Early developmental toxicity of Atlantic salmon exposed to conventional and unconventional oils.

Atlantic salmon is an important species for Canadian culture and economy and its importance extends beyond Canada to Scandinavia and Western Europe. However, it is a vulnerable species facing decline due to habitat contamination and destruction. Existing and new Canadian pipeline projects pose a threat to salmonid habitat. The effects of diluted bitumen (dilbit), the main oil circulating in pipelines, are less studied than those of conventional oils, especially during the critical early embryonic developmental stage occurring in freshwater ecosystems. Therefore, this study aimed to compare the effects of water-accommodated fractions (WAF) of the Clearwater McMurray dilbit and the Lloydminster Heavy conventional oil on Atlantic salmon embryos exposed either from fertilization or from eyed stage. The dilbit contained the highest concentrations of low molecular weight (LMW) compounds (including BTEX and C6-C10), while the conventional oil contained the highest concentrations of PAHs. The Clearwater dilbit caused a higher percentage of mortality and malformations than the conventional oil at similar WAF concentrations. In addition, the embryos exposed from fertilization suffered a higher mortality rate, more developmental delays, and malformations than embryos exposed from the eyed stage, suggesting that early development is the most sensitive developmental stage to oil exposure. Gene expression and enzymatic activity of the detoxification phase I and II enzymes (CYP1A and GST) were measured. Data showed increases in both cyp1a expression and GST activity with increasing WAF concentrations, while gst expression was not affected by the exposures. Also, gene expression of proteins involved in the biotransformation of vitamin A and DNA damage repair were modified by the oil exposures. Overall, this study indicates that Atlantic salmon is mostly affected by oil exposure at the beginning of its development, during which embryos accumulate deformities that may impact their survival at later life stages.

The effects of dissolved petroleum hydrocarbons on benthic organisms: Chironomids and amphipods.

The oil sands industry in Canada, produces heavy unconventional oils, diluted for transport and called diluted bitumen. However, despite advances in our knowledge of the ecotoxicological risk that these products represent, their effects on benthic organisms following a spill are still largely unknown. In order to fill these gaps, this study aims to determine the lethal and sublethal effects of two diluted bitumens (Bluesky and Cold Lake) and one conventional oil (Lloydminster) for two freshwater benthic invertebrates: Chironomus riparius and Hyalella azteca. The objective of this study is to assess the toxicity of dissolved hydrocarbons, resulting from the physical dispersion of oil, immediately after a spill on the benthic invertebrates. To this end, organisms were exposed for 7 days for chironomids and 14 days for amphipods to a fraction containing soluble hydrocarbons (WAF: water accommodated fraction; 10 g/L, 18 h of agitation, followed by 6 h of sedimentation) with natural or artificial sediment. After exposure, the effects of hydrocarbons were determined using size, mortality, and antioxidant capacities. Dissolved hydrocarbons induced mortality for both species, but these hydrocarbons disappeared very quickly from the water column, regardless of the oil type. The amphipods were sensitive to both types of oil while the chironomids were only sensitive to diluted bitumens. The presence of a natural sediment seems to provide a protective role against dissolved hydrocarbons. The antioxidant enzymes measured (CAT, SOD and GPx) do not appear to be relevant biomarkers for the exposure of these organisms to diluted bitumen.

Time-dependent biological responses of juvenile yellow perch (Perca flavescens) exposed in situ to a major urban effluent.

Municipal wastewater treatment plant (WWTP) effluents are significant sources of organic and inorganic pollutants to aquatic ecosystems. Several studies have shown that the health of aquatic organisms can be adversely impacted following exposure to these complex chemical mixtures. The objective of this study was to examine the effects of in situ exposure in the St. Lawrence River (QC, Canada) of juvenile yellow perch (Perca flavescens) to a major WWTP effluent. Perch were caged at a reference site in the St. Lawrence River and downstream of a WWTP effluent-influenced site for one, three, and six weeks. Fish kept in controlled laboratory setting were also examined at the beginning of the experiment to evaluate the potential effect of caging on fish. Liver metabolites and gill oxidative stress biomarkers as well as body condition of perch were investigated at four time points (zero, one, three, and six weeks). Nitrogen (δ15N) and carbon (δ13C) stable isotopes as well as tissue concentrations of halogenated flame retardants and trace metals were also analyzed. Results indicated that body condition of perch caged in the effluent increased after three and six weeks of exposure compared to that of reference fish. Perch caged at the WWTP effluent-influenced site also had higher muscle δ13C and slightly depleted muscle δ15N after three and six weeks of exposure, suggesting differences in sewage-derived nutrient assimilation between sites. Concentrations of Σ34 polybrominated diphenyl ether (PBDE) were 2-fold greater in perch exposed downstream of the WWTP compared to those caged at the reference site. Metal concentrations in kidney of perch after three weeks of exposure were significantly lower at the effluent-influenced site. Kidney concentrations of Cd, Cu, Se, As, Zn and Fe were, however, higher after six weeks of exposure, supporting that metal accumulation is time- and element-specific. The metabolomes of perch from the effluent-influenced and reference sites were similar, but were distinct from the laboratory control fish, suggesting a caging effect on fish. Seven liver metabolites (glucose, malate, fumarate, glutamate, creatinine, histamine, and oxypurinol) were significantly more abundant in perch from cages than in the laboratory control perch. The combination of metabolomics and physiological variables provides a powerful tool to improve our understanding of the mechanisms of action of complex environmental pollutant mixtures in wild fish.

A seasonal comparison of trace metal concentrations in the tissues of Arctic charr (Salvelinus alpinus) in Northern Québec, Canada.

Ecotoxicological research detailing trace metal contamination and seasonal variation in the tissues of northern fishes such as Arctic charr (Salvelinus alpinus) has been poorly represented in the literature beyond examination of mercury. In an effort to address this, anadromous Arctic charr were collected from the Deception River watershed in the late summer and post-winter season, before quantifying seasonal and organotropic variations in dorsal muscle and liver concentrations of arsenic, cadmium, chromium, copper, nickel, lead, and zinc. Potential linkages with biological variables (fork length, age, and somatic condition) and indicators of feeding behavior (δ13C and δ15N) were also assessed. Trace metal organotropism favouring elevation in liver tissue concentrations was exhibited by cadmium, copper, nickel and zinc, while arsenic, chromium and lead exhibited no significant organotropic variation. Seasonal differences in concentrations were metal and tissue dependent, but generally increased in tissues collected from post-winter sampled Arctic charr. Significant correlations with biological and trophic descriptors were also determined to be element and tissue dependent. These parameters, in addition to season, were incorporated into multi-predictor variable models, where variations in trace metal concentration data were often best explained when season, somatic condition, and trophic descriptors were included. These variables were also of greatest relative importance across all considered trace metals and tissue types. These findings suggest that seasonally linked processes have the greatest influence on trace metal concentrations in anadromous Arctic charr. Future metal-related research on Arctic charr and other northern fish species should further consider these variables when evaluating elemental accumulation.

Temperature and metal exposure affect membrane fatty acid composition and transcription of desaturases and elongases in fathead minnow muscle and brain.

In this study, we tested the hypothesis that metal exposure affected the normal thermal response of cell membrane FA composition and of elongase and desaturase gene transcription levels. To this end, muscle and brain membrane FA composition and FA desaturase (fads2, degs2 and scd2) and elongase (elovl2, elovl5 and elovl6) gene transcription levels were analyzed in fathead minnows (Pimephales promelas) acclimated for eight weeks to 15, 25 or 30°C exposed or not to cadmium (Cd, 6µg/l) or nickel (Ni, 450 6µg/l). The response of membrane FA composition to temperature variations or metal exposure differed between muscle and brain. In muscle, an increase of temperature induced a decrease of polyunsaturated FA (PUFA) and an increase of saturated FA (SFA) in agreement with the current paradigm. Although a similar response was observed in brain between 15 and 25°C, at 30°C, brain membrane unsaturation was higher than predicted. In both tissues, metal exposure affected the normal thermal response of membrane FA composition. The transcription of desaturases and elongases was higher in the brain and varied with acclimation temperature and metal exposure but these variations did not generally reflect changes in membrane FA composition. The mismatch between gene transcription and membrane composition highlights that several levels of control other than gene transcription are involved in adjusting membrane FA composition, including post-transcriptional regulation of elongases and desaturases and de novo phospholipid biosynthesis. Our study also reveals that metal exposure affects the mechanisms involved in adjusting cell membrane FA composition in ectotherms.

Transcriptome profile analysis reveals specific signatures of pollutants in Atlantic eels.

Identifying specific effects of contaminants in a multi-stress field context remain a challenge in ecotoxicology. In this context, "omics" technologies, by allowing the simultaneous measurement of numerous biological endpoints, could help unravel the in situ toxicity of contaminants. In this study, wild Atlantic eels were sampled in 8 sites presenting a broad contamination gradient in France and Canada. The global hepatic transcriptome of animals was determined by RNA-Seq. In parallel, the contamination level of fish to 8 metals and 25 organic pollutants was determined. Factor analysis for multiple testing was used to identify genes that are most likely to be related to a single factor. Among the variables analyzed, arsenic (As), cadmium (Cd), lindane (γ-HCH) and the hepato-somatic index (HSI) were found to be the main factors affecting eel's transcriptome. Genes associated with As exposure were involved in the mechanisms that have been described during As vasculotoxicity in mammals. Genes correlated with Cd were involved in cell cycle and energy metabolism. For γ-HCH, genes were involved in lipolysis and cell growth. Genes associated with HSI were involved in protein, lipid and iron metabolisms. Our study proposes specific gene signatures of pollutants and their impacts in fish exposed to multi-stress conditions.

Abnormal ovarian DNA methylation programming during gonad maturation in wild contaminated fish.

There is increasing evidence that pollutants may cause diseases via epigenetic modifications. Epigenetic mechanisms such as DNA methylation participate in the regulation of gene transcription. Surprisingly, epigenetics research is still limited in ecotoxicology. In this study, we investigated whether chronic exposure to contaminants experienced by wild female fish (Anguilla anguilla) throughout their juvenile phase can affect the DNA methylation status of their oocytes during gonad maturation. Thus, fish were sampled in two locations presenting a low or a high contamination level. Then, fish were transferred to the laboratory and artificially matured. Before hormonal treatment, the DNA methylation levels of the genes encoding for the aromatase and the receptor of the follicle stimulating hormone were higher in contaminated fish than in fish from the clean site. For the hormone receptor, this hypermethylation was positively correlated with the contamination level of fish and was associated with a decrease in its transcription level. In addition, whereas gonad growth was associated with an increase in DNA methylation in fish from the clean site, no changes were observed in contaminated fish in response to hormonal treatment. Finally, a higher gonad growth was observed in fish from the reference site in comparison to contaminated fish.

Modeling the geochemical evolution of mine waters during mixing.

This study focuses on assessing the hydrogeochemical processes influencing the mobility of dissolved metal and metalloid species during mine effluent mixing. Field samples were collected to characterize effluents at an active gold mine located in the Abitibi Greenstone belt in western Québec, Canada. Controlled laboratory mixing experiments were further performed with real effluents. In situ physicochemical parameters, concentrations of major dissolved ions and trace elements were analyzed. Mineralogical analyses were also performed on precipitates from the laboratory mixtures. The data were used for statistical analyses and for modeling the geochemical evolution of effluents using PHREEQC with the wateq4f.dat database (with modifications). The results suggest that the formation of secondary minerals such as schwertmannite, Fe(OH)3, and jarosite could significantly affect the concentrations of trace elements in effluents. The precipitation of secondary minerals immobilized trace elements through coprecipitation and sorption processes. The main limitations of the modeling approach used here include the evaluation of the ion balance for low pH samples with high Fe and Al concentrations and the omission of biological processes. The approach provides insights into the geochemical evolution of mine effluents and could be adapted to several mining sites as a tool for improving water management.

Temporal fluctuation of metallic trace elements concentrations in three morphotypes of floating holopelagic Sargassum from the Caribbean coast (Guadeloupe, French West Indies).

Since 2011, the Caribbean coasts have unprecedented stranding of a pelagic brown macroalgae Sargassum inducing damages for coastal ecosystems and economy. This study evaluated the temporal fluctuations of metallic trace elements (MTE) in Sargassum freshly arrived on the Caribbean coast. From May 2020 to September 2021, 12 floating samples of three morphotypes (S. fluitans III and S. natans I and VIII) were regularly collected in the Petit Cul-de-Sac Marin (Guadeloupe, French West Indies). Measured concentrations of 28 metal(loid)s trace elements reveal i) an absence of seasonal patterns in MTE concentrations except for metals Fe and Al during 2020 summer ii) a regular and high As content during the entire survey iii) a similar trend of contamination for each morphotype. The constant and high amount of As implies that stranding management policy and valorization processes of Sargassum must consider As contamination and that this vigilance must be constantly along the year.

Evolutionary change driven by metal exposure as revealed by coding SNP genome scan in wild yellow perch (Perca flavescens).

Pollution can drive rapid evolutionary change in wild populations. This study targets functional polymorphisms of chronically metal-contaminated wild yellow perch (Perca flavescens). A de novo transcriptome scan contrasted subsets of individuals from clean (n = 16) and contaminated (n = 16) lakes to identify 87 candidate annotated coding SNPs. Candidate genotypes and liver [metal] were obtained in 10 populations (n = 1,052) and a genome scan distinguished outliers: one nuclear (cyclin G1 gene) and two mitochondrial (cytochrome b and NADH dehydrogenase subunit 2 genes) also displaying allelic correlation to mean population [cadmium]. Whole mtDNA and 17 kb surrounding cyclin G1 were characterised through 454 sequencing thus revealing two non-synonymous substitutions involving dissimilar amino acids. Based on associated functions and inter-population differentiation, contaminated perch may have been selected for fast life cycle completion (p53 pathway) and memorization impairment mitigation (long-term potentiation pathway). In accordance with predicted evolutionary trajectory for stressed and energy deprived organisms, adapted perch would not compensate for repair mechanism inhibition, instead reallocating energy towards growth and favouring inexpensive impairment mitigation adaptations over costly detoxification. Overall, 85 years of selection could have driven rapid, potentially adaptive evolution by selecting alleles increasing perch fitness in polluted environments.

Thermal modulation of mitochondrial function is affected by environmental nickel in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the combined effects of temperature and nickel (Ni) contamination on liver mitochondria electron transport system (ETS) enzymes, citrate synthase (CS), phospholipid fatty acid composition and lipid peroxidation in rainbow trout (Oncorhynchus mykiss). Juvenile trout were acclimated for two weeks to two different temperatures (5˚C and 15˚C) and exposed to nickel (Ni; 520 µg/L) for three weeks. Using ratios of ETS enzymes and CS activities, our data suggest that Ni and an elevated temperature acted synergistically to induce a higher capacity for reduction status of the ETS. The response of phospholipid fatty acid profiles to thermal variation was also altered under nickel exposure. In control conditions, the proportion of saturated fatty acids (SFA) was higher at 15˚C than at 5˚C, while the opposite was observed for monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA). However, in nickel contaminated fish, the proportion of SFA was higher at 5˚C than at 15˚C, while PUFA and MUFA followed the opposite direction. A higher PUFA ratio is associated with higher vulnerability to lipid peroxidation. Thiobarbituric Acid Reactive Substances (TBARS) content was higher when the PUFA were in higher proportions, except for Ni-exposed, warm-acclimated fish, in which we reported the lowest level of TBARS but the highest proportion of PUFA. We suspect that the interaction of nickel and temperature on lipid peroxidation is due to their synergistic effects on aerobic energy metabolism, as supported by the decrease in the activity of complex IV of the ETS enzyme activity in those fish, or on antioxidant enzymes and pathways. Overall, our study demonstrates that Ni exposure in heat-challenged fish can lead to the remodelling of the mitochondrial phenotype and potentially stimulate alternative antioxidant mechanisms.

Influence of ozone microbubble enhanced oxidation on mine effluent mixes and Daphnia magna toxicity.

The mining industry often must mix different kinds of water on the mine site during pre-treatment or post-treatment before the final discharge of the treated water to the environment. Microbubble ozonation has proven to be efficient in the removal of contaminants of concern from mine water, such as metals, metalloids, and nitrogen compounds, which can persist in the environment and entail toxicity issues. This study evaluated the efficiency of ozone microbubbles combined with lime precipitation on contaminant removal and its impact on toxicity for Daphnia magna with five different mine effluent mixes from an active mine site located in Abitibi-Témiscamingue, QC, Canada. For the non-acidic mixes, two scenarios were tested: first, pre-treatment of metals using lime precipitation and a flocculant was conducted prior to ozonation; and second, ozonation was conducted prior to metals post-treatment using the same precipitation and flocculation technique. Results showed that the NH3-N removal efficiency ranged from 90% for the lower initial concentrations (1.1 mg/L) to more than 99% for the higher initial concentrations (58.4 mg/L). Moreover, ozonation without metals pre-treatment improved NH3-N treatment efficiency in terms of kinetics but entailed abnormal toxicity issues. Results of bioassays conducted on water with metals pre-treatment did not show any toxicity events but showed abnormal toxicity patterns on the mixes treated without metals pre-treatment (diluted effluents were toxic, while undiluted were not). At 50% dilution, the water was toxic, probably due to the potential presence of metal oxide nanoparticles. The confirmation of the source of toxicity requires further investigation.

The effects of winter cold acclimation on acute and chronic cadmium bioaccumulation and toxicity in the banded killifish (Fundulus diaphanus).

Temperate freshwater fishes can experience large seasonal temperature fluctuations that could affect their exposure and sensitivity to trace metals. Yet, temperature effects are overlooked in ecotoxicology studies, especially for cold temperatures typical of the winter. In the present study, the effects of long-term cold acclimation on Cd bioaccumulation and toxicity were investigated in a freshwater fish, the banded killifish (Fundulus diaphanus). Killifish were acclimated to 14 °C or gradually cooled (2 °C/week) to 4 °C and cold acclimated for 6 weeks. Then, both acclimation groups were exposed to environmentally realistic waterborne Cd concentrations (0, 0.5 or 5 µg Cd L-1) for a further 28 d at their respective acclimation temperatures. Tissue metal bioaccumulation, fish survival, condition, and markers of oxidative and ionoregulation stress, were measured after 0, 2, 5 and 28 days of Cd exposure. Cadmium tissue accumulation increased over the exposure duration and was typically lower in cold-acclimated fish. In agreement with this lower bioaccumulation, fewer Cd toxic effects were observed in cold-acclimated fish. There was little evidence of a difference in intrinsic Cd sensitivity between 4 °C- and 14 °C-acclimated fish, as Cd toxicity appeared to closely follow Cd bioaccumulation. Our study suggests that current environmental water quality guidelines would be protective in the winter for the abundant and ecologically-important banded killifish.

Comparison of the accumulation and effects of copper pyrithione and copper sulphate on rainbow trout larvae.

Copper pyrithione (CuPT) is used as a co-biocide in new antifouling paints but its toxicity remains little known. To compare the toxicity of copper-based compounds, rainbow trout (Oncorhynchus mykiss) larvae were exposed for 8-day to CuPT and CuSO4 at equivalent copper concentrations. CuPT exposure led to the greatest accumulation of Cu in larvae. Exposure to 10 µg.L-1 CuPT induced 99% larval mortality but only 4% for CuSO4-exposed larvae. The larval development and growth were affected by CuPT (from 0.5 µg.L-1 Cu) but not by CuSO4. Lipid peroxidation was not induced by either contaminant. The expression of genes involved in oxidative stress defence, detoxification and copper transport was induced in larvae exposed to CuSO4 and CuPT but at higher concentrations for CuPT. This study highlights the marked toxicity of CuPT for early life stages of fish and raises the question of the possible environmental risks of this antifouling compound.

Evidence for metabolic imbalance of vitamin A2 in wild fish chronically exposed to metals.

In a recent study on indigenous yellow perch chronically exposed to metals, we reported a negative correlation between liver metal concentration and liver transcription levels of genes encoding for enzymes involved in the metabolism of retinoids. We therefore speculated that metals, and especially the non-essential metal Cd, could alter the metabolism of retinoids in wild fish. Thus the present field study investigates the impact of in situ metal exposure on retinoid storage. A total of 55 yellow perch (Perca flavescens) were sampled in six lakes representing a metal contamination gradient (8≤N≤10 per lake). Our results show that yellow perch from Cd-contaminated lakes had significantly higher concentrations of liver dehydroretinol and dehydroretinyl esters than did fish from reference lakes. However, the increase in retinyl ester stores with increasing Cd concentrations was quantitatively much more important than the increase in free dehydroretinol. As a result, a significant decrease in the percentage of hepatic free dehydroretinol with increasing renal Cd concentrations was observed. These results suggest that the enzymes and the binding proteins involved in vitamin A homeostasis are inhibited by the presence of Cd. Alternatively, the increase in tissue vitamin A (antioxidant) levels could serve to better counteract the oxidative stress engendered by Cd exposure. Overall our findings illustrate that vitamin A(2) homeostasis can be altered as a consequence of chronic exposure to low Cd concentrations. Thus, in the context of environmental risk assessment, the percentage of liver free dehydroretinol can be considered as a biomarker of for in situ Cd exposure.

Comparative toxicity of conventional and unconventional oils during rainbow trout (Oncorhynchus mykiss) embryonic development: From molecular to health consequences.

Canadian freshwater ecosystems are vulnerable to oil spills from pipelines, which contain mostly diluted bitumen. This study aimed to compare the toxicity of a dilbit and a conventional oil on developing rainbow trout. A total of five exposure scenarios were performed, from 10 to 43 days, using water-accommodated fraction (WAF) with an initial loading of 1:9 oil to water ratio (w/v) in a range of dilutions from 0.32 to 32% WAF, respectively, with TPAH and VOC concentrations from 2.41 to 17.5 µg/L and 7.94-660.99 µg/L, and with or without a recovery period. Following the five exposures, several endpoints were examined, including survivorship, morphometrics, gene expression, and enzymatic activity. Significant mortality rates were measured for the highest WAF concentration of the dilbit in all five exposures (60-100% mortality at 32% WAF). In comparison, the highest WAF concentration of the conventional oil induced significant mortality in three out of the five exposure (from 35 to 100% mortality at 32% WAF). Hatching delays were noted in embryos exposed to both oils. Developmental delays were observed in dilbit-exposed embryos and are suspected to be an indicator of reduced survivorship after hatching. The induced expression of cyp1a remained a reliable biomarker of exposure and of fish malformations, though it did not always predict mortality. Using CYP1A activity in combination with cyp1a may bring more insights in studies of oil risk assessment. This study demonstrates that dilbits are more toxic to early life stages compared to conventional oils and highlights the need to consider the most sensitive stage of development when performing risk assessment studies on oils.

Effects of chronic metal exposure on wild fish populations revealed by high-throughput cDNA sequencing.

Given the inherent variability of aquatic systems, predicting the in situ effects of contaminants on such ecosystems still represents a major challenge for ecotoxicology. In this context, transcriptomic tools can help identify and investigate the mechanisms of toxicity beyond the traditional morphometric, physiological and population-level endpoints. In this study, we used the 454 sequencing technology to examine the in situ effects of chronic metal (Cd, Cu) exposure on the yellow perch (Perca flavescens) transcriptome. Total hepatic mRNA from fish sampled along a polymetallic gradient was extracted, reverse transcribed, labeled with unique barcode sequences and sequenced. This approach allowed us to identify correlations between the transcription level of single genes and the hepatic concentrations of individual metals; 71% of the correlations established were negative. Chronic metal exposure was thus associated with a decrease in the transcription levels of numerous genes involved in protein biosynthesis, in the immune system, and in lipid and energy metabolism. Our results suggest that this marked decrease could result from an impairment of bile acid metabolism by Cd and energy restriction but also from the recruitment of several genes involved in epigenetic modifications of histones and DNA that lead to gene silencing.

Fate and Fathead Minnow Embryotoxicity of Weathering Crude Oil in a Pilot-Scale Spill Tank.

For several years now, the Natural Resources Canada research facility at CanmetENERGY Devon (AB, Canada) has been performing experiments in a pilot-scale spill tank using 1200 L of river water to examine the physical and chemical behaviors of various crude oil/water mixtures under varying water temperature regimes. Because oil toxicity can be modulated by weathering of the petroleum products, the present study aimed to assess changes in fish embryotoxicity to mixed sweet blend crude oil as it weathered at air and water temperatures of 14 °C and 15 °C, respectively, for 28 d. The physicochemical behavior of the oil was also monitored. Water samples were taken from the spill tank 5 times during the 28-d experiment on days 1, 6, 14, 21, and 28 and were used to perform toxicity exposures using fathead minnow embryos (Pimephales promelas). For each water sampling day, newly fertilized embryos were exposed to a serial dilution of the spill tank water, noncontaminated river water (used in the spill tank), and a reconstituted water laboratory control. Embryos were raised until hatching. Although mortality was not significantly altered by the oil contamination over the time period, malformation occurrence and severity showed concentration-dependent responses to all contaminated water collected. The results suggest that days 14, 21, and 28 were the most toxic time periods for the fish embryos, which corresponded to increasing concentrations of unidentified oxidized organic compounds detected by a quadropole-time-of-flight system. The present study highlights a novel area for oil research, which could help us to better understand the toxicity associated with oil weathering for aquatic species. Environ Toxicol Chem 2021;40:127-138. © 2020 Her Majesty the Queen in Right of Canada. Reproduced with the permission of the Minister of Natural Resources Canada.

Lethal and sublethal effects of diluted bitumen and conventional oil on fathead minnow (Pimephales promelas) larvae exposed during their early development.

The increasing extraction of bitumen from the oil sands region in Canada is creating a need for transport. Spills from current and projected pipelines represent a significant environmental risk, especially for freshwater ecosystems. The toxicity of diluted bitumen (dilbit) on freshwater fish is largely unknown. This study assessed the toxicity of two dilbits (Clearwater McMurray and Bluesky) and compared their toxicity to a conventional oil (Lloydminster Heavy) on fathead minnow (Pimephales promelas) larvae. Larvae were exposed to various concentrations of the water-accommodated fraction (WAF) of the oils during 7 days from hatching. In the WAF treatments, the concentrations of volatile organic compounds (VOCs), including benzene, toluene, ethylbenzene, xylene (BTEX), hydrocarbons containing 6 to 10 carbon atoms (C6-10), and polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms were measured. Both dilbits contained higher concentrations of light components, while the conventional oil contained the highest concentrations of PAHs and alkylated PAHs. The Clearwater McMurray dilbit induced a higher mortality, with a maximum of 65.3%, while the other oils induced a similar mortality up to 16.5% and 18.6% for Lloydminster and for Bluesky, respectively. All three oils induced an increase in gene expression of the phase I detoxification enzyme (cyp1a) with increasing total hydrocarbon concentrations. All three exposures induced a similar increase in glutathione S-transferase (GST) activity, but no change in gst gene expression. For the Bluesky and Lloydminster exposures, an increase in malondialdehyde concentration was also observed, suggesting a rate limiting capacity of GST and phase II enzymes to perform the biotransformation of the PAH metabolites. Overall, this study brings new insights on the toxicity of dilbits in comparison to conventional oils on early life stages of North American freshwater fish and demonstrated that dilbits can be more toxic than conventional oils, depending on their composition and diluent proportions.

Comparative developmental toxicity of conventional oils and diluted bitumen on early life stages of the rainbow trout (Oncorhynchus mykiss).

Petroleum hydrocarbons are widely used and transported, increasing the risks of spills to the environment. Although conventional oils are the most commonly produced, the production of unconventional oils (i.e. diluted bitumen or dilbit) is increasing. In this study, we compared the effects of conventional oils (Arabian Light and Lloydminster) and dilbits (Bluesky and Clearwater) on early life stages of a salmonid. To this end, aqueous fractions (WAF: water accommodated fraction) of these oils were extracted using mountain spring water. Rainbow trout (Oncorhynchus mykiss) larvae were exposed to 10 and 50% dilutions of these WAFs from hatching (340 DD; degree days) until yolk sac resorption (541 DD). Exposure to WAFs increased skeletal malformations (both dilbits) and hemorrhage (both conventional oils and Bluesky) and decreased head growth (Arabian Light). In addition, increases in EROD activity and DNA damage were measured for all oils and an increase in cyp1a gene expression was measured for Arabian Light, Bluesky and Clearwater. The PAH and C10C50 concentrations were positively correlated to total larval EROD activity, whereas concentrations of total hydrocarbons, VOCs, PAHs, and C10C50 were positively correlated to cyp1a expression. Total hydrocarbon, VOC, and C10C50 concentrations were also negatively correlated to larval growth. This study supports that petroleum hydrocarbons are toxic to early developmental stages of rainbow trout and show that their degree and spectrum of toxicity depends on their chemical composition.