Evaluating the Biodegradability and Effects of Dispersed Oil using Arctic Test
Species and Conditions: Phase 2 Activities
Kelly M. McFarlin and Robert A. Perkins
Institute of Northern Engineering, University of Alaska Fairbanks
Fairbanks, Alaska, USA
raperkins@alaska.edu
William W. Gardiner and Jack D. Word
NewFields Northwest
Port Gamble, Washington, USA
Abstract
In the event of a marine oil spill, managers must decide on response actions
such as natural attenuation, mechanical recovery, in situ burning, and/or chemical
dispersion. Responders making those decisions need to know the relative toxicity of
physically and chemically dispersed fresh oil and the rates of biodegradation for fresh
and weathered oil. A Joint Industry Program was established in 2008 to collect these
parameters and this paper discusses the second phase of completed research. Phase 1
activities included determining species relevant to the Beaufort and Chukchi Sea
ecosystems, creating and setting up a toxicity and biodegradation laboratory with a
cold room in Barrow, Alaska, developing collection and culture methods for test
organisms, and developing toxicity and biodegradation test protocols. Phase 2 of the
project included the toxicity testing of the local environmentally significant species,
the copepod (C. glacialis), arctic cod (B. saida), and larval sculpin (Myoxocephalus
sp.). WAF and CE-WAF were made with fresh ANS following CROSERF protocols,
modified to represent site-specific conditions of Arctic open waters. Additional
toxicity tests were performed on the dispersant, Corexit 9500. Biodegradation of
chemically and physically dispersed fresh and weathered ANS petroleum was
measured by CO2 production in a respirometer and by GC/MS analysis.
1
Introduction
Dispersants have been used for many years to limit the impact of an oil spill
on various environmental components. While there are many studies that have
evaluated the potential biological effects of dispersants in temperate waters (Aurand
and Coelho, 2005), (Burridge and Shir, 1995), (George-Ares and Clark, 2000), little
information is available about such potential impacts in the cold waters of the Arctic.
A technical workshop was held in March 2008 in Anchorage, Alaska, to facilitate
discussions on various topics specific to the Arctic ( NewFields, 2008). The
discussion topics included the state of knowledge for food webs in the Beaufort and
Chukchi Seas, the behavior of oil dispersed by physical or chemical means in cold
waters, the biodegradation potential of dispersed oil in cold waters, and the potential
toxicity of dispersed oil to cold water species.
Experts in the fields of toxicology, Arctic biology, and petroleum
chemistry/biology from industry, government agencies, academia, and Alaska Native
communities participated in the workshop. As a result, the group identified the
following data gaps: 1) the toxicity of physically and chemically dispersed oil on
pelagic species that are key components of Arctic food-webs, and 2) the
biodegradation of physically and chemically dispersed oil released into pelagic waters
using indigenous Arctic microbes under Arctic conditions. To investigate these key
research areas, a Joint Industry Program (JIP) was formed with Shell, ExxonMobil,
Statoil and ConocoPhillips as the sponsoring members.
Toxicity tests were conducted with the copepod (Calanus glacialis), Arctic
cod (Boreogadus saida) and larval sculpin (Myoxocephalus sp.). The Arctic cod and
copepod were chosen because they are valuable ecosystem components of the Arctic.
These species play an important role at the base of the food webs in the Beaufort and
Chukchi Seas and may potentially come in contact with a spill event during ice free
periods. Larval sculpin were tested because they represented a sensitive life stage of
fish present in the Arctic. Native microbial communities occurring in the Beaufort
and Chukchi Seas were used for the biodegradation experiments.
Phase 1, which began in January 2009, focused on the development of an
appropriate laboratory space and specialized equipment, the development of coldwater protocols, the demonstration of the ability to collect and perform tests, as well
as the specialized training of personnel for safe operations in Arctic environments.
This work is summarized in McFarlin et al., 2010. Phase 2 began in August 2009
and ended in September 2010. This report summarizes the accomplishments of Phase
2.
2
Field Expeditions and Sampling Techniques
All field activities were conducted in either the Chukchi or Beaufort Seas
adjacent to Point Barrow, which is located near the town of Barrow, Alaska.
Figure 1. Location of Barrow and nearby seas.
Field collection of the target test organisms, the copepod (Calanus glacialis)
and the cod (Boreogadus saida), was a major effort during 2009 and 2010. Sculpin
(Myoxocephalus sp.) were captured in early larval stages and were used to provide
data on the relative sensitivity of an early life stage fish. Between July and October,
efforts to collect test organisms were intensified during the four-month window of
open-water. A combination of collection gear was used; a Sameoto net was used to
collect organisms at the surface and plankton nets were deployed at the surface and
subsurface. The nets were modified with a nylon lattice to exclude sea jellies
(medusae) which appeared to interfere with the health of the captured organisms.
Copepods are planktonic organisms that are carried by ocean currents.
Calanus copepods were all caught using small plankton nets. A plankton net fitted
with 80-250 micron mesh was lowered into the water column and then pulled
vertically back through the water column. The plankton net is attached to a bottlelike device at its end called a “cod end” where the sample is concentrated. Under
winter conditions, the plankton net is lowered down a hole drilled in the ice. Currents
are strong enough to pull the net into the water column. Once the drag line is fully
extended (3-5 m), the net is manually pulled up to the surface. Copepods captured
during the winter were primarily obtained in waters approximately 20 m deep. The
majority of copepods captured during the summer were found near the surface of the
water adjacent to icebergs floating over water depths ranging from 20-30 m.
Environmental clues such as the presence of sea birds or whales were used as an
indication of the location of the copepods.
Juvenile Arctic cod (100-130 mm long) were captured using a Fyke net
deployed in Elson lagoon, an area adjacent to an entrance channel near Plover Point
(Barrow, Alaska). Arctic cod seemed to be more abundant at this sampling location
when the winds were greater than 10 mph and blowing for more than 2 days from
directions ranging from north to west. These strong winds appeared to create a
current that pushed the more marine water from the Beaufort Sea into Elson lagoon.
It is hypothesized that the Arctic cod entered the lagoon with this current and then
distributed throughout the lagoon. The use of a Fyke net to capture and retain healthy
Arctic cod was a major improvement during the 2010 field season.
Larval sculpin were also caught in Elson lagoon, in a small bay adjacent to
Plover point. Larval sculpin were collected using beach seines which were ideal for
the capture of large numbers. Collected sculpin were 10-15 mm in length with a 2
week post yolk sac.
2.1
Maintenance of Test Organisms
All juvenile cod and larval sculpin were collected and cared for according to
procedures outlined in the approved UAF Institutional Animal Care and Use
Committee (IACUC) assurance. Prior to collection, 150 L Igloo coolers were filled
with ambient seawater. Collected organisms were immediately transferred to coolers
containing seawater for temporary storage. Coolers were transported to the lab and
were allowed to slowly acclimate to the cold room temperature. Once acclimated, the
organisms were transferred to aerated 10 gallon aquariums containing 80% filtered
(0.45 µm) seawater and 20% fresh non-filtered seawater. Water quality parameters
were measured daily and cultures containing Arctic cod were renewed (50%) with
filtered (0.45 µm) seawater every other day, while copepod cultures were renewed
once a week. Collected copepods were used to feed the arctic cod and sculpin
cultures, while copepods were fed 2 mL of 8-20 µm phytoplankton suspension
(Phytogold-M, Brightwell Aquatics, Catawiss, PA) every four days. Algal growth
within the aquariums was minimal due to the frequent renewals with filtered
seawater.
3
Toxicity Studies
Toxicity tests were conducted using fresh Alaska North Slope (ANS) crude
oil and the dispersant Corexit 9500. All test solutions were prepared in 20-L glass
aspirator bottles containing 16 L of aerated filtered seawater, with a headspace that
was 20% of the overall volume. The mixing velocity was set at a rate such that the
vortex occupied 20% to 25% of the total depth of the bottle. The test solutions were
mixed on stir plates for 18 hours, followed by a 6 hour resting period. All
maintenance and renewal water was 0.45-µm- filtered Chukchi or Beaufort Sea
water. All toxicity testing was conducted in a temperature-controlled cold room
maintained at 2°C (±0.5°C).
Physically dispersed oil preparations included a water-accommodated fraction
(WAF) and a more energetic physical dispersion termed a “breaking wave WAF” or
BW-WAF. The BW-WAF was prepared in a manner similar to the WAF. However,
in this case, the oil-water mixture was manually shaken by gently rocking the bulk
mixture for 30 seconds every 15 minutes during the first two hours of the mixing
period. The chemically-dispersed oil, or chemically-enhanced WAF (CE-WAF), was
prepared in a manner similar to the WAF, but with the addition of dispersant.
Stock solutions of WAF, BW-WAF, and CE-WAF were prepared with a
loading rate of 10 g/L ANS in filtered seawater. For the CE-WAF preparation,
Corexit 9500 was added at a DOR of 1:20. Test concentrations of the WAF, CEWAF, and BW-WAF were prepared by volumetrically diluting the 100% stock
solution in filtered seawater. All stock solutions were removed from the aspirator
bottles through the bottom spigot. Test solutions consisted of dilutions ranging from
1% to 100% of the stock solution and were gently and continuously stirred during
preparation to ensure that test solutions were well mixed. Once mixed, subsamples
were collected for chemical analysis and then aliquots were poured directly into
bioassay test chambers.
Toxicity tests were based upon the Chemical Response to Oil Spills:
Ecological Effects Research Forum (CROSERF) (Aurand and Coelho, 2005)
“spiked” exposure method which included an initial dosing at a given concentration,
followed by dilution using a continuous flow of clean seawater. The spiked exposure
was developed to simulate the spike in oil concentrations followed by dilution that
occurs from vertical and horizontal mixing and transport under natural conditions.
For these tests, spiked exposures were conducted with a half-life of approximately
four hours. In addition, tests were carried out in open systems to allow for
volatilization, as would occur naturally.
Toxicity tests included four replicates for each test concentration, with 10
organisms per chamber for the copepod tests and 5 organisms per chamber for the
fish tests. Initial samples of each dilution were analyzed for TPH by gas
chromatography and mass spectrometry (GC/MS). Daily water quality measurements
and mortality observations were recorded through 96 hours. At 96 hours, test
organisms were transferred to clean seawater and held for an additional observation
period of 8 to 12 days to account for potential delayed responses by the Arctic
organisms to an initial spiked stress (Chapman and Riddle, 2005). The selected
endpoints for evaluation were 4 and 12 days for copepods and 4 days for fish. The
longer endpoint for copepods was included after observing a continued response
following the 4 day extension. Minimal change in fish survival was observed over
the extended holding period.
4
Biodegradation Studies
The biodegradation tests were carried out in general agreement with OECD
(Organization for Economic Cooperation and Development) 301F guidelines for
biodegradation testing, with the exception of a single initial addition of low level
nutrients (0.5-1.0% of the recommended volume of Bushnell Haas Broth) in order to
provided approximately 0.05mM - 0.1mM of biologically available nitrogen and
0.07mM - 0.15mM of biologically available phosphorus. Nutrient addition is
necessary in a closed system in order to provide the small amount of nutrients
required for metabolism. Biodegradation experiments utilized ANS crude oil and the
chemical dispersant Corexit 9500 (Nalco Energy Services, Sugar Land, Texas) as the
sole carbon source.
Manometric respirometry combined with GC/MS analysis was used to
evaluate biodegradation. Manometric respirometry was used to measure oxygen
consumption in sealed treatment flasks provided with oxygen replacement.
Respirometry helps distinguish biological degradation of oil from chemical or
physical losses because it only measures the oxygen consumed from biological
activity. A Co-ordinated Environmental Services (CES) respirometer provided by
ExxonMobil Biomedical Sciences Inc. was used in all biodegradation experiments.
The closed respirometry system consists of twenty electrolytic cells, CO2 traps, and
1 L sample flasks all equipped with a stirring mechanism. As microorganisms
degrade the oil in the flask, they consume oxygen and release carbon dioxide. The
CO2 is absorbed in the sodium hydroxide trap, thus lowering the pressure in the
system. The resulting change in pressure triggers the electrolytic cell to generate a
measurable amount of oxygen by passing an electrical charge between electrodes in a
copper sulfate solution. This oxygen is then transferred to the sample flask and
becomes available to the microorganisms degrading the oil and the cycle restarts.
Two seasonal biodegradation tests were conducted to determine the ability of
indigenous Arctic marine microorganisms to degrade oil that has been dispersed
through physical or chemical means, that is, with or without the addition of a
chemical dispersant. The first experiment tested the biodegradation of fresh and
weathered ANS crude oil under fall conditions (Temperature = 2°C). The second test
represented spring conditions (Temperature = -1°C) and focused on the
biodegradation of weathered ANS crude oil. Positive control tests were also
conducted with sodium benzoate and peptone in order to verify equipment integrity
and strengthen methodological advances. In addition, a negative control comprised
of unamended seawater was setup to account for any background biodegradation.
It is important to note that the concentrations of oil and dispersant in the
respirometer studies are not representative of environmental conditions. In order to
elicit a measureable response in the respirometer, it was necessary for treatments to
contain concentrations that exceeded real-world measurements. Furthermore, the
closed, laminar respirometer system is not representative of natural conditions, as it
does not allow for other processes such as evaporation or high mixing energy due to
wave action. To circumvent the loading issue, a companion study was conducted in
parallel with the respirometer. This study utilized 4 L of sea water, 2.5 mg/L of oil
and Corexit 9500 was added at a dispersant to oil ratio (DOR) of 1:20.
The tests were terminated after 57 days and samples were collected for
chemical analysis. Each sample was extracted three times by a liquid-liquid
methylene chloride open container extraction technique. The extract was
concentrated by evaporation, dried and filtered by passage through a column of
sodium sulfate, and concentrated to a nominal concentration of 10mg/mL (initial oil).
Samples were analyzed by GC/MS as previously described (Douglas et al., 1992).
Hopane (17α(H), 21β(H)-hopane) was used as a conserved internal marker (Prince et
al. 1994), measured as m/z =191. This conserved internal marker approach was
developed during the response to the Exxon Valdez oil spill (Bragg et al., 1994;
Butler et al., 1991), and was subsequently validated (Prince and Douglas, 2005).
While it is true that this molecule can be biodegraded (Huesemann et al., 2003), it is
among the last of the resolved and identified hydrocarbons to be biodegraded. If it
were significantly biodegraded, the resulting amount of calculated biodegradation
would be a conservative underestimate.
5
5.1
Test Results
Toxicity Test Results
The loading rate or nominal concentrations of petroleum in preparations of
WAF, BW-WAF, or CE-WAF did not provide an accurate representation of actual
petroleum constituents or concentrations measured in test solutions. Our results
indicate that chemical analysis of initial concentrations is necessary to obtain accurate
assessments of chemical components in the water and consequent toxicity. Based on
the chemical analysis of the initial concentrations of WAF, BW-WAF, or CE-WAF,
volumetric dilution was then found to provide highly predictable exposure
concentrations in each dilution series.
Toxic responses of calanoid copepods were manifested over a 12-day period
rather than 4 days typical of temperate species. Juvenile and larval fish (Boreogadus
saida and Myoxocephalus sp.) responded over the standard 4-day test period for a
spiked toxicant test. Larval sculpin (Myoxocephalus sp.) were more sensitive to
petroleum mixtures than were Arctic cod juveniles. Early season copepods (JulyAugust) were equally sensitive as larval sculpin; late season copepods (September –
November) were equally sensitive as Arctic cod.
Toxicity in the copepod test varied between the early and late season
copepods (Table 1). For early season copepods, the mean of the CE-WAF
concentration lethal to 50% of the test organisms (LC50) was 22 mg/L TPH. Late
season copepods were less sensitive to CE-WAF with a mean LC50 of 62 mg/L TPH.
LC50s for the WAF were not calculable because there was insufficient TPH (<1.0
mg/L) in the water column to elicit a response. LC50s for the BW-WAF preparations
ranged from 2.2 to >5.5 mg/L TPH, with a mean above 3.7 mg/L TPH.
Table 1. Summary of LC50 Results for Copepod Tests (mg/L TPH).
“Late Season”
“Early Season”
Test
CE-WAF
WAF
Test
CE-WAF WAF
Mean
62
-Mean
22
-(SD)
(21)
(9.5)
BW-WAF
3.7
(1.1)
For larval sculpin the mean LC50 of CE-WAF was 27 mg/L TPH. The sculpin
mean LC50 for BW-WAF was 4.0 mg/L TPH (Table 2). Juvenile Arctic cod were
less sensitive to CE-WAF than larval sculpin with a mean LC50 of 55 mg/L. As with
copepods and sculpin, the Arctic cod LC50s for physically dispersed oil were
substantially lower than those of the chemically dispersed petroleum, with WAF and
BW-WAF LC50s ranging from 1.2 to 5.7 mg/L TPH.
Table 2. Summary of LC50 Results for Fish Studies (mg/L TPH).
Arctic Cod
Sculpin
CEBWCEWAF
WAF
WAF
WAF
WAF
Mean
55
1.6
3.3
Mean
27
2.2
(SD)
(17)
(0.4)
(2.2)
(SD)
(13.5)
(1.0)
BW-WAF
4.0
(1.7)
The degree of lethality demonstrated by Arctic copepods and fish (juveniles
and larvae) is found to be comparable to that of temperate species when exposed to
chemically or physically dispersed oil (Aurand and Coelho, 2005). Experiments with
chemical dispersants (Corexit 9500) did not increase the toxicity of fresh oil in
seawater. Results indicate that chemically dispersed petroleum is less toxic than the
physically dispersed petroleum per unit of measured oil.
5.2
Biodegradation Results
Petroleum biodegradation in pelagic waters was found to occur under Arctic
conditions (-1°C and +2°C) with indigenous Arctic microbes in natural seawater
collected from the Chukchi Sea. The results of the first biodegradation experiment
which tested the biodegradation of fresh ANS crude oil under fall conditions (2°C)
are shown in Table 3. The results of the second biodegradation test which focused on
the biodegradation of weathered ANS crude oil under spring conditions (-1°C) are
shown in Table 4.
Table 3. Comparison of Biodegradation as measured with GC/MS (primary
biodegradation) and respirometry (mineralization) containing 10 mg/L fresh crude
oil, Corexit 9500 (1:20 DOR) and 1% of recommended volume of Bushnell Haas at
2°C.
Treatment
Primary Biodegradation, % Mineralization, %
Fresh Oil
37
12
Fresh Oil + Corexit
56
27
Fresh Oil + Corexit + Nutrients
66
37
Table 4. Comparison of Biodegradation as measured with GC/MS (primary
biodegradation) and respirometry (mineralization) containing 12 mg/L of 20%
weathered crude oil, with and without Corexit 9500 (1:20), at -1°C. All treatments
contained 0.5% of recommended volume of Bushnell Haas.
Treatment
Primary Biodegradation, % Mineralization, %
20% Weathered Oil
46
19
20% Weathered Oil + Corexit
55
19
The results demonstrate that dispersants do not reduce biodegradation, but
rather provided equal or greater amounts of petroleum biodegradation. In both tests,
primary biodegradation exceeds mineralization. This is expected, as primary
biodegradation is the initial oxidation of the detectable hydrocarbons and
mineralization is the complete breakdown of ANS to carbon dioxide and water. The
addition of Corexit 9500 to fresh oil in fall conditions increased the primary
biodegradation and mineralization by 19% and 15%, respectively (Table 1).
Furthermore, the addition of Corexit 9500 and 1% of the recommend volume of
Bushnell Haas increased the primary biodegradation and mineralization of fresh oil
by 29% and 25%, respectively (Table 1). The first test suggests that a low level
nutrient addition is necessary to allow the microorganisms the nutrients to perform
basic metabolic processes, thus preventing nutrient limitations. All subsequent
biodegradation tests included the addition of 0.5% of the recommended volume of
Bushnell Haas. The addition of Corexit 9500 to 20% weathered crude oil in spring
conditions increased the primary biodegradation of TPH by 9% (Table 2). The
addition of Corexit 9500 did not impact the percent of petroleum compounds that
were fully degraded to carbon dioxide and water; both treatments using weathered oil
experienced a mineralization of 19%.
6
Conclusions
Results from the biodegradation studies demonstrated that microbes
indigenous to the Arctic biodegrade both chemically and physically dispersed oil
under Arctic conditions. The experiments also demonstrated that dispersants do not
interfere with the extent of microbial biodegradation. Furthermore, fresh oil that is
chemically dispersed undergoes more complete primary biodegradation and
mineralization than physically dispersed or weathered petroleum over an 8-week
testing period. Results from toxicity testing indicate that chemically dispersed
petroleum is less toxic than physically dispersed petroleum (per measured unit of oil)
indicating that more oil, containing less acutely toxic components, are introduced into
the water column with dispersant application. The key species selected for toxicity
evaluations are comparably sensitive to non-Arctic test species when exposed to
similar contaminant concentrations under similar test conditions.
7
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