Low-level embryonic crude oil exposure disrupts ventricular ballooning and subsequent trabeculation in Pacific herring.

There is a growing awareness that transient, sublethal embryonic exposure to crude oils cause subtle but important forms of delayed toxicity in fish. While the precise mechanisms for this loss of individual fitness are not well understood, they involve the disruption of early cardiogenesis and a subsequent pathological remodeling of the heart much later in juveniles. This developmental cardiotoxicity is attributable, in turn, to the inhibitory actions of crude oil-derived mixtures of polycyclic aromatic compounds (PACs) on specific ion channels and other proteins that collectively drive the rhythmic contractions of heart muscle cells via excitation-contraction coupling. Here we exposed Pacific herring (Clupea pallasi) embryos to oiled gravel effluent yielding ΣPAC concentrations as low as ~ 1 µg/L (64 ng/g in tissues). Upon hatching in clean seawater, and following the depuration of tissue PACs (as evidenced by basal levels of cyp1a gene expression), the ventricles of larval herring hearts showed a concentration-dependent reduction in posterior growth (ballooning). This was followed weeks later in feeding larvae by abnormal trabeculation, or formation of the finger-like projections of interior spongy myocardium, and months later with hypertrophy (overgrowth) of the spongy myocardium in early juveniles. Given that heart muscle cell differentiation and migration are driven by Ca2+-dependent intracellular signaling, the observed disruption of ventricular morphogenesis was likely a secondary (downstream) consequence of reduced calcium cycling and contractility in embryonic cardiomyocytes. We propose defective trabeculation as a promising phenotypic anchor for novel morphometric indicators of latent cardiac injury in oil-exposed herring, including an abnormal persistence of cardiac jelly in the ventricle wall and cardiomyocyte hyperproliferation. At a corresponding molecular level, quantitative expression assays in the present study also support biomarker roles for genes known to be involved in muscle contractility (atp2a2, myl7, myh7), cardiomyocyte precursor fate (nkx2.5) and ventricular trabeculation (nrg2, and hbegfa). Overall, our findings reinforce both proximal and indirect roles for dysregulated intracellular calcium cycling in the canonical fish early life stage crude oil toxicity syndrome. More work on Ca2+-mediated cellular dynamics and transcription in developing cardiomyocytes is needed. Nevertheless, the highly specific actions of ΣPAC mixtures on the heart at low, parts-per-billion tissue concentrations directly contravene classical assumptions of baseline (i.e., non-specific) crude oil toxicity.

Urban stormwater and crude oil injury pathways converge on the developing heart of a shore-spawning marine forage fish.

Understanding how aquatic organisms respond to complex chemical mixtures remains one of the foremost challenges in modern ecotoxicology. Although oil spills are typically high-profile disasters that release hundreds or thousands of chemicals into the environment, there is growing evidence for a common adverse outcome pathway (AOP) for the vulnerable embryos and larvae of fish species that spawn in oiled habitats. Molecular initiating events involve the disruption of excitation-contraction coupling in individual cardiomyocytes, which then dysregulate the form and function of the embryonic heart. Phenanthrenes and other three-ring (tricyclic) polycyclic aromatic hydrocarbons (PAHs) are key drivers for this developmental cardiotoxicity and are also relatively enriched in land-based urban runoff. Similar to oil spills, stormwater discharged from roadways and other high-traffic impervious surfaces contains myriad contaminants, many of which are uncharacterized in terms of their chemical identity and toxicity to aquatic organisms. Nevertheless, given the exceptional sensitivity of the developing heart to tricyclic PAHs and the ubiquitous presence of these compounds in road runoff, cardiotoxicity may also be a dominant aspect of the stormwater-induced injury phenotype in fish early life stages. Here we assessed the effects of traffic-related runoff on the embryos and early larvae of Pacific herring (Clupea pallasii), a marine forage fish that spawns along the coastline of western North America. We used the well-characterized central features of the oil toxicity AOP for herring embryos as benchmarks for a detailed analysis of embryolarval cardiotoxicity across a dilution gradient ranging from 12 to 50% stormwater diluted in clean seawater. These injury indicators included measures of circulatory function, ventricular area, heart chamber looping, and the contractility of both the atrium and the ventricle. We also determined tissue concentrations of phenanthrenes and other PAHs in herring embryos. We find that tricyclic PAHs are readily bioavailable during cardiogenesis, and that stormwater-induced toxicity is in many respects indistinguishable from canonical crude oil toxicity. Given the chemical complexity of urban runoff, non-tricyclic PAH-mediated mechanisms of developmental toxicity in fish remain likely. However, from the standpoint of managing wild herring populations, our results suggest that stormwater-driven threats to individual survival (both near-term and delayed mortality) can be understood from decades of past research on crude oil toxicity. Moreover, Pacific herring embryos are promising sentinels for water quality monitoring in nearshore marine habitats, as in situand sensitive indicators of both toxic runoff and the effectiveness of pollution reduction efforts such as green stormwater infrastructure.

The effects of exposure to crude oil or PAHs on fish swim bladder development and function.

The failure of the swim bladder to inflate during fish development is a common and sensitive response to exposure to petrochemicals. Here, we review potential mechanisms by which petrochemicals or their toxic components (polycyclic aromatic hydrocarbons; PAHs) may affect swim bladder inflation, particularly during early life stages. Surface films formed by oil can cause a physical barrier to primary inflation by air gulping, and are likely important during oil spills. The act of swimming to the surface for primary inflation can be arduous for some species, and may prevent inflation if this behavior is limited by toxic effects on vision or musculature. Some studies have noted altered gene expression in the swim bladder in response to PAHs, and Cytochrome P450 1A (CYP1A) can be induced in swim bladder or rete mirabile tissue, suggesting that PAHs can have direct effects on swim bladder development. Swim bladder inflation failure can also occur secondarily to the failure of other systems; cardiovascular impairment is the best elucidated of these mechanisms, but other mechanisms might include non-inflation as a sequela of disruption to thyroid signaling or cholesterol metabolism. Failed swim bladder inflation has the potential to lead to chronic sublethal effects that are as yet unstudied.

Toxicity of weathered Deepwater Horizon oil to bay anchovy (Anchoa mitchilli) embryos.

The BP-contracted Deepwater Horizon Macondo well blowout occurred on 20 April 2010 and lasted nearly three months. The well released millions of barrels of crude oil into the northern Gulf of Mexico, causing extensive impacts on pelagic, benthic, and estuarine fish species. The bay anchovy (Anchoa mitchilli) is an important zooplanktivore in the Gulf, serving as an ecological link between lower trophic levels and pelagic predatory fish species. Bay anchovy spawn from May through November in shallow inshore and estuarine waters throughout the Gulf. Because their buoyant embryos are a dominant part of the inshore ichthyoplankton throughout the summer, it is likely bay anchovy embryos encountered oil in coastal estuaries during the summer and fall of 2010. Bay anchovy embryos were exposed to a range of concentrations of two field-collected Deepwater Horizon oils as high-energy and low-energy water accommodated fractions (HEWAFs and LEWAFs, respectively) for 48h. The median lethal concentrations (LC50) were lower in exposures with the more weathered oil (HEWAF, 1.48µg/L TPAH50; LEWAF, 1.58µg/L TPAH50) compared to the less weathered oil (HEWAF, 3.87µg/L TPAH50; LEWAF, 4.28µg/L TPAH50). To measure delayed mortality and life stage sensitivity between embryos and larvae, an additional 24h acute HEWAF exposure using the more weathered oil was run followed by a 24h grow-out period. Here the LC50 was 9.71µg/L TPAH50 after the grow-out phase, suggesting a toxic effect of oil at the embryonic or hatching stage. We also found that exposures prepared with the more weathered Slick B oil produced lower LC50 values compared to the exposures prepared with Slick A oil. Our results demonstrate that even relatively acute environmental exposure times can have a detrimental effect on bay anchovy embryos.

Molecular Mechanisms of Crude Oil Developmental Toxicity in Fish.

With major oil spills in Korea, the United States, and China in the past decade, there has been a dramatic increase in the number of studies characterizing the developmental toxicity of crude oil and its associated polycyclic aromatic compounds (PACs). The use of model fish species with associated tools for genetic manipulation, combined with high throughput genomics techniques in nonmodel fish species, has led to significant advances in understanding the cellular and molecular bases of functional and morphological defects arising from embryonic exposure to crude oil. Following from the identification of the developing heart as the primary target of crude oil developmental toxicity, studies on individual PACs have revealed a diversity of cardiotoxic mechanisms. For some PACs that are strong agonists of the aryl hydrocarbon receptor (AHR), defects in heart development arise in an AHR-dependent manner, which has been shown for potent organochlorine agonists, such as dioxins. However, crude oil contains a much larger fraction of compounds that have been found to interfere directly with cardiomyocyte physiology in an AHR-independent manner. By comparing the cellular and molecular responses to AHR-independent and AHR-dependent toxicity, this review focuses on new insights into heart-specific pathways underlying both acute and secondary adverse outcomes to crude oil exposure during fish development.

The Toxicity to Fish Embryos of PAH in Crude and Refined Oils.

Oil spills are a potential threat to the recruitment and production of fish. Polycyclic aromatic hydrocarbons (PAH), particularly 3-5-ringed alkyl PAH, are components of oil that cause chronic embryotoxicity. Toxicity is related to molecular size and octanol-water partition coefficients (Kow), indicating that water-lipid partitioning controls exposure and tissue dose. Nevertheless, more than 25% of the variation in toxicity among congeners is unexplained. Congeners with the same number of rings, alkyl carbon atoms, and Kow, but different molecular shapes, have markedly different toxicities, likely due to differences in interactions with cellular receptors. The potentiation and antagonism of metabolism and toxicity in PAH mixtures suggest that measured effect concentrations for individual PAH are conservative. Because mixture interactions are not well understood, total PAH concentrations >0.1 µg/L following oil spills should be considered hazardous.

Significance of adverse outcome pathways in biomarker-based environmental risk assessment in aquatic organisms.

In environmental risk assessments (ERA), biomarkers have been widely used as an early warning signal of environmental contamination. However, biomarker responses have limitation due to its low relevance to adverse outcomes (e.g., fluctuations in community structure, decreases in population size, and other similar ecobiologically relevant indicators of community structure and function). To mitigate these limitations, the concept of adverse outcome pathways (AOPs) was developed. An AOP is an analytical, sequentially progressive pathway that links a molecular initiating event (MIE) to an adverse outcome. Recently, AOPs have been recognized as a potential informational tool by which the implications of molecular biomarkers in ERA can be better understood. To demonstrate the utility of AOPs in biomarker-based ERA, here we discuss a series of three different biological repercussions caused by exposure to benzo(a)pyrene (BaP), silver nanoparticles (AgNPs), and selenium (Se). Using mainly aquatic invertebrates and selected vertebrates as model species, we focus on the development of the AOP concept. Aquatic organisms are suitable bioindicator species whose entire lifespans can be observed over a short period; moreover, these species can be studied on the molecular and population levels. Also, interspecific differences between aquatic organisms are important to consider in an AOP framework, since these differences are an integral part of the natural environment. The development of an environmental pollutant-mediated AOP may enable a better understanding of the effects of environmental pollutants in different scenarios in the diverse community of an ecosystem.

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.

Scyphomedusae of the Mediterranean: state of the art and future perspectives.

Scyphomedusae (Phylum Cnidaria, Class Scyphozoa) are perceived as a nuisance due to their sudden outbreaks that negatively affect human activities (particularly tourism and fisheries) mainly because of their stings. A brief review of the history of scyphozoan blooms in the Mediterranean and updated information available after 2010 point to an increase in scyphozoan outbreaks. Whilst the negative effects on public health, aquaculture, coastal industrial activities and fisheries operations are undeniable, the effects on the ecosystem are not well defined. We focus on the trophic interactions between scyphomedusae and fish, highlighting that the negative effects of scyphomedusae on fish stocks exerted through direct predation on early life stages of fish and competition for plankton are at present speculative. In favor of a positive effect of scyphomedusae on fish populations, the reports of predation upon scyphozoans are increasing, which suggests that predators may benefit from the availability of scyphozoans by shifting their diet toward jelly prey. Additionally, scyphomedusae may provide nursery habitats to early life stages of ecologically and economically important forage fishes and other organisms which shelter underneath their bells. Together with these ecosystem services, compounds extracted from scyphozoan tissues and venoms are having a variety of biomedical applications and are likely to contribute to treat a growing number of diseases, including cancer. Our analysis highlights that a re-evaluation of the balance between "positive" and "negative" effects of scyphomedusae on the ecosystem and human activities is needed and provides indications on potential directions for future studies.

Toxicity of crude oil chemically dispersed in a wave tank to embryos of Atlantic herring (Clupea harengus).

Tests of crude oil toxicity to fish are often chronic, exposing embryos from fertilization to hatch to oil solutions prepared using standard mixing procedures. However, during oil spills, fish are not often exposed for long periods and the dynamic nature of the ocean is not easily replicated in the lab. Our objective was to determine if brief exposures of Atlantic herring (Clupea harengus) embryos to dispersed oil prepared by standard mixing procedures was as toxic as oil dispersed in a more realistic model system. Embryos were first exposed to chemically dispersed Alaska North Slope crude and Arabian light crude oil for 2.4 h to 14 d from fertilization to determine if exposure time affected toxicity. Toxicity increased with exposure time, but 2.4-h exposures at realistic concentrations of oil induced blue-sac disease and reduced the percentage of normal embryos at hatch; there was little difference in toxicity between the two oils. Secondly, oil was chemically dispersed in a wave tank to determine if the resultant oil solutions were as toxic to herring embryos as laboratory-derived dispersed oil using a single exposure period of 24 h. Samples taken 15 min postdispersion were more toxic than laboratory-prepared solutions, but samples taken at 5, 30, and 60 min postdispersion were less toxic. Overall, the laboratory- and wave tank-derived solutions of dispersed oil provided similar estimates of toxicity despite differences in the methods for preparing test solutions, suggesting that laboratory and wave tank data are a reliable basis for ecological risk assessments of spilled oil.

Potent phototoxicity of marine bunker oil to translucent herring embryos after prolonged weathering.

Pacific herring embryos (Clupea pallasi) spawned three months following the Cosco Busan bunker oil spill in San Francisco Bay showed high rates of late embryonic mortality in the intertidal zone at oiled sites. Dead embryos developed to the hatching stage (e.g. fully pigmented eyes) before suffering extensive tissue deterioration. In contrast, embryos incubated subtidally at oiled sites showed evidence of sublethal oil exposure (petroleum-induced cardiac toxicity) with very low rates of mortality. These field findings suggested an enhancement of oil toxicity through an interaction between oil and another environmental stressor in the intertidal zone, such as higher levels of sunlight-derived ultraviolet (UV) radiation. We tested this hypothesis by exposing herring embryos to both trace levels of weathered Cosco Busan bunker oil and sunlight, with and without protection from UV radiation. Cosco Busan oil and UV co-exposure were both necessary and sufficient to induce an acutely lethal necrotic syndrome in hatching stage embryos that closely mimicked the condition of dead embryos sampled from oiled sites. Tissue levels of known phototoxic polycyclic aromatic compounds were too low to explain the observed degree of phototoxicity, indicating the presence of other unidentified or unmeasured phototoxic compounds derived from bunker oil. These findings provide a parsimonious explanation for the unexpectedly high losses of intertidal herring spawn following the Cosco Busan spill. The chemical composition and associated toxicity of bunker oils should be more thoroughly evaluated to better understand and anticipate the ecological impacts of vessel-derived spills associated with an expanding global transportation network.

Effect of heavy oil on the development of the nervous system of floating and sinking teleost eggs.

Heavy oil (HO) on the sea surface penetrates into fish eggs and prevents the normal morphogenesis. To identify the toxicological effects of HO in the context of the egg types, we performed exposure experiments using floating eggs and sinking eggs. In the course of development, HO-exposed embryos of floating eggs showed abnormal morphology, whereas early larva of the sinking eggs had almost normal morphology. However, the developing peripheral nervous system of sinking eggs showed abnormal projections. These findings suggest that HO exposed fishes have problems in the developing neurons, although they have no morphological malformations. Through these observations, we conclude that HO is strongly toxic to floating eggs in the morphogenesis, and also affect the neuron development in both floating and sinking eggs.

Oil spills and fish health: exposing the heart of the matter.

The chemical complexity of crude oil and its fuel products poses many important challenges for exposure science in marine ecosystems that support productive fisheries throughout the world. Meeting these challenges will enable better decisions on approaches to protecting and restoring these ecosystems.

Toxicity of dispersed weathered crude oil to early life stages of Atlantic herring (Clupea harengus).

Reports of the chronic toxicity of dispersed crude oil to early life stages of fish perpetuate uncertainty about dispersant use. However, realistic exposures to dispersed oil in the water column are thought to be much briefer than exposures associated with chronic toxicity testing. To address this issue, the toxicity of dispersed weathered oil to early life stages of Atlantic herring (Clupea harengus) was tested for short exposure durations, ranging from 1 to 144 h. Toxicity was a function of concentration and duration of exposure, as well as of the life stage exposed. Medium South American crude oil dispersed with Corexit 9500 caused blue sac disease in embryos, but not in free-swimming embryos. The age of embryos was negatively correlated with their sensitivity to oil; those freshly fertilized were most sensitive. Sensitivity increased after hatch, with free-swimming embryos showing signs of narcosis. Gametes were also tested; dispersed oil dramatically impaired fertilization success. For exposures of less than 24 h, gametes and free-swimming embryos were the most sensitive life stages. For those of more than 24 h, young embryos (<1 d old) were most sensitive. The results are presented as statistical models that could assist decisions about dispersant use in the vicinity of fish spawning habitats.

Cardiac arrhythmia is the primary response of embryonic Pacific herring (Clupea pallasi) exposed to crude oil during weathering.

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.

Preliminary investigation of the effects of dispersed Prudhoe Bay Crude Oil on developing topsmelt embryos, Atherinops affinis.

Static exposure experiments were conducted to assess the toxicity of dispersed Prudhoe Bay Crude Oil (PBCO) to embryos of the topsmelt (Atherinops affinis). Treatment with the dispersant COREXIT 9500 resulted in greater hydrocarbon concentrations in chemically enhanced water-accommodated fractions (CEWAFs) of oil, relative to the untreated water-accommodated fractions (WAFs). Topsmelt embryo development and survival to hatching was significantly inhibited in CEWAF tests while minimal effects on embryo-larval survival were observed in WAF tests. Increased hydrocarbon concentrations in the CEWAF tests caused cardiovascular and other abnormalities in developing topsmelt embryos.

Live birth in the Devonian period.

The extinct placoderm fishes were the dominant group of vertebrates throughout the Middle Palaeozoic era, yet controversy about their relationships within the gnathostomes (jawed vertebrates) is partly due to different interpretations of their reproductive biology. Here we document the oldest record of a live-bearing vertebrate in a new ptyctodontid placoderm, Materpiscis attenboroughi gen. et sp. nov., from the Late Devonian Gogo Formation of Australia (approximately 380 million years ago). The new specimen, remarkably preserved in three dimensions, contains a single, intra-uterine embryo connected by a permineralized umbilical cord. An amorphous crystalline mass near the umbilical cord possibly represents the recrystallized yolk sac. Another ptyctodont from the Gogo Formation, Austroptyctodus gardineri, also shows three small embryos inside it in the same position. Ptyctodontids have already provided the oldest definite evidence for vertebrate copulation, and the new specimens confirm that some placoderms had a remarkably advanced reproductive biology, comparable to that of some modern sharks and rays. The new discovery points to internal fertilization and viviparity in vertebrates as originating earliest within placoderms.

Genetic analysis of somite formation in laboratory fish models.

The repeated appearance of somites is one of the most fascinating aspects of vertebrate embryogenesis. Recent studies identified complex regulatory circuits that provide the molecular basis for the "clock and wave front" model, postulated almost 30 years ago by Cooke and Zeeman. The highly coordinated process of somite formation involves several networks of molecular cascades including the Delta/Notch, Wnt, FGF and retinoid signalling pathways. Studies in mouse, Xenopus and especially chicken over the last decade have helped to understand the role and interactions of these pathways in somitogenesis. More recently, this has been supplemented by experiments in zebrafish. This animal model offers the possibility of performing large scale mutagenesis screens to identify novel factors and pathways involved in somitogenesis. Molecular cloning of zebrafish somite mutants mainly resulted in genes that belong to the Delta/Notch pathway and therefore underlined the importance of this pathway during somitogenesis. The fact that other pathways have not yet been identified by genetic screening in this species was assumed to be caused by functional redundancy of duplicated genes in zebrafish. In 2000, a large-scale mutagenesis screen has been initiated in Kyoto, Japan using the related teleost medaka (Oryzias latipes). In this screen, mutants with unique phenotypes have been identified, which have not been described in zebrafish or mouse. In this chapter, we will review the progress that has been made in understanding the molecular control of somite formation in zebrafish and will discuss recent efforts to screen for novel phenotypes using medaka somitogenesis mutants.