Novel adverse outcome pathways revealed by chemical genetics in a developing marine fish.

Crude oil spills are a worldwide ocean conservation threat. Fish are particularly vulnerable to the oiling of spawning habitats, and crude oil causes severe abnormalities in embryos and larvae. However, the underlying mechanisms for these developmental defects are not well understood. Here, we explore the transcriptional basis for four discrete crude oil injury phenotypes in the early life stages of the commercially important Atlantic haddock (Melanogrammus aeglefinus). These include defects in (1) cardiac form and function, (2) craniofacial development, (3) ionoregulation and fluid balance, and (4) cholesterol synthesis and homeostasis. Our findings suggest a key role for intracellular calcium cycling and excitation-transcription coupling in the dysregulation of heart and jaw morphogenesis. Moreover, the disruption of ionoregulatory pathways sheds new light on buoyancy control in marine fish embryos. Overall, our chemical-genetic approach identifies initiating events for distinct adverse outcome pathways and novel roles for individual genes in fundamental developmental processes.

Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development.

Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7-7 µg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish.

Unexpected interaction with dispersed crude oil droplets drives severe toxicity in Atlantic haddock embryos.

The toxicity resulting from exposure to oil droplets in marine fish embryos and larvae is still subject for debate. The most detailed studies have investigated the effects of water-dissolved components of crude oil in water accommodated fractions (WAFs) that lack bulk oil droplets. Although exposure to dissolved petroleum compounds alone is sufficient to cause the characteristic developmental toxicity of crude oil, few studies have addressed whether physical interaction with oil micro-droplets are a relevant exposure pathway for open water marine speices. Here we used controlled delivery of mechanically dispersed crude oil to expose pelagic embryos and larvae of a marine teleost, the Atlantic haddock (Melanogrammus aeglefinus). Haddock embryos were exposed continuously to two different concentrations of dispersed crude oil, high and low, or in pulses. By 24 hours of exposure, micro-droplets of oil were observed adhering and accumulating on the chorion, accompanied by highly elevated levels of cyp1a, a biomarker for exposure to aromatic hydrocarbons. Embryos from all treatment groups showed abnormalities representative of crude oil cardiotoxicity at hatch (5 days of exposure), such as pericardial and yolk sac edema. Compared to other species, the frequency and severity of toxic effects was higher than expected for the waterborne PAH concentrations (e.g., 100% of larvae had edema at the low treatment). These findings suggest an enhanced tissue uptake of PAHs and/or other petroleum compounds from attached oil droplets. These studies highlight a novel property of haddock embryos that leads to greater than expected impact from dispersed crude oil. Given the very limited number of marine species tested in similar exposures, the likelihood of other species with similar properties could be high. This unanticipated result therefore has implications for assessing the ecological impacts of oil spills and the use of methods for dispersing oil in the open sea.