**Preassessment Data Report #10**

**Chronic exposure of seaducks to oil released by the**

***Selendang Ayu* at Unalaska Island**

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Paul L. Flint ^1^

Jason L. Schamber ^1^

Kimberly A Trust ^2^

A. Keith Miles ^3^

Barry W. Wilson ^4^

*^1^ U.S. Geological Survey-Alaska Science Center, 1011 East Tudor Road,
Anchorage, AK 99503*

*^2^ U.S. Fish and Wildlife Service, Anchorage Fish & Wildlife Field
Office, 605 West 4^th^ Avenue, Room G62, Anchorage, AK 99501*

*^3^ U. S. Geological Survey, Western Ecological Research Center, Davis
Field Station, 1 Shields Ave., University of California, Davis, CA 95616
USA*

*^4^ Animal Sciences and Environmental Toxicology, 1 Shields Avenue,
University of California, Davis, CA 95616 USA*

**Executive Summary**

On 8 December 2004, the M/V *Selendang Ayu* ran aground and broke in
half in rough seas off Unalaska Island, Alaska (53°38\'N, 167° 07\'W).
An estimated 354,218 gallons of oil (339,538 gallons of bunker oil \[IFO
380\] and 14,680 gallons of marine diesel and miscellaneous oils) were
released. In the weeks following the spill numerous bird carcasses were
recovered. In addition, aerial surveys assessed the abundance of live
birds using the oiled area. Our goal was to determine the acute and
chronic exposure of these live birds to hydrocarbons associated with
this spill. In 2005 and 2006, harlequin ducks (*Histrionicus
histrionicus*) were captured in three oiled (Skan, Humpback and Portage)
bays and one minimally oiled reference (Chernofski) bay. Liver biopsies
were surgically obtained and birds were released at the capture sites.
The biopsy samples were analyzed for induction of the cytochrome P450 IA
gene (P450), an indicator of hydrocarbon exposure. In 2005 and 2006 the
P450 values of ducks in oiled locations were significantly higher than
those in Chernofski. The results suggest that harlequin ducks were
exposed to hydrocarbons more than a year after the spill. Although it is
not known how long these effects will persist, studies following the
*Exxon Valdez* suggest that they may persist for years. Elevated P450 in
harlequin ducks has previously been associated with reductions in
survival of female ducks following exposure to *Exxon Valdez* oil.

**Introduction**

On 8 December 2004, the *Selendang Ayu* ran aground and broke in half in
rough seas off Unalaska Island, Alaska (53°38\'N, 167° 07\'W). An
estimated 354,218 gallons of oil (339,538 gallons of bunker oil \[IFO
380\] and 14,680 gallons of marine diesel and miscellaneous oils) were
released. In the weeks following the spill numerous bird carcasses were
recovered despite logistical constraints and the large spill zone. In
addition, aerial surveys assessed the abundance of live birds using the
oiled area.

During oil spill response and natural resource damage preassessment
activities, recovery of oiled carcasses and observations of oiled
individuals established that certain bird species were acutely exposed
to *Selendang* oil. Because carcass collection and bird observations
were limited by logistical constraints, poor observation conditions, and
other factors, the trustees initiated an examination of exposure levels
to determine if additional species, such as the threatened Steller's
eider, had been injured. In addition, re-wash of beached oil, subsequent
oil releases from the wreck, and the chance that oil mixed with
sediments was entrained in the nearshore environment necessitated an
examination to determine if bird injuries likely extended beyond those
caused by the initial oil release.

Extensive studies of harlequin ducks in Prince William Sound following
the *Exxon Valdez* oil spill revealed that these ducks have high site
fidelity and feed on intertidal resources making them susceptible to
continuing oil exposure. The research has shown that chronic exposure to
oil can have long-term deleterious effects on survival of harlequin
ducks (*Histrionicus histrionicus*) (Esler et al. 2002). Accordingly,
chronic exposure to sub-lethal levels of oil may have negative effects
on population recovery following an oil spill.

We used Cytochrome P450 induction levels in harlequin duck liver as a
biomarker to determine if seaducks were being exposed to PAH in the
*Selendang Ayu* spill area in the 2 years following the spill. The
results are interpreted in regard to geographic and temporal variation
in exposure.

**Methods**

[Field Capture and Surgery]{.underline}: Harlequin ducks were captured
using mist nets and decoys following standard capture methods (Kaiser et
al. 1995). Birds were captured in four locations: Portage Bay, Skan Bay,
Humpback Bay, and Chernofski Harbor (Figures 1 and 2). According to
shoreline surveys conducted following the spill, these bays differed in
the degree to which they were oiled (Figures 1 and 2). Chernofski Harbor
was originally selected as an unoiled reference site, but was later
found to have been oiled; although to a lesser degree than the other
three bays.

After capture, birds were transported a short distance to a mobile field
laboratory where they were banded and examined. Their weight, sex, and
age (adult or immature) were recorded. A biopsy of liver (approximately
0.05 g) was then extracted from healthy birds according to established
procedure (Mulcahy, DVM, Alaska Science Center, Anchorage, unpublished
guidelines). The liver samples were immediately frozen and stored in
liquid nitrogen and then retained at --80°C until processing. After
suitable recovery, as determined by the veterinarian, birds were
released near the capture site.

[Laboratory Assays:]{.underline} Approximately 4 mg (wet weight) of
liver per bird was analyzed for EROD activity (i.e., P 450 induction) at
the Department of Animal Sciences, University of California, Davis. For
quality assurance and quality control, 5 embryonated mallard (*Anas
platyrhynchos*) duck eggs were injected with 2 mg/egg of
beta-napthoflavone (BNF), a known P450 inducer, and their livers assayed
at 24 hours. Similarly, livers from 5 undosed embryonated mallard eggs
were run as baseline controls. Liver samples were prepared by separating
the hepatic microsomes using differential centrifugation. Microsome
preparations were prepared from livers homogenized in 0.1 M NaPO~4~
buffer at pH 7.4, centrifuged at 100,000 x g for 1 hour, and then
resuspended (ml/g tissue) in 50 mM Tris solution that contained 1 mM
EDTA, 1 mM DTT, and 20% v/v glycerol at pH 7.4. EROD activity was
measured as described by Trust et al. (2000) according to the method of
Bruke and Mayer (1974), adapted to a fluorescence microwell plate
scanner (Melancon 1996). Modifications were determined in triplicate in
a 96 well plate at 25°C using a Packard FluoroCount microplate
fluorometer (Packard Instrument Company, Meriden, CT). Each well
contained 1 μl of microsomes, 159 ul of 2.5 uM final concentration
7-ethoxyresorufin in 50 mM Tris-buffer at pH 8.0. The addition of 40 ul
of 1.34 mM final concentration of NADPH initiated activity. Fluorescence
was measured at excitation wavelength of 530 and emission wavelength of
590 nm at 1-minute intervals for 6 minutes. EROD was expressed as
picomoles per minute per milligram of protein (pmol/min/mg protein).
Protein was determined using the Bradford reagent (Sigma, St. Louis,
MO).

[Analyses:]{.underline} The ratio of EROD activity in dosed versus
control eggs was calculated for each year's sample. If the ratios
differed then we assumed that the absolute values of EROD activity were
not directly comparable among years. The P450 data from wild birds were
highly skewed with a few very large values, so we log transformed those
data prior to analyses to improve normality.

Randomization procedures were used to assess the probability that EROD
activity differed in oiled bays compared to Chernofski. We calculated
the difference in mean log P450 values between oiled and unoiled
locations. We then randomly reassigned individuals to a sampling
location, without replacement, such that each location retained its
original sample size. We re-calculated the difference in mean log P450
values among locations. We repeated this process 1000 times and we
report the *P*-value as the proportion of random trials with greater
differences between oiled and unoiled locations than observed in the
actual data + 1 divided by the number of trial + 1 (Davidson and Hinkley
1997). Each year was analyzed separately. Conditional on the sample of
birds captured, this proportion represents the probability that the
variation observed among sample areas occurred by random chance. All
analyses were conducted using the Poptools Macro within Microsoft Excel.

Harlequin duck captures were attempted in four locations: Portage Bay,
Skan Bay, Humpback Bay, and Chernofski Harbor (Figures 1 and 2).
According to shoreline surveys conducted following the spill, these bays
differed in the degree to which they were oiled (Figures 1 and 2).
Chernofski Harbor was originally selected as an unoiled reference site,
but was later found to have been oiled; although to a lesser degree than
the other three bays. Age and sex ratios as well as mean body mass of
birds captured was compared among birds captured in the oiled bays and
Chernofski using randomization procedures as described above.

**Results**

*Capture:* One hundred-twenty harlequin ducks were captured in and
around the spill zone (Figures 1 and 2). A total of sixty birds were
captured in three bays with moderate to heavy oil (Skan, Humpback and
Portage) and 60 birds were captured in the minimally oiled reference
location Chernofski Harbor. There was no difference in the age and sex
ratios, or body weights, between birds captured in oiled and unoiled
locations in 2005. There was a lower proportion of adult birds captured
in the oiled areas (0.73) compared to Chernofski (0.96) in 2006, but
there were no differences in sex ratios or body weights or age ratios in
2005.

*Cytochrome P450:* The measured enzyme (or P450) response of dosed
mallard eggs varied among years. In 2005 the ratio of EROD activity in
dosed to control eggs was 62. In 2006, the ratio was only 24.2.
Therefore, we concluded that the absolute values of EROD activity were
not directly comparable among years.

Samples analyzed for EROD activity to date include: 60 from oiled areas
(n = 22 from Skan, n = 18 from Humpback, and n = 20 from Portage) and 58
from Chernofski Harbor. We deleted two extreme samples from Chernofski
as outliers: one value was more than double the next nearest value and
the other point was a negative value; both points were suspected to be
laboratory errors and were having a disproportionate effect on the
results. Additionally, two samples from Chernofski were damaged during
preparation; thus statistical analyses for Chernofski were based on 56
samples.

There was a significant difference in log EROD activity between oiled
and unoiled areas in both years (2005: *P* \< 0.001, 2006: *P* \< 0.003)
(Figure 3).

**Discussion**

Because the dosed egg standards did not yield equivalent results across
years we assumed that absolute values of EROD activity were not directly
comparable among years. Thus, all analyses were based on P450 values
between oiled and reference sampling locations within years. This
accounted for any systematic bias in laboratory procedures between
years.

The study was designed to determine sublethal hydrocarbon exposure of
sea ducks within the *Selendang Ayu* spill zone. Harlequin ducks were
observed in all areas and were captured consistently from all study
bays. The study design utilized a control/exposed comparison where birds
captured in oiled area were compared with birds captured in a single
"reference" area within years. However, after the study was initiated,
oil was observed on and around Chernofski Harbor. Thus, birds using this
area may have also been exposed to hydrocarbons from the *Selendang
Ayu*. The effect of such exposure would decrease the relative difference
between the oiled and reference bays in our statistical analyses, thus
making true differences more difficult to detect. Given that higher
levels of P450 induction were still detected in the more heavily oiled
bays compared to the oiled 'reference', sampling birds in an unoiled
reference area would only increase the magnitude of the oiling relative
effect. So while we likely underestimated the true magnitude of the P450
response resulting from exposure to *Selendang Ayu* oil, an effect is
nonetheless clear.

The control/exposed study design assumes that all other potential
factors influencing the variable of interest (i.e., P450 induction in
this case) are equal across sampling areas such that the only difference
between the control and exposed sample is the effects of the *Selendang
Ayu* spill. Evidence of non-*Selendang Ayu* oil was found in both the
oiled bays as well as Chernofski (see source allocation of tarball
samples) and we assumed that these background levels of exposure were
equivalent among areas. The age and sex ratios, as well as body weights,
of birds captured in each area were comparable with the exception that
more hatch year birds were captured in the oiled bays compared to
Chernofski in 2006. Given the comparable sex ratios and body weights
among sampling areas in 2006, we doubt this difference in age ratios
would cause us to reach an erroneous conclusion. In the absence of any
data regarding age and sex specificity in P450 response levels in birds,
we assumed that samples were equivalent across control and exposed
areas.

Harlequin ducks in the spill zone were exposed to hydrocarbons at
significantly higher levels than harlequin ducks in a minimally oiled
bay immediately following the spill (Figure 3). Thus, in addition to
birds killed in the initial spill event (i.e., carcasses found on
beaches), oil from the *Selendang Ayu* spill appears to have continued
to affect other birds using the spill areas. Studies in Prince William
Sound following the *Exxon* *Valdez* spill indicate that exposure
resulting in elevated P450 levels correlate with reduced over-winter
survival of harlequin duck females (Esler et al. 2002). Thus, mortality
estimates based solely on dead birds found immediately after the spill
event likely underestimate the magnitude of the overall injury as birds
chronically exposed to low levels of hydrocarbons may also suffer
increased mortality.

Harlequin ducks can be exposed to oil long after it is no longer visible
on the surface of beaches. For example, harlequin ducks were still being
exposed to *Exxon* *Valdez* oil, sixteen years after the spill (Trust et
al 2000; Esler et al. 2002). More than a year following the wreck of the
*Selendang Ayu*, levels of P450 inductions were still significantly
higher in oiled bays compared to Chernofski. In spite of clean-up
efforts in the oiled bays, birds utilizing these habitats were
apparently still being exposed to hydrocarbons from the *Selendang Ayu*
more than a year following the spill. Given the known effects of oil
exposure on survival of harlequin ducks (Esler et al. 2002), we
hypothesize that harlequin ducks were still dying as a result of
exposure to hydrocarbons released by the *Selendang Ayu* more than a
year after the spill. Although it is not known how long harlequin ducks
in the spill area will be exposed to *Selendang* oil, based on studies
conducted in response to other spills, and depending on factors such as
the geomorphology of beaches and oil toxicity, the spill effects may
persist for years.

**Acknowledgements**

We thank our field crew: Tyson Alteri, Scott Byrne, Joseph Connor, Ellen
Lance, Beth Pattinson, John Reed, and Joseph Seyfried. Alaska Wildlife
and Wildfowl Conservation conducted the surgeries and biopsies. We also
thank the captain and crew of the M/V *Exito* in 2005 and the M/V
*Kittiwake II* in 2006 for safe passage through bays of Unalaska Island.
We thank Dr. Deborah Rocque and Dr. Joel Reynolds for comments on this
manuscript.

**References**

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assay of a microsomal O-dealkylation which is preferentially inducible
by 3-methylcholanthrene. Drug Metabolism and Disposition 2:583-588.

Davidson, A. C., and D. V. Hinkley. 1997. Bootstrap methods and their
application. Cambridge University Press.

Esler, D., J. A. Schmutz, R. L. Jarvis and D. M. Mulcahy. 2002. Winter
survival of adult female Harlequin Ducks in relation to history of
contamination by the Exxon Valdez oil spill. Journal of Wildlife
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Kaiser, G. W., A. E. Derosher, S. Crawford, M. J. Gill, and I. A.
Manley. 1995. A capture technique for Marbled Murrelets in coastal
inlets. Journal of Field Ornithology 66:321-333.

Melancon M. J. 1996. Development of cytochrome P450 in avian species as
a biomarker for environmental contaminant exposure and effect.
Procedures and baseline values. In: Bengston, D.A. and Hensel, D.S.
(eds) Environmental toxicology and risk assessment: biomarkers and risk
assessment. Vol. 5.

Trust, K. A., D. Esler, B. R. Woodin and J. J. Stegeman. 2000.
Cytochrome P450 1A induction in sea ducks inhabiting nearshore areas of
Prince William Sound, Alaska. Marine Pollution Bulletin 40:397-403.

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Figure 3: P450 induction was significantly higher in oiled locations
than in Chernofski Harbor ("reference") (P \< 0.003) in both 2005 and
2006. Due to differences in results for laboratory standards, absolute
values of P450 inductions are not directly comparable among years.