The objective of the present study was to assess the assumptions requisite for the interstitial toxic water hypothesis, that water leaching from oil deposits on the beaches adjacent to pink salmon streams was lethal to incubating embryos. We assessed the requisite assumptions by placing eggs in artificial redds in beaches known to harbor oil deposits and also by placing eggs in direct contact with those deposits. If eggs incubating in streams could be threatened by leachate from the adjacent oiled beach, as suggested in the interstitial toxic water hypothesis, then eggs placed on those beaches and away from the flushing freshwater stream flow should absorb TPAH concentrations that clearly represent toxic levels to incubating embryos. Moreover, if the oil deposits become effectively more toxic to embryos as HPAH increase in concentration over time from weathering, then the concentrations of HPAH in the developing eggs should be greater than they were when the oil deposits were less weathered. Thus, placing the eggs directly in oil deposits exposed on the oiled beaches is a worst-case test of the effects of weathered crude oil on pink salmon embryos.
Bioavailability and tissue TPAH concentrations
Concentrations of TPAH in eggs at most oiled sites were <40 ppb, well below those known to be lethal (6,000 ppb ; >7,100 ppb ). Excluding oil deposits at Sleepy Bay and Bay of Isles, there was no relationship between tissue and sediment TPAH concentrations. Mean tissue TPAH concentrations were not significantly higher at oiled over nonoiled beaches, there was no significant correlation between tissue and sediment TPAH, and there was no evidence of increased tissue loads in oiled over nonoiled beaches. Unless the eggs were in direct contact with the oil deposit, the effect of dissolution of the deposit on embryos even in close proximity to the source (15 cm-1.5 m) was not apparent in embryo tissue TPAH concentrations. Consequently, there was no evidence of high TPAH concentrations in interstitial waters surrounding the oil deposit and thus no evidence supporting the concept that oil deposits were a threat to eggs associated with oiled beaches. These data suggest that HPAH compounds are relatively insoluble and that the large volume of marine water flushing the beach sediments during daily tidal cycles dilute any aqueous TPAH emanating from such deposits.
Tissue TPAH did increase when directly in contact with oil deposits but did not elevate to lethal concentrations. At Sleepy Bay, tissue TPAH levels did not exceed 625 ppb even though the eggs were in contact with sediment TPAH concentrations of 23,149 ppb. Similar results were observed for eggs placed on other beaches with high sediment TPAH concentrations. At Bay of Isles, tissue TPAH levels did not exceed 515 ppb, despite eggs having been placed in the mudflats within an oil/sediment sheen, where sediment TPAH concentrations reached 6,608 ppb. East Herring Bay was also revealing because the pink salmon spawned naturally over buried oil deposits. Despite 17,673 ppb TPAH in the buried oil beneath the redds, only low TPAH levels of 11.7 and 13.8 ppb were present in eggs resting in redds less than 15 cm above the deposit and under the influence of vertical cyclic tidal infiltration.
Composition analysis and the interstitial toxic water hypothesis
Contrary to previous understanding of oil toxicity, Auke Bay Laboratory investigators have suggested that it is not the LPAH components of oil that pose the greatest long-term risk to pink salmon and the aqueous environment but rather the exposure to the HPAH compounds . Rice et al.  refer to this as the oil toxicity paradigm shift from short-term LC50 (lethal concentrations to 50%) determinations and from acute effects to long-term effects and from parts-per-million to parts-per-billion toxicity. Their hypothesis is based on interpretation of laboratory results of Heintz et al. , where toxicity was thought to have occurred at very low concentrations of HPAH, and the theory was promoted that toxic levels of HPAH leaching into the interstitial water account for embryo mortality years after the spill [9,10,19] as an explanation of the egg mortality reported by Bue et al. .
The interstitial toxic water hypothesis relies on the two premises: First, HPAH homologues become more bioavailable (and thus more toxic) over time as the crude oil weathers, and, second, the dissolved HPAH are carried with pore water into adjacent intertidal salmon streams at concentrations high enough to harm the developing salmon embryos. Both of these two premises must occur or the hypothesis is invalid.
Our results are not consistent with the first premise regarding bioavailability of toxic dissolved TPAH concentrations. In the laboratory studies by Brannon et al. , the sediment TPAH approaching the lethal threshold of laboratory-weathered oil was 8,300 ppb, in which naphthalenes were 2,890 ppb, making up 35% of the total. In contrast, HPAH (all PAH compounds from fluoranthenes to benzoperylenes) were 747 ppb, or 9% of the total, representing a ratio of 4:1 between naphthalenes and HPAH. In the corresponding laboratory embryo tissue TPAH of 7,100 ppb, naphthalenes were 2,631 ppb, 37% of the TPAH, and HPAH at 251 ppb, 3.5% of the total, or a ratio of 10:1, respectively.
However, in the Sleepy Bay oil deposit, the sediment TPAH was 23,149 ppb, and because of LPAH losses due to greater solubility during weathering, the naphthalenes had diminished to 2,882 ppb, 12% of the TPAH, much lower than concentrations of HPAH at 5,183 ppb, 22% of the total, and a ratio of approximately 1:2, respectively. The HPAH compounds were accumulating in the oil deposit because of their much lower solubility.
The most compelling evidence of low HPAH solubility was apparent in the embryo tissue. Naphthalenes (280 ppb) made up 45% of the tissue TPAH (625 ppb), while HPAH (46 ppb) were only 7% of the total. This was the circumstance even though the HPAH concentration in the sediment was nearly twice that of naphthalenes.
Bioavailability of petroleum hydrocarbons of well-weathered oil is low because the remaining oil mass is dominated by HPAH homologues that are relatively insoluble. Consequently, the HPAH were less in eggs (46 ppb) in contact with the well-weathered oil deposit (TPAH 23,149 ppb) than HPAH in eggs (251 ppb) exposed to lower laboratory concentrations (TPAH 8,300 ppb) of less weathered oil . Because of their relative insolubility, HPAH compounds are mostly what remain in the asphalt debris on the beaches, and that material will persist for a long time integrated within the other beach substrates.
These were noteworthy results because in the presence of much higher concentrations of naturally weathered oil the total tissue TPAH was much reduced compared with laboratory exposures of much lower oil concentrations , and the tissue HPAH concentrations were nominal because of the limited availability of HPAH. The pertinent point of this discussion is that the sediment TPAH, composed largely of HPAH, did not transfer to the embryo tissue as the new oil toxicity paradigm predicts . Therefore, the first premise on the increased bioavailability of HPAH is unsupported by evidence from the present study. The evidence shows that weathered oil deposits in beaches are not a source of readily available toxic compounds.
Our results also refute the second premise, that HPAH concentrations in weathered oil deposits are transferred in pore water through the beach substrate at toxic levels to incubating embryos in adjacent streams. It is noted that the tissue load of embryos in the laboratory study by Brannon et al.  was at a level close to or higher in relative concentration than that initially in the sediment TPAH. For example, in the laboratory, the tissue TPAH of 7,100 ppb was approximately 85% of the gravel TPAH of 8,300 ppb in the 1,500-ppm oil-on-gravel test lots, and in most other test concentrations, the ratio of tissue to sediment TPAH is even higher. However, the tissue load in eggs on Sleepy Bay beach, but not immersed in the oil deposit (<19 ppb), was at a level <0.1% of the adjacent highest sediment TPAH (23,149 ppb) and <0.3% of the lethal toxic threshold (>7,100 ppb) of weathered oil measured in the laboratory .
Similarly, mean tissue TPAH concentrations in eggs buried or naturally spawned in close proximity to oil-contaminated sediments at Bay of Isles (6,608 ppb sediment TPAH) and east Herring Bay (17,673 ppb sediment TPAH) were approximately 216 and 13 ppb, respectively, <4% and <0.1% of the respective sediment TPAH and well below the toxic dose. Therefore, while the dissolved TPAH available for absorption by embryos in the field was only a fraction of what is available under laboratory conditions, no interstitial transport of TPAH levels above background control concentrations was apparent even on beaches away from diluting freshwater flows. Consequently, there is no evidence supporting the second premise that transport of pore water from weathered oil deposits poses a toxic risk to incubating pink salmon embryos.
A critical point exposed by these field data is that in order for the interstitial toxic water hypothesis to be viable, the pore water must have extreme toxicity of bioavailable HPAH homologues in order to overcome the diluting and flushing effects of stream flow and tidal cycles. However, the enrichment of HPAH in oil-contaminated sediments, with their corresponding much reduced presence in the embryo tissue TPAH, demonstrates that the high molecular weight and putatively the most toxic analytes [1,9] remain undissolved and unavailable to eggs except for a little more than trace amounts. At all three oiled sites, Sleepy Bay, Bay of Isles, and Herring Bay, the tissue TPAH compositions were dominated by naphthalenes (Fig. 2b). Since naphthalenes were the primary components partitioned in the embryo tissues, even when they were very low in the contaminated sediments, it indicates that naphthalenes dominate uptake over the more prevalent HPAH compounds in weathered oil deposits because of limited solubility of HPAH analytes.
We emphasize that these field studies were conducted on the oiled beaches with only tidal flushing and without freshwater flow over the artificial redds, which was the most extreme exposure of eggs to interstitial toxic water emanating from the known oil deposits at those sites. Moreover, the weathered oil deposits were high in HPAH composition, which should have increased the dissolved HPAH availability, if they were soluble . However, not only did the eggs show low tissue TPAH levels, but the HPAH homologues were minor contributors. We suggest, therefore, that with the major dilution of soluble analytes around oil deposits by marine tidal cycles, as well as the freshwater flushing of the incubation substrate, the risk to incubating pink salmon embryos in streams adjacent to weathered oil deposits on the beach is extremely low.