Dietary Exposure to Environmentally Relevant Levels of Chemical Contaminants Reduces Growth and Survival in Juvenile Chinook Salmon.

Chemical pollution can degrade aquatic ecosystems. Chinook salmon in contaminated habitats are vulnerable to health impacts from toxic exposures. Few studies have been conducted on adverse health outcomes associated with current levels and mixtures of contaminants. Fewer still address effects specific to the juvenile life-stage of salmonids. The present study evaluated contaminant-related effects from dietary exposure to environmentally relevant concentrations and mixture profiles in juvenile Chinook salmon from industrialized waterways in the U.S. Pacific Northwest using two end points: growth assessment and disease susceptibility. The dose and chemical proportions were reconstituted based on environmental sampling and analysis using the stomach contents of juvenile Chinook salmon recently collected from contaminated, industrialized waterways. Groups of fish were fed a mixture with fixed proportions of 10 polychlorinated biphenyls (PCBs), 3 dichlorodiphenyltrichloroethanes (DDTs), and 13 polycyclic aromatic hydrocarbons (PAHs) at five concentrations for 35 days. These contaminant compounds were selected because of elevated concentrations and the widespread presence in sediments throughout industrialized waterways. Fork length and otolith microstructural growth indicators were significantly reduced in fish fed environmentally relevant concentrations of these contaminants. In addition, contaminant-exposed Chinook salmon were more susceptible to disease during controlled challenges with the pathogen Aeromonas salmonicida. Our results indicate that dietary exposure to contaminants impairs growth and immune function in juvenile Chinook salmon, thereby highlighting that current environmental exposure to chemicals of potential management concern threatens the viability of exposed salmon.

Biological Responses of Pacific Herring Embryos to Crude Oil Are Quantifiable at Exposure Levels Below Conventional Limits of Quantitation for PAHs in Water and Tissues.

Pacific herring (Clupea pallasii), a cornerstone of marine food webs, generally spawn on marine macroalgae in shallow nearshore areas that are disproportionately at risk from oil spills. Herring embryos are also highly susceptible to toxicity from chemicals leaching from oil stranded in intertidal and subtidal zones. The water-soluble components of crude oil trigger an adverse outcome pathway that involves disruption of the physiological functions of cardiomyocytes in the embryonic herring heart. In previous studies, impaired ionoregulation (calcium and potassium cycling) in response to specific polycyclic aromatic hydrocarbons (PAHs) corresponds to lethal embryolarval heart failure or subtle chamber malformations at the high and low ends of the PAH exposure range, respectively. Sublethal cardiotoxicity, which involves an abnormal outgrowth (ballooning) of the cardiac ventricular chamber soon after hatching, subsequently compromises juvenile heart structure and function, leading to pathological hypertrophy of the ventricle and reduced individual fitness, measured as cardiorespiratory performance. Previous studies have not established a threshold for these sublethal and delayed-in-time effects, even with total (∑)PAH exposures as low as 29 ng/g of wet weight (tissue dose). Here, we extend these earlier findings showing that (1) cyp1a gene expression provides an oil exposure metric that is more sensitive than typical quantitation of PAHs via GC-MS and (2) heart morphometrics in herring embryos provide a similarly sensitive measure of toxic response. Early life stage injury to herring (impaired heart development) thus occurs below the quantitation limits for PAHs in both water and embryonic tissues as a conventional basis for assessing oil-induced losses to coastal marine ecosystems.

Characterizing the Chemical Profile of Biological Decline in Stormwater-Impacted Urban Watersheds.

Chemical contamination is an increasingly important conservation issue in urban runoff-impacted watersheds. Regulatory and restoration efforts typically evaluate limited conventional parameters and pollutants. However, complex urban chemical mixtures contain hundreds to thousands of organic contaminants that remain unidentified, unregulated, and poorly understood. This study aimed to develop broadly representative metrics of water quality impairment corresponding to previously documented biological degradation along gradients of human impacts. Stream samples (n = 65, baseflow/rainfall conditions, 2017-2018) were collected from 15 regional watersheds (Puget Sound, WA, USA) across an urbanization gradient defined by landscape characteristics. Surface water chemical composition characterized via non-targeted high-resolution mass spectrometry (7068 detections) was highly correlated with landscape-based urbanization gradient (p < 0.01) and season (p < 0.01). Landscape-scale changes in chemical composition closely aligned with two anchors of biological decline: coho salmon (Oncorhynchus kisutch) mortality risk (p < 0.001) and loss of stream macroinvertebrate diversity and abundance (p < 0.001). We isolated and identified 32 indicators for urban runoff impacts and corresponding receiving water ecological health, including well-known anthropogenic contaminants (e.g., caffeine, organophosphates, vehicle-derived chemicals), two related environmental transformation products, and a novel (methoxymethyl)melamine compound. Outcomes support data-directed selection of next-generation water quality indicators for prioritization and evaluation of watershed management efforts intended to protect aquatic ecosystems.

Decreased Growth Rate Associated with Tissue Contaminants in Juvenile Chinook Salmon Out-Migrating through an Industrial Waterway.

The industrial waterway in Portland Harbor, Oregon, is a migration corridor for a distinct population segment of Chinook Salmon (Upper Willamette River) currently protected by the U.S. Endangered Species Act. Juveniles are exposed to a suite of contaminants during outmigration including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethanes. We collected natural origin subyearling Chinook salmon from sites in and around the industrial harbor to evaluate growth (otolith microstructural analysis) in relation to measured chemical concentrations in tissue. A reduced growth rate was associated with higher tissue contaminant concentrations, particularly mixtures represented by PAHs and certain PCBs, which were elevated in juvenile Chinook collected throughout sites within Portland Harbor relative to those captured upstream. First-year growth is an established predictor of individual survival and eventual reproductive success in Chinook salmon. Therefore, our results indicate that legacy pollution may be limiting the population abundance of threatened Willamette River Chinook salmon, and future habitat remediation or restoration actions may benefit ongoing species recovery efforts.

Treading Water: Tire Wear Particle Leachate Recreates an Urban Runoff Mortality Syndrome in Coho but Not Chum Salmon.

Tire tread wear particles (TWP) are increasingly recognized as a global pollutant of surface waters, but their impact on biota in receiving waters is rarely addressed. In the developed U.S. Pacific Northwest, acute mortality of adult coho salmon (Oncorhynchus kisutch) follows rain events and is correlated with roadway density. Roadway runoff experimentally triggers behavioral symptoms and associated changes in blood indicative of cardiorespiratory distress prior to death. Closely related chum salmon (O. keta) lack an equivalent response. Acute mortality of juvenile coho was recently experimentally linked to a transformation product of a tire-derived chemical. We evaluated whether TWP leachate is sufficient to trigger the acute mortality syndrome in adult coho salmon. We characterized the acute response of adult coho and chum salmon to TWP leachate (survival, behavior, blood physiology) and compared it with that caused by roadway runoff. TWP leachate was acutely lethal to coho at concentrations similar to roadway runoff, with the same behaviors and blood parameters impacted. As with runoff, chum salmon appeared insensitive to TWP leachate at concentrations lethal to coho. Our results confirm that environmentally relevant TWP exposures cause acute mortalities of a keystone aquatic species.

Cardiac remodeling in response to embryonic crude oil exposure involves unconventional NKX family members and innate immunity genes.

Cardiac remodeling results from both physiological and pathological stimuli. Compared with mammalian hearts, fish hearts show a broader array of remodeling changes in response to environmental influences, providing exceptional models for dissecting the molecular and cellular bases of cardiac remodeling. We recently characterized a form of pathological remodeling in juvenile pink salmon (Oncorhynchus gorbuscha) in response to crude oil exposure during embryonic cardiogenesis. In the absence of overt pathology (cardiomyocyte death or inflammatory infiltrate), cardiac ventricles in exposed fish showed altered shape, reduced thickness of compact myocardium and hypertrophic changes in spongy, trabeculated myocardium. Here, we used RNA sequencing to characterize molecular pathways underlying these defects. In juvenile ventricular cardiomyocytes, antecedent embryonic oil exposure led to dose-dependent upregulation of genes involved in innate immunity and two NKX homeobox transcription factors not previously associated with cardiomyocytes, nkx2.3 and nkx3.3 Absent from mammalian genomes, the latter is largely uncharacterized. In zebrafish embryos, nkx3.3 demonstrated a potent effect on cardiac morphogenesis, equivalent to that of nkx2.5, the primary transcription factor associated with ventricular cardiomyocyte identity. The role of nkx3.3 in heart growth is potentially linked to the unique regenerative capacity of fish and amphibians. Moreover, these findings support a cardiomyocyte-intrinsic role for innate immune response genes in pathological hypertrophy. This study demonstrates how an expanding mechanistic understanding of environmental pollution impacts - i.e. the chemical perturbation of biological systems - can ultimately yield new insights into fundamental biological processes.

Using High-Resolution Mass Spectrometry to Identify Organic Contaminants Linked to Urban Stormwater Mortality Syndrome in Coho Salmon.

Urban stormwater is a major threat to ecological health, causing a range of adverse, mostly sublethal effects. In western North America, urban runoff is acutely lethal to adult coho salmon ( Oncorhynchus kisutch) that spawn each fall in freshwater creeks. Although the mortality syndrome is correlated to urbanization and attributed to road runoff contaminant(s), the causal agent(s) remain unknown. We applied high-resolution mass spectrometry to isolate a coho mortality chemical signature: a list of nontarget and identified features that co-occurred in waters lethal to coho spawners (road runoff from controlled exposures and urban receiving waters from two field observations of symptomatic coho). Hierarchical cluster analysis indicated that tire wear particle (TWP) leachates were most chemically similar to the waters with observed toxicity, relative to other vehicle-derived sources. Prominent road runoff contaminants in the signature included two groups of nitrogen-containing compounds derived from TWP, polyethylene glycols, octylphenol ethoxylates, and polypropylene glycols. A (methoxymethyl)melamine compound family, previously unreported in North America, was detected in road runoff and urban creeks at concentrations up to ∼9 and ∼0.3 µg/L, respectively. The results indicate TWPs are an under-appreciated contaminant source in urban watersheds and should be prioritized for fate and toxicity assessment.

Roads to ruin: conservation threats to a sentinel species across an urban gradient.

Urbanization poses a global challenge to species conservation. This is primarily understood in terms of physical habitat loss, as agricultural and forested lands are replaced with urban infrastructure. However, aquatic habitats are also chemically degraded by urban development, often in the form of toxic stormwater runoff. Here we assess threats of urbanization to coho salmon throughout developed areas of the Puget Sound Basin in Washington, USA. Puget Sound coho are a sentinel species for freshwater communities and also a species of concern under the U.S. Endangered Species Act. Previous studies have demonstrated that stormwater runoff is unusually lethal to adult coho that return to spawn each year in urban watersheds. To further explore the relationship between land use and recurrent coho die-offs, we measured mortality rates in field surveys of 51 spawning sites across an urban gradient. We then used spatial analyses to measure landscape attributes (land use and land cover, human population density, roadways, traffic intensity, etc.) and climatic variables (annual summer and fall precipitation) associated with each site. Structural equation modeling revealed a latent urbanization gradient that was associated with road density and traffic intensity, among other variables, and positively related to coho mortality. Across years within sites, mortality increased with summer and fall precipitation, but the effect of rainfall was strongest in the least developed areas and was essentially neutral in the most urbanized streams. We used the best-supported structural equation model to generate a predictive mortality risk map for the entire Puget Sound Basin. This map indicates an ongoing and widespread loss of spawners across much of the Puget Sound population segment, particularly within the major regional north-south corridor for transportation and development. Our findings identify current and future urbanization-related threats to wild coho, and show where green infrastructure and similar clean water strategies could prove most useful for promoting species conservation and recovery.

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

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

Severe Coal Tar Sealcoat Runoff Toxicity to Fish Is Prevented by Bioretention Filtration.

Coal tar sealcoats applied to asphalt surfaces in North America, east of the Continental Divide, are enriched in petroleum-derived compounds, including polycyclic aromatic hydrocarbons (PAHs). The release of PAHs and other chemicals from sealcoat has the potential to contaminate nearby water bodies, reducing the resiliency of aquatic communities. Despite this, relatively little is known about the aquatic toxicology of sealcoat-derived contaminants. We assessed the impacts of stormwater runoff from sealcoated asphalt on juvenile coho salmon (Oncorhynchus kisutch) and embryo-larval zebrafish (Danio rerio). We furthermore evaluated the effectiveness of bioretention as a green stormwater method to remove PAHs and reduce lethal and sublethal toxicity in both species. We applied a coal tar sealcoat to conventional asphalt and collected runoff from simulated rainfall events up to 7 months postapplication. Whereas sealcoat runoff was more acutely lethal to salmon, a spectrum of cardiovascular abnormalities was consistently evident in early life stage zebrafish. Soil bioretention effectively reduced PAH concentrations by an order of magnitude, prevented mortality in juvenile salmon, and significantly reduced cardiotoxicity in zebrafish. Our findings show that inexpensive bioretention methods can markedly improve stormwater quality and protect fish health.

Confirmation of Stormwater Bioretention Treatment Effectiveness Using Molecular Indicators of Cardiovascular Toxicity in Developing Fish.

Urban stormwater runoff is a globally significant threat to the ecological integrity of aquatic habitats. Green stormwater infrastructure methods such as bioretention are increasingly used to improve water quality by filtering chemical contaminants that may be harmful to fish and other species. Ubiquitous examples of toxics in runoff from highways and other impervious surfaces include polycyclic aromatic hydrocarbons (PAHs). Certain PAHs are known to cause functional and structural defects in developing fish hearts. Therefore, abnormal heart development in fish can be a sensitive measure of clean water technology effectiveness. Here we use the zebrafish experimental model to assess the effects of untreated runoff on the expression of genes that are classically responsive to contaminant exposures, as well as heart-related genes that may underpin the familiar cardiotoxicity phenotype. Further, we assess the effectiveness of soil bioretention for treating runoff, as measured by prevention of both visible cardiac toxicity and corresponding gene regulation. We find that contaminants in the dissolved phase of runoff (e.g., PAHs) are cardiotoxic and that soil bioretention protects against these harmful effects. Molecular markers were more sensitive than visible toxicity indicators, and several cardiac-related genes show promise as novel tools for evaluating the effectiveness of evolving stormwater mitigation strategies.

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

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

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

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

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

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

Structure-activity relationships for alkyl-phenanthrenes support two independent but interacting synergistic models for PAC mixture potency.

Multiple lines of evidence at whole animal, cellular and molecular levels implicate polycyclic aromatic compounds (PACs) with three rings as drivers of crude oil toxicity to developing fish. Phenanthrene (P0) and its alkylated homologs (C1- through C4-phenanthrenes) comprise the most prominent subfraction of tricyclic PACs in crude oils. Among this family, P0 has been studied intensively, with more limited detail available for the C4-phenanthrene 1-methyl-7-isopropyl-phenanthrene (1-M,7-IP, or retene). While both compounds are cardiotoxic, P0 impacts embryonic cardiac function and development through direct blockade of K+ and Ca2+ currents that regulate cardiomyocyte contractions. In contrast, 1-M,7-IP dysregulates aryl hydrocarbon receptor (AHR) activation in developing ventricular cardiomyocytes. Although no other compounds have been assessed in detail across the larger family of alkylated phenanthrenes, increasing alkylation might be expected to shift phenanthrene family member activity from K+/Ca2+ ion current blockade to AHR activation. Using embryos of two distantly related fish species, zebrafish and Atlantic haddock, we tested 14 alkyl-phenanthrenes in both acute and latent developmental cardiotoxicity assays. All compounds were cardiotoxic, and effects were resolved into impacts on multiple, highly specific aspects of heart development or function. Craniofacial defects were clearly linked to developmental cardiotoxicity. Based on these findings, we suggest a novel framework to delineate the developmental toxicity of petrogenic PAC mixtures in fish, which incorporates multi-mechanistic pathways that produce interactive synergism at the organ level. In addition, relationships among measured embryo tissue concentrations, cytochrome P4501A mRNA induction, and cardiotoxic responses suggest a two-compartment toxicokinetic model that independently predicts high potency of PAC mixtures through classical metabolic synergism. These two modes of synergism, specific to the sub-fraction of phenanthrenes, are sufficient to explain the high embryotoxic potency of crude oils, independent of as-yet unmeasured compounds in these complex environmental mixtures.

Interactive neurobehavioral toxicity of diazinon, malathion, and ethoprop to juvenile coho salmon.

In western North America, mixtures of current use pesticides have been widely detected in streams and other aquatic habitats for threatened and endangered Pacific salmon and steelhead (Oncorhynchus sp.). These include organophosphate insecticides that inhibit acetylcholinesterase (AChE) enzyme activity in the salmon nervous system, thereby disrupting swimming and feeding behaviors. Several organophosphates have been shown to interact as mixtures to produce synergistic AChE inhibition at concentrations near or above the upper range of surface water detections in freshwater systems. To evaluate potential synergism at lower concentrations (near or below 1 part per billion), juvenile coho (Oncorhynchus kisutch) were exposed to a range of mixtures of diazinon-malathion and ethoprop-malathion below a cumulative 0.05 of the predicted EC50 for AChE inhibition, as determined from single chemical concentration-response curves. Brain enzyme inhibition was concentration-dependent, with a 90% reduction and a significant decrease in spontaneous swimming speed at the highest binary mixture concentrations evaluated (diazinon-malathion at 2.6 and 1.1 µg/L, respectively; ethoprop-malathion at 2.8 and 1.2 µg/L, respectively). Brain enzyme activity gradually recovered over six weeks. Our findings extend earlier observations of organophosphate synergism in salmon and reveal an unusually steep concentration-response relationship across a mere 2-fold increase in mixture concentration.

Bioretention filtration prevents acute mortality and reduces chronic toxicity for early life stage coho salmon (Oncorhynchus kisutch) episodically exposed to urban stormwater runoff.

As the human population of western North America continues to expand, widespread patterns of urban growth pose increasingly existential threats to certain wild stocks of Pacific salmon and steelhead (Oncorhynchus sp.). Rainfall previously absorbed into the soils of forests and grasslands falls instead on pavement and other hardened surfaces. This creates stormwater runoff that carries toxic metals, oil, and many other contaminants into salmon-bearing habitats. These include freshwater streams where coho salmon (O. kisutch) spawn in gravel beds. Coho salmon embryos develop within a thick eggshell (chorion) for weeks to months before hatching as alevins and ultimately emerging from the gravel as fry. Untreated urban runoff is highly toxic to older coho salmon (freshwater-resident juveniles and adult spawners), but the vulnerability of the earliest life stages remains poorly understood. To address this uncertainty, we fertilized eggs and raised them under an episodic stormwater exposure regimen, using runoff collected from a high-traffic arterial roadway from 15 discrete storm events. We monitored survival and morphological development, as well as molecular markers for contaminant exposure and cardiovascular stress. We also evaluated the benefit of treating runoff with green infrastructure (bioretention filtration) on coho salmon health and survival. Untreated runoff caused subtle sublethal toxicity in pre-hatch embryos with no mortality, followed by high rates of mortality from exposure at hatch. Bioretention filtration removed most measured contaminants (bacteria, dissolved metals, and polycyclic aromatic hydrocarbons), and the treated effluent was considerably less toxic - notably preventing mortality at the alevin stage. Our findings indicate that untreated urban runoff poses an important threat to early life stage coho salmon, in terms of both acute and delayed-in-time mortality. Moreover, while inexpensive management strategies involving bioinfiltration are promising, future green infrastructure effectiveness research should emphasize sublethal metrics for contaminant exposure and adverse health outcomes in salmonids.

Low-level copper exposures increase visibility and vulnerability of juvenile coho salmon to cutthroat trout predators.

Copper contamination in surface waters is common in watersheds with mining activities or agricultural, industrial, commercial, and residential human land uses. This widespread pollutant is neurotoxic to the chemosensory systems of fish and other aquatic species. Among Pacific salmonids (Oncorhynchus spp.), copper-induced olfactory impairment has previously been shown to disrupt behaviors reliant on a functioning sense of smell. For juvenile coho salmon (O. kisutch), this includes predator avoidance behaviors triggered by a chemical alarm cue (conspecific skin extract). However, the survival consequences of this sublethal neurobehavioral toxicity have not been explored. In the present study juvenile coho were exposed to low levels of dissolved copper (5-20 microg/L for 3 h) and then presented with cues signaling the proximity of a predator. Unexposed coho showed a sharp reduction in swimming activity in response to both conspecific skin extract and the upstream presence of a cutthroat trout predator (O. clarki clarki) previously fed juvenile coho. This alarm response was absent in prey fish that were exposed to copper. Moreover, cutthroat trout were more effective predators on copper-exposed coho during predation trials, as measured by attack latency, survival time, and capture success rate. The shift in predator-prey dynamics was similar when predators and prey were co-exposed to copper. Overall, we show that copper-exposed coho are unresponsive to their chemosensory environment, unprepared to evade nearby predators, and significantly less likely to survive an attack sequence. Our findings contribute to a growing understanding of how common environmental contaminants alter the chemical ecology of aquatic communities.

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

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

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.