John P. Incardona

Aryl Hydrocarbon Receptor-Independent Toxicity of Weathered Crude Oil during Fish Development

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.

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish

Significance The 2010 Deepwater Horizon (MC252) disaster in the northern Gulf of Mexico released more than 4 million barrels of crude oil. Oil rose from the ocean floor to the surface where many large pelagic fish spawn. Here we describe the impacts of field-collected oil samples on the rapidly developing embryos of warm-water predators, including bluefin and yellowfin tunas and an amberjack. For each species, environmentally relevant MC252 oil exposures caused serious defects in heart development. Moreover, abnormalities in cardiac function were highly consistent, indicating a broadly conserved developmental crude oil cardiotoxicity. Losses of early life stages were therefore likely for Gulf populations of tunas, amberjack, swordfish, billfish, and other large predators that spawned in oiled surface habitats. The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1–15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

Fish embryos are damaged by dissolved PAHs, not oil particles

To distinguish the toxicity of whole oil droplets from compounds dissolved in water, responses of zebrafish embryos exposed to particulate-laden, mechanically dispersed Alaska North Slope crude oil (mechanically dispersed oil (MDO)) were compared to those of embryos protected from direct oil droplet contact by an agarose matrix. Most polycyclic aromatic hydrocarbons (PAHs) in MDO were contained in oil droplets; about 16% were dissolved. The agarose precluded embryo contact with particulate oil but allowed diffusive passage of dissolved PAHs. The incidence of edema, hemorrhaging, and cardiac abnormalities in embryos was dose-dependent in both MDO and agarose and the biological effects in these compartments were identical in character. Although mean total PAH (TPAH) concentrations in MDO were about 5-9 times greater than in agarose, dissolved PAH concentrations were similar in the two compartments. Furthermore, mean differences in paired embryo responses between compartments were relatively small (14-23%, grand mean 17%), typically with a larger response in embryos exposed to MDO. Therefore, the embryos reacted only to dissolved PAHs and the response difference between compartments is explained by diffusion. Averaged over 48 h, the estimated mean TPAH concentration in agarose was about 16% less than the dissolved TPAH concentration in MDO. Thus, PAHs dissolved from oil are toxic and physical contact with oil droplets is not necessary for embryotoxicity.

Sublethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish

Exposure to high concentrations of crude oil produces a lethal syndrome of heart failure in fish embryos. Mortality is caused by cardiotoxic polycyclic aromatic hydrocarbons (PAHs), ubiquitous components of petroleum. Here, we show that transient embryonic exposure to very low concentrations of oil causes toxicity that is sublethal, delayed, and not counteracted by the protective effects of cytochrome P450 induction. Nearly a year after embryonic oil exposure, adult zebrafish showed subtle changes in heart shape and a significant reduction in swimming performance, indicative of reduced cardiac output. These delayed physiological impacts on cardiovascular performance at later life stages provide a potential mechanism linking reduced individual survival to population-level ecosystem responses of fish species to chronic, low-level oil pollution.

Crude Oil Impairs Cardiac Excitation-Contraction Coupling in Fish

Crude oil from the Deepwater Horizon spill is cardiotoxic to tuna species that spawn in the Gulf of Mexico. Crude oil is known to disrupt cardiac function in fish embryos. Large oil spills, such as the Deepwater Horizon (DWH) disaster that occurred in 2010 in the Gulf of Mexico, could severely affect fish at impacted spawning sites. The physiological mechanisms underlying such potential cardiotoxic effects remain unclear. Here, we show that crude oil samples collected from the DWH spill prolonged the action potential of isolated cardiomyocytes from juvenile bluefin and yellowfin tunas, through the blocking of the delayed rectifier potassium current (IKr). Crude oil exposure also decreased calcium current (ICa) and calcium cycling, which disrupted excitation-contraction coupling in cardiomyocytes. Our findings demonstrate a cardiotoxic mechanism by which crude oil affects the regulation of cellular excitability, with implications for life-threatening arrhythmias in vertebrates. Oil Is Bad for the Heart Crude oil, which commonly makes up the largest proportion of spilled oil, is cardiotoxic to fish embryos. To better understand the processes involved, Brette et al. (p. 772) exposed captive young tuna to oil samples collected from the Deep Water Horizon spill site to determine the mode of cardiotoxicity. Crude oil prolonged the action potential of cardiomyocytes and disrupted the excitation-contraction coupling in these cells, functionally disrupting cellular excitability and creating the potential for cardiac arrhythmias. Such cardiac impacts may be more broadly distributed in vertebrates exposed to crude oil.

Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development

Petroleum-derived compounds, including polycyclic aromatic hydrocarbons (PAHs), commonly occur as complex mixtures in the environment. Recent studies using the zebrafish experimental model have shown that PAHs are toxic to the embryonic cardiovascular system, and that the severity and nature of this developmental cardiotoxicity varies by individual PAH. In the present study we characterize the toxicity of the relatively higher molecular weight 5-ring PAHs benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), and benzo[k]fluoranthene (BkF). While all three compounds target the cardiovascular system, the underlying role of the ligand-activated aryl hydrocarbon receptor (AHR2) and the tissue-specific induction of the cytochrome p450 metabolic pathway (CYP1A) were distinct for each. BaP exposure (40μM) produced AHR2-dependent bradycardia, pericardial edema, and myocardial CYP1A immunofluorescence. By contrast, BkF exposure (4-40μM) caused more severe pericardial edema, looping defects, and erythrocyte regurgitation through the atrioventricular valve that were AHR2-independent (i.e., absent myocardial or endocardial CYP1A induction). Lastly, exposure to BeP (40μM) yielded a low level of CYP1A+ signal in the vascular endothelium of the head and trunk, without evident toxic effects on cardiac function or morphogenesis. Combined with earlier work on 3- and 4-ring PAHs, our findings provide a more complete picture of how individual PAHs may drive the cardiotoxicity of mixtures in which they predominate. This will improve toxic injury assessments and risk assessments for wild fish populations that spawn in habitats altered by overlapping petroleum-related human impacts such as oil spills, urban stormwater runoff, or sediments contaminated by legacy industrial activities.

Exxon Valdez to Deepwater Horizon: comparable toxicity of both crude oils to fish early life stages

The 2010 Deepwater Horizon disaster in the Gulf of Mexico was the largest oil spill in United States history. Crude oils are highly toxic to developing fish embryos, and many pelagic fish species were spawning in the northern Gulf in the months before containment of the damaged Mississippi Canyon 252 (MC252) wellhead (April-July). The largest prior U.S. spill was the 1989 grounding of the Exxon Valdez that released 11 million gallons of Alaska North Slope crude oil (ANSCO) into Prince William Sound. Numerous studies in the aftermath of the Exxon Valdez spill defined a conventional crude oil injury phenotype in fish early life stages, mediated primarily by toxicity to the developing heart. To determine whether this type of injury extends to fishes exposed to crude oil from the Deepwater Horizon - MC252 incident, we used zebrafish to compare the embryotoxicity of ANSCO alongside unweathered and weathered MC252 oil. We also developed a standardized protocol for generating dispersed oil water-accommodated fractions containing microdroplets of crude oil in the size range of those detected in subsurface plumes in the Gulf. We show here that MC252 oil and ANSCO cause similar cardiotoxicity and photo-induced toxicity in zebrafish embryos. Morphological defects and patterns of cytochrome P450 induction were largely indistinguishable and generally correlated with polycyclic aromatic compound (PAC) composition of each oil type. Analyses of embryos exposed during different developmental windows provided additional insight into mechanisms of crude oil cardiotoxicity. These findings indicate that the impacts of MC252 crude oil on fish embryos and larvae are consistent with the canonical ANSCO cardiac injury phenotype. For those marine fish species that spawned in the northern Gulf of Mexico during and after the Deepwater Horizon incident, the established literature can therefore inform the assessment of natural resource injury in the form of potential year-class losses.

Acute embryonic or juvenile exposure to Deepwater Horizon crude oil impairs the swimming performance of mahi-mahi (Coryphaena hippurus).

The Deepwater Horizon incident likely resulted in exposure of commercially and ecologically important fish species to crude oil during the sensitive early life stages. We show that brief exposure of a water-accommodated fraction of oil from the spill to mahi-mahi as juveniles, or as embryos/larvae that were then raised for ∼25 days to juveniles, reduces their swimming performance. These physiological deficits, likely attributable to polycyclic aromatic hydrocarbons (PAHs), occurred at environmentally realistic exposure concentrations. Specifically, a 48 h exposure of 1.2 ± 0.6 μg L(-1) ΣPAHs (geometric mean ± SEM) to embryos/larvae that were then raised to juvenile stage or a 24 h exposure of 30 ± 7 μg L(-1) ΣPAHs (geometric mean ± SEM) directly to juveniles resulted in 37% and 22% decreases in critical swimming velocities (Ucrit), respectively. Oil-exposed larvae from the 48 h exposure showed a 4.5-fold increase in the incidence of pericardial and yolk sac edema relative to controls. However, this larval cardiotoxicity did not manifest in a reduced aerobic scope in the surviving juveniles. Instead, respirometric analyses point to a reduction in swimming efficiency as a potential alternative or contributing mechanism for the observed decreases in Ucrit.

Dissolved copper triggers cell death in the peripheral mechanosensory system of larval fish

Dissolved copper is an increasingly common non-point source contaminant in urban and urbanizing watersheds. In the present study, we investigated the sublethal effects of dissolved copper on the peripheral mechanosensory system, or lateral line, of larval zebrafish (Danio rerio). Zebrafish larvae were exposed to copper (0-65 microg/L), and the cytotoxic responses of individual lateral line receptor neurons were examined using a combination of in vivo fluorescence imaging, confocal microscopy, scanning electron microscopy, and conventional histology. Dissolved copper triggered a dose-dependent loss of neurons in identified lateral line neuromasts at concentrations > or = 20 microg/L. The onset of cell death in the larval mechanosensory system was rapid (< 1 h). When copper-exposed zebrafish were transferred to clean water, the lateral line regenerated over the course of 2 d. In contrast, the lateral line of larvae exposed continuously to dissolved copper (50 microg/L) for 3 d did not recover. Collectively, these results show that peripheral mechanosensory neurons are vulnerable to the neurotoxic effects of copper. Consequently, dissolved copper in non-point source storm-water runoff has the potential to interfere with rheotaxis, schooling, predator avoidance, and other mechanosensory-mediated behaviors that are important for the migration and survival of fish.

Unexpectedly high mortality in Pacific herring embryos exposed to the 2007 Cosco Busan oil spill in San Francisco Bay

In November 2007, the container ship Cosco Busan released 54,000 gallons of bunker fuel oil into San Francisco Bay. The accident oiled shoreline near spawning habitats for the largest population of Pacific herring on the west coast of the continental United States. We assessed the health and viability of herring embryos from oiled and unoiled locations that were either deposited by natural spawning or incubated in subtidal cages. Three months after the spill, caged embryos at oiled sites showed sublethal cardiac toxicity, as expected from exposure to oil-derived polycyclic aromatic compounds (PACs). By contrast, embryos from the adjacent and shallower intertidal zone showed unexpectedly high rates of tissue necrosis and lethality unrelated to cardiotoxicity. No toxicity was observed in embryos from unoiled sites. Patterns of PACs at oiled sites were consistent with oil exposure against a background of urban sources, although tissue concentrations were lower than expected to cause lethality. Embryos sampled 2 y later from oiled sites showed modest sublethal cardiotoxicity but no elevated necrosis or mortality. Bunker oil contains the chemically uncharacterized remains of crude oil refinement, and one or more of these unidentified chemicals likely interacted with natural sunlight in the intertidal zone to kill herring embryos. This reveals an important discrepancy between the resolving power of current forensic analytical chemistry and biological responses of keystone ecological species in oiled habitats. Nevertheless, we successfully delineated the biological impacts of an oil spill in an urbanized coastal estuary with an overlapping backdrop of atmospheric, vessel, and land-based sources of PAC pollution.