Fathead Minnows Exposed to Organic Compounds from Oil Sands Tailings as Embryos Have Reduced Survival, Impaired Development, and Altered Behaviors That Persist into Larval Stages.

Our study evaluated whether exposure to naphthenic acid fraction compounds (NAFCs) extracted from oil sands process-affected waters (OSPW) has adverse effects on fish embryos that persist into later life. We exposed fathead minnow (Pimephales promelas) embryos to concentrations of NAFCs found in OSPW (2.5-54 mg/L) for 7 days (1 day postfertilization to hatch), then raised surviving larvae in outdoor mesocosms of uncontaminated lake water for 1 month. Embryos exposed to NAFCs were more likely to exhibit malformations (by up to 8-fold) and had slower heart rates (by up to 24%) compared to controls. Fish raised in uncontaminated lake water following exposure to NAFCs as embryos, were 2.5-fold less likely to survive during the larval stage than control fish. These fish also showed up to a 45% decrease in swim activity and a 36% increase in swim burst events during behavioral tests relative to controls. We conclude that exposure to NAFCs during the embryonic stage can have lasting effects on fish survival, physiology, and behavior that persist at least through the larval stage. These findings of delayed mortalities and persistent sublethal effects of embryonic NAFC exposure are relevant to informing the development of regulations on treated OSPW releases from mining operations. Environ Toxicol Chem 2022;41:1319-1332. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects on Trout Alevins of Chronic Exposures to Chemically Dispersed Access Western Blend and Cold Lake Blend Diluted Bitumens.

The present study assessed the chronic toxicity of 2 chemically enhanced water accommodated fractions (CEWAFs) of diluted bitumens (dilbits), Access Western Blend (AWB) and Cold Lake Blend (CLB), to rainbow trout alevins. Chemical dispersion was used to overcome the resistance to dispersion of dilbits and to generate test solutions that contained more and smaller oil droplets for increased partitioning of petroleum hydrocarbons into water. Test solutions were characterized by fluorescence spectroscopy, a rapid and inexpensive analytical tool to compare toxicity endpoints measured by fluorescence (total petroleum hydrocarbons measured by fluorescence [TPH-F]). Cumulative mortality and the prevalence and severity of malformations increased following exposure of alevins to dispersed dilbits. Toxicity curves overlapped for AWB and CLB when expressed as TPH-F and 22- to 24-d median lethal and effect concentrations ranged from 0.36 to 1.5 mg/L. Gene expression in alevins was also altered following exposure to dispersed dilbit, with relative cytochrome P450-1A mRNA levels increasing up to 170-fold for AWB and up to 240-fold for CLB. Access Western Blend and CLB caused similar toxicity to rainbow trout alevins as light to medium conventional crude oils, and rainbow trout alevins were more sensitive than yellow perch, Japanese medaka, and fathead minnow embryos exposed to dispersed AWB and CLB. The present study is the first to assess the embryotoxicity of dilbits to a Canadian freshwater salmonid species. Environ Toxicol Chem 2020;39:1620-1633. © 2020 SETAC.

Transcriptional responses in newly-hatched Japanese medaka (Oryzias latipes) associated with developmental malformations following diluted bitumen exposure.

Japanese medaka embryos were exposed to water accommodated fractions (WAF) and chemically-enhanced WAF of two types of diluted bitumen (dilbit) at concentrations bracketing the EC50s for developmental malformations. Within these treatments, fish were grouped based on the presence or absence of developmental malformations (e.g., blue sac disease (BSD)), and analyzed for novel transcriptomic responses. Microarray analyses identified novel biomarkers and gene networks in dilbit-exposed malformed embryos that were not evident in dilbit-exposed fish without BSD or in controls without dilbit. The top differentially expressed genes (DEGs) included cytochrome P450 transcripts (cyp1) in fish from all dilbit treatments (malformed and non-malformed fish), as well as: fibroblast growth factor (fgf7), AHR repressor (ahrr), and squalene monooxygenase (sqle). In dilbit-exposed fish that did not develop BSD, the only reported individual DEG was eukaryotic translation initiation factor 3 subunit D (eif3d). However, a number of other pathways were enriched, including melatonin effects on circadian clock and the antioxidant response, estrogen and androgen metabolism as well as many receptor signaling pathways. Pathways associated with hedgehog, steroid biosynthesis, and Wnt signaling were significantly altered between low and high concentrations of dilbit exposure. An effect of the dispersant control on swim bladder development was observed at concentrations 10-fold higher than those used to disperse dilbit, and a number of gene targets unique to fish in this comparison were affected. This suggests that the toxic effects of dispersant may involve alternative mechanisms to dilbit, but cause similar phenotypic responses. This study identified novel biomarkers in fish exposed to dilbit, with or without visual malformations, that can be used to assess the risks of dilbit to aquatic ecosystem health.

Comparative toxicity of two diluted bitumens to developing yellow perch (Perca flavescens).

Increasing demand for diluted bitumen (dilbit) has led to the development of the oil sands industry and the expansion of transcontinental pipelines. Dilbit is an unresolved complex mixture with variable diluent and bitumen composition. Thus, it is important to understand the effects of the two most transported dilbits in Canada, Access Western Blend (AWB) and Cold Lake Blend (CLB) on a North America native and freshwater fish species, the yellow perch (Perca flavescens). Fertilized embryos were exposed to both dilbits for 16 days, from <24 h post-fertilization until hatch. The treatment regime was a static daily renewal of water accommodated fractions (WAF) and chemically-enhanced water accommodated fractions (CEWAF) at concentrations ranging from 0.01 to 21.3 µg/L of total polycyclic aromatic compounds (TPACs). Hatched embryos were assessed for malformations and changes in the expression of genes associated with phase I and II detoxification and oxidative stress. The prevalence of developmental malformations increased significantly at the highest concentrations of all treatments, with WAF treatments yielding a higher prevalence than CEWAF. The EC50s for AWB and CLB WAF and CEWAF solutions ranged from 9.8 to 24 µg/L TPACs, with the CEWAF of AWB being the least toxic. Relative mRNA levels of cyp1a showed induction by up to 18-fold in WAF and 50-fold in CEWAF treatments at similar concentrations of measured dilbit in solution. Complementary DNA methylation analysis was assessed and fish embryos exposed to AWB CEWAF and CLB WAF showed decreased DNA methylation profiles with increasing exposure to dilbit, suggesting that global gene expression is increasing in these treatments. With recent approvals of pipelines in North America, these data will support site-specific risk assessments and monitoring of Canadian ecosystems should a pipeline spill occur.

Cold Lake Blend diluted bitumen toxicity to the early development of Japanese medaka.

Diluted bitumen (dilbit) from Alberta oil sands (Canada) is transported across major continental watersheds, yet little is known about its toxicity to fish if spilled into aquatic environments. The toxicity of Cold Lake (CLB) dilbit was assessed for medaka embryos (Oryzias latipes) exposed to water accommodated fractions (WAF) and chemically-enhanced WAF (CEWAF) using Corexit®EC9500A as dispersant. The effects of CLB toxicity were similar to conventional crude oils and Access Western Blend (AWB) dilbit. The prevalence of malformations and cyp1a mRNA synthesis in hatched fish increased monotonically with concentration during WAF and CEWAF treatments and provided a novel indicator of dilbit PAH toxicity. Apart from nfe2 (an antioxidant transcription factor), there were no statistically significant monotonic exposure-responses of ahr, arnt2, cat, sod, gpx, gst, gsr, g6pdh, p53, and hsp70 transcripts at total polycyclic aromatic hydrocarbons (TPAH) concentrations bracketing EC50s for embryotoxicity (WAF ≅ 3 µg/L; CEWAF ≅ 0.1 µg/L TPAH). Based on measured TPAH concentrations in exposure test solutions, CLB dilbit was 6-10 fold more toxic to medaka than AWB during chronic exposures. Lack of direct monotonic gene transcription responses to increasing oil concentrations during exposures that were embryotoxic suggests that the capacity of the oxidative stress response is limited in earlier lifestages or that differences exist among species in mechanisms of toxicity. This study provides a comparative framework for identifying suitable biomarkers and toxicity methods for those fish species in sensitive lifestages at highest risk of Canadian oil sands dilbit exposure following a spill in the freshwater environment.

Diluted bitumen causes deformities and molecular responses indicative of oxidative stress in Japanese medaka embryos.

This study characterized the toxicity and physiological effects of unweathered diluted bitumen (Access Western Blend dilbit; AWB) to fish. Embryos of Japanese medaka (Oryzias latipes) were exposed for 17 days to dilutions of physically-dispersed (water accommodated fraction; WAF) and chemically-dispersed (chemically-enhanced WAF; CEWAF) dilbit. AWB dilbit exposure was not lethal to medaka, but resulted in a high prevalence of blue sac disease (BSD), impaired development, and abnormal or un-inflated swim bladders at hatch. Physiological effects were indicated by the relative mRNA levels of key genes associated with, among others, cell cycling and the response to mutations (p53), xenobiotic metabolism (ahr, arnt2), phase I (cyp1a) and II processes associated with oxidative stress (cat, g6pdh, hsp70, gst, gpx, gsr, nfe2, and sod). AWB dilbit treatment increased p53 and cyp1a transcript levels (1.5-fold and >15-fold, respectively), with significant, but less pronounced changes in indicators of oxidative stress and metabolism. The exposure-related changes in embryotoxicity and mRNA synthesis were consistent with metabolism of polycyclic aromatic hydrocarbons (PAHs) to reactive and toxic metabolites. Medaka embryos responded similarly to WAF and CEWAF treatments, but CEWAF was about 100 times more efficient in delivering toxic concentrations of PAHs. The toxicity of chemically-dispersed nujol, a non-toxic mineral oil used as an experimental control, suggested that a portion of the observed effects of AWB could be attributed to excess dispersant in solution. This first study of the physiological effects of dilbit toxicity to fish embryos provides a baseline to compare toxicity between dilbit and conventional crude oils, and the groundwork for the development of molecular biomarkers of the sensitivity and level of risk of native Canadian fish species to dilbit exposure.

Chronic cortisol and the regulation of food intake and the endocrine growth axis in rainbow trout.

To gain a better understanding of the mechanisms by which cortisol suppresses growth during chronic stress in fish, we characterized the effects of chronic cortisol on food intake, mass gain, the expression of appetite-regulating factors, and the activity of the GH/IGF axis. Fish given osmotic pumps that maintained plasma cortisol levels at ∼70 or 116 ng/ml for 34 days were sampled 14, 28 and 42 days post-implantation. Relative to shams, the cortisol treatments reduced food intake by 40-60% and elicited marked increases in liver leptin (lep-a1) and brain preoptic area (POA) corticotropin-releasing factor (crf) mRNA levels. The cortisol treatments also elicited 40-80% reductions in mass gain associated with increases in pituitary gh, liver gh receptor (ghr), liver igfI and igf binding protein (igfbp)-1 and -2 mRNA levels, reduced plasma GH and no change in plasma IGF1. During recovery, while plasma GH and pituitary gh, liver ghr and igfI gene expression did not differ between treatments, the high cortisol-treated fish had lower plasma IGF1 and elevated liver igfbp1 mRNA levels. Finally, the cortisol-treated fish had higher plasma glucose levels, reduced liver glycogen and lipid reserves, and muscle lipid content. Thus, our findings suggest that the growth-suppressing effects of chronic cortisol in rainbow trout result from reduced food intake mediated at least in part by increases in liver lep-a1 and POA crf mRNA, from sustained increases in hepatic igfbp1 expression that reduce the growth-promoting actions of the GH/IGF axis, and from a mobilization of energy reserves.

Duress without stress: Cryptobia infection results in HPI axis dysfunction in rainbow trout.

Despite clear physiological duress, rainbow trout (Oncorhynchus mykiss) infected with the pathogenic haemoflagellate Cryptobia salmositica do not appear to mount a cortisol stress response. Therefore, we hypothesized that the infection suppresses the stress response by inhibiting the key effectors of the hypothalamic-pituitary-interrenal (HPI) axis. To test this, we characterized the basal activity of the HPI axis and the cortisol response to air exposure in saline- and parasite-injected fish. All fish were sampled at 4 and 6 weeks post-injection (wpi). While both the treatment groups had resting plasma cortisol levels, the parasite-infected fish had lower levels of plasma ACTH than the control fish. Relative to the control fish, the infected fish had higher mRNA levels of brain pre-optic area corticotrophin-releasing factor (CRF) and pituitary CRF receptor type 1, no change in pituitary POMC-A1, -A2 and -B gene expression, higher and lower head kidney melanocortin 2 receptor mRNA levels at 4 and 6 wpi respectively and reduced gene expression of key proteins regulating interrenal steroidogenesis: StAR, cytochrome P450scc and 11ß-hydroxylase. The parasite-infected fish also had a reduced plasma cortisol response to a 60-s air exposure stressor. Superfusion of the head kidney tissues of the parasite-infected fish led to significantly lower ACTH-stimulated cortisol release rates than that observed in the control fish. These novel findings show that infection of rainbow trout with C. salmositica results in complex changes in the transcriptional activity of both central and peripheral regulators of the HPI axis and in a reduction in the interrenal capacity to synthesize cortisol.

Haematological responses of acute nitrite exposure in walleye (Sander vitreus).

Nitrite (NO2-) is a toxic intermediary of the bacterial oxidation of nitrogenous wastes (e.g. ammonia) in an aquatic environment. It becomes most lethal when oxygen becomes limited due to high fish densities or in the presence of high bacterial activity due to waste build-up-both situations commonly found in intensive aquaculture. To date however, little is known about how this toxin affects the physiology of walleye, an intended culture species, particularly in intensive re-circulating systems. This study aims to define threshold concentrations of nitrite that affect haemoglobin-oxygen affinity and carrying capacity in walleye. During in vivo tests, fish (N=20) were subjected to a medium effective concentration (EC50) of nitrite (0.9 mmol L(-1)) for 48 h while the effects of nitrite accumulation on blood properties were measured. The effects of oxygenation state on red blood cell (RBC) nitrite uptake and metHb formation was further investigated by in vitro tonometry. In vitro nitrite exposure to 3 mmol L(-1) resulted in a significantly higher methaemoglobin formation in 50% air saturated than 100% air saturated RBCs. Both cell water content and haematocrit decreased with time in 50% air saturated treatments, whereas total Hb remained constant, suggesting a reduction in RBC volume. Similar effects were observed during 48 h in vivo and in vitro nitrite exposure tests, indicating the reduction in RBC volume likely was not the result of a catecholamine response. Walleye were found to be tolerant to an accumulation of blood-NO2- levels similar to common carp, a highly Mean Cellular Volume (MCV) tolerant species, before succumbing to methaemoglobinemia. The elevated tolerance to nitrite of walleye is a beneficial characteristic for successful rearing in a culture setting, where reduced oxygen and elevated MCV levels are prevalent. The findings from this study may be used in developing guidelines for species-specific management of nitrogenous wastes in aquaculture.