More than 200 million gallons of crude oil were released from the Macondo Well in the Gulf of Mexico during the 2010 Deepwater Horizon oil spill [1
]. The oil gushed from approximately 5,000 feet below the surface creating underwater plumes of oil and slicks on both the ocean floor and surface. The oil's total vertical exposure to the water column suggested it had the potential to impact a diversity of ecosystems in the Gulf of Mexico and Atlantic Ocean from coastal wetlands to coral reefs to deep-water benthic communities [3
]. In fact, many thousands of invertebrate and vertebrate carcasses have been discovered and attributed to the Macondo oil, and due to the wide-spread and hidden nature of this disaster, species mortality rates are hypothesized to be at least 50 times underrepresented [4
Of particular concern is how embryos and larvae will be affected by this oil spill, as organisms in these early life stages are typically unable to flee a contaminated area and lack the tissue mass and mature detoxification systems necessary to withstand toxic insults. Furthermore, loss of whole clutches of embryos and larvae can have catastrophic consequences for population dynamics over long time periods [6
]. The National Oceanic and Atmospheric Administration (NOAA) reported an unusually high number of stillborn and newborn dolphins that began washing up on the Gulf shores starting in January 2011 [7
]. While only a small number of these dead dolphins had obvious signs of oil exposure, the timing suggests an association between the gestational period of dolphin embryonic and fetal development with the Deepwater Horizon blowout and oil spill [4
]. There are 52 taxa of native fish species also known to spawn in the Gulf of Mexico such as tuna, red snapper and yellowfin groupers [8
], and unfortunately little is known about how the Macondo crude oil might directly impact early development of dolphins or these fish species. It will be critically important to identify the molecular targets of crude oil toxicants that effect embryonic development to provide greater predictive power in the risk assessment of this and future spills.
Exposure to crude oil and/or the water-soluble, polycyclic aromatic hydrocarbons (PAH) that make up much of the oil have been shown to cause cardiovascular abnormalities and pericardial edema in topsmelt and Pacific herring embryos, as well as additional defects in jaw and spinal cord development in the crimson-spotted rainbow fish [9
]. Whether oil from the Macondo well can cause similar defects in embryonic development needs to be determined. The majority of research using native Gulf fish species to determine the effects of crude oil on embryonic development has been limited to the analysis of mortality and gross morphological changes. However, the relatively recent characterization of zebrafish (Danio rerio
) as a laboratory fish model system has revealed significant insights into the developmental mechanisms governing embryogenesis [12
]. Zebrafish provide researchers with the consistent accessibility of genetically defined lines of embryos and a battery of reliable genetic, molecular and cellular techniques for higher resolution analysis, benefits that are providing significant advantages to the use of zebrafish in toxicology. The small adult fish size, large embryo clutches, ex utero
development, and transparent embryo and larval stages of zebrafish enable cost effective maintenance of many fish, reproducible sample sizes, simple application of toxin treatments, and easy evaluation of end-point toxicity [14
]. The use of zebrafish to assay drug and pollutant toxicity has already provided insights into the developmental and molecular mechanistic roles of metals [19
], dioxins [21
], pesticides [25
], endocrine disruptors [27
], alcohols [29
], chemotherapies [32
] and specific pharmaceutical compounds many of which were assessed via high-throughput screening [35
Examination of the effect of crude oil and its more common PAHs on zebrafish embryos has shown developmental abnormalities that include cardiac edema, reduced jaw development, curvature of the spine, hemorrhaging, reduced larval heart rate and cardiac arrhythmia [6
]. While these varied deformations are clearly attributed to crude oil exposure, it is unknown what early embryonic processes at the cellular or molecular levels are directly affected. Further investigations utilizing zebrafish to elucidate the molecular mechanisms that interact with and mediate crude oil effects could guide future preventative measures to oil spills and their clean up procedures, as well as yield new strategies to combat the teratogenesis associated with oil spill exposure.
In the present study, we sought to determine whether water-soluble components of the crude oil from the Deepwater Horizon oil spill could disrupt normal embryonic and larval development in zebrafish. Embryos were exposed to water accumulated fractions (WAFs) made from crude oil sampled from the riser insertion tube attached to the Macondo well during the 2010 oil spill. Using an array of high resolution assays to characterize a variety of discrete developmental processes, we show that the Macondo oil does not cause general and wide-spread toxicity but rather disrupts specific developmental processes, some of which have never been reported previously for any crude oil source. We show that WAF-induced deformations in cardiovascular and craniofacial development, observed in this and likely other studies [6
], are associated with reductions in specific rostral migratory streams of cranial neural crest cells. Surprisingly, Macondo oil exposure also generated novel phenotypes in apoptosis, sensory and motor axon pathfinding, somitogenesis and muscle fiber type development, which provide causative evidence for the locomotor swimming behaviors exhibited by WAF-treated embryos. Lastly, we demonstrate that dose and timing of WAF exposure are equally important for the induction of cell death and muscle specific phenotypes. Our results suggest that crude oil components may interact with important molecular mechanisms to influence embryogenesis. The severe teratogenic effects in zebrafish associated with the Deepwater Horizon oil are cause for concern if native fish species in the Gulf of Mexico responded similarly. Finally, our findings provide a more comprehensive phenotypic map to assess pollution effects on development.