Abstract-Pacific herring eggs were exposed for 16 d to weathered Alaska North Slope crude oil. Exposure to an initial aqueous concentration of 0.7 parts per billion (ppb) polynuclear aromatic hydrocarbons (PAHs) caused malformations, genetic damage, mortality, and decreased size and inhibited swimming. Total aqueous PAH concentrations as low as 0.4 ppb caused sublethal responses such as yolk sac edema and immaturity consistent with premature hatching. Responses to less weathered oil, which had relatively lower proportions of high molecular weight PAH, generally paralleled those of more weathered oil, but lowest observed effective concentrations (LOECs) were higher (9.1 ppb), demonstrating the importance of composition. The LOEC for more weathered oil (0.4 ppb) was similar to that observed in pink salmon (1.0 ppb), a species with a very different development rate; by inference, other species may be similarly sensitive to weathered oil. Our methods simulated conditions observed in Prince William Sound (PWS) following the Exxon Valdez oil (EVO) spill. Biological effects were identical to those observed in embryolarval herring from PWS in 1989 and support the conclusion that EVO caused significant damage to herring in PWS. Previous demonstration by our laboratory that most malformed or precocious larvae die corroborates the decreased larval production measured after the spill.

Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.

Teleost embryos develop a syndrome characterized by edema when exposed to water that weathers substrates contaminated with crude oil. Previous studies using zebrafish demonstrated that crude oil exposure causes cardiogenic edema, and that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils (tricyclic fluorenes, dibenzothiophenes, and phenanthrenes) are cardiotoxic, causing arrhythmia through a pathway that does not require activation of the aryl hydrocarbon receptor (AHR). We demonstrate here for Pacific herring, a species impacted by the Exxon Valdez oil spill, that the developing heart is the primary target of crude oil exposure. Herring embryos exposed to the effluent of oiled gravel columns developed dose-dependent edema and irregular cardiac arrhythmia soon afterthe heartbeat was established. At a dose that produced cardiac dysfunction in 100% of exposed embryos, tissue levels of tricyclic PAHs were below 1 micromol/kg, suggesting a specific, high affinity target in the heart. These findings have implications for understanding the mechanism of tricyclic PAH cardiotoxicity, the development of biomarkers for the effects of PAH exposure in fish, and understanding the long-term impacts of oil spills and other sources of PAH pollution in aquatic environments.

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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

The photoenhanced toxicity of weathered Alaska North Slope crude oil (ANS) was investigated in the eggs and larvae of Pacific herring (Clupea pallasi) with and without the chemical dispersant Corexit 9527. Oil alone was acutely toxic to larvae at aqueous concentrations below 50 microg/L total polycyclic aromatic hydrocarbons (tPAH), and median lethal (LC50s) and effective concentrations (EC50s) decreased with time after initial oil exposure. Brief exposure to sunlight (approximately 2.5 h/d for 2 d) significantly increased toxicity 1.5- to 48-fold over control lighting. Photoenhanced toxicity only occurred when oil was present in larval tissue and increased with increasing tPAH concentration in tissue. Ultraviolet radiation A (UVA) treatments were less potent than natural sunlight, and UVA + sunlight caused greater toxicity than sunlight alone. The toxicity of chemically dispersed oil was similar to oil alone in control and UVA treatments, but oil + dispersant was significantly more toxic in the sunlight treatments. The chemical dispersant appeared to accelerate PAH dissolution into the aqueous phase, resulting in more rapid toxicity. In oil + dispersant exposures, the 96-h no-observed-effect concentrations in the UVA + sunlight treatment were 0.2 microg/L tPAH and 0.01 microg/g tPAH. Exposure of herring eggs to oil caused yolk sac edema, but eggs were not exposed to sun and UVA treatment did not cause phototoxicity. These results are consistent with the hypothesis that weathered ANS is phototoxic and that UV can be a significant and causative factor in the mortality of early life stages of herring exposed to oil and chemically dispersed oil.

Pacific herring eggs were exposed for 16 d to weathered Alaska North Slope crude oil. Exposure to an initial aqueous concentration of 0.7 parts per billion (ppb) polynuclear aromatic hydrocarbons (PAHs) caused malformations, genetic damage, mortality, and decreased size and inhibited swimming. Total aqueous PAH concentrations as low as 0.4 ppb caused sublethal responses such as yolk sac edema and immaturity consistent with premature hatching. Responses to less weathered oil, which had relatively lower proportions of high molecular weight PAH, generally paralleled those of more weathered oil, but lowest observed effective concentrations (LOECs) were higher (9.1 ppb), demonstrating the importance of composition. The LOEC for more weathered oil (0.4 ppb) was similar to that observed in pink salmon (1.0 ppb), a species with a very different development rate; by inference, other species may be similarly sensitive to weathered oil. Our methods simulated conditions observed in Prince William Sound (PWS) following the Exxon Valdez oil (EVO) spill. Biological effects were identical to those observed in embryolarval herring from PWS in 1989 and support the conclusion that EVO caused significant damage to herring in PWS. Previous demonstration by our laboratory that most malformed or precocious larvae die corroborates the decreased larval production measured after the spill.

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