574 (CUL-1117-742070) Behavioral effects on the salt marsh fiddler crab, Uca pugnax, chronically exposed to petroleum hydrocarbons.
Start time: 1:50 PM
Culbertson, J¹, Valiela, I¹, Peacock, E², Reddy, C², Carter, A³, ¹ Boston University Marine Program/Marine Biological Laboratory, Woods Hole, MA, United States² Woods Hole Oceanographic Institution, Woods Hole, MA, United States³ East Tennessee State University, Johnson City, TN, United States
Over thirty years after the barge Florida spilled approximately 700,000 L of No. 2 fuel there is still substantial undegraded residue at a depth of 10-14 cm. within salt marsh sediments of Wild Harbor, MA. Although the salt marsh vegetation seems to be recovered, fiddler crabs, Uca pugnax, which burrow into the sediments, could be exposed to the petroleum residue. To test for possible effects of exposure to the buried oil, we assessed feeding rates of populations of U. pugnax from Wild Harbor and a control site, Great Sippewissett marsh. Crabs from both marshes were exposed to contaminated sediments for two weeks, and we then measured fecal pellet production (as a proxy for feeding rate). Fecal pellet production was significantly lower with exposure to oiled sediments. Another indication of the response of crabs to the oil was that casts of fiddler crab burrows indicated significantly abnormal burrow construction in oiled areas versus non-oiled areas. Burrows from Wild Harbor were, on average, 45% shorter in length then those from Great Sippewissett, and often turned laterally at depths greater than 10 cm., sometimes even turning upwards. Many years after the spill, the residual oil still appears to have measurable effects on fiddler crab populations.

575 (BOE-1118-205730) Combined Analytical and Comprehensive Sampling Approaches to Determine Sources of PAHs in Prince William Sound.
Start time: 2:10 PM
Boehm, Paul¹, Page, David², Bence, A. Edward³, ¹ Exponent, Inc., Maynard, MA, USA² Bowdoin College, Brunswick, ME, USA³ ExxonMobil Upstream Research, Houston, TX, USA
The NRDA CERCLA Regulation describes how oil spill injury assessment must be performed. They establish the rationale for conducting chemical fingerprinting and considering all hydrocarbon sources other than the just the spill. Fingerprinting methodology is both qualitative (source recognition) and quantitative (source allocation). Here we discuss the evolution of this methodological approach as applied to the Exxon Valdez oil spill (EVOS) investigations. Hydrocarbon fingerprinting was used to recognize the spill oil at various weathering states, to define the pre-existing baseline, and to quantitatively resolve multiple sources contributing to environmental samples. Steps included determining: a) composition of the oil and weathering trends; b) identification of the non-EVOS natural petrogenic background; c) identification of anthropogenic sources not related to the spill. Analysis of hundreds of samples of potential sources and environmental assemblages of PAHs formed the backbone of the effort. Source apportionment methodology (Principal Components Analysis; Partial Least Squares; TOC methodology apportionment from multiple sources) were used to analyze the data. These analyses supported the overall exposure assessment which involved: discrimination of sources of any exposure; evaluation of the bioavailability of the oil compared to background; evaluation the dose-response paradigm (e.g., sediment toxicity); and overall to relate to the biological effects of the spill. Results support the conclusions that weathering and removal rates of oil and the toxic fraction of the PAHs were rapid from the vast majority of shorelines in the Sound; toxicity of any oil residues rapidly declined as the oil weathered and became insignificant; bioavailability of EVOS oil residues decreased rapidly over time and by 1998 the background levels of non-EVOS bioavailable PAHs was the dominant factor in the Sound; any possible chemical exposure from EVOS could not be distinguished from that of the background. These results further support the likely explanation of bile FAC/CYP1A studies as performed by numerous investigators. Such non-source specific measures of exposure can be largely attributed to the non-EVOS background in the Sound and not to the residual minor amounts of buried EVOS oil.

576 (AAA-1117-571215) Predicting and Comparing the Narcotic Potential of Neat and Weathered Crude Oil: Is A New Paradigm Needed?
Start time: 2:30 PM
Di Toro, D^{1, 2}, McGrath, J², Stubblefield, W³, ¹ University of Delaware² HydroQual, Inc³ Oregon State University
The toxicity of the components of oils can be understood using the concept of narcotic potential. It is the toxicity of a solution at the water solubility of each component of the oil. Using the target lipid model (TLM) of narcotic toxicity, and the observed relationship of the solubility of oil components to log (K_OW), it is demonstrated that components with lower log (K_OW) have greater narcotic potential than those with higher log (K_OW). The total toxic unit concentration in an aqueous phase in equilibrium with the oil is shown to be weighted sum of the narcotic potentials of the components, where the weights are the component mole fraction concentrations in the oil. Weathering removes the lower log (K_OW) chemicals and the higher log (K_OW) chemicals remain. The replacement of more narcotically potent compounds with less narcotically potent compounds in the oil lowers the toxicity of the aqueous phase. The idea that weathering increases toxicity is based on the erroneous use of total petroleum hydrocarbon concentrations as though it were a single chemical compound that can be used to gauge the toxicity of a mixture, regardless of it makeup. The use of narcotic potential and toxic units eliminates this confusion, puts all the chemicals on the same footing, and allows an intuitive understanding of the effects of weathering. Case studies using laboratory and field data will demonstrate that weathered oil is less toxic than neat oil. This miss-application of total PAH concentration has lead to the proposal of a new paradigm of PAH toxicity. In fact no new paradigm is needed to explain the observed toxicity of PAH mixtures.

577 (SAR-1117-860889) A method for selective extraction of CYP1A inducing components in Alaskan North Slope crude oil.
Start time: 2:50 PM
SARAVANABHAVAN, G¹, KHAN, C², HODSON, P², BROWN, R¹, ¹ Department of Chemistry and School of Environmental Studies, Queens University, Kingston, Ontario, Canada² Department of Biology and School of Environmental Studies, Queens University, Kingston, Ontario, Canada
In recent times, several scientific reports have been published concerning the toxicity of crude oil to the early life stages of aquatic organisms. Crude oil contains a wide variety of chemical compounds whose relative toxicities are generally unknown. In several instances, the toxicity of metabolites has been found to be more severe than that of parent compounds. Hence the activation of the CYP450 enzyme system is considered as an important indicator of toxicity. Earlier studies from our group have shown that the exposure of trout (Oncorhynchus mykiss) to ANS crude oil and its sub-fractions (coal tar and wax fractions) showed higher CYP1A induction. The ′coal tar′ fraction is rich in PAHs and also contains a considerable amount of waxes. The ′wax′ fraction predominantly contains asphaltenes and waxes, but also some PAHs. The PAHs present in these fractions are considered to be responsible for CYP1A induction. To test this hypothesis, we have developed a low temperature solvent extraction method to isolate the PAHs from other components. Operating parameters of the method such as the choice of the solvent, volume, and extraction temperature were optimized for this study. Extraction of the coal tar used acetone for the precipitation of wax components. The wax fraction was extracted with pentane to isolate PAHs while precipitating asphaltenes. The CYP1A induction of the extract was potent, which correlated with the high PAH content. The wax or asphaltene residues did not induce CYP1A appreciably. The PAHs in the extracts were further fractionated using semi-preparative HPLC on a silica column into five fractions, based on the number of rings in the PAH structure. Results of CYP1A induction studies on these fractions and the detailed chemical analysis will be reported.

(58814) Break.
Start time: 3:10 PM

578 (RIC-1117-874827) Corroboration and significance of elevated CYP1A in Sea Otters and Harlequin ducks from chronic exposure to the Exxon Valdez spill in Prince William Sound.
Start time: 3:50 PM
Rice, S¹, Short, J¹, Carls, M¹, Springman, K², Bodkin, J ³, Ballachey, B³, ¹ NOAA, NMFS, Auke Bay Lab, Juneau, Alaska, USA² U C Davis, Center for Health and Environment, Davis, CA, USA³ USGS, Anchorage, Alaska, USA
Elevated levels of cytochrome P4501A (CYP1A) have been observed in several species in post-spill areas of Prince William Sound, site of the 1989 Exxon Valdez oil spill. In later years, as the oil level diminished, the conclusion that the Exxon Valdez oil spill was responsible for the elevated CYP1A response was challenged by Exxon contractors, who attributed the elevated levels of this enzyme to other sources. Much of the controversy was stimulated by findings 10-15 years after the spill: (1) significant pockets of sub-surface Exxon Valdez oil in the middle- to lower-intertidal zone areas, where mussels, clams, and other prey species for intertidal predators are found; (2) elevated CYP1A levels of intertidal predators (Sea Otters and Harlequin ducks); and (3) slow population recovery of these predators in the areas with greatest oil persistence. Other possible sources of P4501A inducers have been suggested, but the recent evidence from large scale field deployment of SPMDs in 2004 accumulating large quantities of PAH from Exxon Valdez oil spill sites, and neither PAH or POPs from non-oiled sites, including historical human use sites, corroborates the conclusion that Exxon Valdez oil is responsible for long term impacts to these species. Further, CYP1A was induced from the extracts of the SPMDs from oiled sites, but was not at other sites, and corroborates that lingering oil was responsible for the elevated CYP1A in Sea Otters and Harlequin ducks from oiled areas. Not only does elevated levels of CYP1A indicate continued chronic exposure to Exxon Valdez oil in Prince William Sound, chronic elevated levels also predict population level impacts in Sea Otters and Harlequin ducks.

579 (SPR-1117-832569) Induction of CYP1A in rainbow trout from bioavailable Exxon Valdez oil: fifteen years and still counting.
Start time: 4:10 PM
Springman, K¹, Short, J², Lindeberg, M², Khan, C³, Larsen, M², Hodson, P³, Rice, S², ¹ University of California, Davis, Davis, California, USA² NOAA/NMFS Auke Bay Laboratory, Juneau, Alaska, USA³ Queen's University, Kingston, Ontario, Canada
Assessing complex mixture toxicity by summing their concentrations may not be sufficiently realistic to evaluate or predict the consequences of exposure. An effective alternative involves the modification of a well-documented form of mimetic chemistry, the semi-permeable membrane device (SPMD), and the exposure of test animals to its contents. This method allows the evaluation of the effects of those bioavailable compounds present, including those whose analysis is difficult with their breakdown products and metabolites, as encountered in situ. This approach facilitates an evaluation of toxicity for each sample and site as a unit. We evaluated the CYP1A induction potential found in intertidal areas in Prince William Sound, some of which were oiled in 1989 from the Exxon Valdez oil spill, using SPMDs. The SPMDs were deployed for the standard 28-day deployment period, processed, and the extracts injected in juvenile rainbow trout (Oncorhynchus mykiss) which were sacrificed after 2 or 7 days, the livers excised and examined with the ethoxyresorufin-o-deethylase (EROD) bioassay. The results demonstrate that even after fifteen years, there is enough bioavailable oil in formerly oiled intertidal habitats of Prince William Sound to elicit a marked induction of CYP1A. The induction potential from oiled sites are comparable to those from a boat harbor (hot control), and are significantly elevated above environmental controls from sites that were not oiled. These results indicate that oil bioavailability is real, and can be evaluated with this technique.

580 (SHO-1117-853248) Lingering Exxon Valdez oil remains the dominant cause of CYP1A induction in Prince William Sound, Alaska.
Start time: 4:30 PM
Short, Jeffrey¹, Rice, Stanley¹, Springman, Kathrine², Sloan, Catherine³, Kahn, Colin⁴, Hodson, Peter⁴, ¹ Alaska Fisheries Science Center, National Marine Fisheries Service, NOAA, Juneau, Alaska, USA² Center for Health and the Environment, University of California, Davis, Davis, California, USA³ Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, Seattle, Washington, USA⁴ Queen's University, Kingston, Ontario, Canada
We measured the induction of cytochrome P450 (CYP1A) in rainbow trout injected with extracts from semi-permeable membrane devices (SPMD) deployed in Prince William Sound, Alaska, to compare the potency of lingering oil from the 1989 Exxon Valdez oil spill (EVOS) with pollutants from alternative sources. Arrays of SPMD were deployed at intertidal sites where EVOS oil remains, at other intertidal sites impacted by present or historical human activity, at salmon streams to assess pollutants imported to PWS by migrating salmon, at Constantine Harbor where a suite of natural petrogenic hydrocarbons is present in intertidal sediments, and at randomly-selected sites to assess inputs from atmospheric transport or from ambient seawater. CYP1A induction was measured by the EROD assay applied to homogenized rainbow trout livers two days following injection. SPMD extracts were also analyzed for polycyclic aromatic hydrocarbons (PAH) and for a suite of persistent organic pollutants (POP) including chlorinated pesticides and PCBs. The magnitude of CYP1A induction caused by SPMD extracts from the EVOS sites ranged from 28 - 72 pmol/mg/min, much greater than elsewhere (1.5 - 6.5 pmol/mg/min; median 2.5). The CYP1A induction from the oiled sites was significantly (P < 0.01) related to total PAH concentrations of the extracts, and these all fingerprinted to EVO. Of the 9 human activity sites (hatcheries, old mine sites), only one current use site registered significant loads of PAH and stimulated a CYP1A response. At 45 un-impacted sites (salmon streams, non-oiled areas, random marine sites), background concentrations of PAH and POP stimulated a weak (< 6.5 pmol/mg/min) to negligible CYP1A response. These results indicate that POPs are negligible as CYP1A induction agents in PWS, as are PAH associated with historical human use sites (except at Sawmill Bay), whereas oil from the EVOS remains a potent CYP1A induction agent.

581 (CAR-1117-740747) Cytochrome P4501A induction in oil-exposed pink salmon embryos predicts reduced survival potential.
Start time: 4:50 PM
Carls, M¹, Heintz, R¹, Marty, G², Rice, S¹, ¹ NOAA, NMFS, Auke Bay Lab, Juneau, Alaska, USA² U C Davis, School of Veternary Medicine, Davis, California, USA
Cytochrome P4501A (CYP1A) induction in pink salmon (Oncorhynchus gorbuscha) embryos exposed to crude oil is linked to adverse effects at cellular, organismal, and population levels and can be used to predict these responses. When combined with the results of this experiment, designed to examine CYP1A induction during embryonic stages and growth after emergence, results from a series of experiments spanning four other brood years demonstrate that CYP1A induction is related to a variety of lethal and sublethal effects, including poorer marine survival, reduced growth, and abnormalities. The lowest observed effective concentration of total polynuclear aromatic hydrocarbons in water that caused significant physiological responses, including reduced size 6 mo after exposure ended (about 1 ppb PAH, was less than the lowest concentration that caused significant CYP1A induction (about 3.7 ppb PAH). Thus, CYP1A induction is not only a biomarker, it can be considered a bioindicator; induction in early life stages implies long-term negative consequences for the individual and the population.

582 (NEF-1117-820442) Effects of the Exxon Valdez Oil Spill on the Pink Salmon Population: an Ecological Risk Assessment.
Start time: 5:10 PM
Neff, J.¹, Brannon, E.², Maki, A.³, ¹ Neff & Associates LLC, Duxbury, MA, 02332² Universith of Idaho, Moscow, ID, USA³ Exxon Mobil Corp., Anchorage, AK, USA
Most wild-run pink salmon (Oncorhynchus gorbuscha) returning to Prince William Sound, AK, (PWS) spawn in tidal reaches of streams; eggs and alevins of these salmon were potentially at risk of exposure to crude oil from the March 24, 1989, Exxon Valdez oil spill (EVOS), particularly in the spring and fall of 1989 when shoreline oiling was heaviest. Emergent fry spend a short time in shallow coastal waters before migrating to the ocean and were potentially at risk of exposure to oil in the PWS water column during the summer of 1989. We have reviewed the many studies of effects of the spill on pink salmon and used ecological risk assessment principals to evaluate relationships between exposure to EVOS oil hydrocarbons and effects on the pink salmon population in PWS. Only a small fraction of wild run salmon embryos and fry was at risk of exposure to harmful amounts of EVOS oil. About 30 (2.5% of the total) salmon streams in PWS were oiled and about 70% of the alevins were in the tidal reaches of streams in the spring of 1989; < 2% of wild-run alevins were at risk of oil exposure in the spring of 1989. In the fall of 1989 and subsequent years, a smaller fraction of the eggs deposited in the streams during spawning was at risk of exposure to residual oil because natural processes and cleanup had removed most of the oil from stream bed and shoreline sediments. There was no effect of the spill on pre-emergent alevin survival in the spring of 1989. Eggs deposited in oiled streams in the fall of 1989 and in subsequent years bioaccumulated small amounts of petroleum polycyclic aromatic hydrocarbons (PAH); survival was slightly depressed in the most heavily oiled streams in 1989-90 but not in subsequent years. Growth of juvenile salmon in coastal waters in 1989 and 1990 was similar in spill path and non-spill path areas of PWS. Direct observations demonstrate that wild-stock escapements and spawner densities have been similar in oiled and reference streams every year from 1989 through 2004. Although some individual fish were harmed by the spill, there were no long-term effects on the PWS pink salmon population.