The 1989 Exxon Valdez disaster exposed embryos of pink salmon and Pacific herring to crude oil in shoreline spawning habitats throughout Prince William Sound, Alaska. The herring fishery collapsed four years later. The role of the spill, if any, in this decline remains one of the most controversial unanswered questions in modern natural resource injury assessment. Crude oil disrupts excitationcontraction coupling in fish heart muscle cells, and we show here that salmon and herring exposed as embryos to trace levels of crude oil grow into juveniles with abnormal hearts and reduced cardiorespiratory function, the latter a key determinant of individual survival and population recruitment. Oil exposure during cardiogenesis led to specific defects in the outflow tract and compact myocardium, and a hypertrophic response in spongy myocardium, evident in juveniles 7 to 9 months after exposure. The thresholds for developmental cardiotoxicity were remarkably low, suggesting the scale of the Exxon Valdez impact in shoreline spawning habitats was much greater than previously appreciated. Moreover, an irreversible loss of cardiac fitness and consequent increases in delayed mortality in oil-exposed cohorts may have been important contributors to the delayed decline of pink salmon and herring stocks in Prince William Sound.The year 2014 marked the 25 th anniversary of the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska. At the time, the spill was the largest in U.S. history, with extensive oiling of shoreline spawning habitats for Pacific herring (Clupea pallasi) and pink salmon (Oncorhynchus gorbuscha), the two most important commercial fish species in the Sound. Herring larvae sampled in proximity to oil were visibly abnormal 1 , and mortality rates were higher for pink salmon embryos at oiled sites 2 . The herring fishery collapsed 3-4 years after the spill 3,4 when the cohort spawned in oiled areas would have reached reproductive maturity 5 . The contribution of the spill to the herring population collapse, if any, was never determined and remains controversial.In the early 1990s little was known about the effects of low-level crude oil exposures on fish early life stages. In the ensuing years, the syndrome of developmental defects and mortality documented in field-collected herring and salmon larvae was linked to polycyclic aromatic hydrocarbons (PAHs), an abundant fraction of most crude oils [6][7][8] . Subsequent research using the zebrafish model showed that the etiology of the syndrome was a disruption of embryonic cardiac function and morphogenesis 9,10 . This cardiotoxicity was specifically attributed to three-ringed PAHs 9,10 , and it was further shown that normal-appearing zebrafish embryos surviving trace crude oil exposures grow into adults with malformed hearts and reduced cardiorespiratory performance 11 . It is now established that crude oils from different geological sources disrupt heart development 12,13 in a diversity of fish species 14,15 , by a mechanism

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A competitive enzyme-linked immunosorbent assay was developed to quantitate vitellogenin (VTG) in plasma and serum of coho (Oncorhynchus kisutch) and chinook (O. tshawytscha) salmon. The working range of the assay was 9 to 313 ng/ml (80-20% binding), with 50% binding at 54 ng/ml. The intra-assay and interassay variations at approximately 50% binding were 8.1% (n = 9) and 9.0% (n = 9), respectively. Dilution curves of plasma or serum from coho and chinook females and estrogen-treated males were parallel to the purified coho VTG standard curve. Male plasma samples could be assayed at a minimum dilution of 1:40 (chinook) or 1:75 (coho) without assay interference because of high sample concentration, whereas minimum acceptable dilutions of male serum samples were 1:200 (chinook) or 1:600 (coho). Identification of proper techniques for preserving VTG integrity in plasma and serum samples showed that VTG from both species was robust; both sample types required no protease inhibitor despite subjection to two freeze-thaw cycles. To test its applicability, this assay was used to measure VTG in out-migrating juvenile chinook that were collected from urban and nonurban areas in Puget Sound, Washington, USA. Results showed a small but significant plasma VTG elevation at two urban sites, suggesting that these juveniles may be exposed to environmental estrogens at an early life stage. Also, wild fish tended to have higher plasma VTG levels than hatchery fish collected in the field. Elevation of mean VTG levels was similar to that previously reported in male English sole from the same area, where both males and females exhibited alterations in timing of spawning.