High Energy Water Accommodated Fraction Research Articles

Uptake of polycyclic aromatic hydrocarbons via high-energy water accommodated fraction (HEWAF) by beach hoppers (Amphipoda, Talitridae) using different sandy beach exposure pathways

Sandy beach ecosystems are highly dynamic coastal environments subject to a variety of anthropogenic pressures and impacts. Pollution from oil spills can damage beach ecosystems through the toxic effects of hydrocarbons on organisms and the disruptive nature of large-scale clean-up practices. On temperate sandy beaches, intertidal talitrid amphipods are primary consumers of macrophyte wrack subsidies and serve as prey for higher trophic level consumers, such as birds and fish. These integral organisms of the beach food web can be exposed to hydrocarbons by direct contact with oiled sand through burrowing and by the consumption of oiled wrack. We experimentally evaluated the primary polycyclic aromatic hydrocarbon (PAH) exposure pathway via high-energy water accommodated fraction (HEWAF) for a species of talitrid amphipod (Megalorchestia pugettensis). Our results indicated that tissue PAH concentrations in talitrids were six-fold higher in treatments that included oiled sand compared to those with only oiled kelp and the controls.

Acute exposure of early-life stage zebrafish (Danio rerio) to Deepwater Horizon crude oil impairs glomerular filtration and renal fluid clearance capacity.

The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any defects in kidney development and function are likely associated with a decreased ability to osmoregulate. Previous work has shown that early-life stage (ELS) zebrafish (Danio rerio) acutely exposed to Deepwater Horizon (DWH) crude oil exhibit transcriptional changes in key genes involved in pronephros development and function, as well as pronephric morphological defects and whole-animal osmoregulatory impairment. The objective of this study was to examine the acute effects of crude oil exposure during zebrafish ELS on pronephros function by assessing its fluid clearance capacity and glomerular filtration integrity. Following a 72-h exposure to control conditions, 20% or 40% dilutions of high-energy water-accommodated fractions (HEWAF) of DWH crude oil, zebrafish were injected into the common cardinal vein either with fluorescein-labeled (FITC) 70-kDa dextran to assess glomerular filtration integrity or with FITC-inulin to assess pronephric clearance capacity. Fluorescence was quantified after the injections at predetermined time intervals by fluorescence microscopy. The results demonstrated a diminished pronephric fluid clearance capacity and failed glomerular perfusion when larvae were exposed to 40% HEWAF dilutions, whereas only a reduced glomerular filtration selectivity was observed in zebrafish previously exposed to the 20% HEWAF dilution.

The Influence of Oil-in-Water Preparations on the Toxicity of Crude Oil to Marine Invertebrates and Fish Following Short-Term Pulse and Continuous Exposures.

Following an oil spill, accurate assessments of the ecological risks of exposure to compounds within petroleum are required, as is knowledge regarding how those risks may change with the use of chemical dispersants. Laboratory toxicity tests are frequently used to assess these risks, but differences in the methods for preparation of oil-in-water solutions may confound interpretation, as may differences in exposure time to those solutions. In the present study, we used recently developed modifications of standardized ecotoxicity tests with copepods (Acartia sinjiensis), sea urchins (Heliocidaris tuberculata), and fish embryos (Seriola lalandi) to assess their response to crude oil solutions and assessed whether the oil-in-water preparation method changed the results. We created a water-accommodated fraction, a chemically enhanced water-accommodated fraction, and a high-energy water-accommodated fraction (HEWAF) using standard approaches using two different dispersants, Corexit 9500 and Slickgone NS. We found that toxicity was best related to total polycyclic aromatic hydrocarbon (TPAH) concentrations in solution, regardless of the preparation method used, and that the HEWAF was the most toxic because it dispersed the highest quantity of oil into solution. The TPAH composition in water did not vary appreciably with different preparation methods. For copepods and sea urchins, we also found that at least some of the toxic response could be attributed to the chemical oil dispersant. We did not observe the characteristic cardiac deformities that have been previously reported in fish embryos, most likely due to the use of unweathered oil, and, as a consequence, the high proportion of naphthalenes relative to cardiotoxic polycyclic aromatic hydrocarbon in the overall composition. The present study highlights the need to characterize both the TPAH composition and concentration in test solutions when assessing oil toxicity. Environ Toxicol Chem 2022;41:2580-2594. © 2022 SETAC.

Early life-stage Deepwater Horizon crude oil exposure induces latent osmoregulatory defects in larval red drum (Sciaenops ocellatus)

Crude oil is known to induce developmental defects in teleost fish exposed during early-life stages (ELSs). A recent study has demonstrated that zebrafish (Danio rerio) larvae acutely exposed to Deepwater Horizon (DHW) crude oil showed transcriptional changes in key genes involved in early kidney (pronephros) development and function, which were coupled with pronephric morphological defects. Given the osmoregulatory importance of the kidney, it is unknown whether ELS effects arising from short-term crude exposures result in long-term osmoregulatory defects, particularly within estuarine fishes likely exposed to DWH oil following the spill. To address this knowledge gap, an acute 72 h exposure to red drum (Sciaenops ocellatus) larvae was performed using high-energy water-accommodated fractions (HEWAFs) of DWH weathered oil to analyze transcriptional changes in genes involved in pronephros development and function by quantitative PCR. To test the latent effects of oil exposure on osmoregulation ability, red drum larvae were first exposed to HEWAF for 24 h. Larvae were then reared in clean seawater for two weeks and a 96 h acute osmotic challenge test was performed by exposing the fish to waters with varying salinities. Latent effects of ELS crude oil exposure on osmoregulation were assessed by quantifying survival during the acute osmotic challenge test and analyzing transcriptional changes at 14 dpf. Results demonstrated that ELS crude oil exposure reduced survival of red drum larvae when challenged in hypoosmotic waters and that latent transcriptional changes in some target pronephric genes were evident, indicating that an affected kidney likely contributed to the increased mortality.

Metabolic responses to crude oil during early life stages reveal critical developmental windows in the zebrafish (Danio rerio)

Morphological effects of crude oil exposure on early development in fishes have been well documented, but crude oil's metabolic effects and when in early development these effects might be most prominent remains unclear. We hypothesized that zebrafish (Danio rerio) exposed to crude oil as a high energy water accommodated fraction (HEWAF) would show increased routine oxygen consumption (ṀO2) and critical oxygen tension (PCrit) and this effect would be dependent upon day of HEWAF exposure, revealing critical windows of development for exposure effects. Zebrafish were exposed to 0%, 10%, 25%, 50% or 100% HEWAF for 24 h during one of the first six days post-fertilization (dpf). Survival rate, body mass, routine ṀO2, and PCrit were then measured at 7 dpf. Survival rate and especially body mass were both decreased based on both exposure concentration and day of crude oil exposure, with the largest decrease when HEWAF exposure occurred at 3 dpf. HEWAF effects on routine ṀO2 also differed depending upon exposure day. The largest effect occurred at 3 dpf, when ṀO2 increased significantly by ~60% from 10.1 ± 0.8 μmol O2/g/h compared to control group value of 6.3 ± 0.4 μmol O2/g/h. No significant effects of HEWAF exposure on any day were evident for PCrit (85 ± 4 mmHg in the control population). Overall, the main effects on body mass and ṀO2 measured at 7 dpf occurred when HEWAF exposures occurred at ~3 dpf. This critical window for metabolism in zebrafish larvae coincides with time of hatching, which may represent an especially vulnerable period in development.

Exposure of zebrafish larvae to water accommodated fractions of weathered crude oil alters steroid hormone concentrations with minimal effect on cholesterol.

Crude oil has multiple toxic effects in fish, particularly during their early life stages. Recent transcriptomics studies have highlighted a potential effect on cholesterol homeostasis and biosynthesis, but have not investigated effects on steroid hormones, which are biosynthetically downstream metabolites of cholesterol. We exposed zebrafish (Danio rerio) embryos and larvae to 3 concentrations of a high energy water accommodated fraction (HEWAF) of crude oil and measured effects on cholesterol and steroid hormones at 48 and 96h post fertilization (hpf). HEWAF exposure caused a small decrease in cholesterol at 96 hpf but not 48 hpf. HEWAF-exposed larvae had higher levels of androstenedione, testosterone, estradiol, cortisol, corticosterone, and progesterone at 96 hpf compared to controls, while effects at 48 hpf were more modest or not present. 2-Methoxyestradiol was lower following HEWAF exposure at both time points. Dihydrotestosterone was elevated in one HEWAF concentration at 48 hpf only. Our results suggest that hormone imbalance may be an important toxic effect of oil HEWAF exposure despite no major effect on their biosynthetic precursor cholesterol.

The developing zebrafish kidney is impaired by Deepwater Horizon crude oil early-life stage exposure: A molecular to whole-organism perspective

Crude oil is known to induce developmental defects in teleost fish exposed during early life stages (ELSs). While most studies in recent years have focused on cardiac endpoints, evidence from whole-animal transcriptomic analyses and studies with individual polycyclic aromatic hydrocarbons (PAHs) indicate that the developing kidney (i.e., pronephros) is also at risk. Considering the role of the pronephros in osmoregulation, and the common observance of edema in oil-exposed ELS fish, surprisingly little is known regarding the effects of oil exposure on pronephros development and function. Using zebrafish (Danio rerio) ELSs, we assessed the transcriptional and morphological responses to two dilutions of high-energy water accommodated fractions (HEWAF) of oil from the Deepwater Horizon oil spill using a combination of qPCR and whole-mount in situ hybridization (WM-ISH) of candidate genes involved in pronephros development and function, and immunohistochemistry (WM-IHC). To assess potential functional impacts on the pronephros, three 24 h osmotic challenges (2 hypo-osmotic, 1 near iso‐osmotic) were implemented at two developmental time points (48 and 96 h post fertilization; hpf) following exposure to HEWAF. Changes in transcript expression level and location specific to different regions of the pronephros were observed by qPCR and WM-ISH. Further, pronephros morphology was altered in crude oil exposed larvae, characterized by failed glomerulus and neck segment formation, and straightening of the pronephric tubules. The osmotic challenges at 96 hpf greatly exacerbated edema in both HEWAF-exposed groups regardless of osmolarity. By contrast, larvae at 48 hpf exhibited no edema prior to the osmotic challenge, but previous HEWAF exposure elicited a concentration-response increase in edema at hypo-osmotic conditions that appeared to have been largely alleviated under near iso‐osmotic conditions. In summary, ELS HEWAF exposure impaired proper pronephros development in zebrafish, which coupled with cardiotoxic effects, most likely reduced or inhibited pronephros fluid clearance capacity and increased edema formation.

Multi-stressor Effects of Ultraviolet Light, Temperature, and Salinity on Louisiana Sweet Crude Oil Toxicity in Larval Estuarine Organisms.

When oil is spilled into the environment its toxicity is affected by abiotic conditions. The cumulative and interactive stressors of chemical contaminants and environmental factors are especially relevant in estuaries where tidal fluctuations cause wide variability in salinity, temperature, and ultraviolet (UV) light penetration, which is an important modifying factor for polycyclic aromatic hydrocarbon (PAH) toxicity. Characterizing the interactions of multiple stressors on oil toxicity will improve prediction of environmental impacts under various spill scenarios. This study examined changes in crude oil toxicity with temperature, salinity, and UV light. Oil exposures included high-energy, water-accommodated fractions (HEWAFs) and thin oil sheens. Larval (24-48h post hatch) estuarine species representing different trophic levels and habitats were evaluated. Mean 96h LC50 values for oil prepared as a HEWAF and tested under standard conditions (20 ppt, 25°C, No-UV) were 62.5µg/L tPAH50 (mud snails), 198.5µg/L (grass shrimp), and 774.5µg/L (sheepshead minnows). Thin oil sheen 96h LC50 values were 5.3µg/L tPAH50 (mud snails), 14.7µg/L (grass shrimp), and 22.0µg/L (sheepshead minnows) under standard conditions. UV light significantly increased the toxicity of oil in all species tested. Oil toxicity also was greater under elevated temperature and lower salinity. Multi-stressor (oil combined with either increased temperature, decreased salinity, or both) LC50 values were reduced to 3µg/L tPAH50 for HEWAFs and < 1.0µg/L tPAH50 for thin oil sheens. Environmental conditions at the time of an oil spill will significantly influence oil toxicity and organismal response and should be taken into consideration in toxicity testing and oil spill damage assessments.

Effects of polycyclic aromatic hydrocarbons and abiotic stressors on Fundulus grandis cardiac transcriptomics

Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Many studies have identified the toxicological effects of PAHs in estuarine and marine fishes, however, only recently has work begun to identify the combinatorial effect of PAHs and abiotic environmental factors such as hypoxia, salinity, and temperature. This study aims to characterize the combined effects of abiotic stressors and PAH exposure on the cardiac transcriptomes of developing Fundulus grandis larvae. In this study, F. grandis larvae were exposed to varying environmental conditions (dissolved oxygen (DO) 2, 6 ppm; temperature 20, 30 °C; and salinity 3, 30 ppt) as well as to a single concentration of high energy water accommodated fraction (HEWAF) (∑PAHs 15 ppb). Whole larvae were sampled for RNA and transcriptional changes were quantified using RNA-Seq followed by qPCR for a set of target genes. Analysis revealed that exposure to oil and abiotic stressors impacts signaling pathways associated with cardiovascular function. Specifically, combined exposures appear to reduce development of the systemic vasculature as well as strongly impact the cardiac musculature through cardiomyocyte proliferation resulting in inhibited cardiac function and modulated blood pressure maintenance. Results of this study provide a holistic view of impacts of PAHs and common environmental stressors on the cardiac system in early life stage estuarine species. To our knowledge, this study is one of the first to simultaneously manipulate oil exposure with abiotic factors (DO, salinity, temperature) and the first to analyze cardiac transcriptional responses under these co-exposures.

Effect of Louisiana sweet crude oil on a Pacific coral, Pocillopora damicornis

Recent oil spill responses such as the Deepwater Horizon event have underscored the need for crude oil ecotoxicological threshold data for shallow water corals to assist in natural resource damage assessments. We determined the toxicity of a mechanically agitated oil-seawater mixture (high-energy water-accommodated fraction, HEWAF) of a sweet crude oil on a branched stony coral, Pocillopora damicornis. We report the results of two experiments: a 96 h static renewal exposure experiment and a “pulse-chase” experiment of three short-term exposure durations followed by a recovery period in artificial seawater. Five endpoints were used to determine ecotoxicological values: 1) algal symbiont chlorophyll fluorescence, 2) a tissue regeneration assay and a visual health metric with three endpoints: 3) tissue integrity, 4) tissue color, and 5) polyp behavior. The sum of 50 entrained polycyclic aromatic hydrocarbons (tPAH50) was used as a proxy for oil exposure. For the 96 h exposure dose response experiment, dark-adapted maximum quantum yield (Fv/Fm) of the dinoflagellate symbionts was least affected by crude oil (EC50 = 913 μg/L tPAH50); light-adapted effective quantum yield (EQY) was more sensitive (EC50 = 428 μg/L tPAH50). In the health assessment, polyp behavior (EC50 = 27 μg/L tPAH50) was more sensitive than tissue integrity (EC50 = 806 μg/L tPAH50) or tissue color (EC50 = 926 μg/L tPAH50). Tissue regeneration proved to be a particularly sensitive measurement for toxicity effects (EC50 = 10 μg/L tPAH50). Short duration (6−24 h) exposures using 503 μg/L tPAH50 (average concentration) resulted in negative impacts to P. damicornis and its symbionts. Recovery of chlorophyll a fluorescence levels for 6−24 h oil exposures was observed in a few hours (Fv/Fm) to several days (EQY) following recovery in fresh seawater. The coral health assessments for tissue integrity and tissue color were not affected following short-term oil exposure durations, but the 96 h treatment duration resulted in significant decreases for both. A reduction in polyp behavior (extension) was observed for all treatment durations, with recovery observed for the short-term (6−24 h) exposures within 1–2 days following placement in fresh seawater. Wounded and intact fragments exposed to oil treatments were particularly sensitive, with significant delays observed in tissue regeneration. Estimating ecotoxicological values for P. damicornis exposed to crude oil HEWAFs provides a basis for natural resource damage assessments for oil spills in reef ecosystems. These data, when combined with ecotoxicological values for other coral reef species, will contribute to the development of species sensitivity models.

Exposure to Oil and Hypoxia Results in Alterations of Immune Transcriptional Patterns in Developing Sheepshead Minnows (Cyprinodon variegatus)

The area and timing of the Deepwater Horizon oil spill highlight the need to study oil and hypoxia exposure in early life stage fishes. Though critical to health, little research has targeted the effect of oil and hypoxia exposure on developing immune systems. To this end, we exposed sheepshead minnows (Cyprinodon variegatus) at three early life stages: embryonic; post-hatch; and post-larval, to a high energy water accommodated fraction (HEWAF) of oil, hypoxia, or both for 48 hours. We performed RNAseq to understand how exposures alter expression of immune transcripts and pathways. Under control conditions, the embryonic to post-hatch comparison (first transition) had a greater number of significantly regulated immune pathways than the second transition (post-hatch to post-larval). The addition of oil had little effect in the first transition, however, hypoxia elicited changes in cellular and humoral immune responses. In the second transition, oil exposure significantly altered many immune pathways (43), and while hypoxia altered few pathways, it did induce a unique signature of generally suppressing immune pathways. These data suggest that timing of exposure to oil and/or hypoxia matters, and underscores the need to further investigate the impacts of multiple stressors on immune system development in early life stage fishes.

Parental exposure to Deepwater Horizon oil in different environmental scenarios alters development of sheepshead minnow (Cyprinodon variegatus) offspring

The explosion of the Deepwater Horizon (DWH) oil exploration platform on April 20, 2010 began a catastrophic leak of approximately 640 million liters crude oil into the northern Gulf of Mexico (GOM), affecting more than 2100 km of coastline, including wetlands and estuaries that provide habitat and nursery for many aquatic species. Estuaries of the GOM are dynamic environments, with constant fluctuations in salinity and dissolved oxygen, including large hypoxic zones during summer months. Spawning fish in northern GOM estuaries following the DWH incident were at significant risk of oil exposure, and adverse environmental conditions at the time of exposure, such as hypoxia and low salinity, could have exacerbated developmental effects in the offspring. The present study investigated the effects of F0 parental oil exposure in different environmental scenarios on development of F1 sheepshead minnow (SHM) offspring. Adult SHM were exposed to the high-energy water accommodated fraction (HEWAF) of crude oil in three environmental scenarios: normoxic (NORM), hypoxic (HYP), and hypoxic with low salinity (HYP-LS). Parental HEWAF exposure in the NORM scenario resulted in developmental effects in F1 offspring, including altered heart rate, decreased length at hatch, and impaired prey capture. Co-exposure of F0 SHM to HEWAF and adverse environmental conditions altered HEWAF effects on F1 heart rate, hatch rate, prey capture, and survival. Time to hatch was not significantly impacted by parental HEWAF in any environmental scenario. The present study demonstrates that parental exposure to HEWAF results in developmental changes in F1 embryos, and co-exposure to adverse environmental conditions altered the effects for several developmental endpoints. These data suggest that SHM exposed to oil in estuaries experiencing hypoxia or low salinity may produce offspring with worsened outcomes. These developmental effects, in addition to previously reported reproductive effects in adult fish, could lead to long-term population level impacts for SHM.

The combined effects of salinity, hypoxia, and oil exposure on survival and gene expression in developing sheepshead minnows, Cyprinodon variegatus

The 2010 Deepwater Horizon oil spill released approximately 780 million liters of crude oil contaminating coastal habitats from Texas to Florida which are important habitats for many fish species during early life stages. These diverse habitats are also prone to rapid fluctuations in water quality, such as dissolved oxygen concentration and salinity. The consequence of combined exposure to crude oil and suboptimal environmental conditions during early life stage development of fish is still largely unknown. The objective of this project was to investigate the impacts of exposure to crude oil in combination with varying environmental stressors on developing Cyprinodon variegatus survival, growth, and gene expression. Three life stages (embryonic, post-hatch, and post-larval) were exposed to four nominal concentrations (6.25%, 12.5%, 50% and 100% with actual polycyclic aromatic hydrocarbon (PAH) concentrations ranging from 0 to 512 μg/L) of high energy water accommodated fractions (HEWAF) under different oxic (2.0 or >5.0 mg/L) and salinity (10 or 30 ppt) regimes at 30 °C for 48 h. We found that the post-larval developmental stage was the most sensitive to oil toxicity. Median lethal concentrations during the post-larval exposures followed a treatment-dependent pattern with the highest mortality observed under hypoxic-high salinity conditions (64.55 μg/L). Real-time PCR analysis identified down regulation of target genes, encoding cytochrome P450-1α (cyp1a1), erythropoietin (epo), and the aryl hydrocarbon receptor nuclear translocator (arnt1) only when oil exposure occurred under hypoxic-high salinity conditions in treatments with PAH concentrations greater than 226 μg/L. The target genes measured in this experiment are involved in the aryl hydrocarbon receptor signaling pathway which modulates metabolism of PAHs (a major component of crude oil), and the hypoxia inducible 1-α signaling pathway which is responsible for resilience to hypoxic stress, and it is known that disruption of these pathways can lead to an array of acute and chronic effects. Our results indicated that sheepshead minnow are most sensitive to oil exposure during the post-larval developmental stage. Survival data from this age-stage also indicate that oil toxicity response is exacerbated in hypoxic and high salinity environments. The increased mortality observed during the post-larval developmental stage might be attributed to the suppression of the aryl hydrocarbon receptor signaling and the hypoxia inducible 1-α signaling pathways which is evident in by the down-regulated expression of cyp1a1, epo, and arnt1. These findings provide more information about interactions between oil and abiotic factors which enable us to make better assumptions of the ecological impacts of DWH on coastal estuaries.

Blood-brain barrier function, cell viability, and gene expression of tight junction-associated proteins in the mouse are disrupted by crude oil, benzo[a]pyrene, and the dispersant COREXIT

Exposure to crude oil, its components, and oil dispersants during a major crude oil spill, such as the Deepwater Horizon Oil Spill, can elicit behavioral changes in animals and humans. However, the underlying mechanisms by which oil spill-related compounds alters behavior remains largely unknown. A major cause of behavioral changes generally is dysfunction of the blood-brain barrier (BBB). We investigated the impact of a crude oil high energy water accommodated fraction (HEWAF), benzo[a] pyrene (BaP; a major component of crude oil), and the oil dispersant COREXIT, on BBB function. BBB function was assessed by measuring transendothelial electrical resistance (TEER) of mouse brain microvascular endothelial cells (BMECs). Within 3 h after treatment, TEER was significantly reduced by exposure to high concentrations of all test compounds. TEER remained reduced in response to COREXIT after 48 h, but this effect waned in BMECs treated with HEWAF and BaP, with low-mid range concentrations inducing increased TEER compared to vehicle controls. At 48 h of treatment, BMEC viability was significantly reduced in response to 2% HEWAF, but was increased in response to BaP (25 and 50 μM). BMEC viability was increased with 80 ppm COREXIT, but was reduced with 160 ppm. Gene expression of tight junction-associated proteins (claudin-5 and tight junction protein-1), and cell adhesion receptor (vascular cell adhesion molecule-1) was reduced in response to HEWAF and COREXIT, but not BaP. Taken together, these data suggest that oil spill-related compounds markedly affect BBB function, and that these changes may underlie the observed behavioral changes due to crude oil exposure.

Hypoxia and reduced salinity exacerbate the effects of oil exposure on sheepshead minnow (Cyprinodon variegatus) reproduction

Estuaries of the northern Gulf of Mexico are dynamic environments, with fluctuations in salinity and dissolved oxygen, including areas of seasonal hypoxia. Fish that reside and reproduce in these estuaries, including sheepshead minnow (Cyprinodon variegatus; SHM), were at significant risk of oil exposure following the Deepwater Horizon oil spill. It is poorly understood how differences in environmental conditions during oil exposure impact its toxicity. The present study investigated the effects of crude oil high-energy water accommodated fraction (HEWAF) on SHM reproduction in three environmental scenarios (normoxic, hypoxic, and hypoxic with low salinity) to determine if differences in salinity (brackish vs low salinity) and dissolved oxygen (normoxia vs hypoxia) could exacerbate the effects of HEWAF-derived polycyclic aromatic hydrocarbons (PAHs). We observed that HEWAF exposure significantly increased liver somatic index of SHM compared to control, but this effect was not exacerbated by hypoxia or low salinity. HEWAF exposure also significantly decreased egg production and egg fertilization rate, but only in the hypoxic and hypoxic with low salinity scenarios. A significant correlation existed between body burdens of PAHs and reproductive endpoints, providing substantial evidence that oil exposure reduced reproductive capacity in SHM, across a range of environmental conditions. These data suggest that oil spill risk assessments that fail to consider other environmental stressors (i.e. hypoxia and salinity) may be underestimating risk.

Blood‐Brain Barrier as a Novel Link between Crude Oil Exposure and Behavioral Dysfunctions?

Low levels of exposure to crude oil and/or its derivatives are associated with numerous changes in coping responses from molecular to organismal levels in vertebrates ranging from fishes to humans. Less understood are behavioral implications of exposure, with a handful of studies suggesting that crude oil leads to behavior that is “riskier” and more aggressive. Moreover, the mechanisms underlying such behavioral changes are largely unknown. Given that blood‐brain barrier (BBB) disruption is implicated in many behavioral abnormalities, a likely, although uninvestigated, link between crude oil and associated behavioral changes is blood‐brain barrier dysfunction. We hypothesize that alterations in the integrity of the BBB is correlated with oil‐induced behavioral changes. In this poster we describe our programmatic approach consisting of three phases to test this hypothesis. Our strategy, based on three experimental approaches, tests the effects of crude oil exposure in the form of High Energy Water Accommodated Fractions (HEWAFs). 1) We have quantified behavioral changes in animal models (zebrafish, betta fish, and mouse). 2) We have assessed BBB integrity by recording real‐time transendothelial electrical resistance (TEER) of in vitro “BBBs” formed by a confluent layer of brain microvascular endothelial cells (BMECs), and assessed gene expression in these cultured endothelial cells. 3) We will correlate in vivo with in vitro findings. In the first phase of our study, initial in vivo assessment using both Siamese fighting fish and zebrafish as models, indicates that fish exposed to very low levels of crude oil HEWAF show significantly increased aggressive behavior, and reduced reproductive and other behaviors, without otherwise showing morphological or growth effects. The second phase involved high‐throughput in vitro assessment using mouse BMECs as a simplified model of the BBB. These experiments indicated that acute exposure to crude oil extracts, as well as environmentally relevant concentrations ofbenzo(a)pyrene (one of the components of crude oil), resulted in significant decreases in TEER within the first 3 to 6 h of exposure. These barrier changes were accompanied by changes in expression of tight junction‐associated proteins such as significantly reduced expression in BMECs of the cell adhesion protein Cldn5, 48h after exposure to 8% HEWAF. Expression of Tjp1, a scaffolding protein that links tight junction (TJ) transmembrane proteins, was also decreased after exposure to just 2% HEWAF. The third, future phase will correlate in vivo with in vitro findings by first inducing behavioral dysfunction in animal models using crude oil HEWAF exposure, and then isolating and culturing BMECs of these animals to determine if in vivo exposure results in the same persistent changes in BBB leakiness and reduced BBB viability that was observed in the phase two in vitro studies. By applying our multi‐faceted experimental approach, we will be able to more fully elucidate the impact of crude oil on the blood‐brain barrier, and discover how this can, in turn, affect behavior in animals and man.Support or Funding InformationGulf of Mexico Research Initiative U.S. Army/Department of DefenseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transgenerational effects of polycyclic aromatic hydrocarbon exposure on sheepshead minnows (Cyprinodon variegatus).

The Deepwater Horizon oil spill resulted in the release of over 640 million L of crude oil into the Gulf of Mexico, affecting over 2000 km of shoreline, including estuaries that serve as important habitats and nurseries for aquatic species. Cyprinodon variegatus (sheepshead minnow) are small-bodied fish that inhabit northern Gulf of Mexico estuaries, are easily adaptable to laboratory conditions, and are commonly used in toxicological assessment studies. The purpose of the present study was to determine the somatic, reproductive, and developmental effects of an environmentally relevant polycyclic aromatic hydrocarbon (PAH) mixture, the oil high-energy water accommodated fraction (HEWAF), on experimentally exposed sheepshead minnow (F0 ) as well as 2 generations of offspring (F1 and F2 ) without additional exposure. The F0 generation exposed to HEWAF had increased liver somatic indices, altered egg production, and decreased fertilization. Several developmental endpoints in the F1 were altered by F0 HEWAF exposure. As adults, low HEWAF-exposed F1 females demonstrated decreased weight and length. Both the F1 and F2 generations derived from high HEWAF-exposed F0 had deficits in prey capture compared to control F1 and F2 , respectively. Correlations between endpoints and tissue PAHs provide evidence that the physiological effects observed were associated with hydrocarbon exposure. These data demonstrate that PAHs were capable of causing physiological changes in exposed adult sheepshead minnow and transgenerational effects in unexposed offspring, both of which could have population-level consequences. Environ Toxicol Chem 2019;38:638-649. © 2018 SETAC.

Sublethal effects of oil-contaminated sediment to early life stages of the Eastern oyster, Crassostrea virginica

The explosion of the Deepwater Horizon (DWH) oil drilling rig resulted in the release of crude oil into the Gulf of Mexico. This event coincided with the spawning season of the Eastern oyster, Crassostrea virginica. Although oil bound to sediments constitutes an important source of polycyclic aromatic hydrocarbon (PAH) exposure to benthic organisms, toxicity of sediment-associated DWH oil has not been investigated in any bivalve species. Here, we evaluated the sublethal effects of acute exposure of gametes, embryos and veliger larvae of the Eastern oyster to different concentrations of unfiltered elutriates of sediment contaminated with DWH oil. Our results suggest that gametes, embryos and veliger larvae are harmed by exposure to unfiltered elutriates of contaminated sediment. Effective concentrations for fertilization inhibition were 40.6 μg tPAH50 L−1 and 173.2 μg tPAH50 L−1 for EC201h and EC501h values, respectively. Embryo exposure resulted in dose-dependent abnormalities (EC20 and EC50 values were 77.7 μg tPAH50 L−1 and 151 μg tPAH50 L−1, respectively) and reduction in shell growth (EC2024h value of 1180 μg tPAH50 L−1). Development and growth of veliger larvae were less sensitive to sediment-associated PAHs compared to embryos. Fertilization success and abnormality of larvae exposed as embryos were the most sensitive endpoints for assessing the toxicity of oil-contaminated sediment. Bulk of measured polycyclic aromatic hydrocarbons were sediment-bound and caused toxic effects at lower tPAH50 concentrations than high energy water accommodated fractions (HEWAF) preparations from the same DWH oil. This study suggests risk assessments would benefit from further study of suspended contaminated sediment.

Combined effects of Deepwater Horizon crude oil and environmental stressors on Fundulus grandis embryos.

In the present study, we examined how sensitivity to oil changes in combination with environmental stressors in Fundulus grandis embryos. We exposed embryos (<24 h post fertilization) to a range of high-energy water accommodated fraction (HEWAF) concentrations (0-50 parts per billion [ppb] total polycyclic aromatic hydrocarbons [PAHs]) made from Macondo crude oil in conjunction with various environmental conditions (temperature: 20 and 30 °C; salinity: 3, 7, and 30 practical salinity units [PSU]; and dissolved oxygen: 2 and 6 mg/L). Endpoints included mortality, hatching rates, and expression of cytochrome p450 1a and 1c (cyp1a, cyp1c) in hatched larvae. There was 100% mortality for all fish under the 2 parts per million (ppm) dissolved oxygen regimes. For the 6 mg/L dissolved oxygen treatments, mortality and median lethal time (LT50) were generally higher in the 30 °C treatments versus the 20 °C treatments. Oil increased mortality in fish exposed to the highest concentration in the 20-3-6 (°C-PSU-mg/L), 25-7-6, and 30-30-6 conditions. Hatching was driven by environmental conditions, with oil exposure having a significant impact on hatching in only the 25-7-6 and 30-30-6 groups at the greatest HEWAF exposure. Expression of cyp1a was up-regulated in most treatment groups versus the controls, with cyp1c expression exhibiting a similar pattern. These data suggest interactive effects among temperature, salinity, and PAHs, highlighting a need to further assess the effects of oil exposure under various environmental conditions. Environ Toxicol Chem 2018;37:1916-1925. © 2018 SETAC.

Evaluation of toxicity of Deepwater Horizon slick oil on spat of the oyster Crassostrea virginica.

The 2010 explosion of the Deepwater Horizon (DWH) oil rig generated the largest marine oil spill in US history with millions of barrels of crude oil released in the Gulf of Mexico (GoM). The eastern oyster, Crassostrea virginica, is an ecologically and economically important species in the northern GoM. Due to its biological characteristics (sessile, filter feeding), juvenile oysters may have been affected. This study investigated the effects of surface-collected DWH oil prepared as high-energy water-accommodated fraction (HEWAF) on the survival of 2-month-old oyster spat, and evaluated the potential impacts of HEWAF on particle clearance rate and spat tissue. Exposure of oysters to a range of oil/HEWAF (0-7-66-147-908-3450μg tPAH50 (sum of 50 polycyclic aromatic hydrocarbons)L-1) resulted in non-dose-dependent mortalities and reduced clearance rates of algal food (Tisochrysis lutea). A morphometric study of the digestive tubules (DGTs) indicated a dose-dependent response to oil exposure on lumen dilation, on epithelium thinning of the DGT, and a significant change in DGT synchrony (LOEC=66μg tPAH50L-1). This finding suggests that structural changes occurred in the digestive gland of exposed oysters most likely due to an oil-related stress. In addition, histological observations showed that tissues in contact with HEWAF (gills, palp, connective tissue, digestive gland) were adversely impacted at ≥ 7μg tPAH50L-1, and exhibited pathological symptoms typical of an inflammatory response (e.g., hemocyte diapedesis and infiltration, syncytia, epithelium sloughing).