 12), which fell well outside the path of the slick and represented a site of low hydrocarbon contamination. The small boat harbour in Cordova (site 39), a site with chronic exposure to freshly spilled hydrocarbons, served as a site of known contamination and high populations of hydrocarbon degraders. In the summer of 1990 (Fig. 2(B)), hydrocarbon degrader populations in shoreline sediments were still high at many study sites (> 10 000 cells g-t sediment) relative to the reference sites (19, 30 and 34). By the summer of 1991 (Fig. 2(C)) all of the sites sampled had populations of hydrocarbon degraders below 10 000 cells g-1 of sediment. A different pattern was observed in the surface sediments collected at 40 m depth offshore (Fig. 3(A)-(C)). In sediments collected from 40 m in the summer of 1989 (Fig. 3(A)) numbers of hydrocarbon degraders in sediments were usually less than 100 cells g-~ of sediment. By 1990 many of these same sites had between 100 and 10 000 hydrocarbon degraders g-1 of sediment. In 1991 (Fig. 3(C)) populations of hydrocarbon degraders in surface sediments collected at 40 m were back below 100 cells g-~ of sediment at all but one of the sites sampled. These trends are reflected in median numbers of hydrocarbon-degrading organisms for all sites at each depth on a given cruise (Fig. 4; Table 3). The numbers of hydrocarbon degraders were highest in shoreline sediments in 1989, generally 128

diminishing with time. Hydrocarbon degrader populations at 20 m increased from less than 20 cells g-~ of sediment in summer 1989 to about 100 cells g-~ of sediment in subsequent cruises in 1990, declining again by 1991 to approximately 10 cells g-~ of sediment. Fewer data were collected for sediments from the 40 and 100 m depths offshore; however, it appears that there was also a peak in the numbers in summer 1990. The data from each cruise were analysed for statistical differences between values obtained at the study sites and at designated reference sites. The data from the summer of 1989 could not be analysed in this manner as fewer replicate samples were collected on this first cruise. Analysis of replicate samples collected at a given depth and site exhibited variation which was small compared with that inherent in a most probable number enumeration (Koch, 1994). The number of sites and depths where populations of hydrocarbondegrading microorganisms were significantly higher than the reference sites (ct=0.05, Mann-Whitney U Test; Zar, 1984) generally decreased with time after the spill (Table 4). For example, in fall 1989 all (100%) the shoreline sediments collected had greater numbers of hydrocarbon degraders than the reference sites; by the summer of 1991 only about half the sites had numbers greater than the reference sites. At all depths the proportion of oiled sites with populations greater than the reference sites were always higher in the summer of 1990 than in the summer of 1991.

Discussion
Microorganisms capable of degrading a variety of petroleum hydrocarbons are widespread in marine environments (Bartha & Atlas, 1987; Leahy & Colwell, 1990). Several studies have shown a positive correlation between the number of hydrocarbon-degrading microorganisms and oil pollution in marine systems contaminated by hydrocarbons (Colwell et al., 1978; Roubal and Atlas, 1978; Ward et al., 1980; Atlas et al., 1981; Lizarraga-Partida et al., 1991). We found that the numbers of hydrocarbon-degrading microorganisms vary by several orders of magnitude among sites sampled after the E x x o n Valdez oil spill and vary

Volume 30/Number 2/February 1995

/A

.I/

14-t"W

~ VALDEZ

IA

//

,,;0.
(~

oV,LO~
610N -

Ii0 Milel

I_+~ ~ _ 2 - I ~ ~" ..~' ;'"'0""'" L~_ ~ > ~ ~ ~ . . e , X ~ ~

ii ~:~

I_Y ~ ~<~~ ' ~ - "

I_~Q>~
/ / /11/~/~

~
~ I,

~7=.e,x~~
WILLIAM

(~ ~o.,........

,~

3=

AI ,~

,=
I

I0

WILLIAM

.=,N==-'- <-//X~.,
t8

CORDOV"~eA,~

~1~ lO

CORDOV~d

,,
5
"v 9

_---,0,5.@-5 ~..

~L..J

i919

~J'~

ill

~0.-

/B

/ /

,~

14-~'W
o

~ VALDEZ
p .~,..
0 Kilometerl

IB

/'/'/

147W

f,, VALDEZ

I~ Q6 ~
~ 5
,~.~-2o r

~.::~-~
souND .~-'~_X '

/~f)., , (5 ~
25

14~.~"20 t

,o

~,,,/,M

S OU N D

co.oo-~.
/ ju

3 8 . 1 "x ~ " .,, "

/C

/'/

' 14;'W

VALDEZ

/C

/7/

t4-tW

_ 0 vALDEZ

1 0'~1:2'2': r$

ldv/ft
/ =F~ ~

,,

.
~'~
,514"Le-2""
22

'

.~_P

WILLIAM
souND
18

CO,O0~
~ / > "

~_~ .e_~' 36.~-o,~//," "~ 1991 ~'/~,~, ~-~ Ic,,,,,g,,,,w,.s,,-,.

~f /S ~, =_17

A< > : ,~

'] Cells/g dry2wt" sed" '

I
v I

,o.-,o.

E~

~'%=~'4"

~'~
,

I ,o?,e i: " > l O

.<,o

.o..,

Fig. 2 Mean numbers of hydrocarbon degraders in shoreline sediments from Prince William Sound during the summer cruises in (A) 1989; (B) 1990; and (C) 1991. Reference site numbers are circled.

Fig. 3 Mean numbers of hydrocarbon degraders in subtidal surface sediments collected at 40 m during the summer cruises in (A) 1989; (B) 1990; and (C) 1991. Reference site numbers are circled.

129

Marine Pollution Bulletin

Summer 1989 Shore 3 I l l 6 m Winter 1989 l l l l Spring 1990 m l lmml
m
m

Summer 1990 1 / 1

Fall 1990 1 = l =
n
m

Summer 1991 1 mm
m
m

~ g
.C

20
40

< 1.3

~. Q Q

< 1.3

100

< 1.3 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

1 1 2 3 4 5 1 2 3 4

1 8 1 2 3 4 S

Logt0 median hydrocarbon d e g r a d e r s / g dry weight sediment

Fig. 4 Median most probable numbers of hydrocarbon degraders in surface sediments collected at non-reference sites in Prince William Sound during six seasonal cruises. Certain depths were only sampled on some cruises (indicated by the absence of a bar). TABLE 3 Summary of log medians of the most probable number (MPN) of hydrocarbon degraders for all study (non-reference) sites in Prince William Sound. Depth (m) Shore Cruise Summer 1989 (range) Winter 1989 (range) Spring 1990 (range) Summer 1990 (range) Fall 1990 (range) Summer 1991 (range) 4.2 ( < 1.3-6.3) 4.7 (3.3-5.8) 2.5 ( < 1.3-4.7) 3.1 (1.6-5.1) 2.3 ( < 1.3-5.7) 1.8 ( < 1.3-3.2) 3 6 20 40 100 Log median MPN hydrocarbon degraders g-I dry wt sediment 2.2 ( < 1.3-4.3) 3.5 (2.2-4.6) 2.4 ( < 1.3-3.8) 2.2 ( < 1.3-3.7) 2.4 ( < 1.3-3.9) 1.8 ( < 1.3-3.2) 1.9 ( < 1.3-3.6) 1.8 (1.5-2.8) 2.1 ( < 1.3-5.0) 2.4 ( < 1.3-4.3) 1.7 ( < 1.3-2.5) < 1.3 ( < 1.3-3.0) 2.3 ( < 1.3-3.3) 2.2 ( < 1.3-4.1) 2.5 ( < 1.3-3.4) < 1.3 ( < 1.3-2.5) < 1.3 ( < 1.3-2.0) < 1.3 ( < 1.3-1.5) 2.5 ( < 1.3-3.9) 1.8 ( < 1.3-3.8) < 1.3 ( < 1.3-2.7) < 1.3 ( < 1.3-3.0)

The ranges of the log MPN are displayed parenthetically below the median. The number of sites used in calculating the medians are found in Table 4. TABLE 4 Cruise summary data of sites and depths in Prince William Sound where the most probable number (MPN) of hydrocarbon-oxidizing microorganisms was significantly higher than that found at the reference sites (a = 0.05, Mann-Whitney U Test; see Methods). Depth Shore 3 6 20 40 100 Number of sites significantly higher/total number of sites sampled Cruise Winter 1989 Spring 1990 Summer 1990 Fall 1990 Summer 1991 % 100 15 69 73 58 (n) (19) (13) (16) (15) (12) % 89 56 86 31 33 (n) (18) (9) (14) (13) (12) % . 25 75 41 42 (n) . (4) (16) (17) (12) . % . 44 69 46 17 (n) . (9) (16) (13) (12) . % . 63 0 (n) (16) (12) % 47 0 (n)

(15) (12)

The numbers in the table are the percent of study sites which had values greater than the reference sites visited during that cruise. The number of study sites (n) included in each calculation is also shown.

between samples collected at study sites within the path of the oil slick and samples collected at reference sites. Microbial studies in Alaskan coastal sediments conducted before the Exxon Valdez oil spill are limited in number. Samples collected during a 1975-1977 survey of Cook Inlet and Gulf of Alaska sediments for 130

hydrocarbon-utilizing microorganisms produced values of about 0.6-12 colony forming units (CFU) g-1 of sediment. The highest mean numbers of hydrocarbon degraders measured were 8.4103 CFU g-1 of sediment at a site in upper Cook Inlet near several oil wells (Roubal & Atlas, 1978). These authors

Volume 30/Number 2/February 1995

hypothesized that sediments containing 103-104 oil degraders g-1 probably had a previous history of oil exposure from either biogenic or polluting sources. At the time of this 1978 study there were already several oil platforms producing oil in upper Cook Inlet where the high numbers of hydrocarbon oxidizers were found. Enrichment of the proportion of hydrocarbonutilizing bacteria in the microbial community was apparent after the Amoco Cadiz oil spill. Ward et al. (1980) found that, of a total of 107-108 bacteria g-1 of sediment, there were 104-107 hydrocarbon-utilizing bacteria g-i of sediment in oiled areas vs < 5 102 cells g-~ of sediment in unoiled areas. We found similar numbers for the relative proportion of hydrocarbon degraders in this study (see Table 2). The total numbers of microorganisms in sediments were relatively constant among sites for at least several months after contamination by oil from the Exxon Valdez spill, even though the number of hydrocarbon-degrading microorganisms in many of the sediment samples increased following exposure to oil (based on data from summer 1989). These results likely reflect shifts in the microbial population toward utilization of hydrocarbons following exposure to oil but could also be a result of other factors such as increased grazing of a more active bacterial population by protozoans. The numbers of hydrocarbon-degrading microorganisms vary by several orders of magnitude among sites and dates sampled after the Exxon Valdez oil spill (Figs 2 and 3). In the Summer of 1989 11 shoreline sites in PWS exceeded the maximum value for hydrocarbon degraders found in 1978 (Roubal & Atlas, 1978) as well as values measured at the reference sites for that cruise. In fall 1989, all of the oiled shoreline sites sampled had high numbers of hydrocarbon degraders, ranging from 3.6 X 103 to 5.5 105 cells g-~ of sediment; reference sites ranged from 20 to 70 cells g-~ of sediment. Statistically significant higher numbers of hydrocarbon degraders were observed at these oiled sites than at the reference sites (Table 4). Several oiled shorelines in PWS were sampled intensively during the summer of 1990 in a study designed to monitor the effectiveness of bioremediation techniques in stimulating microbial activity (Lindstrom et aL, 1991). The populations of hydrocarbon degraders measured on these shorelines ranged from about 104 to 106 cells g-~ of sediment from both treated and untreated plots. These values compare favourably with populations from shorelines measured in our study in 1990 (see Fig. 2(B)). Median numbers of hydrocarbon-degrading microorganisms on the shorelines in PWS decreased from 1989 to 1991 (Fig. 4). However, there were still several shorelines in the summer of 1991 that had > 103 hydrocarbon degraders g-I of sediment. In the summer of 1989, numbers of hydrocarbon degraders in subtidal surface sediments at depths > 6 m were below the detection limits of the assay ( < 20 cells g-~ of sediment). However, at some sites by the summer of 1990, there were measurable numbers of hydrocarbon degraders at all depths (shore through 100 in). These data indicate that hydrocarbon-utilizing microorganisms increased in surface sediments at depth over

time and provide indirect evidence of the mobilization of residual labile hydrocarbons to these sediments. Data from the summer of 1991 show a trend toward much lower total numbers of hydrocarbon degraders for all sites and depths, implying that conditions are no longer favourable for biodegradation or that biodegradable hydrocarbons are no longer present. It has been recently proposed that substantial quantities of hydrocarbons are naturally found in PWS derived from seeps in the eastern Gulf of Alaska (Page et al., 1993) and from refined products originating in California (Kvenvolden et al., 1993). This oil can be distinguished from Exxon Valdez oil by very expensive and time-consuming advanced chemical hydrocarbon fingerprinting techniques. These additional sources of oil in PWS confound the use of standard chemical analyses alone in the collection of data for damage assessment purposes. If this older weathered oil is present (in surface sediments) at our study sites then our data indicate that the older oil has not led to increased populations of hydrocarbon degraders. The response of microbial populations may then be a useful complement to hydrocarbon chemistry data in defining the extent of recent pollution occurring from a spill. In addition to measuring changes in the numbers of hydrocarbon-degrading microorganisms after an oil spill it is also important to assess the hydrocarbon biodegradation activity level of those populations. The pattern of microbial activity and population distributions can yield convincing evidence for in situ biodegradation (Madsen et al., 1991), a process otherwise difficult to document. The mineralization potential of a given hydrocarbon fraction by the natural microbial population is one measure of such activity (Bartha & Atlas, 1987; Leahy & Colwell, 1990). Many of the abiotic factors that can affect hydrocarbon mineralization can be standardized in the laboratory so that the relative importance of microbial biomass and acclimation of the community can be assessed. When physical and chemical factors are optimized, an increase in the number of hydrocarbon-degrading microorganisms and their level of acclimation to a given substrate will represent an increased potential for biodegradation of hydrocarbon contaminants (Bartha & Atlas, 1987). We measured mineralization potentials for several hydrocarbon fractions in conjunction with measuring microbial population numbers during this study with a procedure described by Brown et al. (1991). These data are published in a research report (Braddock, 1993). Additionally, another research group (NOAA/National Marine Fisheries Service Auke Bay, Alaska Laboratory) involved in documenting the extent of oil contamination collected sediments from the same sites for hydrocarbon chemistry data. We are currently working on merging these extensive data sets. The objective of this study was to document the effect of the Exxon Valdez oil spill on hydrocarbondegrading microorganism populations in PWS sediments. We found elevated numbers of hydrocarbon degraders in sediments collected in the path of the Exxon Valdez spill. In general, the populations were greatest in surface sediments collected from shoreline 131

Marine Pollution Bulletin

and nearshore shallow subtidal sites in 1989. In surface sediments collected at greater depths further offshore, maximum numbers were observed about 1 year after the spill (in 1990). As has been observed in other spills, the increase in numbers of hydrocarbon degraders compared with likely pre-spill values also provides evidence of rapid acclimation of naturally occurring microbial populations to biodegradation of these compounds in most sediments. The numbers and activity of these microorganisms are good indicators of exposure of PWS sediments to labile hydrocarbons and may be useful indicators of the mobilization of hydrocarbons with time. Microbial population and activity patterns and hydrocarbon levels should be measured periodically for several years following other major oil spill events to monitor transport of petroleum compounds in the environment.
The authors would like to acknowledge Tom Yeager, Brian Rasley, Greg Winter, and Mike Brockman for technical assistance and Doug Braddock for graphics. This work was supported by the National Oceanic and Atmospheric Administration contract 50-DSNC-8-00141 to Science Applications International Corporation and contracts from the Alaska Department of Environmental Conservation through the Natural Resource Damage Assessment process mandated under the Comprehensive Environmental Response, Compensation and Liability Act of 1980. We also acknowledge the support of the Water Research Center and Institute of Arctic Biology at the University of Alaska for their support as a service to the State of Alaska.

Atlas, R. M., Boehm, P. D. & Calder, J. A. (1981). Chemical and biological weathering of oil, from the Amoco Cadiz spillage, within the littoral zone. Estuar. Coastal ShelfSci. 12,589-608. Bartha, R. & Atlas, R. M. (1987). Transport and transformation of petroleum: biological processes. In The Long-term Effects of Offshore Oil and Gas Development: an Assessment and a Research Strategy (D. M. Boesch & N. N. Rabalais, eds), pp. 287-341. Elsevier Applied Science, New York. Braddock, J. F., Rasley, B. T., Yeager, T. R., Lindstrom, J. E. & Brown, E. J. (1993). Hydrocarbon mineralization potentials and microbial populations in marine sediments following the Exxon Valdez oil spill. Alaska Department of Environmental Conservation, Report, Anchorage, AK. Brown, E. J. & Braddock, J. E (1990). Sheen Screen, a miniaturized most probable number method for enumeration of oil-degrading microorganisms. Appl. Environ. Microbiol. 56, 3895-3896. Brown, E. J., Resnick, S. M., Rebstock, C., Luong, H. V. & Lindstrom, J. E. (1991). UAF radiorespirometric protocol for assessing hydro-

carbon mineralization potential in environmental samples. Biodegradation 2, 121-127. Bunch, J. N. (1987). Effects of petroleum releases on bacterial numbers and microheterotrophic activity in the water and sediment of an arctic marine ecosystem. Arctic 40, 172-183. Colwell, R. R., Mills, A. L., Walker, J. D., Garcia-Tello, E & Campos-E, V. (1978). Microbial ecology studies of the Metula spill in the Straits of Magellan. Z Ftsh. Res. Board Can. 35,573-580. Gait, J. A., Lehr, W. J. & Payton, D. L. (1991). Fate and transport of the Exxon Valdez oil spill. Environ. Sci. Technol. 25, 202-209. Karl, D. M. (1992). The grounding of the Bahia Paraiso: microbial ecology of the 1989 Antarctic spill. Microbiol. Ecol. 24, 77-89. Kirchman, D., Sigda, J., Kapuscinski, R. & Mitchell, R. (1982). Statistical analysis of the direct count method for enumerating bacteria. Appl. Environ. Microbiol. 44,376-382. Koch, A. L. (1994). Growth measurement. In Methods for General and Molecular Bacteriology (P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg, eds), pp. 248-277. American Society for Microbiology, Washington, DC. Kvenvolden, K. A., Hostettler, F. D., Rapp, J. B. & Carlson, P. R. (1993). Hydrocarbons in oil residues on beaches of islands of Prince William Sound, Alaska. Mar. Pollut. Bull. 26, 24-29. Leahy, J. G. & Colwell, R. R. (1990). Microbial degradation of hydrocarbons in the environment. Microbiol. Rev. 54, 305-315. Lindstrom, J. E., Prince, R. C., Clark, J. C., Grossman, M. J., Yeager, T. R., Braddock, J. E & Brown, E. J. (1991). Microbial populations and hydrocarbon biodegradation potentials in fertilized shoreline sediments affected by the T/V Exxon Valdez oil spill. Appl. Environ. Microbiol. 57, 2514-2522. Lizarraga-Partida, M. L., Izquierdo-Vicuna, E B. & Wong-Chang, I. (1991). Marine bacteria on the Campeche Bank oil field. Mar. Pollut. Bull. 22, 401-405. Madsen, E. L., Sinclair, J. L. & Ghiorse, W. C. (1991). In situ biodegradation: microbiological patterns in a contaminated aquifer. Science 252,830-834. Montagna, P. A. (1982). Sampling design and enumeration statistics for bacteria extracted from marine sediments. Appl. Environ. Microbiol. 43, 1366-1372. Page, D. S., Boehm, P. D., Douglas, G. S. & Bence, A. E. (1993). The natural petroleum hydrocarbon background in subtitdal sediments of Prince William Sound, Alaska. 089, p. 37 Abst 14th Annu. Meet. Soc. Environ. Toxic. Chem. 1993. Porter, K. G. & Feig, Y. S. (1980). The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25,943-948. Roubal, G. & Atlas, R. M. (1978). Distribution of hydrocarbonutilizing microorganisms and hydrocarbon biodegradation potentials in Alaska continental shelf areas. Appl. Environ. Microbiol. 35,897905. Royer, T. C., Vermersch, J. A., Weingartner, T. J., Niebauer, H. J. & Muench, R. D. (1990). Ocean circulation influencing the Exxon Valdez oil spill. Oceanography 3, 3-10. Ward, D. M., Atlas, R. M., Boehm, P. D. & Calder, J. A. (1980). Microbial biodegradation and chemical evolution of oil from the Amoco spill. Ambio 9, 277-283. Zar, J. H. (1984). Biostatistical analysis, 2nd edn. Prentice-Hall, Englewood Cliffs, NJ.

132