Transcriptomic profiling of miR-203a inhibitor and miR-34b-injected zebrafish (Danio rerio) validates oil-induced neurological, cardiovascular and eye toxicity response pathways.

The global sequencing of microRNA (miRNA; miR) and integration to downstream mRNA expression profiles in early life stages (ELS) of fish following exposure to crude oil determined consistently dysregulated miRNAs regardless of the oil source or fish species. The overlay of differentially expressed miRNAs and mRNAs into in silico software determined that the key roles of these miRNAs were predicted to be involved in cardiovascular, neurological and visually-mediated pathways. Of these, altered expression of miRNAs, miR-203a and miR-34b were predicted to be primary targets of crude oil. To better characterize the effect of these miRNAs to downstream transcript changes, zebrafish embryos were microinjected at 1 h post fertilization (hpf) with either a miR-203a inhibitor or miR-34b. Since both miRs have been shown to be associated with aryl hydrocarbon receptor (AhR) function, benzo(a)pyrene (BaP), a potent AhR agonist, was used as a potential positive control. Transcriptomic profiling was conducted on injected and exposed larvae at 7 and 72 hpf, and eye morphology assessed following exposure at 72 hpf. The top predicted physiological system disease and functions between differentially expressed genes (DEGs) shared with miR-203a inhibitor-injected and miR-34b-injected embryos were involved in brain formation, and the development of the central nervous system and neurons. When DEGs of miR-203a inhibitor-injected embryos were compared with BaP-exposed DEGs, alterations in nervous system development and function, and abnormal morphology of the neurosensory retina, eye and nervous tissue were predicted, consistent with both AhR and non-AhR pathways. When assessed morphologically, the eye area of miR-203a inhibitor and miR-34b-injected and BaP-exposed embryos were significantly reduced. These results suggest that miR-203a inhibition and miR-34b overexpression contribute to neurological, cardiovascular and eye toxicity responses that are caused by oil and PAH exposure in ELS fish, and are likely mediated through both AhR and non-AhR pathways.

Transcriptomic responses and apoptosis in larval red drum (Sciaenops ocellatus) co-exposed to crude oil and ultraviolet (UV) radiation.

Ultraviolet (UV) radiation can significantly increase the toxicity of polycyclic aromatic hydrocarbons (PAHs) in crude oil to early life stage (ELS) fishes through photo-induced /photo-enhanced toxicity. However, little is known about the sub-lethal effects and mechanisms of photo-induced PAH toxicity in ELS fishes. The present study investigated apoptosis and global transcriptomic effects in larval red drum (Sciaenops ocellatus) (24-72 h post-fertilization) following co-exposure to oil (0.29-0.30 µg/L ∑PAH50) and UV. Apoptosis was quantified using the TUNEL assay, and transcriptomic effects were assessed using RNA sequencing analysis. Apoptotic fluorescence was greatest in the eyes and skin following 24 and 48 h co-exposure to oil and UV, indicating photo-induced toxicity. Consistent with these phenotypic responses, pathways associated with phototransduction, eye development, and dermatological disease were among the top predicted pathways impacted. The present study is the first to provide global transcriptomic analysis of UV and oil co-exposure in an ELS fish.

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 96 h 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.

A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

Effects of Dissolved Organic Carbon, Ultraviolet Light and their Co-Exposure on Deepwater Horizon crude oil acute toxicity to larval red drum (Sciaenops ocellatus).

In the aquatic environment, ubiquitous natural factors such as ultraviolet light (UV) and dissolved organic carbon (DOC) are likely to influence crude oil toxicity. The present study examined the interactive effects of DOC, UV, and DOC-UV co-exposure on the acute toxicity of Deepwater Horizon crude oil in larval red drum (Sciaenops ocellatus). Although DOC alone did not influence crude oil toxicity, it mildly reduced UV photo-enhanced toxicity. Environ Toxicol Chem 2020;39:2509-2515. © 2020 SETAC.

Exposure to Crude Oil Induces Retinal Apoptosis and Impairs Visual Function in Fish.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil are known to impair visual development in fish. However, the underlying mechanism of PAH-induced toxicity to the visual system of fish is not understood. Embryonic zebrafish (Danio rerio) at 4 h post fertilization were exposed to weathered crude oil and assessed for visual function using an optokinetic response, with subsequent samples taken for immunohistochemistry and gene expression analysis. Cardiotoxicity was also assessed by measuring the heart rate, stroke volume, and cardiac output, as cardiac performance has been proposed to be a contributing factor to eye-associated malformations following oil exposure. Larvae exposed to the highest concentrations of crude oil (89.8 µg/L) exhibited an increased occurrence of bradycardia, though no changes in stroke volume or cardiac output were observed. However, genes important in eye development and phototransduction were downregulated in oil-exposed larvae, with an increased occurrence of cellular apoptosis, reduced neuronal connection, and reduced optokinetic behavioral response in zebrafish larvae.

Changes in microRNA-mRNA Signatures Agree with Morphological, Physiological, and Behavioral Changes in Larval Mahi-Mahi Treated with Deepwater Horizon Oil.

In this study, we performed a systematic evaluation of global microRNA-mRNA interactions associated with the developmental toxicity of Deepwater Horizon oil using a combination of integrated mRNA and microRNA deep sequencing, expression profiling, gene ontology enrichment, and functional predictions by a series of advanced bioinformatic tools. After exposure to water accommodated fraction (WAF) of both weathered slick oil (0.5%, 1%, and 2%) and source oil (0.125%, 0.25%, and 0.5%) from the Deep Water Horizon oil spill, four dose-dependent miRNAs were identified, including three up-regulated (miR-23b, miR-34b, and miR-181b) and one down-regulated miRNAs (miR-203a) in mahi-mahi hatchings exposed from 6 h postfertilization (hpf) to 48 hpf. Consistent with morphological, physiological, and behavioral changes, the target genes of these miRNAs were largely involved in the development of the cardiovascular, visual, nervous system and associated toxicity pathways, suggesting that miRNAs play an essential role in regulating the responses to oil exposure. The results obtained from this study improve our understanding of the role of miRNAs and their target genes in relation to dose-dependent oil toxicity and provide the potential of using miRNAs as novel biomarkers in future oil studies.

Effects of Deepwater Horizon crude oil on ocular development in two estuarine fish species, red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil have been shown to cause the dysregulation of genes important in eye development and function, as well as morphological abnormalities of the eye. However, it is not currently understood how these changes in gene expression are manifested as deficits in visual function. Embryonic red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus) were exposed to water accommodated fractions (WAFs) of weathered crude oil and assessed for visual function using an optomotor response assay in early life-stage larvae, with subsequent samples taken for histological analysis of the eyes. Larvae of both species exposed to increasing concentrations of oil exhibited a reduced optomotor response. The mean diameters of retinal layers, which play an important role in visual function and image processing, were significantly reduced in oil-exposed sheepshead larvae, though not in red drum larvae. The present study provides evidence that weathered crude oil has a significant effect on visual function in early life-stage fishes.

Estimating incident ultraviolet radiation exposure in the northern Gulf of Mexico during the Deepwater Horizon oil spill.

Millions of barrels of oil were released into the Gulf of Mexico following the 2010 explosion of the Deepwater Horizon oil rig. Polycyclic aromatic hydrocarbons (PAHs) are toxic components of crude oil, which may become more toxic in the presence of ultraviolet (UV) radiation, a phenomenon known as photo-induced toxicity. The Deepwater Horizon spill impacted offshore and estuarine sites, where biota may be co-exposed to UV and PAHs. Penetration of UV into the water column is affected by site-specific factors. Therefore, measurements and/or estimations of UV are necessary when one is assessing the risk to biota posed by photo-induced toxicity. We describe how estimates of incident UV were determined for the area impacted by the Deepwater Horizon oil spill, using monitoring data from radiometers near the spill, in conjunction with reference spectra characterizing the composition of solar radiation. Furthermore, we provide UV attenuation coefficients for both near- and offshore sites in the Gulf of Mexico. These estimates are specific to the time and location of the spill, and fall within the range of intensities utilized during photo-induced toxicity tests performed in support of the Deepwater Horizon Natural Resource Damage Assessment (NRDA). These data further validate the methodologies and findings of phototoxicity tests included in the Deepwater Horizon NRDA, while underscoring the importance of considering UV exposure when assessing possible risks following oil spills. Environ Toxicol Chem 2018;37:1679-1687. © 2018 SETAC.

Photo-induced toxicity in early life stage fiddler crab (Uca longisignalis) following exposure to Deepwater Horizon oil.

The 2010 explosion of the Deepwater Horizon (DWH) oil rig led to the release of millions of barrels of oil in the Gulf of Mexico. Oil in aquatic ecosystems exerts toxicity through multiple mechanisms, including photo-induced toxicity following co-exposure with UV radiation. The timing and location of the spill coincided with both fiddler crab reproduction and peak yearly UV intensities, putting early life stage fiddler crabs at risk of injury due to photo-induced toxicity. The present study assessed sensitivity of fiddler crab larvae to photo-induced toxicity during co-exposure to a range of environmentally relevant dilutions of high-energy water accommodated fractions of DWH oil, and either <10, 50, or 100% ambient sunlight, achieved with filters that allowed for variable UV penetration. Solar exposures (duration: 7-h per day) were conducted for two consecutive days, with a dark recovery period (duration: 17-h) in between. Survival was significantly decreased in treatments the presence of >10% UV and relatively low concentrations of oil. Results of the present study indicate fiddler crab larvae are sensitive to photo-induced toxicity in the presence of DWH oil. These results are of concern, as fiddler crabs play an important role as ecosystem engineers, modulating sediment biogeochemical processes via burrowing action. Furthermore, they occupy an important place in the food web in the Gulf of Mexico.

Photo-induced toxicity following exposure to crude oil and ultraviolet radiation in 2 Australian fishes.

Some polycyclic aromatic hydrocarbons (PAHs), components of crude oil, are known to cause increased toxicity when organisms are co-exposed with ultraviolet radiation, resulting in photo-induced toxicity. The photodynamic characteristics of some PAHs are of particular concern to places like Australia with high ultraviolet radiation levels. The objective of the present study was to characterize the photo-induced toxicity of an Australian North West Shelf oil to early life stage yellowtail kingfish (Seriola lalandi) and black bream (Acanthopagrus butcheri). The fish were exposed to high-energy water accommodated fractions for 24 to 36 h. During the exposure, the fish were either co-exposed to full-intensity or filtered natural sunlight and then transferred to clean water. At 48 h, survival, cardiac effects, and spinal deformities were assessed. Yellowtail kingfish embryos co-exposed to oil and full-spectrum sunlight exhibited decreased hatching success and a higher incidence of cardiac arrhythmias, compared with filtered sunlight. A significant increase in the incidence of pericardial edema occurred in black bream embryos co-exposed to full-spectrum sunlight. These results highlight the need for more studies investigating the effects of PAHs and photo-induced toxicity under environmental conditions relevant to Australia. Environ Toxicol Chem 2018;37:1359-1366. © 2018 SETAC.

Larval Red Drum (Sciaenops ocellatus) Sublethal Exposure to Weathered Deepwater Horizon Crude Oil: Developmental and Transcriptomic Consequences.

The Deepwater Horizon (DWH) incident resulted in extensive oiling of the pelagic zone and shoreline habitats of many commercially important fish species. Exposure to the water-accommodated fraction (WAF) of oil from the spill causes developmental toxicity through cardiac defects in pelagic fish species. However, few studies have evaluated the effects of the oil on near-shore estuarine fish species such as red drum (Sciaenops ocellatus). Following exposure to a certified weathered slick oil (4.74 µg/L ∑PAH50) from the DWH event, significant sublethal impacts were observed ranging from impaired nervous system development [average 17 and 22% reductions in brain and eye area at 48 h postfertilization (hpf), respectively] to abnormal cardiac morphology (100% incidence at 24, 48, and 72 hpf) in red drum larvae. Consistent with the phenotypic responses, significantly differentially expressed transcripts, enriched gene ontology, and altered functions and canonical pathways predicted adverse outcomes in nervous and cardiovascular systems, with more pronounced changes at later larval stages. Our study demonstrated that the WAF of weathered slick oil of DWH caused morphological abnormalities predicted by a suite of advanced bioinformatic tools in early developing red drum and also provided the basis for a better understanding of molecular mechanisms of crude oil toxicity in fish.

Combined effects of oil exposure, temperature and ultraviolet radiation on buoyancy and oxygen consumption of embryonic mahi-mahi, Coryphaena hippurus.

The Deepwater Horizon oil spill occurred in the summer of 2010 and coincided with the spawning window of the ecologically and economically important pelagic fish mahi-mahi (Coryphaena hippurus). During summer months, early life stage mahi-mahi were likely also exposed to other naturally occurring stressors such as increased temperature and ultraviolet radiation (UV). Previous research has shown that co-exposure to oil and additional natural stressors can affect the timing and duration of negative buoyancy in mahi-mahi embryos. The current study aimed to elucidate the factors affecting the onset of negative buoyancy and to also explore possible mechanisms behind buoyancy change. Embryos co-exposed to oil and/or increased temperature and UV radiation displayed early onset of negative buoyancy with concurrent increases in oxygen consumption and sinking rates, which are normally only seen during the period directly preceding hatch. Results also suggest a behavioral response in which embryos avoid UV radiation by sinking down the water column but reestablish positive buoyancy once the UV radiation is removed. These findings imply that embryos can dynamically change their position in the water column in response to external cues and thus may have much greater control over buoyancy than previously thought.

Co-exposure to sunlight enhances the toxicity of naturally weathered Deepwater Horizon oil to early lifestage red drum (Sciaenops ocellatus) and speckled seatrout (Cynoscion nebulosus).

The 2010 Deepwater Horizon oil spill resulted in the accidental release of millions of barrels of crude oil into the Gulf of Mexico. Photo-induced toxicity following co-exposure to ultraviolet (UV) radiation is 1 mechanism by which polycyclic aromatic hydrocarbons (PAHs) from oil spills may exert toxicity. Red drum and speckled seatrout are both important fishery resources in the Gulf of Mexico. They spawn near-shore and produce positively buoyant embryos that hatch into larvae in approximately 24 h. The goal of the present study was to determine whether exposure to UV as natural sunlight enhances the toxicity of crude oil to early lifestage red drum and speckled seatrout. Larval fish were exposed to several dilutions of high-energy water-accommodated fractions (HEWAFs) from 2 different oils collected in the field under chain of custody during the 2010 spill and 3 gradations of natural sunlight in a factorial design. Co-exposure to natural sunlight and oil significantly reduced larval survival compared with exposure to oil alone. Although both species were sensitive at PAH concentrations reported during the Deepwater Horizon spill, speckled seatrout demonstrated a greater sensitivity to photo-induced toxicity than red drum. These data demonstrate that even advanced weathering of slicks does not ameliorate the potential for photo-induced toxicity of oil to these species. Environ Toxicol Chem 2017;36:780-785. © 2016 SETAC.

Exposure to ultraviolet radiation late in development increases the toxicity of oil to mahi-mahi (Coryphaena hippurus) embryos.

The Deepwater Horizon oil spill in 2010 overlapped with the spawning of many pelagic fish species in the Gulf of Mexico, including mahi-mahi (Coryphaena hippurus). Polycyclic aromatic hydrocarbons (PAHs) released during the spill have been shown to cause photo-induced toxicity under ultraviolet (UV) radiation. In the present study, mahi-mahi embryos were exposed to high-energy water accommodated fractions of source and naturally weathered oils for up to 48 h. The timing of co-exposure with UV radiation varied between an early development exposure for 8 h or a late development exposure for 8 h. The UV co-exposure had a photo-induced toxic effect on hatching success for all oil types and exposure scenarios. A more sensitive developmental window to photo-induced toxicity was observed when UV exposure occurred late in development. Source Oil B was over 6-fold more toxic, and Massachusetts source oil was 1.6-fold more toxic when the embryos were co-exposed to UV light late in development. Furthermore, weathered oil from the surface co-exposure with UV late in development resulted in bradycardia in the mahi-mahi. The present study provides evidence that the developmental window when UV co-exposure occurs has a significant effect on the degree of photo-induced toxicity of oil and that UV co-exposure may exacerbate long-term cardiac effects in developing fish. Environ Toxicol Chem 2017;36:1592-1598. © 2016 SETAC.

A novel system for embryo-larval toxicity testing of pelagic fish: Applications for impact assessment of Deepwater Horizon crude oil.

Key differences in the developmental process of pelagic fish embryos, in comparison to embryos of standard test fish species, present challenges to obtaining sufficient control survival needed to successfully perform traditional toxicity testing bioassays. Many of these challenges relate to the change in buoyancy, from positive to negative, of pelagic fish embryos that occurs just prior to hatch. A novel exposure system, the pelagic embryo-larval exposure chamber (PELEC), has been developed to conduct successful bioassays on the early life stages (ELSs; embryos/larvae) of pelagic fish. Using this unique recirculating upwelling system, it was possible to significantly improve control survival in pelagic fish ELS bioassays compared to commonly used static exposure methods. Results demonstrate that control performance of mahi-mahi (Coryphaena hippurus) embryos in the PELEC system, measured as percent survival after 96-hrs, significantly outperformed agitated static exposure and static exposure systems. Similar significant improvements in 72-hr control survival were obtained with yellowfin tuna (Thunnus albacares). The PELEC system was subsequently used to test the effects of photo-induced toxicity of crude oil to mahi-mahi ELSs over the course of 96-hrs. Results indicate a greater than 9-fold increase in toxicity of Deepwater Horizon (DWH) crude oil during co-exposure to ambient sunlight compared to filtered ambient sunlight, revealing the importance of including natural sunlight in 96-hr DWH crude oil bioassays as well as the PELEC system's potential application in ecotoxicological assessments.

Ultraviolet Radiation Enhances the Toxicity of Deepwater Horizon Oil to Mahi-mahi (Coryphaena hippurus) Embryos.

The 2010 Deepwater Horizon oil spill resulted in the accidental release of millions barrels of crude oil into the Gulf of Mexico. Photoinduced toxicity following coexposure to ultraviolet (UV) radiation is one mechanism by which polycyclic aromatic hydrocarbons (PAHs) from oil spills may exert toxicity. Mahi-mahi (Coryphaena hippurus), an important fishery resource, have positively buoyant, transparent eggs. These characteristics may result in mahi-mahi embryos being at particular risk from photoinduced toxicity. The goal of this study was to determine whether exposure to ultraviolet radiation as natural sunlight enhances the toxicity of crude oil to embryonic mahi-mahi. Mahi-mahi embryos were exposed to several dilutions of water accommodated fractions (WAF) from slick oil collected during the 2010 spill and gradations of natural sunlight in a fully factorial design. Here, we report that coexposure to natural sunlight and WAF significantly reduced percent hatch in mahi-mahi embryos. Effect concentrations of PAH in WAF were within the range of surface PAH concentrations reported in the Gulf of Mexico during the Deepwater Horizon spill. These data suggest that laboratory toxicity tests that do not include UV may underestimate the toxicity of oil spills to early lifestage fish species.

Photo-induced toxicity of Deepwater Horizon slick oil to blue crab (Callinectes sapidus) larvae.

The 2010 Deepwater Horizon oil spill resulted in the accidental release of approximately 700 million L of crude oil into the Gulf of Mexico. Photo-induced toxicity after co-exposure to ultraviolet (UV) radiation is 1 mechanism by which polycyclic aromatic hydrocarbons (PAHs) from oil spills may exert toxicity. Blue crab are an important commercial and ecological resource in the Gulf of Mexico, and their largely transparent larvae may make them sensitive to PAH photo-induced toxicity. The goal of the present study was to examine the sensitivity of early lifestage blue crab (Callinectes sapidus) zoea to slick oil collected during the Deepwater Horizon spill. Blue crab zoea were exposed to 1 of several dilutions of water accommodated fractions from 1 of 2 sources of oil and gradations of natural sunlight in a factorial design. Two 7-h solar exposures were carried out with a recovery period (dark) in between. Survival was found to be UV- and PAH-dependent. Toxicity was observed within the range of surface PAH concentrations reported in the Gulf of Mexico during the Deepwater Horizon spill. These findings indicate that early lifestage blue crab are sensitive to photo-induced toxicity from Deepwater Horizon slick oil.

Treatment with coated layer double hydroxide clays decreases the toxicity of copper-contaminated water.

Copper is a common pollutant found in watersheds that exerts toxic effects on both invertebrates and vertebrates. Layer double hydroxide (LDH) clays are able to adsorb a wide range of contaminants through ion-exchange mechanisms. Coating LDH clays with various materials alters the aggregation of clay particles into the nano-size range, thus increasing relative surface area and offering great potential for contaminant remediation. The goal of this study was to determine if treatment with coated LDH clays decreases the toxicity of copper-containing solutions to Daphnia magna. Four LDH clays with different coatings used to alter hydrophobicity were as follows: used: Na(+) montmorillonite, Zn-Al LDH-nitrate, Zn-Al LDH-stearate, and Zn-Al LDH-carbonate. It was determined that coated LDH clays decreased copper toxicity by decreasing bioavailability and that smaller aggregate sizes decreased bioavailability the most. 96 h LC50 values increased by as much as 4.2 times with the treatment of the solutions with 100 mg/L LDH clay. Copper analysis of the clay and solutions indicated that the clays work by decreasing copper bioavailability by way of a binding mechanism. Coated LDH clays hold promise as a small-scale remediation tool or as an innovative tool for toxicity identification and evaluation characterization of metals.