Interspecies Differences in 6PPD-Quinone Toxicity Across Seven Fish Species: Metabolite Identification and Semiquantification.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire antidegradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations that surpass those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semiquantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkii lewisi), and nonfry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might detoxify 6PPD-Q more effectively. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semiquantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being valuable to environmental monitoring and risk assessment.

Retinoic acid signaling regulates late stages of semicircular canal morphogenesis and otolith maintenance in the zebrafish inner ear.

BACKGROUND: The vitamin A derivative all-trans retinoic acid (RA) regulates early stages of inner ear development. As the early disruption of the RA pathway results in profound mispatterning of the developing inner ear, this confounds analyses of specific roles in later stages. Therefore, we used the temporal-specific exposure of all-trans RA or diethylaminobenzaldehyde to evaluate RA functions in late otic development. RESULTS: Perturbing late RA signaling causes behavioral defects analogous to those expected in larvae suffering from vestibular dysfunction. These larvae also demonstrate malformations of the semi-circular canals, as visualized through (a) use of the transgenic strain nkhspdmc12a, a fluorescent reporter expressed in otic epithelium; and (b) injection of the fluorescent lipophilic dye DiI. We also noted the altered expression of genes encoding ECM proteins or modifying enzymes. Other malformations of the inner ear observed in our work include the loss or reduced size of the utricular and saccular otoliths, suggesting a role for RA in otolith maintenance. CONCLUSION: Our work has identified a previously undescribed late phase of RA activity in otic development, demonstrating that vestibular defects observed in human patients in relation to perturbed RA signaling are not solely due to its early disruption in otic development.

Early-life exposure to weathered, unweathered and dispersed oil has persisting effects on ecologically relevant behaviors in sheepshead minnow.

The Deepwater Horizon oil spill released 3.19 million barrels of crude oil into the Gulf of Mexico, making it the largest oil spill in U.S. history. Weathering and the application of dispersants can alter the solubility of compounds within crude oil, thus modifying the acute toxicity of the crude oil to aquatic life. The primary aim of our study was to determine the lasting impact of early-life stage sheepshead minnow (Cyprinodon variegatus variegatus) exposure to weathered, unweathered and dispersed crude oil on prey capture, male aggression, novel object interaction and global DNA methylation. Embryos were exposed from 1 to 10 dpf to water accommodations of crude oil and were raised to adulthood in artificial seawater. Our results suggest exposure to crude oil did not result in lasting impairment of complex behavioral responses of male sheepshead minnow. Exposure to dispersed weathered oil, however, decreased border dwelling in response to a novel object (i.e. decreased anxiety). Principal component analysis revealed that exposure to weathered oil had no overarching effect, but that unweathered crude oil increased variability in exploratory behaviors but decreased variability in anxiety-associated behaviors. Further work is needed to understand the effects of oil exposure on fish behavior and the potential ecological impact of subtle behavioral changes in fishes.

Comparison of Diluted Bitumen (Dilbit) and Conventional Crude Oil Toxicity to Developing Zebrafish.

To facilitate pipeline transport of bitumen, it is diluted with natural gas condensate, and the resulting mixture, "dilbit", differs greatly in chemical composition to conventional crude oil. Despite the risk of accidental dilbit release, the effects of dilbit on aquatic animals are largely unknown. In this study, we compared the toxicity of water accommodated fractions (WAFs) of dilbit and two conventional crude oils, medium sour composite and mixed sweet blend, to developing zebrafish. Mortality and pericardial edema was lowest in dilbit WAF-exposed embryonic zebrafish but yolk sac edema was similar in all exposures. Shelter-seeking behavior was decreased by dilbit and conventional crude WAF exposures, and continuous swimming behavior was affected by all tested WAF exposures. Regardless of WAF type, monoaromatic hydrocarbon content (largely made up of benzene, toluene, ethylbenzene, and xylene (BTEX)) was a more accurate predictor of lethality and pericardial edema than polycyclic aromatic hydrocarbon (PAH) content. Our results suggest that the toxicity of dilbit to a model fish is less than or similar to that of conventional crudes.

The lethal and sublethal impacts of two tire rubber-derived chemicals on brook trout (Salvelinus fontinalis) fry and fingerlings.

Recent toxicity studies of stormwater runoff implicated N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) as the contaminant responsible for the mass mortality of coho salmon (Oncorhynchus kisutch). In the wake of this discovery, 6PPD-quinone has been measured in waterways around urban centers, along with other tire wear leachates like hexamethoxymethylmelamine (HMMM). The limited data available for 6PPD-quinone have shown toxicity can vary depending on the species. In this study we compared the acute toxicity of 6PPD-quinone and HMMM to Brook trout (Salvelinus fontinalis) fry and fingerlings. Our results show that fry are ∼3 times more sensitive to 6PPD-quinone than fingerlings. Exposure to HMMM ≤6.6 mg/L had no impact on fry survival. These results highlight the importance of conducting toxicity tests on multiple life stages of fish species, and that relying on fingerling life stages for species-based risk assessment may underestimate the impacts of exposure. 6PPD-quinone also had many sublethal effects on Brook trout fingerlings, such as increased interlamellar cell mass (ILCM) size, hematocrit, blood glucose, total CO2, and decreased blood sodium and chloride concentrations. Linear relationships between ILCM size and select blood parameters support the conclusion that 6PPD-quinone toxicity is an outcome of osmorespiratory challenges imposed by gill impairment.

Changes in Temperature Alter the Toxicity of Polycyclic Aromatic Compounds to American Lobster (Homarus americanus) Larvae.

Polycyclic aromatic compounds (PACs) present in the water column are considered to be one of the primary contaminant groups contributing to the toxicity of a crude oil spill. Because crude oil is a complex mixture composed of thousands of different compounds, oil spill models rely on quantitative structure-activity relationships like the target lipid model to predict the effects of crude oil exposure on aquatic life. These models rely on input provided by single species toxicity studies, which remain insufficient. Although the toxicity of select PACs has been well studied, there is little data available for many, including transformation products such as oxidized hydrocarbons. In addition, the effect of environmental influencing factors such as temperature on PAC toxicity is a wide data gap. In response to these needs, in the present study, Stage I lobster larvae were exposed to six different understudied PACs (naphthalene, fluorenone, methylnaphthalene, phenanthrene, dibenzothiophene, and fluoranthene) at three different relevant temperatures (10, 15, and 20 °C) all within the biological norms for the species during summer when larval releases occur. Lobster larvae were assessed for immobilization as a sublethal effect and mortality following 3, 6, 12, 24, and 48 h of exposure. Higher temperatures increased the rate at which immobilization and mortality were observed for each of the compounds tested and also altered the predicted critical target lipid body burden, incipient median lethal concentration, and elimination rate. Our results demonstrate that temperature has an important influence on PAC toxicity for this species and provides critical data for oil spill modeling. More studies are needed so oil spill models can be appropriately calibrated and to improve their predictive ability. Environ Toxicol Chem 2023;42:2389-2399. © 2023 SETAC.

Calibration of an acute toxicity model for the marine crustacean, Artemia franciscana, nauplii to support oil spill effect assessments.

Oil spill risk and impact assessments rely on time-dependent toxicity models to predict the hazard of the constituents that comprise crude oils and petroleum substances. Dissolved aromatic compounds (ACs) are recognized as a primary driver of aquatic toxicity in surface spill exposure scenarios. However, limited time-dependent toxicity data are available for different classes of ACs to calibrate such models. This study examined the acute toxicity of 14 ACs and 3 binary AC mixtures on Artemia franciscana nauplii at 25 °C. Toxicity tests for 3 ACs were also conducted at 15 °C to evaluate the role of temperature on toxicity. The ACs investigated represented parent and alkylated homocyclic and nitrogen-, sulfur- and oxygen-containing heterocyclic structures with octanol-water partition coefficients (log Kow) ranging from 3.2 to 6.6. Passive dosing was used to expose and maintain concentrations in toxicity tests which were confirmed using fluorometry, and independently validated for 6 ACs using GC-MS analysis. Mortality was assessed at 6, 24, and 48 h to characterize the time course of toxicity. No mortality was observed for the most hydrophobic AC tested, 7,12-dimethylbenz[a]anthracene, due to apparent water solubility constraints. Empirical log LC50 s for the remaining ACs were fit to a linear regression with log Kow to derive a critical target lipid body burden (CTLBB) based on the target lipid model. The calculated 48 h CTLBB of 47.1 ± 8.1 µmol/g octanol indicates that Artemia nauplii exhibited comparable sensitivity to other crustaceans. A steep concentration-response was found across all compounds as evidenced by a narrow range (1.0-3.1) in the observed LC50 /LC10 ratio. Differences in toxicokinetics were noted, and no impacts of temperature-dependence of AC toxicity were found. Toxicity data obtained for individual ACs yielded acceptable predictions of observed binary AC mixture toxicity. Results from this study advance toxicity models used in oil spill assessments.

Comparing the effects of unconventional and conventional crude oil exposures on zebrafish and their progeny using behavioral and genetic markers.

Diluted bitumen, also known as dilbit, is transported by rail and pipeline across Canada and the United States. Due to the fewer number of studies characterizing the toxicity of dilbit, a dilbit spill poses an unknown risk to freshwater aquatic ecosystems. In the following study, we compared the impact of early-life exposure to conventional and unconventional crude oils on the optomotor behavior, reproductive success, and transgenerational differences in gene expression in zebrafish and their progeny. For exposures, water accommodated fractions (WAFs) of crude oil were generated using a 1:1000 oil to water ratio for 3 different crudes; mixed sweet blend (MSB), medium sour composite (MSC) and dilbit. All three oils generated unique volatile organic compound (VOC) and polycyclic aromatic compound (PAC) profiles. Of the WAFs tested, only dilbit decreased the eye size of 2 dpf larvae, and only MSB exposed larvae had an altered behavioral response to a visual simulation of a predator. Early-life exposure to crude oil had no lasting impact on reproductive success of adult fish; however, each oil had unique impacts on the basal gene expression of the somatically exposed offspring. In this study, the biological effects differed between each of the oils tested, which implied chemical composition plays a critical role in determining the sublethal toxicity of conventional and unconventional crude oils in freshwater ecosystems.

Assessing the Toxicity of Individual Aromatic Compounds and Mixtures to American Lobster (Homarus americanus) Larvae Using a Passive Dosing System.

Aquatic exposures to aromatic compounds (ACs) may be important contributors to biological effects of oil spills. The present study examined the acute toxicity of 11 ACs and 3 binary AC mixtures on stage 1 American lobster larvae using a passive dosing test design. The ACs investigated covered a range of classes and log octanol-water partition coefficient values (KOW ; 2.5-5.5). Silicone O-rings were used to partition ACs into seawater and maintain stable exposures. Exposed lobster larvae were assessed for mobility and survival at 3, 6, 12, 24, 36, and 48 h. Fluorometry and gas chromatography-mass spectrometry measurements confirmed well-defined substance exposures. Expressing lethality in terms of chemical activities yielded values between 0.01 and 0.1, consistent with a baseline mode of action. Analysis of time-dependent median lethal/effect concentration (L/EC50) values were used to determine incipient values. An expected linear relationship between the incipient log L/EC50 and log KOW was fit to the empirical toxicity data to derive critical target lipid body burdens for immobilization and lethality endpoints. These values indicate that American lobster larvae fall on the sensitive end of the acute species sensitivity distribution. We used AC toxicity data to successfully predict toxicity of binary mixtures assuming additive toxicity. The observed time-dependent toxicity was inversely related to log KOW and occurred more quickly than reported previously. The results contribute to improving models for predicting oil spill impacts on American lobster larvae populations. Environ Toxicol Chem 2021;40:1379-1388. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A burning issue: The effect of organic ultraviolet filter exposure on the behaviour and physiology of Daphnia magna.

Ultraviolet (UV) filters are compounds utilized in many manufacturing processes and personal care products such as sunscreen to protect against UV-radiation. These highly lipophilic compounds are emerging contaminants of concern in aquatic environments due to their previously observed potential to bioaccumulate and exert toxic effects in marine ecosystems. Currently, research into the toxic effects of UV filter contamination of freshwater ecosystems is lacking, thus the present study sought to model the effects of acute and chronic developmental exposures to UV filters avobenzone, oxybenzone and octocrylene as well as a mixture of these substances in the freshwater invertebrate, Daphnia magna, at environmentally realistic concentrations. Median 48-hour effect and lethal concentrations were determined to be in the low mg/L range, with the exception of octocrylene causing 50% immobilization near environmental concentrations. 48-hour acute developmental exposures proved to behaviourally impair daphnid phototactic response; however, recovery was observed following a 19-day post-exposure period. Although no physiological disruptions were detected in acutely exposed daphnids, delayed mortality was observed up to seven days post-exposure at 200 µg/L of avobenzone and octocrylene. 21-day chronic exposure to 7.5 µg/L octocrylene yielded complete mortality within 7 days, while sublethal chronic exposure to avobenzone increased Daphnia reproductive output and decreased metabolic rate. 2 µg/L oxybenzone induced a 25% increase in metabolic rate of adult daphnids, and otherwise caused no toxic effects at this dose. These data indicate that UV filters can exert toxic effects in freshwater invertebrates, therefore further study is required. It is clear that the most well-studied UV filter, oxybenzone, may not be the most toxic to Daphnia, as both avobenzone and octocrylene induced behavioural and physiological disruption at environmentally realistic concentrations.

Field-collected crude oil, weathered oil and dispersants differentially affect the early life stages of freshwater and saltwater fishes.

The Deepwater Horizon (DWH) oil spill was the biggest in US history and released 3.19 million barrels of light crude oil into the Gulf of Mexico. In this study, we compared the toxicity of water accommodated fractions (WAFs) of naturally weathered crude oils, source oil, and source oil with dispersant mixtures and their effects on developing sheepshead minnow and zebrafish. Although a freshwater fish, zebrafish has been used as a model for marine oil spills owing to the molecular and genetic tools available and their amenability to lab care. Our study not only aimed to determine the effect of crude oil on early life stages of these two fish species, but also aimed to determine whether dissolved crude oil constituents were similar in fresh and saltwater, and if freshwater fish might be a suitable model to study marine spills. Weathering and dispersant had similar effects on WAF composition in both fresh and saltwater, except that the saltwater source oil + dispersant WAF had markedly higher PAH levels than the freshwater equivalent. WAF exposure differentially affected survival, as the LC50 values in %WAF for the zebrafish and sheepshead minnow exposures were 44.9% WAF (95% confidence interval (C.I.) 42.1-47.9) and 16.8% WAF (95% C.I. 13.7-20.5); respectively. Exposure increased heart rate of zebrafish embryos, whereas in sheepshead, source oil exposure had the opposite effect. WAF exposure altered mRNA expression of biotransformation makers, vitellogenin and neurodevelopment genes in both species. Muscle deformations were only found in oil-exposed zebrafish. This is one of the most comprehensive studies to date on crude oil toxicity, and highlights the species-specific differences in cardiotoxicity, estrogenic effects, biotransformation enzyme induction and potential neurotoxicity of crude oil exposure.

Persistent and transgenerational effects of raw and ozonated oil sands process-affected water exposure on a model vertebrate, the zebrafish.

Exposure to oil sands process-affected water (OSPW), a by-product of Canadian oil sands mining operations, can cause both acute and chronic adverse effects in aquatic life. Ozonation effectively degrades naphthenic acids in OSPW, mitigating some of the toxicological effects of exposure. In this study we examined the effect of developmental exposure to raw and ozonated OSPW had on the breeding success, prey capture, and alarm cue response in fish months/years after exposure and the transgenerational effect exposure had on gene expression, global DNA methylation, and larval basal activity. Exposure to raw and ozonated OSPW had no effect on breeding success, and global DNA methylation. Exposure altered the expression of vtg and nkx2.5 in the unexposed F1 generation. Exposure to both raw and ozonated OSPW had a transgenerational impact on larval activity levels, anxiety behaviors, and maximum swim speed compared to the control population. Prey capture success was unaffected, however, the variability in the behavioral responses to the introduction of prey was decreased. Fish developmentally exposed to either treatment were less active before exposure and did not have an anxiety response to the alarm cue hypoxanthine-3-n-oxide. Though ozonation was able to mitigate some of the effects of OSPW exposure, further studies are needed to understand the transgenerational effects and the implications of exposure on complex fish behaviors.

Assessment of raw and ozonated oil sands process-affected water exposure in developing zebrafish: Associating morphological changes with gene expression.

With the ever-increasing amounts of oil sands process-affected water (OSPW) accumulating from Canada's oil sands operations, its eventual release must be considered. As OSPW has been found to be both acutely and chronically toxic to aquatic organisms, remediation processes must be developed to lower its toxicity. Ozone treatment is currently being studied as a tool to facilitate the removal of organic constituents associated with toxicity. Biomarkers (e.g. gene expression) are commonly used when studying the effects of environmental contaminants, however, they are not always indicative of adverse effects at the whole organism level. In this study, we assessed the effects of OSPW exposure on developing zebrafish by linking gene expression to relevant cellular and whole organism level endpoints. We also investigated whether or not ozone treatment decreased biomarkers and any associated toxicity observed from OSPW exposure. The concentrations of classical naphthenic acids in the raw and ozonated OSPW used in this study were 16.9 mg/L and 0.6 mg/L, respectively. Ozone treatment reduced the total amount of naphthenic acids (NAs) in the OSPW sample by 92%. We found that exposure to both raw and ozonated OSPW had no effect on the survival of zebrafish embryos. The expression levels of biotransformation genes CYP1A and CYP1B were induced by raw OSPW exposure, with CYP1B being more highly expressed than CYP1A. In contrast, ozonated OSPW exposure did not increase the expression of CYP1A and only slightly induced CYP1B. A decrease in cardiac development and function genes (NKX2.5 and APT2a2a) was not associates with large changes in heart rate, arrhythmia or heart size. We did not find any indications of craniofacial abnormalities or of increased occurrence of apoptotic cells. Overall, our study found that OSPW was not overtly toxic to zebrafish embryos.

Growth and recovery of zebrafish embryos after developmental exposure to raw and ozonated oil sands process-affected water.

Due to the increasing volume of oil sands process-affect water (OSPW) and its toxicity to aquatic organisms, it is important to fully understand its effects and study remediation processes that will enable its release to the environment. Ozone treatment is currently being considered as a tool to expedite remediation, as it is known to degrade toxic organic compounds present in OSPW. In this study, we aimed to measure the effects of OSPW exposure on the growth, development and recovery of zebrafish (Danio rerio) embryos. We also used ozone-treated OSPW to determine whether ozonation negated any effects of raw OSPW exposure. As biomarkers of exposure, we assessed the expression of genes involved in neurodevelopment (ngn1, neuroD), estrogenicity (vtg), oxidative stress (sod1), and biotransformation (cyp1a, cyp1b). Our study found that exposure to both raw and ozonated OSPW did not impair growth of zebrafish embryos, however, otoliths of exposed embryos were smaller than those of control embryos. The expression levels of both cyp1a and cyp1b were induced by raw OSPW exposure. However, after the exposure period, expression levels of these genes returned to control levels within two days of residence in clean water. We found no changes in the expression levels of ngn1, neuroD and vtg genes with exposure to treated or untreated OSPW. Overall, our study found that raw OSPW exposure did not have many negative effects on zebrafish embryos and embryos appeared to recover relatively quickly after exposure ended. Furthermore, ozone treatment decreased the induction of cyp1a and cyp1b.