A model naphthenic acid decouples oxidative phosphorylation through selective inhibition of mitochondrial complex activity.

The naphthenic acid fraction compound (NAFC), 3,5-dimethyladamantane-1-acetic acid, was tested for its ability to uncouple mitochondrial oxidative phosphorylation. Mitochondria isolated from rainbow trout (Oncorhynchus mykiss) liver were exposed to 3,5-dimethyladamantane-1-acetic acid in state 3 and 4 respiration, and mitochondrial membrane potential were quantified. Electron transport chain (ETC) protein complexes were isolated using pharmacological agents and inhibitors, and their activities measured. The NAFC compound completely inhibited states 3 and 4 respiration with an IC50 of 0.77 and 1.01 mM, respectively. The NAFC compound partially uncoupled mitochondrial membrane potential in state 3 and 4 respiration with an IC50 of 2.19 and 1.73 mM, respectively. The NAFC impaired the activities of ETC protein complexes with a 9.5-fold range in sensitivity. The relative inhibitory effect of the ETC protein complexes to NAFC was CIV≥CI>CIII>CII. The impairment of mitochondrial oxidative phosphorylation by adamantane 3,5-dimethyladamantane-1-acetic acid is mediated via its inhibition of ETC protein complexes.

Internal nutrients dominate load and drive hypoxia in a eutrophic estuary.

The hypothesis that local hypoxia and chlorophyll concentration are spatially tethered to local, sediment-driven nutrient release was examined in a small, nutrient-impacted estuary in the Southern Gulf of St. Lawrence, Canada. Sediment reactor core samples were taken at 10 locations between 0.25 and 100% of the estuary area in spring and fall (2019) and used to estimate nitrogen and phosphate flux. Sediment organic matter, carbonate, percent nitrogen, percent carbon, δ13C, and δ15N were measured from the reactor core stations. Oxygen was recorded continually using oxygen loggers while chlorophyll and salinity were measured bi-weekly. A hydrodynamic model was used to determine water renewal time at each station. The most severe eutrophication effects were in the upper one-fifth of the estuary. There were strong local relationships between sediment biogeochemistry, hypoxia, and chlorophyll metrics but not with water renewal time. Internal nutrient loading represented 65% and 69% of total N loading, and 98% and 89% of total P loading to the estuary in June and September, respectively. Sediment nitrogen flux was highly predictable from a range of local sediment variables that reflect either nutrient content, or organic carbon enrichment in general. Percent nitrogen and percent carbon were highly correlated but sediment P flux was poorly predicted from sediment parameters examined. The highest correlations were with percent nitrogen and percent carbon. These results indicate that incorporating internal nutrient loading into nutrient monitoring programs is a critical next step to improve predictive capacity for eutrophication endpoints and to mitigate nutrient effects.

Exposure of American lobster (Homarus americanus) to the pesticide chlorpyrifos results in changes in gene expression.

Chlorpyrifos is an organophosphate that is currently used to reduce arthropod pests for the protection of agricultural crops. Coastal marine ecosystems may be exposed to agricultural pesticides via runoff and pesticide exposure can impact the health and survival of non-target species such as the American lobster (Homarus americanus). In the current study, the gene expression changes of H. americanus stage IV larvae were evaluated to understand the physiological mechanisms affected by exposure to sublethal concentrations of chlorpyrifos. After 48 h chlorpyrifos exposure, surviving lobsters were processed for Illumina RNA sequencing (RNA-seq). Genes of interest that showed significant changes using RNA-seq were verified using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Analysis of RNA-seq and the confirmation of gene expression patterns via RT-qPCR found altered expression in genes related to stress response (glutathione peroxidase 3 and heat shock protein 60), hypoxia response (hairy, astakine 2, hemocyanin), moulting (cytochrome P450 307a1 and chitinase), and immunity (astakine 2) pathways. Changes to gene expression were most notable in lobsters exposed to 0.57 µg/L chlorpyrifos.

The Differential Effects of Salinity Level on Chlorpyrifos and Imidacloprid Toxicity to an Estuarine Amphipod.

Agricultural activity within coastal watersheds results in estuaries becoming the receiving environment for pesticide inputs. In estuaries, salinity can alter insecticide responses of exposed crustaceans. The acute toxicity of environmentally relevant doses of chlorpyrifos and imidacloprid were examined using the euryhaline amphipod Gammarus lawrencianus at 20 and 30 Practical Salinity Units (PSU). Responses were recorded every 24 h until an incipient (threshold) L(E)C50 was reached. For chlorpyrifos, LC50 ranged from 0.1 to 0.5 µg/L and was two-fold higher at 30 vs. 20 PSU at all time-points over the 96 h exposure. Imidacloprid immobility EC50 ranged from 4 to 40 µg/L over the 144 h exposure. An effect of salinity was only observed at 48 h and the EC50 values showed 1.4 times more potency at 20 PSU compared to 30 PSU. Measured concentrations of both compounds did not differ between salinities. Acetylcholinesterase activity in chlorpyrifos exposed amphipods showed no salinity effect at 96 h. We conclude that salinity level alters G. lawrencianus susceptibility to chlorpyrifos exposure, but not imidacloprid.

Assessing reproductive effects on fish populations: an evaluation of methods to predict the reproductive strategy of fishes.

Many environmental monitoring programs include an assessment of the health of fish populations using a sentinel species and include an indicator of reproductive potential. Knowledge of the reproductive strategy of the fish species is critical for data interpretation but is not always known. The reproductive strategy of a species can be determined from detailed histological analyses of ovaries throughout the reproductive cycle; however, these studies can be costly and can delay the implementation of a monitoring program. Three quick and cost-effective methods of predicting the reproductive strategy (annual single spawning or annual multiple spawning) are evaluated in this study using predicted probabilities from binary logistic regression models as a means of classifying the reproductive strategies of 18 different fish species in Atlantic Canada. The first method was based on the hypothesis that the variability in the ovary weight-body weight relationship in prespawning females is higher in multiple spawners. This method did not have a good classification rate due to some multiple spawners having low variability. The other two methods involved predictor variables representing the proportion of oocytes in different stages of development and predictor variables representing the distribution of oocyte sizes during the prespawning season for 111 fish (25 different samples for species). Predicted probabilities from these regression models could be used to correctly classify the reproductive strategies of all 25 samples (development stage model) and all but one sample (oocyte size distribution model). These models can be used to estimate the reproductive strategy of a species from a single sample of fish collected during the prespawning period to support species selection and data interpretation in environmental monitoring programs.

Effects of Sublethal Chlorpyrifos Exposure on Postlarval American Lobster (Homarus americanus).

The organophosphate pesticide chlorpyrifos has been introduced to the marine environment via adsorption to agricultural soil runoff or as spray drift. Chlorpyrifos affects the survival of some larval decapod crustaceans, but no data exist on the impacts to the American lobster, Homarus americanus. The purpose of the present study was to assess the levels at which chlorpyrifos affects the survival of postlarval H. americanus. Using acute saltwater exposures, the 24- and 48-h median lethal concentrations were established for stage IV H. americanus (1.56 and 1.33 µg/L, respectively). Movement, acetylcholinesterase activity, intermoult period, specific growth rate, and moult increment were measured during exposure to sublethal concentrations. Movement patterns were assessed to establish a 48-h median inhibition concentration for cessation of normal movement (0.66 µg/L). Acetylcholinesterase activity was found to be inhibited immediately post-exposure to 0.50, 0.57, and 0.82 µg/L chlorpyrifos but could be recovered within a period (9-15 d) in clean seawater. Sublethal growth effects of increased intermoult period, decreased specific growth rate, and decreased moult increment were observed during exposure to an environmentally relevant concentration (0.82 µg/L). The present study suggests that H. americanus stage IV larvae were marginally less sensitive to chlorpyrifos compared with other decapods and that acute lethality of H. americanus postlarvae is not likely to occur with chlorpyrifos concentrations previously reported from aquatic environments. Environ Toxicol Chem 2019;38:1294-1301. © 2019 SETAC.

Oil Sands Derived Naphthenic Acids Are Oxidative Uncouplers and Impair Electron Transport in Isolated Mitochondria.

Naphthenic acids (NAs) are predominant compounds in oil sands influenced waters. These acids cause numerous acute and chronic effects in fishes. However, the mechanism of toxicity underlying these effects has not been fully elucidated. Due to their carboxylic acid moiety and the reported disruption of cellular bioenergetics by similar structures, we hypothesized that NAs would uncouple mitochondrial respiration with the resultant production of reactive oxygen species (ROS). Naphthenic acids were extracted and purified from 17-year-old oil sands tailings waters yielding an extract of 99% carboxylic acids with 90% fitting the classical O2-NA definition. Mitochondria were isolated from rainbow trout liver and exposed to different concentrations of NAs. Mitochondrial respiration, membrane potential, and ROS emission were measured using the Oroboros fluorespirometry system. Additionally, mitochondrial ROS emission and membrane potential were evaluated with real-time flow cytometry. Results showed NAs uncoupled oxidative phosphorylation, inhibited respiration, and increased ROS emission. The effective concentration (EC50) and inhibition concentration (IC50) values for the end points measured ranged from 21.0 to 157.8 mg/L, concentrations similar to tailings waters. For the same end points, EC10/IC10 values ranged from 11.8 to 66.7 mg/L, approaching concentrations found in the environment. These data unveil mechanisms underlying effects of NAs that may contribute to adverse effects on organisms in the environment.

An empirical model using dissolved oxygen as an indicator for eutrophication at a regional scale.

Simple empirical models can sometimes capture salient patterns without sacrificing predictive capacity when compared to more complex models. Herein we examine dissolved oxygen as an indicator of eutrophication status for shallow estuaries. Dissolved oxygen was measured hourly in the upper estuary of 15 watersheds along a nutrient-loading and geographic gradient. Metrics describing hypoxia and supersaturation were devised and then analyzed using multivariate statistics. Results revealed independent responses for hypoxia and supersaturation with hypoxia-related metrics correlating strongly with water residence. A metric integrating hypoxia and supersaturation effectively discriminated between seagrass and algae-dominated habitats and was significantly correlated with both water residence and nitrate-N loading. Chlorophyll, measured bi-weekly, was not correlated with our predictor variables likely because it does not account for benthic production. Over 70% of the variability in hypoxia was explained by water residence and nitrate-N loading indicating that this model can be of use for managers.

Opportunistic disease in yellow perch in response to decadal changes in the chemistry of oil sands-affected waters.

Oil sands-affected water from mining must eventually be incorporated into the reclaimed landscape or treated and released. However, this material contains petrogenic organic compounds, such as naphthenic acids and traces of polycyclic aromatic hydrocarbons. This has raised concerns for impacts of oil sands process-affected waters on the heath of wildlife and humans downstream of receiving environments. The objective of this study was to evaluate the temporal association of disease states in fish with water chemistry of oil sands-affected waters over more than a decade and determine the pathogens associated with disease pathologies. Yellow perch (Perca flavescens) captured from nearby lakes were stocked into two experimental ponds during 1995-1997 and 2008-2010. South Bison Pond is a drainage basin that has received unextracted oil sands-contaminated material. Demonstration Pond is a constructed pond containing mature fine tailings capped with fresh water. Two disease pathologies, fin erosion for which a suspected bacterial pathogen (Acinetobacter Iwoffi) is identified, and lymphocystis (confirmed using a real-time PCR) were associated with oil sands-affected water exposure. From 1995 to 1997 pathologies were most prevalent in the South Bison Pond; however, from 2008 to 2009, disease was more frequently observed in the Demonstration Pond. CYP1A activity was 3-16 fold higher in fish from experimental ponds as compared to reference populations and this pattern was consistent across all sampling years. Bile fluorescence displayed a gradient of exposure with experimental ponds being elevated over local perch populations. Naphthenic acids decreased in the Bison Pond from approximately 12 mg/L to <4 mg/L while naphthenic acids increased in the Demonstration Pond from 6 mg/L to 12 mg/L due to tailings densification. Temporal changes in naphthenic acid levels, CYP1A activity and bile fluorescent metabolites correlate positively with incidence of disease pathologies whereas all inorganic water quality changes (major ions, pH, metals) were not associated with disease responses.

Transcriptional response in rainbow trout (Oncorhynchus mykiss) B cells and thrombocytes following in vivo exposure to benzo[a]pyrene.

Immune toxicity of polycyclic aromatic hydrocarbons (PAHs) in fishes has been frequently reported but the reasons for differential cell toxicity remains unclear. Rainbow trout were exposed in vivo with a single intraperitoneal injection of corn oil or 100mg/kg of the immunotoxic PAH benzo[a]pyrene (B[a]P) in corn oil. Leukocytes were harvested from head kidney, spleen and blood after 14days, the optimal time for B cell depletion found in a previous study. The mRNA expression of five cytochrome P450 (CYP) enzymes, the aryl hydrocarbon receptor (AhR), and an intrinsic pathway apoptosis checkpoint (p53) in B cells and thrombocytes were examined. Transcript levels were measured in immunomagnetically-isolated B cells and thrombocytes from those tissues as well as in liver as B cells had been previously shown to be responsive the BaP whereas thrombocytes were not. There was induction of CYP1A1 in liver, blood B cells, and blood and spleen thrombocytes; CYP1B1 in blood B cells, blood and spleen thrombocytes; CYP1A3 in liver, blood and spleen B cells, and blood thrombocytes; CYP1C1 in liver; and AhR in liver and spleen thrombocytes. There was no change in CYP1C2, or p53 mRNA levels across tissues or cell type. Induction in mRNA was observed 14 d after exposure, indicating a prolonged physiological effect of a single B[a]P injection. CYP1A1 and CYP1A3 were the most abundantly expressed CYP genes and CYP1B1 was generally least abundant. B[a]P-induced thrombocytes had a significantly different pattern of CYP expression than either liver or B cells. Given the importance of metabolites in the toxicity of PAHs, differences in CYP expression between tissues may explain differences in toxicity previously observed between B cells and thrombocytes.

Are floating algal mats a refuge from hypoxia for estuarine invertebrates?

Eutrophic aquatic habitats are characterized by the proliferation of vegetation leading to a large standing biomass that upon decomposition may create hypoxic (low-oxygen) conditions. This is indeed the case in nutrient impacted estuaries of Prince Edward Island, Canada, where macroalgae, from the genus Ulva, form submerged ephemeral mats. Hydrological forces and gases released from photosynthesis and decomposition lead to these mats occasionally floating to the water's surface, henceforth termed floating mats. Here, we explore the hypothesis that floating mats are refugia during periods of sustained hypoxia/anoxia and examine how the invertebrate community responds to it. Floating mats were not always present, so in the first year (2013) sampling was attempted monthly and limited to when both floating and submerged mats occurred. In the subsequent year sampling was weekly, but at only one estuary due to logistical constraints from increased sampling frequency, and was not limited to when both mat types occurred. Water temperature, salinity, and pH were monitored bi-weekly with dissolved oxygen concentration measured hourly. The floating and submerged assemblages shared many of the same taxa but were statistically distinct communities; submerged mats tended to have a greater proportion of benthic animals and floating mats had more mobile invertebrates and insects. In 2014, sampling happened to occur in the weeks before the onset of anoxia, during 113 consecutive hours of sustained anoxia, and for four weeks after normoxic conditions returned. The invertebrate community on floating mats appeared to be unaffected by anoxia, indicating that these mats may be refugia during times of oxygen stress. Conversely, there was a dramatic decrease in animal abundances that remained depressed on submerged mats for two weeks. Cluster analysis revealed that the submerged mat communities from before the onset of anoxia and four weeks after anoxia were highly similar to each other, indicating recovery. When mobile animals were considered alone, there was an exponential relationship between the percentage of animals on floating mats, relative to the total number on both mat types, and hypoxia. The occupation of floating mats by invertebrates at all times, and their dominance there during hypoxia/anoxia, provides support for the hypothesis that floating mats are refugia.

The Relationship between Organic Loading and Effects on Fish Reproduction for Pulp Mill Effluents across Canada.

This study builds upon the work of a multiagency consortium tasked with determining cost-effective solutions for the effects of pulp mill effluents on fish reproduction. A laboratory fathead minnow egg production test and chemical characterization tools were used to benchmark 81 effluents from 20 mills across Canada, representing the major pulping, bleaching, and effluent treatment technologies. For Kraft and mechanical pulp mills, effluents containing less than 20 mg/L BOD5 were found to have the greatest probability of having no effects. Organic loading, expressed as the total detected solvent-extractable components by gas chromatography/mass spectrometry (GC/MS), also correlated with decreased egg laying. Exceptions were found for specific Kraft, mechanical, and sulfite mills, suggesting yet unidentified causative agents are involved. Recycled fiber mill effluents, tested for the first time, were found to have little potential for reproductive effects despite large variations in BOD5 and GC/MS profiles. Effluent treatment systems across all production types were generally efficient, achieving a combined 82-98% BOD5 removal. Further reductions of final effluent organic loadings toward the target of less than 20 mg/L are recommended and can be realized through biotreatment optimization, the reduction of organic losses associated with production upsets and selecting best available technologies that reduce organic loadings to biotreatment.

Population impacts in white sucker (Catostomus commersonii) exposed to oil sands-derived contaminants in the Athabasca River.

Biological and chemical endpoints were measured in white sucker collected downstream of Athabasca oil sands developments (AB, Canada) and compared with those at Calling Lake (AB, Canada), a reference location upstream of the Athabasca oil sands deposit. Naphthenic acid concentrations were also measured at 14 sites in the Athabasca River watershed. Concentrations of naphthenic acids were elevated in tributaries adjacent to oil sands mining developments. Tributary naphthenic acid profiles were more similar to aged oil sands process water than samples from the Athabasca River, suggesting an influence of tailings in the tributaries. White sucker showed higher energy storage in the Athabasca River as indicated by significantly higher condition and liver size. White sucker were not investing that energy into reproductive effort as measured by gonad size and fecundity, which were significantly reduced relative to the reference location. White sucker showed increased exposure to polycyclic aromatic hydrocarbons as indicated by hepatic cytochrome P4501A (CYP1A) activity and fluorescent bile metabolites, as well as higher concentrations of naphthenic acids in bile. Cadmium, copper, nickel, and selenium were also elevated in white sucker liver tissue compared with the reference location. Based on the exposure profile and response pattern observed, effects on energy storage and utilization in white sucker from the Athabasca River most likely resulted from exposure to polycyclic aromatic hydrocarbons derived from petrogenic and pyrolytic sources. Environ Toxicol Chem 2017;36:2058-2067. © 2017 SETAC.

Monitoring stream sediment loads in response to agriculture in Prince Edward Island, Canada.

Increased agricultural land use leads to accelerated erosion and deposition of fine sediment in surface water. Monitoring of suspended sediment yields has proven challenging due to the spatial and temporal variability of sediment loading. Reliable sediment yield calculations depend on accurate monitoring of these highly episodic sediment loading events. This study aims to quantify precipitation-induced loading of suspended sediments on Prince Edward Island, Canada. Turbidity is considered to be a reasonably accurate proxy for suspended sediment data. In this study, turbidity was used to monitor suspended sediment concentration (SSC) and was measured for 2 years (December 2012-2014) in three subwatersheds with varying degrees of agricultural land use ranging from 10 to 69 %. Comparison of three turbidity meter calibration methods, two using suspended streambed sediment and one using automated sampling during rainfall events, revealed that the use of SSC samples constructed from streambed sediment was not an accurate replacement for water column sampling during rainfall events for calibration. Different particle size distributions in the three rivers produced significant impacts on the calibration methods demonstrating the need for river-specific calibration. Rainfall-induced sediment loading was significantly greater in the most agriculturally impacted site only when the load per rainfall event was corrected for runoff volume (total flow minus baseflow), flow increase intensity (the slope between the start of a runoff event and the peak of the hydrograph), and season. Monitoring turbidity, in combination with sediment modeling, may offer the best option for management purposes.

Reproductive strategies and seasonal changes in the somatic indices of seven small-bodied fishes in Atlantic Canada in relation to study design for environmental effects monitoring.

Small-bodied fishes are more commonly being used in environmental effects monitoring (EEM) studies. There is a lack of understanding of the biological characteristics of many small-bodied species, which hinders study designs for monitoring studies. For example, 72% of fish population surveys in Canada's EEM program for pulp and paper mills that used small-bodied fishes were conducted outside of the reproductive period of the species. This resulted in an inadequate assessment of the EEM program's primary effect endpoint (reproduction) for these studies. The present study examined seasonal changes in liver size, gonad size, and condition in seven freshwater and estuarine small-bodied fishes in Atlantic Canada. These data were used to examine differences in reproductive strategies and patterns of energy storage among species. Female gonadal recrudescence in all seven species began primarily in the 2-month period in the spring before spawning. Male gonadal development was concurrent with females in five species; however, gonadal recrudescence began in the fall in male three-spined stickleback (Gasterosteus aculeatus) and slimy sculpin (Cottus cognatus). The spawning period for each species was estimated from the decline in relative ovary size after its seasonal maximum value in spring. The duration of the spawning period reflected the reproductive strategy (single vs multiple spawning) of the species. Optimal sampling periods to assess reproductive impacts in each species were determined based on seasonal changes in ovary size and were identified to be during the prespawning period when gonads are developing and variability in relative gonad size is at a minimum.

Zinc and calcium modulate mitochondrial redox state and morphofunctional integrity.

Zinc and calcium have highly interwoven functions that are essential for cellular homeostasis. Here we first present a novel real-time flow cytometric technique to measure mitochondrial redox state and show it is modulated by zinc and calcium, individually and combined. We then assess the interactions of zinc and calcium on mitochondrial H2O2 production, membrane potential (ΔΨm), morphological status, oxidative phosphorylation (OXPHOS), complex I activity, and structural integrity. Whereas zinc at low doses and both cations at high doses individually and combined promoted H2O2 production, the two cations individually did not alter mitochondrial redox state. However, when combined at low and high doses the two cations synergistically suppressed and promoted, respectively, mitochondrial shift to a more oxidized state. Surprisingly, the antioxidants vitamin E and N-acetylcysteine showed pro-oxidant activity at low doses, whereas at high antioxidant doses NAC inhibited OXPHOS and dyscoupled mitochondria. Individually, zinc was more potent than calcium in inhibiting OXPHOS, whereas calcium more potently dissipated the ΔΨm and altered mitochondrial volume and ultrastructure. The two cations synergistically inhibited OXPHOS but antagonistically dissipated ΔΨm and altered mitochondrial volume and morphology. Overall, our study highlights the importance of zinc and calcium in mitochondrial redox regulation and functional integrity. Importantly, we uncovered previously unrecognized bidirectional interactions of zinc and calcium that reveal distinctive foci for modulating mitochondrial function in normal and disease states because they are potentially protective or damaging depending on conditions.

In vitro assessment of endocrine disrupting potential of naphthenic Acid fractions derived from oil sands-influenced water.

Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast androgen screen. The mixtures were characterized by high resolution mass spectrometry, (1)H nuclear magnetic resonance, and attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed. Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent antiandrogen or antiestrogens.

Sublethal effects of aged oil sands-affected water on white sucker (Catostomus commersonii).

To investigate impacts of proposed oil sands aquatic reclamation techniques on benthic fish, white sucker (Catostomus commersonii Lacépède, 1803) were stocked in 2 experimental ponds-Demonstration Pond, containing aged fine tailings capped with fresh water, consistent with proposed end-pit lake designs, and South Bison Pond, containing aged unextracted oil sands material-to examine the effects of unmodified hydrocarbons. White sucker were stocked from a nearby reservoir at both sites in May 2010 and sampled 4 mo later to measure indicators of energy storage and utilization. Comparisons were then made with the source population and 2 reference lakes in the region. After exposure to aged tailings, white sucker had smaller testes and ovaries and reduced growth compared with the source population. Fish introduced to aged unextracted oil sands material showed an increase in growth over the same period. Limited available energy, endocrine disruption, and chronic stress likely contributed to the effects observed, corresponding to elevated concentrations of naphthenic acids, aromatic compounds in bile, and increased CYP1A activity. Because of the chemical and biological complexity of these systems, direct cause-effect relationships could not be identified; however, effects were associated with naphthenic acids, polycyclic aromatic hydrocarbons, ammonia, and high pH. Impacts on growth have not been previously observed in pelagic fishes examined in these systems, and may be related to differences in sediment interaction.

The effects of benzo[a]pyrene on leucocyte distribution and antibody response in rainbow trout (Oncorhynchus mykiss).

Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds with immunotoxic and carcinogenic potential that may pose a threat to fish populations. This study aims to utilize a newly developed fish immunotoxicology model to determine the immune tissue/cell population level effects of PAHs on rainbow trout, using benzo[a]pyrene (BaP) as a representative immunotoxic PAH. Intraperitoneal injection of 25 or 100mg/kg BaP resulted in sustained exposure as indicated by biliary fluorescence at BaP wavelengths for up to 42 days. A new flow cytometry method for absolute counts of differential leucocyte distributions in spleen, blood, and head kidney was developed by combining absolute quantitative counts of total leukocytes in the tissue (3,3'-dihexyloxacarbocyanine iodide (DiOC6) dye) with relative differential counts using monoclonal antibodies for B cells, T cells, myeloid cells, and thrombocytes. Experiments indicated dose- and time-dependent decreases in the absolute number of B cells, myeloid cells, or T cells in blood, spleen, or head kidney after 7, 14 or 21 d of exposure. There was no change in the absolute numbers of erythrocytes or thrombocytes in any tissue. When rainbow trout were exposed to inactivated Aeromonas salmonicida after a 21 d exposure to 100mg/kg BaP, circulating antibody concentrations were decreased by 56%. It was concluded that BaP has a cell lineage-specific toxic effect on some immune cells of rainbow trout, and causes a decrease in circulating antibody levels.