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.

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.

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.