93.Kennedy, CJ. (2021) P-glycoprotein induction and its energetic costs in rainbow trout (Oncorhynchus mykiss).Fish Physiol. Biochem. 47: 265-279 P-glycoprotein induction and its energetic costs in rainbow trout (Oncorhynchus mykiss)
P-glycoprotein; Induction; Energetic costs; Respiration; Oxygen consumption; Fish; Rainbow trout
Biological organisms are constantly challenged by xenobiotics and have evolved mechanisms to reduce, neutralize, or repair toxic outcomes. The various chemical defenses all utilize energy, but their specific costs and impacts on energy budgets are currently unknown. In this study, the energetic costs associated with the induction and substrate transport of the efflux transporter P-glycoprotein (P-gp [ABCB1, MDR1]) were examined in rainbow trout. An intraperitoneal injection of the P-gp inducer clotrimazole (0, 0.1, 1.0, and 10 mg/kg) increased P-gp activity (as measured by a competitive rhodamine 123 transport assay in hepatocytes) in a dose-dependent manner reaching a maximum induction of 2.8-fold. Maximum P-gp induction occurred at 50 h post-administration with the highest dose; significant induction of P-gp activity remained elevated over constitutive values until the last sampling time point (168 h). In vitro measurements of hepatocyte respiration indicated that basal P-gp activity transporting R123 as a substrate did not significantly increase respiration rates (range 18.0 to 23.2 ng O-2/min/10(6) cells); however, following the induction of P-gp by clotrimazole and exposure to the P-gp substrate R123, respiration rates increased significantly (3.52-fold) over baseline values. Using whole animal respirometry, it was shown that respiration rates in fish exposed to R123 only or induced with clotrimazole were not different from controls (range 1.2 to 2.1 mg O-2/kg/min); however, respiration rates were significantly increased in fish with induced P-gp levels and also exposed to R123. This work indicates that basal and induced levels of P-gp activity do not incur significant energetic costs to fish; however, upon induction of P-gp and concomitant substrate exposures, energetic costs can increase and could pose challenges to organisms facing limited energy resources. DOI PubMed
92. Lin, F; Baillon, L; Langlois, VS; Kennedy, CJ. (2021) Environmental modulators of diluted bitumen effects in juvenile pink salmon (Oncorhynchus gorbuscha).Mar. Environ. Res. 169 Environmental modulators of diluted bitumen effects in juvenile pink salmon (Oncorhynchus gorbuscha)
Diluted bitumen; Pink salmon; Salinity; Temperature; Toxicity
Recent and potential expansions in the transportation of diluted bitumen (dilbit) through marine terminals in coastal regions of British Columbia require the examination of potential risks to estuarine species such as Pacific salmon. The estuarine habitat of out-migrated pink salmon (Oncorhynchus gorbuscha) exhibits dynamic temperature and salinity regimes, possibly modifying dilbit exposure, bioavailability and/or its effects. To examine dilbit toxicity and its modification by environmental stressors, juvenile pinks were subchronically exposed for 3 months to the water-accommodated fraction (WAF) of Cold Lake Blend dilbit (winter) in seawater at three salinities (7, 14, and 28%o [temperature 12.5 degrees C]) and three temperatures (8.5, 12.5, and 16.5 degrees C [salinity of 28%o]). Temperature and salinity alone did not affect any measured endpoints in control fish. Dilbit exposure induced higher mortality at high (16.5 degrees C) and low temperatures (8.5 degrees C) as well as at higher salinity (28%o) in fish exposed to the highest dilution of WAF [total polycyclic aromatic compounds (TPAC) = 128.9 mu g/L]. A concentration-dependent reduction of growth was evident in fish exposed to the medium (TPAC = 97.3 mu g/L) and high dilution of WAF at higher temperatures (12.5 and 16.5 degrees C) and high salinity (28%o). At 28%o, swimming performance (Uburst) was decreased in fish exposed to the highest concentration of dilbit at all 3 temperatures. Gill Na+-K+-ATPase activity, white muscle lactate, glycogen, and triglyceride concentrations were altered by dilbit exposure and modified by temperature and salinity. In addition, gene expression associated with phase I biotransformation, energy metabolism, mitochondrial activity, and inflammation showed significant upregulation with exposure and temperature stress. Dilbit exposure at PAC concentrations in the ppb range, affected pink salmon at the molecular, biochemical, and whole organism level; effects that were exacerbated by environmental temperature and salinity. DOI PubMed
91. Love, RC; Osachoff, HL; Kennedy, CJ. (2021) Short communication: Tissue-specific transcript expression of P-glycoprotein isoforms abcb1a and abcb1b in rainbow trout (Oncorhynchus mykiss) following induction with clotrimazole.Comp. Biochem. Physiol. B-Biochem. Mol. Biol. 252 Short communication: Tissue-specific transcript expression of P-glycoprotein isoforms abcb1a and abcb1b in rainbow trout (Oncorhynchus mykiss) following induction with clotrimazole
P-glycoprotein; Rainbow trout; ABC transporter; Clotrimazole; Induction
P-glycoprotein (P-gp) plays a pivotal role in cellular defense, aimed at reducing xenobiotic accumulation. As a member of the ABC family of proteins, expression of this protein confers the multixenobiotic resistant (MXR) phenotype in aquatic organisms, including fish. To identify tissues protected by or contributing to the elimination of xenobiotics via P-gp, tissue-specific P-gp isoforms abcb1a and abcb1b transcript expression were measured in rainbow trout (Oncorhynchus mykiss). Tissues investigated included the proximal and distal intestines, liver, head kidney, gills, gonads, and 5 regions of the brain: olfactory lobe, cerebrum, optic lobe, cerebellum and medulla. Abcb1a transcript was more widely expressed across tissues and generally showed higher transcript expression than abcb1b. Deviation from this trend occurred in the gills, cerebrum and head kidney, where transcript levels were relatively equal between abcb1a and abcb1b. Intestinal tissues had greater abcb1a expression than abcblb (3 orders of magnitude). Abcb1b was absent from liver tissue indicating that abcb1a is relied upon for hepatic defense. This study suggests that abcb1b acts to protect sensitive organs from compounds in the systemic circulation (brain and gonad), whereas abcb1a acts primarily in an elimination role in organs such as liver and intestine. To determine if P-gp induction alters transcript responses, the antifungal mammalian Pregnane-X-Receptor (PXR) agonist clotrimazole (CTZ) was used. CTZ-treated rainbow trout showed significantly increased abcb1b transcript expression in the optic lobe and distal intestine, providing evidence that trout PXR exhibits a similar substrate base as mammalian PXR, albeit selectively in regions of the brain and intestine. DOI PubMed
90. Mill, K; Kennedy, CJ. (2021) Lethal and sublethal effects of the anti-sea lice formulation Salmosan (R) on the Pacific spot prawn (Pandalus platyceros).J. World Aquacult. Soc.Lethal and sublethal effects of the anti-sea lice formulation Salmosan (R) on the Pacific spot prawn (Pandalus platyceros)
aquaculture; azamethiphos; behavior; chemotherapeutant; molt; pesticide; sea lice; toxicity
The effects of the aquaculture chemotherapeutant Salmosan (R) (active ingredient [a.i.]: azamethiphos) were examined in Pacific spot prawns (Pandalus platyceros) at three temperatures (5, 11, and 17 degrees C). Post-molt prawns were more sensitive to Salmosan (R) than intermolt prawns; repeated (3x) 1-hr LC50 values for post-molt prawns ranged from 17 (9.3-31 95% confident intervals) to 40 (25-63) mu g/L a.i. while intermolt prawns survived 3 x 1-hr exposures up to 100 mu g/L a.i. Using LC50 values, Salmosan (R) was approximately 2.4 times more toxic at 17 versus 5 degrees C. Temperature significantly altered chemosensory and locomotory behaviors in intermolt prawns with the highest activity at the intermediate temperature. Significant decreases in antennule flicking (84 and 104% over controls) were seen at 17 degrees C after 3 x 1-hr pulse exposures to 50 and 100 mu g/L a.i., respectively. Temperature, but not Salmosan (R), affected molting success: at 17 degrees C significantly lower survival was seen during ecdysis (60% of those at 5 degrees C) and at 5 degrees C, molt time was longer (41 +/- 3 days) compared to 11 degrees C (34 +/- 4 days) or 17 degrees C (21 +/- 4 days). Life stage (molt status) and environmental parameters (temperature) alter the effects of Salmosan (R) to non-target spot prawns. DOI
89. Mill, K; Sahota, C; Hayek, K; Kennedy, CJ. (2021) Effects of sea louse chemotherapeutants on early life stages of the spot prawn (Pandalus platyceros).Aquac. Res.Effects of sea louse chemotherapeutants on early life stages of the spot prawn (Pandalus platyceros)
azamethiphos; chemotherapeutants; cypermethrin; deltamethrin; emamectin benzoate; hydrogen peroxide; spot prawn
The effects of five salmon aquaculture anti-sea louse chemotherapeutant active ingredients [azamethiphos (AZ), hydrogen peroxide (HP), emamectin benzoate (EB), cypermethrin (CP) and deltamethrin (DM)] were examined in five life stages (egg, Stage I, Stage III, Stage V and juvenile) of Pacific spot prawn (Pandalus platyceros) for two exposure durations (1-h and 3-h). Endpoints affected by exposures to chemotherapeutants included morbidity in all life stages, egg hatch success, hatchling fitness, latency to moult between successive stages and survival to the juvenile stage. Across life stages, 1-h and 3-h median lethal effect concentrations on morbidity ranged from 73 to 809 mg/L HP; 10-236 mu g/L AZ; 321 to >1200 mu g/L EB; 0.1 to >5 mu g/L CP; and 12 to >1000 ng/L DM. Across endpoints, DM was the most toxic, followed by CP, AZ and EB, followed by HP, which was generally observed to be the least toxic regardless of endpoint examined. The embryo and juvenile life stages were generally more resilient to effects than any larval life stage. Longer duration (3-h) exposures were generally more potent than shorter (1-h) exposures, with up to a threefold greater magnitude of effect when these exposure time frames were compared. Considering predicted environmental concentrations, the estimated greatest risk to ELS spot prawns was observed to be posed by DM, followed by CP, AZ and then EB, with a variable relative risk of HP across different endpoints/life stages. DOI
88. Sahota, C; Hayek, K; Surbey, B; Kennedy, CJ. (2021) Lethal and sublethal effects in Pink salmon (Oncorhynchus gorbuscha) following exposure to five aquaculture chemotherapeutants.EcotoxicologyLethal and sublethal effects in Pink salmon (Oncorhynchus gorbuscha) following exposure to five aquaculture chemotherapeutants
Chemotherapeutants; Hydrogen peroxide; Azamethiphos; Cypermethrin; Deltamethrin; Emamectin benzoate
Early life stages of Pink salmon (Oncorhynchus gorbuscha) are at risk of exposure to the active ingredients of chemotherapeutant formulations (hydrogen peroxide [HP], azamethiphos [AZ], emamectin benzoate [EB], cypermethrin [CP] and deltamethrin [DM]) used to control sea lice in salmon aquaculture. LC50 values (95% confidence intervals) for acute 48-h water exposures in order of least to most toxic to seawater-adapted pink salmon fry were: HP (227 [138-418] mg/L), EB (1090 [676-2006] mu g/L), AZ (80 [52-161] mu g/L), CP (5.1 [3.0-10.5] mu g/L), and DM (980 [640-1800] ng/L), and in subchronic 10-d lethality sediment exposure tests: EB (2065 [1384-3720] mu g/kg), CP (97 [58-190] mu g/kg), and DM (1035 [640-2000] ng/kg). Alterations in behaviour varied between chemicals; no chemical attracted pink salmon fry; fish avoided HP to a limited extent at 50 mg/L), as well as EB (300 mu g/L), and AZ (50 mu g/L). Significant concentration-dependent decreases in olfactory responsiveness to food extract were seen following AZ, CP and DM exposures that occurred at lower concentrations with longer exposure periods (10 mu g/L, 0.5 mu g/L and 100 ng/L thresholds at 7 d). Following 10-d sediment exposures, olfaction was only affected by CP exposure at 50 mu g/kg. Significant decreases in swimming performance (Ucrit) occured for HP, AZ, CP and DM at concentrations as low as 100 mg/L, 10 mu g/L, 2 mu g/L and 200 ng/L, respectively. This study provides comprehensive data on the lethal and sublethal effects of aquaculture chemotherapeutant exposure in early life stage pink salmon. DOI PubMed
87. Strachan, F; Kennedy, CJ. (2021) The environmental fate and effects of anti-sea lice chemotherapeutants used in salmon aquaculture.Aquaculture 544 The environmental fate and effects of anti-sea lice chemotherapeutants used in salmon aquaculture
Chemotherapeutants; Pesticides; Sea lice; Fate; Toxicity; Salmon
The active ingredients from 5 chemotherapeutant formulations (Slice (R) [active ingredient [AI] emamectin benzoate), Salmosan (R) (AI azimethiphos), Alphamax (R) (AI deltamethrin), Excis (R) (AI cypermethrin), and Interox (R) Paramove 50 (AI hydrogen peroxide) that have been used or continue to be used to treat sea lice in-festations in salmon aquaculture were examined to generate data on their environmental partitioning and persistence in water-sediment microcosms, as well as their acute and subchronic toxicity to representative classes of marine organisms. Emamectin benzoate, cypermethrin and deltamethrin partitioned mainly to the sediment phase; azimethiphos and hydrogen peroxide remained mainly in the water phase. The persistence of chemicals in water was: CP > DM > AZ > HP (half-lives: 19.8, 17.9, 12.7 d, and 8.9 h, respectively). In sediments, the following trend in calculated half-lives was observed: CP > EB > DM (half-lives: 557, 230 and 45 d, respectively). Toxicity test results with a wide variety of marine organisms (macroalgae, echinoderms, bivalves, crustaceans and fish) showed no susceptibility trend for any species, or inherent toxicity trend for any chemical, although DM tended to be the most toxic and HP the least toxic to the majority of species. This information is useful for identifying risks; specifically, toxicological parameters calculated for several of the non-target marine organisms examined, indicate that recommended treatment concentrations could result in non-target organism toxicity following release in the immediate vicinity of aquaculture sites before significant dilution. This study provides valuable data on the environmental fate and associated risks of chemotherapeutant use to non-target marine organisms whose habitat coincides with salmon aquaculture sites. DOI
86. Alderman, SL; Dilkumar, CM; Avey, SR; Farrell, AP; Kennedy, CJ; Gillis, TE. (2020) Effects of diluted bitumen exposure and recovery on the seawater acclimation response of Atlantic salmon smolts.Aquat. Toxicol. 221 Effects of diluted bitumen exposure and recovery on the seawater acclimation response of Atlantic salmon smolts
Polycyclic aromatic hydrocarbons; PAH; Crude oil; Dilbit; Aquatic contamination; Gill; Kidney; Euryhaline fish
Petrogenic chemicals are common and widespread contaminants in the aquatic environment. In Canada, increased extraction of bitumen from the oil sands and transport of the major crude oil export product, diluted bitumen (dilbit), amplifies the risk of a spill and contamination of Canadian waterways. Fish exposed to sublethal concentrations of crude oil can experience a variety of adverse physiological effects including osmoregulatory dysfunction. As regulation of water and ion balance is crucial during the seawater transition of anadromous fish, the hypothesis that dilbit impairs seawater acclimation in Atlantic salmon smolts (a fish at risk of exposure in Canada) was tested. Smolts were exposed for 24 d to the water-soluble fraction of dilbit in freshwater, and then transferred directly to seawater or allowed a 1 wk depuration period in uncontaminated freshwater prior to seawater transfer. The seawater acclimation response was quantified at 1 and 7 d post-transfer using established hematological, tissue, and molecular endpoints including gill Na+/K+-ATPase gene expression (nka). All smolts, irrespective of dilbit exposure, increased serum Na+ concentrations and osmolality within 1 d of seawater transfer. The recovery of these parameters to freshwater values by 7 d post-transfer was likely driven by the increased expression and activity of Na+/K+-ATPase in the gill. Histopathological changes in the gill were not observed; however, CYP1A-like immunoreactivity was detected in the pillar cells of gill lamellae of fish exposed to 67.9 mu g/L PAC. Concentration-specific changes in kidney expression of a transmembrane water channel, aquaporin 3, occurred during seawater acclimation, but were resolved with 1 wk of depuration and were not associated with histopathological changes. In conclusion, apart from a robust CYP response in the gill, dilbit exposure did not greatly impact common measures of seawater acclimation, suggesting that significant osmoregulatory dysfunction is unlikely to occur if Atlantic salmon smolts are exposed sub-chronically to dilbit. DOI PubMed
85. Avey, SR; Kennedy, CJ; Farrell, AP; Gillis, TE; Alderman, SL. (2020) Effects of diluted bitumen exposure on Atlantic salmon smolts: Molecular and metabolic responses in relation to swimming performance.Aquat. Toxicol. 221 Effects of diluted bitumen exposure on Atlantic salmon smolts: Molecular and metabolic responses in relation to swimming performance
Crude oil; PAH; Fish; Heart; Muscle; Aerobic metabolism
Canada's oil sands industry continues to expand and the volume of diluted bitumen (dilbit) transported across North America is increasing, adding to spill risk and environmental contamination. Dilbit exposure is known to cause adverse effects in fish, but linking molecular and cellular changes with ecologically-relevant individual performance metrics is needed to better understand the potential consequences of a dilbit spill into the aquatic environment. Therefore, this study examined the effects of dilbit exposure on subcellular responses in cardiac and skeletal muscle in relation to swimming performance in a migratory fish species at risk of exposure, Atlantic salmon. Smolts were exposed subchronically to environmentally relevant concentrations of the water-soluble fraction of dilbit (WSFd) for 24 d, and then a subset of exposed fish underwent a depuration period of 7 or 14 d, for a total of 3 experimental time points. At each time point, repeat swimming performance was assessed using sequential critical swimming speed tests (U-crit) separated by a 24 h rest period, and then several tissues were collected to determine biotransformation enzyme activation, energetic responses, and gene expression changes. U-crit was unaffected in fish exposed to 67.9 mu g/L total initial polycyclic aromatic compounds (PAC), but fish showed a decreased reliance on lipid metabolism for adenosine triphosphate (ATP) in the heart that was maintained through 7 d depuration. In contrast, U-crit increased in fish exposed to 9.65 mu g/L PAC, corresponding to an increased reliance on anaerobic metabolic pathways in cardiac and red skeletal muscle, with partial recovery after 7 d depuration. As expected, at both concentrations WSFd hepatic cyp 1A-mediated biotransformation reactions increased, as measured by EROD activity, which remained elevated for 7 d but not after 14 d depuration. Transcript abundance of cyp1a was also increased in muscle tissue and recovered by 14 d depuration. The expression of other stress-related genes increased in white muscle of dilbit-exposed fish, but were largely unchanged in cardiac and red muscle. The transcriptional profile of cardiac tissue was compared to that of sockeye salmon similarly exposed to WSFd in a previous experiment, and is provided in supplemental text. Combined, these results demonstrate that dilbit exposure alters gene expression and enzyme activities related to xenobiotic exposure, cellular stress, and muscle energetics in juvenile Atlantic salmon without impairing swimming performance, and that most of these changes are recoverable within 14 d depuration. DOI PubMed
84. Lin, F; Osachoff, HL; Kennedy, CJ. (2020) Physiological disturbances in juvenile sockeye salmon (Oncorhynchus nerka) exposed to the water-soluble fraction of diluted bitumen.Aquat. Toxicol. 220 Physiological disturbances in juvenile sockeye salmon (Oncorhynchus nerka) exposed to the water-soluble fraction of diluted bitumen
Oil sands; Diluted bitumen; Crude oil; Toxicity; Fish; Sockeye salmon; Stress; Osmoregulation; Immune system; Physiology
Current and proposed transcontinental pipelines for the transport of diluted bitumen (dilbit) from the Canadian oil sands traverse the coastal watersheds of British Columbia, habitat essential to Pacific salmonids. To determine the potential risks posed to these keystone species, juvenile sockeye (Oncorhynchus nerka; 1+ parr) were acutely (24-96 h) or subchronically (21-42 d) exposed to 4 concentrations of the water-soluble fraction (WSF) of unweathered Cold Lake Blend dilbit (initial total PAC concentrations: 0, 13.7, 34.7 and 124.5 mu g/L) in a flow-through system. Dilbit effects on iono-osmoregulation, the physiological stress response, and the immune system were assessed by both biochemical and functional assays. Hydrocarbon bioavailability was evidenced by a significant induction of liver ethoxyresorufin-O-deethylase (EROD) activity in exposed fish. Acute and subchronic exposure significantly reduced gill Na+-K+-ATPase activity and resulted in lower plasma osmolality, Cl-, and Na+ concentrations. Acute exposure to dilbit resulted in a classic physiological stress response, however at 21 d of exposure, plasma cortisol remained elevated while other measured parameters had returned to baseline values. A compromised immune system was demonstrated by a 29.5 % higher mortality in fish challenged with Vibrio (Listonella) anguillarum following dilbit exposure compared to unexposed controls. Exposure of juvenile salmonids to the WSF of dilbit (at TPAC concentrations at the ppb level) resulted in sublethal effects that included a classic physiological stress response, and alterations in iono-osmoregulatory homeostasis and immunological performance. DOI PubMed
83. Marlatt, VL; Leung, TYG; Calbick, S; Metcalfe, C; Kennedy, C. (2019) Sub-lethal effects of a neonicotinoid, clothianidin, on wild early life stage sockeye salmon (Oncorhynchus nerka).Aquat. Toxicol. 217 Sub-lethal effects of a neonicotinoid, clothianidin, on wild early life stage sockeye salmon (Oncorhynchus nerka)
Growth; Development; Estradiol; Testosterone; Gene expression; Glucocorticoid receptor
One of the categories of environmental contaminants possibly contributing to declining sockeye salmon (Oncorhynchus nerka) in the Fraser River, British Columbia, Canada is pesticides. In this 4-month study, the effects of environmentally relevant concentrations of a waterborne neonicotinoid, clothianidin (0.15, 1.5, 15 and 150 mu g/L), on embryonic, alevin and early swim-up fry sockeye salmon derived from four unique genetic crosses of the Pitt River, BC stock were investigated. There were no significant effects of clothianidin on survival, hatching, growth or deformities, although genetic variation significantly affected these endpoints. Clothianidin caused a significant 4.7-fold increase in whole body 17 beta-estradiol levels in swim-up fry after exposure to 0.15 mu g/L, but no effects were observed on testosterone levels. In addition, hepatic expression of the gene encoding glucocorticoid receptor 2 was also impacted at the highest concentration of clothianidin tested, and was found to be similar to 4-fold lower compared to the sockeye reared in control water. These results indicate additional examination of clothianidin and its effects on salmonid gonad development and the reproductive and stress endocrine axes in general, is warranted. DOI PubMed
82. Alderman, SL; Lin, F; Gillis, TE; Farrell, AP; Kennedy, CJ. (2018) Developmental and latent effects of diluted bitumen exposure on early life stages of sockeye salmon (Oncorhynchus nerka).Aquat. Toxicol. 202 Developmental and latent effects of diluted bitumen exposure on early life stages of sockeye salmon (Oncorhynchus nerka)
Bitumen; Crude oil; Fish; Development; Toxicity; Morphogenesis
The early life stages of Pacific salmon are at risk of environmental exposure to diluted bitumen (dilbit) as Canada's oil sands industry continues to expand. The toxicity and latent effects of dilbit exposure were assessed in sockeye salmon (Oncorhynchus nerka) exposed to water-soluble fractions of dilbit (WSFd) from fertilization to the swim-up stage, and then reared in clean water for 8 months. Mortality was significantly higher in WSFd-exposed embryos, with cumulative mortality up to 4.6-fold higher in exposed relative to unexposed embryos. The sublethal effects of WSFd exposure included transcriptional up-regulation of cypla, a concentration-dependent delay in the onset and progression of hatching, as well as increased prevalence of developmental deformities at total polycyclic aromatic hydrocarbon (TPAH) concentrations >= 35 mu g L-1. Growth and body composition were negatively affected by WSFd exposure, including a concentration-specific decrease in soluble protein concentration and increases in total body lipid and triglyceride concentrations. Mortality continued during the first 2 months after transferring fish to clean water, reaching 53% in fish exposed to 100 mu g L-1 TPAH; but there was no latent impact on swimming performance, heart mass, or heart morphology in surviving fish after 8 months. A latent effect of WSFd exposure on brain morphology was observed, with fish exposed to 4 mu g L-1 TPAH having significantly larger brains compared to other treatment groups after 8 months in clean water. This study provides comprehensive data on the acute, sub-chronic, and latent impacts of dilbit exposure in early life stage sockeye, information that is critical for a proper risk analysis of the impact of a dilbit spill on this socioeconomically important fish species. DOI PubMed
81. Trowell, JJ; Gobas, FAPC; Moore, MM; Kennedy, CJ. (2018) Estimating the Bioconcentration Factors of Hydrophobic Organic Compounds from Biotransformation Rates Using Rainbow Trout Hepatocytes.Arch. Environ. Contam. Toxicol. 75 Estimating the Bioconcentration Factors of Hydrophobic Organic Compounds from Biotransformation Rates Using Rainbow Trout Hepatocytes
Determining the biotransformation potential of commercial chemicals is critical for estimating their persistence in the aquatic environment. In vitro systems are becoming increasingly important as screening methods for assessing the potential for chemical metabolism. Depletion rate constants (k (d)) for several organic chemicals with high octanol-water partition coefficient (K (ow)) values (9-methylanthracene, benzo(a)pyrene, chrysene, and PCB-153) in rainbow trout hepatocytes were determined to estimate biotransformation rate constants (k (MET)) that were used in fish bioconcentration factor (BCF) models. Benzo[a]pyrene was rapidly biotransformed when incubated singly; however, its depletion rate constant (k (d)) declined 79% in a mixture of all four chemicals. Chrysene also exhibited significant biotransformation and its depletion rate constant declined by 50% in the mixture incubation. These data indicate that biotransformation rates determined using single chemicals may overestimate metabolism in environments containing chemical mixtures. Incubations with varying cell concentrations were used to determine whether cell concentration affected k (d) estimates. No statistically significant change in depletion rate constants were seen, possibly due to an increase in nonspecific binding of hydrophobic chemicals as cell density increased, decreasing overall biotransformation. A new model was used to estimate BCFs from k (MET) values calculated from empirically derived k (d) values. The inclusion of k (MET) in models resulted in significantly lower BCF values (compared k (MET) = 0). Modelled BCF values were consistent with empirically derived BCF values from the literature. DOI PubMed
80. Alderman, SL; Dindia, LA; Kennedy, CJ; Farrell, AP; Gillis, TE. (2017) Proteomic analysis of sockeye salmon serum as a tool for biomarker discovery and new insight into the sublethal toxicity of diluted bitumen.Comp. Biochem. Physiol. D-Genomics Proteomics 22: 157-166 Proteomic analysis of sockeye salmon serum as a tool for biomarker discovery and new insight into the sublethal toxicity of diluted bitumen
Crude oil; Bitumen; Proteomics; Biomarker; iTRAQ; Blood; Creatine kinase; Oil sands; Fish; Exercise
Pipelines carrying diluted bitumen (dilbit) from Canada's oil sands traverse North America, including the freshwater habitat of Pacific salmon, posing a risk of environmental release and aquatic exposure. Swimming performance is impacted in juvenile sockeye (Oncorhynchus nerka) exposed to dilbit; therefore biomarkers of dilbit exposure will be valuable for monitoring at-risk salmon stocks. This study characterized changes in the serum proteome of sockeye exposed to a sub-lethal and environmentally relevant concentration of dilbit using isobaric tags for relative and absolute quantitation (iTRAQ), and included a range of experimental conditions to permit identification of biomarkers that are robust across time (1 and 4 wk) and exercise level (at rest and following a swim test). Over 500 proteins were identified and quantified in sockeye serum, with dilbit exposure significantly altering the abundance of 24 proteins irrespective of time and exercise, including proteins associated with immune and inflammatory responses, coagulation, and iron homeostasis. An increase in creatine kinase (CK) activity in serum of dilbit-exposed salmon confirmed the higher CK protein abundance measured using iTRAQ. The combination of 4 wk dilbit exposure and a swim test had a greater effect on the serum proteome than either treatment alone, including a marked increase in tissue leakage proteins, suggesting that aerobic exercise exacerbates the serum proteome response to dilbit, and the increased cellular damage could impede exercise recovery. This study provides a foundation for the development of bio-monitoring tools for salmon stock assessments, and offers new insights into the sub-lethal toxicity of crude oil exposure in fish. DOI
79. Alderman, SL; Lin, F; Farrell, AP; Kennedy, CJ; Gillis, TE. (2017) Effects of diluted bitumen exposure on juvenile sockeye salmon: From cells to performance.Environmental Toxicology and Chemistry 36: 354-360 Effects of diluted bitumen exposure on juvenile sockeye salmon: From cells to performance
Bitumen; Polycyclic aromatic hydrocarbon; Heart; Fish; Crude oil
Diluted bitumen (dilbit; the product of oil sands extraction) is transported through freshwater ecosystems critical to Pacific salmon. This is concerning, because crude oil disrupts cardiac development, morphology, and function in embryonic fish, and cardiac impairment in salmon can have major consequences on migratory success and fitness. The sensitivity of early life-stage salmon to dilbit and its specific cardiotoxic effects are unknown. Sockeye salmon parr were exposed to environmentally relevant concentrations of the water-soluble fraction (WSF) of dilbit for 1wk and 4wk, followed by an examination of molecular, morphological, and organismal endpoints related to cardiotoxicity. We show that parr are sensitive to WSF of dilbit, with total polycyclic aromatic hydrocarbon (PAH) concentrations of 3.5 mu g/L sufficient to induce a liver biomarker of PAH exposure, and total PAH of 16.4 mu g/L and 66.7 mu g/L inducing PAH biomarkers in the heart. Furthermore, WSF of dilbit induces concentration-dependent cardiac remodeling coincident with performance effects: fish exposed to 66.7 mu g/L total PAH have relatively fewer myocytes and more collagen in the compact myocardium and impaired swimming performance at 4wk, whereas the opposite changes occur in fish exposed to 3.5 mu g/L total PAH. The results demonstrate cardiac sensitivity to dilbit exposure that could directly impact sockeye migratory success. Environ Toxicol Chem 2017;36:354-360. (c) 2016 SETAC DOI
78. Du Gas, LM; Ross, PS; Walker, J; Marlatt, VL; Kennedy, CJ. (2017) Effects of atrazine and chlorothalonil on the reproductive success, development, and growth of early life stage Sockeye Salmon (Oncorhynchus nerka).Environmental Toxicology and Chemistry 36: 1354-1364 Effects of atrazine and chlorothalonil on the reproductive success, development, and growth of early life stage Sockeye Salmon (Oncorhynchus nerka)
Salmon; Pesticides; Commercial formulations; Emergence; Hatch
The effects of 2 currently used commercial pesticide formulations on Pacific sockeye salmon (Oncorhynchus nerka), from fertilization to emergence, were evaluated in a gravel-bed flume incubator that simulated a natural streambed. Embryos were exposed to atrazine at 25 mg/L (low atrazine) or atrazine at 250 mg/L (high atrazine) active ingredient (a.i.), and chlorothalonil at 0.5 mu g/L (low chlorothalonil) or chlorothalonil at 5 mg/L a. i. (high chlorothalonil) and examined for effects on developmental success and timing, as well as physical and biochemical growth parameters. Survival to hatch was reduced in the high chlorothalonil group (55% compared with 83% in controls), accompanied by a 24% increase in finfold deformity incidence. Reduced alevin condition factor (2.9-5.4%) at emergence and elevated triglyceride levels were seen in chlorothalonil-exposed fish. Atrazine exposure caused premature hatch (average high atrazine time to 50% hatch [H50] = 100 d postfertilization [dpf]), and chlorothalonil exposure caused delayed hatch (high chlorothalonil H50 = 108 dpf; controls H50 = 102 dpf). All treatments caused premature emergence (average time to 50% emergence [E50]: control E50 = 181 dpf, low chlorothalonil E50 = 175 dpf, high chlorothalonil E50 = 174 dpf, high atrazine E50 = 175 dpf, low atrazine E50 = 174 dpf), highlighting the importance of using a gravel-bed incubator to examine this subtle, but critical endpoint. These alterations indicate that atrazine and chlorothalonil could affect survival of early life stages of sockeye salmon in the wild. (C) 2017 SETAC DOI
77. Jackson, JS; Kennedy, CJ. (2017) Regulation of hepatic abcb4 and cyp3a65 gene expression and multidrug/multixenobiotic resistance (MDR/MXR) functional activity in the model teleost, Danio rerio (zebrafish).Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 200: 34-41 Regulation of hepatic abcb4 and cyp3a65 gene expression and multidrug/multixenobiotic resistance (MDR/MXR) functional activity in the model teleost, Danio rerio (zebrafish)
Fish; Multi-xenobiotic resistance; P-glycoprotein; Efflux; Biotransformation; CYP450; Pxr; Ketoconazole; PCN
Multidrug/multixenobiotic resistance (MDR/MXR) confers resistance to a diverse range of potentially toxic pharmaceuticals and environmental contaminants through a cellular response that involves the coordinated induction and activity of the ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) and the Phase I metabolizing enzyme cytochrome P450 3A (CYP3A). In mammals, ligand-mediated pregnane X receptor (PXR) transcriptional activity regulates the induction of P-gp and CYP3A; however, this mechanism has not been well characterized in piscine species. Zebrafish (Danio rerio) treated with the Pxr agonist pregnenolone 16 alpha-carbonitrile (PCN) showed decreased P-gp (zebrafish Abcb4) and CYP3A (zebrafish Cyp3a65) mRNA levels after 48 h expoure; however, treatment with PCN also resulted in increased hepatic MDR/MXR functional activity (i.e. increased Rhodamine 123 efflux) in vivo. Consistent with mammalian-like MDR/MXR regulated by PXR, the PCN-mediated modulation of hepatic Abcb4 and Cyp3a65 mRNA levels and MDR/MXR functional activity was attenuated by co-treatment with PCN and the mammalian PXR antagonist, ketoconazole (KTC). These results provide evidence that zebrafish Pxr may play a role in MDR/MXR through transcriptional regulation of abcb4 and cyp3a65 gene expression. DOI
76. Lee, YS; Lo, JC; Otton, SV; Moore, MM; Kennedy, CJ; Gobas, FAPC. (2017) IN VITRO TO IN VIVO EXTRAPOLATION OF BIOTRANSFORMATION RATES FOR ASSESSING BIOACCUMULATION OF HYDROPHOBIC ORGANIC CHEMICALS IN MAMMALS.Environmental Toxicology and Chemistry 36: 1934-1946 IN VITRO TO IN VIVO EXTRAPOLATION OF BIOTRANSFORMATION RATES FOR ASSESSING BIOACCUMULATION OF HYDROPHOBIC ORGANIC CHEMICALS IN MAMMALS
Biotransformation; Bioaccumulation; In vitro to in vivo extrapolation; Bioaccumulation modeling
Incorporating biotransformation in bioaccumulation assessments of hydrophobic chemicals in both aquatic and terrestrial organisms in a simple, rapid, and cost-effective manner is urgently needed to improve bioaccumulation assessments of potentially bioaccumulative substances. One approach to estimate whole-animal biotransformation rate constants is to combine in vitro measurements of hepatic biotransformation kinetics with in vitro to in vivo extrapolation (IVIVE) and bioaccumulation modeling. An established IVIVE modeling approach exists for pharmaceuticals (referred to in the present study as IVIVE-Ph) and has recently been adapted for chemical bioaccumulation assessments in fish. The present study proposes and tests an alternative IVIVE-B technique to support bioaccumulation assessment of hydrophobic chemicals with a log octanol-water partition coefficient (K-OW) >= 4 in mammals. The IVIVE-B approach requires fewer physiological and physiochemical parameters than the IVIVE-Ph approach and does not involve interconversions between clearance and rate constants in the extrapolation. Using in vitro depletion rates, the results show that the IVIVE-B and IVIVE-Ph models yield similar estimates of rat whole-organism biotransformation rate constants for hypothetical chemicals with log K-OW >= 4. The IVIVE-B approach generated in vivo biotransformation rate constants and biomagnification factors (BMFs) for benzo[a] pyrene that are within the range of empirical observations. The proposed IVIVE-B technique may be a useful tool for assessing BMFs of hydrophobic organic chemicals in mammals. (C) 2016 SETAC DOI
75. Marlatt, VL; Sherrard, R; Kennedy, CJ; Elphick, JR; Martyniuk, CJ. (2016) Application of molecular endpoints in early life stage salmonid environmental biomonitoring.Aquatic Toxicology 173: 178-191 Application of molecular endpoints in early life stage salmonid environmental biomonitoring
Environmental monitoring; Gene expression; Metals; PAH; Salmonid; Development
Molecular endpoints can enhance existing whole animal bioassays by more fully characterizing the biological impacts of aquatic pollutants. Laboratory and field studies were used to examine the utility of adopting molecular endpoints for a well-developed in situ early life stage (eyed embryo to onset of swim-up fry) salmonid bioassay to improve diagnostic assessments of water quality in the field. Coastal cutthroat trout (Oncorhynchus clarki clarki) were exposed in the laboratory to the model metal (zinc, 40 mu g/L) and the polycyclic aromatic hydrocarbon (pyrene, 100 mu g/L) in water to examine the resulting early life stage salmonid responses. In situ field exposures and bioassays were conducted in parallel to evaluate the water quality of three urban streams in British Columbia (two sites with anthropogenic inputs and one reference site). The endpoints measured in swim-up fry included survival, deformities, growth (weight and length), vitellogenin (vtg) and metallothionein (Mt) protein levels, and hepatic gene expression (e.g., metallothioneins [mta and mtb], endocrine biomarkers [vtg and estrogen receptors, esr] and xenobiotic-metabolizing enzymes [cytochrome P4501A3, cyp1a3 and glutathione transferases, gstk]). No effects were observed in the zinc treatment, however exposure of swim-up fry to pyrene resulted in decreased survival, deformities and increased estrogen receptor alpha (er1) mRNA levels. In the field exposures, xenobiotic-metabolizing enzymes (cyp1a3, gstk) and zinc transporter (zntBigM103) mRNA were significantly increased in swim-up fry deployed at the sites with more anthropogenic inputs compared to the reference site. Cluster analysis revealed that gene expression profiles in individuals from the streams receiving anthropogenic inputs were more similar to each other than to the reference site. Collectively, the results obtained in this study suggest that molecular endpoints may be useful, and potentially more sensitive, indicators of site-specific contamination in real-world, complex exposure scenarios in addition to whole body morphometric and physiological measures. (C) 2016 Elsevier B.V. All rights reserved. DOI
74. Osachoff, HL; Brown, LLY; Tirrul, L; van Aggelen, GC; Brinkman, FSL; Kennedy, CJ. (2016) Time course of hepatic gene expression and plasma vitellogenin protein concentrations in estrone-exposed juvenile rainbow trout (Oncorhynchus mykiss).Comparative Biochemistry and Physiology D-Genomics & Proteomics 19: 112-119 Time course of hepatic gene expression and plasma vitellogenin protein concentrations in estrone-exposed juvenile rainbow trout (Oncorhynchus mykiss)
Estrone; QPCR; RNA-Seq; Rainbow trout; Vitellogenin; Vitelline envelope proteins; Estrogen receptor; Transcriptome
Estrone (E1), a natural estrogen hormone found in sewage effluents and surface waters, has known endocrine disrupting effects in fish, thus, it is a contaminant of emerging concern. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to an environmentally-relevant concentration of E1 (24 ng/L E1 [0.1 nM]) for 7 d and then placed in clean water for a 9 d recovery period. RNA sequencing showed transcripts from numerous affected biological processes (e.g. immune, metabolic, apoptosis, clotting, and endocrine) were altered by E1 after 4 d of treatment. The time course of E1-inducible responses relating to vitellogenesis was examined daily during the two phases of exposure. Hepatic gene expression alterations evaluated by quantitative polymerase chain reaction (QPCR) were found during the treatment period for vitellogenin (VTG), vitelline envelope proteins (VEPs) alpha, beta and gamma, and estrogen receptor alpha 1 (ER alpha 1) transcripts. ER alpha 1 was the only transcript induced each day during the treatment phase, thus it was a good indicator of E1 exposure. Gradual increases occurred in VEP beta and VEP gamma transcripts, peaking at d7. VTG transcript was only elevated at d4, making it less sensitive than VEPs to this low-level E1 treatment. Inductions of ER alpha 1, VEP alpha, VEP beta and VEP gamma transcripts ceased 1 d into the recovery phase. Plasma VTG protein concentrations were not immediately elevated but peaked 7 d into the recovery phase. Thus, elevated vitellogenesis-related gene expression and protein production occurred slowly but steadily at this concentration of E1, confirming the sequence of events for transcripts and VTG protein responses to xenoestrogen exposure. (C) 2016 Published by Elsevier Inc. DOI
73.Kennedy, CJ; Smyth, KR. (2015) Disruption of the rainbow trout reproductive endocrine axis by the polycyclic aromatic hydrocarbon benzo[a]pyrene.General and Comparative Endocrinology 219: 102-111 Disruption of the rainbow trout reproductive endocrine axis by the polycyclic aromatic hydrocarbon benzo[a]pyrene
Reproduction; Endocrine disruption; Steroids; Benzo[a]pyrene; Fish
Successful reproduction in salmonids depends on a complex and highly regulated interplay between the pharmacokinetics and pharmacodynamics of naturally circulating sex steroids. The effects of a single intraperitoneal administration of the model PAH benzo[a]pyrene (B[a]P) on the kinetics of circulating levels of estradiol and testosterone through 7 d post-injection in mature male and female rainbow trout (Oncorhynchus mykiss) in pre-spawning and spawning condition were investigated. Detailed measurements of the time course of injected E2 and excretion into the bile followed by pharmacokinetic modeling techniques were used to aid in identifying the potential mechanism of ED caused by B[a]P exposure. Plasma E2 and T concentrations were reduced significantly in both male and female trout. Administration of the GnRH analogue des-Gly(10)[D-Ala(6)]LH-RH-ethylamide, to induce spawning steroid profiles increased plasma E2 concentrations in control females, but not in B[a]P-treated fish. The mechanism underlying reductions in sex steroids in pre-spawning and spawning salmonids appears to be unrelated to the induction of P450 and related biotransformation enzymes by B[a]P. Induced biotransformation enzyme activities did not result in altered [H-3]estradiol pharmacokinetics (e.g. terminal half-life) or elimination of steroid in bile, suggesting that B[a]P alters plasma E2 and T concentrations by other ED mechanisms in an anti-estrogenic manner. (C) 2015 Elsevier Inc. All rights reserved. DOI
72. Lo, BP; Elphick, JR; Bailey, HC; Baker, JA; Kennedy, CJ. (2015) The effect of sulfate on selenate bioaccumulation in two freshwater primary producers: A duckweed (Lemna minor) and a green alga (Pseudokirchneriella subcapitata).Environmental Toxicology and Chemistry 34: 2841-2845 The effect of sulfate on selenate bioaccumulation in two freshwater primary producers: A duckweed (Lemna minor) and a green alga (Pseudokirchneriella subcapitata)
Selenium; Sulfate; Bioaccumulation; Duckweed; Algae
Predicting selenium bioaccumulation is complicated because site-specific conditions, including the ionic composition of water, affect the bioconcentration of inorganic selenium into the food web. Selenium tissue concentrations were measured in Lemna minor and Pseudokirchneriella subcapitata following exposure to selenate and sulfate. Selenium accumulation differed between species, and sulfate reduced selenium uptake in both species, indicating that ionic constituents, in particular sulfate, are important in modifying selenium uptake by primary producers. Environ Toxicol Chem 2015;34:2841-2845. (c) 2015 SETAC DOI
71. Lo, JC; Campbell, DA; Kennedy, CJ; Gobas, FAPC. (2015) Somatic and gastrointestinal in vivo biotransformation rates of hydrophobic chemicals in fish.Environmental Toxicology and Chemistry 34: 2282-2294 Somatic and gastrointestinal in vivo biotransformation rates of hydrophobic chemicals in fish
Biotransformation; Bioaccumulation; In vivo; Hydrophobic chemicals
To improve current bioaccumulation assessment methods, a methodology is developed, applied, and investigated for measuring in vivo biotransformation rates of hydrophobic organic substances in the body (soma) and gastrointestinal tract of the fish. The method resembles the Organisation for Economic Co-operation and Development (OECD) 305 dietary bioaccumulation test but includes reference chemicals to determine both somatic and gastrointestinal biotransformation rates of test chemicals. Somatic biotransformation rate constants for the test chemicals ranged between 0 d(-1) and 0.38 (standard error [SE] 0.03)/d(-1). Gastrointestinal biotransformation rate constants varied from 0 d(-1) to 46 (SE 7) d(-1). Gastrointestinal biotransformation contributed more to the overall biotransformation in fish than somatic biotransformation for all test substances but 1. Results suggest that biomagnification tests can reveal the full extent of biotransformation in fish. The common presumption that the liver is the main site of biotransformation may not apply to many substances exposed through the diet. The results suggest that the application of quantitative structure-activity relationships (QSARs) for somatic biotransformation rates and hepatic in vitro models to assess the effect of biotransformation on bioaccumulation can underestimate biotransformation rates and overestimate the biomagnification potential of chemicals that are biotransformed in the gastrointestinal tract. With some modifications, the OECD 305 test can generate somatic and gastrointestinal biotransformation data to develop biotransformation QSARs and test in vitro-in vivo biotransformation extrapolation methods. Environ Toxicol Chem 2015;34:2282-2294. (c) 2015 SETAC DOI PubMed
70.Kennedy, CJ; Tierney, KB; Mittelstadt, M. (2014) Inhibition of P-glycoprotein in the blood-brain barrier alters avermectin neurotoxicity and swimming performance in rainbow trout.Aquatic Toxicology 146: 176-185 Inhibition of P-glycoprotein in the blood-brain barrier alters avermectin neurotoxicity and swimming performance in rainbow trout
Blood brain barrier; P-glycoprotein; Ivermectin; Emamectin benzoate; Neurotoxicity; Swimming; Fish; Rainbow trout
The importance of the blood brain barrier (BBB) and the contribution to its function by the efflux transporter P-glycoprotein (P-gp) in teleosts were examined using the P-gp substrates and central nervous system neurotoxins ivermectin (22,23-dihydroavermectin B-1a + 22,23-dihydroavermectin B-1b) [IVM) and emamectin benzoate (4"-deoxy-49"epimethylaminoavermectin B1 benzoate [EB]). Trout were injected intraperitoneally with 0.01-1.0 and 1-50 mg/kg of IVM or EB, respectively either alone or in combination with cyclosporin A (CsA: a P-gp substrate) at 1 mg/kg. IVM affected the swimming performance (critical swimming speed, burst swimming distance, and schooling) at significantly lower concentrations than EB. When fish were exposed to IVM or EB in the presence of CsA, alterations to swimming were increased, suggesting that competition for P-gp in the BBB by CsA increased IVM and EB penetration into the CNS and decreased swimming capabilities. The effect of co-administration of CsA on swimming-related toxicity was different between IVM and EB-treated fish; EB toxicity was increased to a greater extent than IVM toxicity. The greater chemosensitization effect of EB vs. IVM was examined using a P-gp competitive inhibition assay in isolated trout hepatocytes with rhodamine 123 as a substrate. At the cellular level, IVM was a more potent inhibitor of P-gp than EB, which allowed for a greater accumulation of R123 in hepatocytes. These results provide evidence for a role of P-gp in the BBB of fish, and suggest that this protein protects fish from environmental neurotoxins. (C) 2013 Elsevier B.V. All rights reserved. DOI
69. Lee, YS; Lee, DHY; Delafoulhouze, M; Otton, SV; Moore, MM; Kennedy, CJ; Gobas, FAPC. (2014) IN VITRO BIOTRANSFORMATION RATES IN FISH LIVER S9: EFFECT OF DOSING TECHNIQUES.Environmental Toxicology and Chemistry 33: 1885-1893 IN VITRO BIOTRANSFORMATION RATES IN FISH LIVER S9: EFFECT OF DOSING TECHNIQUES
In vitro biotransformation; Bioaccumulation; Sorbent-phase dosing; Ethylene vinyl acetate thin film
In vitro biotransformation assays are currently being explored to improve estimates of bioconcentration factors of potentially bioaccumulative organic chemicals in fish. The present study compares thin-film and solvent-delivery dosing techniques as well as single versus multiple chemical dosing for measuring biotransformation rates of selected polycyclic aromatic hydrocarbons in rainbow trout (Oncorhynchus mykiss) liver S9. The findings show that biotransformation rates of very hydrophobic substances can be accurately measured in thin-film sorbent-dosing assays from concentration-time profiles in the incubation medium but not from those in the sorbent phase because of low chemical film-to-incubation-medium mass-transfer rates at the incubation temperature of 13.5 degrees C required for trout liver assays. Biotransformation rates determined by thin-film dosing were greater than those determined by solvent-delivery dosing for chrysene (octanol-water partition coefficient [K-OW] = 10(5.60)) and benzo[a]pyrene (K-OW = 10(6.04)), whereas there were no statistical differences in pyrene (K-OW = 10(5.18)) biotransformation rates between the 2 methods. In sorbent delivery-based assays, simultaneous multiple-chemical dosing produced biotransformation rates that were not statistically different from those measured in single-chemical dosing experiments for pyrene and benzo[a]pyrene but not for chrysene. In solvent-delivery experiments, multiple-chemical dosing produced biotransformation rates that were much smaller than those in single-chemical dosing experiments for all test chemicals. While thin-film sorbent-phase and solvent delivery-based dosing methods are both suitable methods for measuring biotransformation rates of substances of intermediate hydrophobicity, thin-film sorbent-phase dosing may be more suitable for superhydrophobic chemicals. (c) 2014 SETAC DOI PubMed
68. Marlatt, VL; Sun, JY; Curran, CA; Bailey, HC; Kennedy, CK; Elphick, JR; Martyniuk, CJ. (2014) Molecular responses to 17 beta-estradiol in early life stage salmonids.General and Comparative Endocrinology 203: 203-214 Molecular responses to 17 beta-estradiol in early life stage salmonids
Rainbow trout; Estrogen receptor; Eyed embryo; Gene expression; 17 beta-estradiol; Vitellogenin
Environmental estrogens (EE) are ubiquitous in many aquatic environments and biological responses to EEs in early developmental stages of salmonids are poorly understood compared to juvenile and adult stages. Using 17 beta-estradiol (E2) as a model estrogen, waterborne exposures were conducted on early life stage rainbow trout (Oncorhynchus mykiss; egg, alevin, swim-up fry) and both molecular and physiological endpoints were measured to quantify the effects of E2. To investigate developmental stage-specific effects, laboratory exposures of 1 mu g/L E2 were initiated pre-hatching as eyed embryos or post-hatching upon entering the alevin stage. High mortality (similar to 90%) was observed when E2 exposures were initiated at the eyed embryo stage compared to the alevin stage (similar to 35% mortality), demonstrating stage-specific sensitivity. Gene expression analyses revealed that vitellogenin was detectable in the liver of swim-up fry, and was highly inducible by 1 mu g/L E2 (>200-fold higher levels compared to control animals). Experiments also confirmed the induction of vitellogenin protein levels in protein extracts isolated from head and tail regions of swim-up fry after E2 exposure. These findings suggest that induction of vitellogenin, a well-characterized biomarker for estrogenic exposure, can be informative measured at this early life stage. Several other genes of the reproductive endocrine axis (e.g. estrogen receptors and androgen receptors) exhibited decreased expression levels compared to control animals. In addition, chronic exposure to E2 during the eyed embryo and alevin stages resulted in suppressive effects on growth related genes (growth hormone receptors, insulin-like growth factor 1) as well as premature hatching, suggesting that the somatotropic axis is a key target for E2-mediated developmental and growth disruptions. Combining molecular biomarkers with morphological and physiological changes in early life stage salmonids holds considerable promise for further defining estrogen action during development, and for assessing the impacts of endocrine disrupting chemicals in vivo in teleosts. (C) 2014 Elsevier Inc. All rights reserved. DOI PubMed
67. Osachoff, HL; Mohammadali, M; Skirrow, RC; Hall, ER; Brown, LLY; van Aggelen, GC; Kennedy, CJ; Helbing, CC. (2014) Evaluating the treatment of a synthetic wastewater containing a pharmaceutical and personal care product chemical cocktail: Compound removal efficiency and effects on juvenile rainbow trout.Water Research 62: 271-280 Evaluating the treatment of a synthetic wastewater containing a pharmaceutical and personal care product chemical cocktail: Compound removal efficiency and effects on juvenile rainbow trout
Synthetic wastewater; Conventional activated sludge; Vitellogenin protein; Oncorhynchus rnykiss
Pharmaceutical and personal care products (PPCPs) can evade degradation in sewage treatment plants (STPs) and can be chronically discharged into the environment, causing concern for aquatic organisms, wildlife, and humans that may be exposed to these bioactive chemicals. The ability of a common STP process, conventional activated sludge (CAS), to remove PPCPs (caffeine, di(2-ethylhexyl)phthalate, estrone, 17 alpha-ethinylestradiol, ibuprofen, naproxen, 4-nonylphenol, tonalide, triclocarban and triclosan) from a synthetic wastewater was evaluated in the present study. The removal of individual PPCPs by the laboratory-scale CAS treatment plant ranged from 40 to 99.6%. While the efficiency of removal for some compounds was high, remaining quantities have the potential to affect aquatic organisms even at low concentrations. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to influent recreated model wastewater with methanol (IM, solvent control) or with PPCP cocktail (IC), or CAS-treated effluent wastewater with methanol (EM, treated control) or with PPCP cocktail (EC). Alterations in hepatic gene expression (evaluated using a quantitative nuclease protection plex assay) and plasma vitellogenin (VTG) protein concentrations occurred in exposed fish. Although there was partial PPCP removal by CAS treatment, the 20% lower VTG transcript levels and 83% lower plasma VTG protein concentration found in EC-exposed fish compared to IC-exposed fish were not statistically significant. Thus, estrogenic activity found in the influent was retained in the effluent even though typical percent removal levels were achieved raising the issue that greater reduction in contaminant load is required to address hormone active agents. (C) 2014 Elsevier Ltd. All rights reserved. DOI PubMed
66. Osachoff, HL; Osachoff, KN; Wickramaratne, AE; Gunawardane, EK; Venturini, FP; Kennedy, CJ. (2014) Altered burst swimming in rainbow trout Oncorhynchus mykiss exposed to natural and synthetic oestrogens.Journal of Fish Biology 85: 210-227 Altered burst swimming in rainbow trout Oncorhynchus mykiss exposed to natural and synthetic oestrogens
17 alpha-ethinyl oestradiol; 17 beta-oestradiol; chloride; erythrocytes; glucose; osmoregulation
Juvenile rainbow trout Oncorhynchus mykiss were exposed to two concentrations each of 17 beta-oestradiol (E2; natural oestrogen hormone) or 17 alpha-ethinyl oestradiol (EE2; a potent synthetic oestrogen hormone) to evaluate their potential effects on burst-swimming performance. In each of six successive burst-swimming assays, burst-swimming speed (U-burst) was lower in fish exposed to 0.5 and 1 mu g l(-1) E2 and EE2 for four days compared with control fish. A practice swim (2 days prior to exposure initiation) in control fish elevated initial U-burst values, but this training effect was not evident in the 1 mu g l(-1) EE2-exposed fish. Several potential oestrogen-mediated mechanisms for U-burst reductions were investigated, including effects on metabolic products, osmoregulation and blood oxygen-carrying capacity. Prior to burst-swimming trials, fish exposed to E2 and EE2 for 4 days had significantly reduced erythrocyte numbers and lower plasma glucose concentrations. After six repeated burst-swimming trials, plasma glucose, lactate and creatinine concentrations were not significantly different among treatment groups; however, plasma Cl- concentrations were significantly reduced in E2- and EE2-treated fish. In summary, E2 and EE2 exposure altered oxygen-carrying capacity ([erythrocytes]) and an osmoregulatory-related variable ([Cl-]), effects that may underlie reductions in burst-swimming speed, which will have implications for fish performance in the wild. (C) 2014 The Fisheries Society of the British Isles DOI PubMed
65. Goulding, AT; Shelley, LK; Ross, PS; Kennedy, CJ. (2013) Reduction in swimming performance in juvenile rainbow trout (Oncorhynchus mykiss) following sublethal exposure to pyrethroid insecticides.Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 157: 280-286 Reduction in swimming performance in juvenile rainbow trout (Oncorhynchus mykiss) following sublethal exposure to pyrethroid insecticides
PERMETHRIN NRDC-143; SALMO-GAIRDNERI; SEA LICE; CYPRINUS-CARPIO; TOXICITY; WATER; DELTAMETHRIN; FISH; KISUTCH; GILL
While the lethal toxicity of pyrethroid insecticides to fish is well documented, their sublethal physio-behavioral effects remain poorly characterized. Known pyrethroid-associated changes to insect neuromuscular function may translate into similar effects in fish, thereby altering swimming ability and affecting foraging, predator avoidance, and migration. Three experiments were conducted using critical (U-crit) and burst (U-max) swimming speeds to assess the sublethal effects of the pyrethroids permethrin and deltamethrin in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to deltamethrin (100, 200, or 300 ng/L) or permethrin (1, 2, or 3 mu g/L) in water for 4 d, and assessed for swimming performance. Deltamethrin (200 and 300 ng/L) reduced U-crit, but not U-max, while both swim performance measurements were unaffected by permethrin. Subsequent experiments used only U-crit to assess deltamethrin exposure. In a time course experiment, deltamethrin (300 ng/L) reduced U-crit after 1 and 4 d of exposure, but after 7 d of exposure U-crit was fully recovered. Finally, deltamethrin (1, 2, or 3 mu g/L) reduced U-crit after I h bath exposures similar to recommended protocols for deltamethrin based sea-lice treatment in aquaculture. The real-world implications of the revealed pyrethroid-associated swimming ability reductions in salmon may be important in areas close to aquaculture facilities. (C) 2013 Published by Elsevier Inc. DOI
64. Marlatt, VL; Lo, BP; Ornostay, A; Hogan, NS; Kennedy, CJ; Elphick, JR; Martyniuk, CJ. (2013) The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas).Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 157: 24-32 The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas)
Anti-androgen; Linuron; Steroidogenic genes; Vitellogenin; Endocrine disruption
Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21 days to dechlorinated water, a solvent control, 17 beta-estradiol (E2; 0.1 mu g/L), dihydrotestosterone (DHT; 100 mu g/L), linuron (1, 10, 100 mu g/L) and one co-treatment of DHT (100 mu g/L) and linuron (100 mu g/L). There were no effects of linuron on egg hatching, 7 day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100 mu g/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr26, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction. (C) 2012 Elsevier Inc. All rights reserved. DOI
63. Osachoff, HL; Shelley, LK; Furtula, V; van Aggelen, GC; Kennedy, CJ. (2013) Induction and Recovery of Estrogenic Effects After Short-Term 17 beta-Estradiol Exposure in Juvenile Rainbow Trout (Oncorhynchus mykiss).Archives of Environmental Contamination and Toxicology 65: 276-285 Induction and Recovery of Estrogenic Effects After Short-Term 17 beta-Estradiol Exposure in Juvenile Rainbow Trout (Oncorhynchus mykiss)
VITELLOGENIN MESSENGER-RNA; MINNOWS PIMEPHALES-PROMELAS; SEWAGE-TREATMENT PLANTS; FLOUNDER PLATICHTHYS-FLESUS; CARP CYPRINUS-CARPIO; GENE-EXPRESSION; FATHEAD MINNOWS; IN-VIVO; DIFFERENTIAL EXPRESSION; ENVIRONMENTAL ESTROGENS
Estrogenic compounds found in the aquatic environment include natural and synthetic estrogen hormones as well as other less potent estrogenic xenobiotics. In this study, a comprehensive approach was used to examine effects on fish endocrine system endpoints during a short-term xenoestrogen exposure as well as after post-exposure recovery. Rainbow trout (Oncorhynchus mykiss) were exposed to an aqueous 17 beta-estradiol (E2) concentration of 0.473 mu g l(-1) for 2 and 7 days (d) followed by a 14-d recovery period. At d2 and d7, plasma E2 concentrations in treated fish were 458- and 205-fold higher than in control fish and 23- and 16-fold higher than the exposure water concentration. E2 treatment resulted in significant increases in hepatosomatic index (HSI), plasma vitellogenin (VTG) protein concentrations, and liver VTG and estrogen receptor alpha mRNA levels. All of these parameters, with the exception of plasma VTG protein, returned to baseline values during the recovery period. Plasma cortisol concentrations were unaffected by treatment. This research shows varied time frames of the estrogen-responsive molecular-, biochemical-, and tissue-level alterations, as well as their persistence, in juvenile rainbow trout treated with aqueous E2. These results have implications for feral rainbow trout exposed to xenoestrogens and indicate the importance of evaluating a comprehensive suite of endpoints in assessing the impact of this type of environmental contaminant. DOI
62. Osachoff, HL; van Aggelen, GC; Mommsen, TP; Kennedy, CJ. (2013) Concentration-response relationships and temporal patterns in hepatic gene expression of Chinook salmon (Oncorhynchus tshawytscha) exposed to sewage.Comparative Biochemistry and Physiology D-Genomics & Proteomics 8: 32-44 Concentration-response relationships and temporal patterns in hepatic gene expression of Chinook salmon (Oncorhynchus tshawytscha) exposed to sewage
BIPHASIC DOSE RESPONSES; MINNOWS PIMEPHALES-PROMELAS; TREATMENT-PLANT EFFLUENTS; ROACH RUTILUS-RUTILUS; RAINBOW-TROUT; SEXUAL-DIFFERENTIATION; ENDOCRINE DISRUPTORS; BIOMARKER RESPONSES; AQUATIC ENVIRONMENT; STEROID ESTROGEN
Changes in liver gene expression were examined in juvenile Chinook salmon (Oncorhynchus tshawytscha) exposed in vivo for 8 d to seawater (control) or one of 5 concentrations of sewage (environmentally-relevant dilutions of 0.05%, 0.1%, and 0.7%; 2%, 5% or 10%) and subsequently transferred to clean seawater for an 8-d recovery period. Livers were sampled on days 1, 4, 8 (sewage-exposed) and 16 (8 d of sewage exposure plus 8 d of recovery). A custom cDNA microarray using a universal DNA reference design was used to examine trends of altered gene expression across sewage concentrations, across timepoints, and at the end of the recovery period. Alterations in gene expression followed four distinct concentration-dependent patterns: (I) concentration response (e.g. estrogen receptor alpha), (2) inverse-concentration response (e.g. insulin receptor beta), U-shaped (e.g. mineralocorticoid receptor), (3) inverse U-shaped (e.g. benzodiazepine receptor), and (4) concentration-independent responses (e.g. ubiquitin). Temporal trends included: (1) peak gene expression at one of the sewage exposure timepoints with recovery to baseline levels after the deputation phase (e.g. vitelline envelope protein beta), (2) gene expression alterations that did not recover (e.g. glucose transporter 3), and (3) delayed gene expression alterations initiated only at the recovery timepoint (e.g. insulin-like growth factor 2). In summary, patterns in gene expression changes were found across sewage concentrations and exposure timepoints. This study is the first to show gene expression trends of this nature. (C) 2012 Elsevier Inc. All rights reserved. DOI
61. Ross, PS; Kennedy, CJ; Shelley, LK; Tierney, KB; Patterson, DA; Fairchild, WL; Macdonald, RW. (2013) The trouble with salmon: relating pollutant exposure to toxic effect in species with transformational life histories and lengthy migrations.Canadian Journal of Fisheries and Aquatic Sciences 70: 1252-1264 The trouble with salmon: relating pollutant exposure to toxic effect in species with transformational life histories and lengthy migrations
JUVENILE CHINOOK SALMON; PERSISTENT ORGANIC POLLUTANTS; ARYL-HYDROCARBON RECEPTOR; FRESH-WATER FISH; DIDECYLDIMETHYLAMMONIUM CHLORIDE DDAC; SOLE PLEURONECTES-VETULUS; ADULT SOCKEYE-SALMON; FRASER-RIVER SYSTEM; SHORT-TERM EXPOSURE; ONCORHYNCHUS-NERKA
The control of point-source contaminants and regulations designed for specific waste discharges have reduced incidents of fish kills. These actions, however, do not protect fish like salmon, which encounter many different contaminants during extensive migrations. Attempts to document pollutant-associated toxicity is challenging in migratory salmon, although a few laboratory and field studies have produced a convincing body of evidence that lifelong contaminant exposure can contribute to the demise of fish. The case of the decline of Fraser River sockeye salmon (Oncorhynchus nerka) in British Columbia, Canada, brought into sharp relief the difficulty of assigning a specific cause (e. g., climate, disease, or contaminants) to a diffuse problem (i.e., low fish returns). Determining the effects that pollutants have on wild salmon requires study designs that consider life history, habitat, and the real world of complex contaminant exposures. In the absence of evidence from such study designs, the future survival of salmon may hinge on the application by managers of the precautionary approach to stressors that are within immediate jurisdictional control, such as toxic chemicals. DOI
60. Shelley, LK; Osachoff, HL; van Aggelen, GC; Ross, PS; Kennedy, CJ. (2013) Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17 beta-estradiol exposed rainbow trout (Oncorhynchus mykiss).General and Comparative Endocrinology 180: 24-32 Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17 beta-estradiol exposed rainbow trout (Oncorhynchus mykiss)
PERIPHERAL-BLOOD LEUKOCYTES; FLOW-CYTOMETRIC ANALYSIS; PERSONAL CARE PRODUCTS; GENE-EXPRESSION; IN-VITRO; CYPRINUS-CARPIO; ENVIRONMENTAL ESTROGENS; DISEASE RESISTANCE; TERM EXPOSURE; COMMON CARP
While the endocrine system is known to modulate immune function in vertebrates, the role of 17 beta-estradiol (E2) in cellular immune function of teleosts is poorly understood. The cellular and molecular responses of juvenile rainbow trout (Oncorhynchus mykiss) to E2 treatment were evaluated by exposing fish to 0.47 +/- 0.02 mu g/L E2 (mean +/- SEM) for either 2 or 7 d, with a subsequent 14 d recovery period. After 2 and 7 d of exposure to E2, hematocrit was significantly lower than in control fish. Lipopolysaccharide-induced lymphocyte proliferation was elevated on day 2 and concanavalin A-induced lymphocyte proliferation was reduced following 7 d of E2 exposure. Four estrogen receptor (ER) transcripts were identified in purified trout head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL). While the mRNA abundance of ER beta 1 and ER beta 2 was unaffected by treatment, ER alpha 1 was up-regulated in HKL and PBL following 7 d of E2 exposure. ER alpha 2 was up-regulated in HKL after 7 d of E2 exposure, but down-regulated in PBL after 2 and 7 d of treatment. All parameters that were altered during the E2 exposure period returned to baseline levels following the recovery period. This study reports the presence of the full repertoire of ERs in purified HKL for the first time, and demonstrates that ER alpha transcript abundance in leukocytes can be regulated by waterborne E2 exposure. It also demonstrated that physiologically-relevant concentrations of E2 can modulate several immune functions in salmonids, which may have widespread implications for xenoestrogen-associated immunotoxicity in feral fish populations inhabiting contaminated aquatic environments. (C) 2012 Elsevier Inc. All rights reserved. DOI
59. Furtula, V; Liu, J; Chambers, P; Osachoff, H; Kennedy, C; Harkness, J. (2012) Sewage Treatment Plants Efficiencies in Removal of Sterols and Sterol Ratios as Indicators of Fecal Contamination Sources.Water Air and Soil Pollution 223: 1017-1031 Sewage Treatment Plants Efficiencies in Removal of Sterols and Sterol Ratios as Indicators of Fecal Contamination Sources
Sterol ratios; Sewage treatment plant; Influent; Effluent; Human fecal contamination
This study assessed the efficiency of sewage treatment plants (STPs) in removing sterols based on chemical analyses of both influents and effluents. Samples from 3s and three tertiary plants were collected and analyzed by gas chromatography mass spectrometry for 23 individual sterols including mestranol, norethindrone, equol, estrone, equilin, norgestrel, 17 alpha-ethinylestradiol, 17 alpha-estradiol, 17 beta-estradiol, estriol, dihydrocholesterol (cholestanol), coprostanol, epicoprostanol, cholesterol, desmosterol, campesterol, stigmasterol, beta-sitosterol, coprostanone, cholestanone, epicholestanol, stigmastanol, and 24-ethylcoprostanol. The percentage of sterols remaining in effluent samples (compared to influent samples) ranged from 0% to 80% and varied among sterol compounds and with STP location and treatment type. Differences in the efficiency of sterol removal for secondary and tertiary STPs were statistically significant. Although the concentration of sterol compounds differed between influents and effluents, sterol abundances remained the same. The most abundant sterol detected was cholesterol, followed by the fecal sterol coprostanol, and the plant sterols 24-ethylcoprostanol and beta-sitosterol. For three STPs, the hormone estrone was detected in effluents at concentrations of 0.03-0.05 mu g L-1. Ten sterol ratios specific for human fecal contamination and eight sterol ratios for differentiating among multiple sources of fecal contamination were calculated and showed that 12 ratios for influent and nine ratios for effluent were successful for human fecal source tracking. Based on sterol ratio values in this study, new criteria for identification of human fecal contamination were suggested. DOI
57.Kennedy, CJ; Picard, C. (2012) Chronic low pH exposure affects the seawater readiness of juvenile Pacific sockeye salmon.Fish Physiology and Biochemistry 38: 1131-1143 Chronic low pH exposure affects the seawater readiness of juvenile Pacific sockeye salmon
pH; Hydrogen ion concentration; Sockeye salmon; Oncorhynchus nerka; Growth; Bioenergetics; Stress; Seawater challenge
Chronic exposure to water of low pH during the freshwater life stage of Pacific salmonids is presently the cause for concern due to its potential to reduce subsequent performance in the marine environment. Sockeye fry (0+) were raised under sublethal long-term, low pH conditions (pH 4.8-6.8) in soft water and assessed for effects on freshwater growth, stress physiology, and seawater tolerance following smoltification. Fish gained significantly lower mass (average 46% of control [pH 6.8] values) and had lower condition factor and liver somatic index values than control fish following a 126-days exposure to water at pH 5.0. Liver glycogen concentrations (49% of control values) and whole-body lipid content (65% of control values) were also significantly lower. Low pH exposure also resulted in a sustained organismal stress response that included significant and substantial increases in plasma cortisol concentrations. Fish exposed to pH 5.0 in freshwater for 30 days exhibited an average of 14% mortality in a seawater challenge, as well as a significant osmoregulatory stress measured by increases in plasma Na+ and Cl- concentrations as well as osmolality compared to controls. Significantly lower critical swimming speed values (U-crit) were also seen (22% reductions compared to controls). The data generated indicate that sockeye salmon are sensitive and do not acclimate to low pH under long-term exposure conditions, potentially decreasing the probability of survival in the marine environment. DOI
56. Lee, YS; Otton, SV; Campbell, DA; Moore, MM; Kennedy, CJ; Gobas, FAPC. (2012) Measuring In Vitro Biotransformation Rates of Super Hydrophobic Chemicals in Rat Liver S9 Fractions Using Thin-Film Sorbent-Phase Dosing.Environmental Science & Technology 46: 410-418 Measuring In Vitro Biotransformation Rates of Super Hydrophobic Chemicals in Rat Liver S9 Fractions Using Thin-Film Sorbent-Phase Dosing
Methods for rapid and cost-effective assessment of the biotransformation potential of very hydrophobic and potentially bioaccumulative chemicals in mammals are urgently needed for the ongoing global evaluation of the environmental behavior of commercial chemicals. We developed and tested a novel solvent-free, thin-film sorbent-phase in vitro dosing system to measure the in vitro biotransformation rates of very hydrophobic chemicals in male Sprague-Dawley rat liver S9 homogenates and compared the rates to those measured by conventional solvent-delivery dosing. The thin-film sorbent-phase dosing system using ethylene vinyl acetate coated vials was developed to eliminate the incomplete dissolution of very hydrophobic substances in largely aqueous liver homogenates, to determine biotransformation rates at low substrate concentrations, to measure the unbound fraction of substrate in solution, and to simplify chemical analysis by avoiding the difficult extraction of test chemicals from complex biological matrices. Biotransformation rates using sorbent-phase dosing were 2-fold greater than those measured using solvent-delivery dosing. Unbound concentrations of very hydrophobic test chemicals were found to decline with increasing S9 and protein concentrations, causing measured biotransformation rates to be independent of S9 or protein concentrations. The results emphasize the importance of specifying both protein content and unbound substrate fraction in the measurement and reporting of in vitro biotransformation rates of very hydrophobic substances, which can be achieved in a thin-film sorbent-phase dosing system. DOI PubMed
55. Shelley, LK; Ross, PS; Kennedy, CJ. (2012) Immunotoxic and cytotoxic effects of atrazine, permethrin and piperonyl butoxide to rainbow trout following in vitro exposure.Fish & Shellfish Immunology 33: 455-458 Immunotoxic and cytotoxic effects of atrazine, permethrin and piperonyl butoxide to rainbow trout following in vitro exposure
Rainbow trout; Pesticide; Immunotoxicity; Lymphocyte proliferation; Cytotoxicity
For many current use pesticides, limited information exists on their cytotoxicity and immunotoxicity in non-target organisms such as fish. We examined the effects of atrazine, permethrin and piperonyl butoxide (PBO) exposure, in vitro, on rainbow trout (Oncorhynchus mykiss) lymphocyte viability and proliferation. Purified rainbow trout peripheral blood leukocytes (PBLs) were exposed in vitro to the test chemicals (0, 0.01, 0.1, 1 and 10 mu M) for 96 h, with and without the mitogen lipopolysaccharide. All three chemicals caused a decrease in both lymphocyte viability and proliferation at 10 mu M, while atrazine also suppressed proliferation of PBLs at 1 mu M. The in vitro toxicity of these chemicals to this salmonid underscores the need for further investigation using in vivo studies and host resistance models. (c) 2012 Elsevier Ltd. All rights reserved. DOI
54. Shelley, LK; Ross, PS; Kennedy, CJ. (2012) The effects of an in vitro exposure to 17 beta-estradiol and nonylphenol on rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes.Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 155: 440-446 The effects of an in vitro exposure to 17 beta-estradiol and nonylphenol on rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes
Estradiol; Estrogen receptor; Immune system; Nonylphenol; Teleost; Xenoestrogen
While xenoestrogens are routinely detected in the aquatic environment, there is little understanding of the immunotoxicological consequences of exposure to these chemicals in fish, or of the mechanisms through which these effects are mediated. This study was conducted to determine if estrogen receptors (ERs) are present in fish leukocytes and to characterize the effects of 17 beta-estradiol (E2) and the xenoestrogen nonylphenol (NP) on immune system endpoints in rainbow trout (Oncorhynchus mykiss). Q-PCR was used to confirm that freshly isolated peripheral blood leukocytes (PBLs) express ERs. Following 96-h incubations with E2 or NP (1 nM to 10 mu M), PBL ER transcription was again examined using Q-PCR and lipopolysaccharide (LPS)-stimulated proliferation was assessed using flow cytometry. While the transcription of all four forms of rainbow trout ER was unaffected by treatment with E2 or NP, transcription of ER alpha 1 and ER alpha 2 was down-regulated following LPS stimulation. Both E2 and NP, at concentrations of >= 100 nM and 10 nM respectively, suppressed leukocyte proliferation. This first report of ERs in rainbow trout PBLs suggests a mechanism through with E2 and other xenoestrogens can modulate immune function. These results highlight the potential for xenoestrogens to impact host resistance to pathogens in wild fish populations. (C) 2011 Elsevier Inc. All rights reserved. DOI
53. Shelley, LK; Ross, PS; Miller, KM; Kaukinen, KH; Kennedy, CJ. (2012) Toxicity of atrazine and nonylphenol in juvenile rainbow trout (Oncorhynchus mykiss): Effects on general health, disease susceptibility and gene expression.Aquatic Toxicology 124: 217-226 Toxicity of atrazine and nonylphenol in juvenile rainbow trout (Oncorhynchus mykiss): Effects on general health, disease susceptibility and gene expression
Immunotoxicity; Atrazine; Nonylphenol; Rainbow trout; Microarray; Host resistance challenge; Leukocyte differential count
Atrazine (ATZ) and nonylphenol (NP) are commonly identified contaminants in aquatic habitats; however, few studies have considered the impact of these endocrine disrupters on immune function and resistance to disease. This study examined the immunotoxicological effects of ATZ and NP at multiple levels of biological organization. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to a solvent control (0.00625%, v/v anhydrous ethanol), or sub-lethal concentrations of ATZ (59 mu g/L and 555 mu g/L) or NP (2.3 mu g/L or 18 mu g/L) for 4 d. At the end of exposure, fish were assessed for a number of physiological endpoints, including a host resistance challenge, and liver gene expression was assessed using a salmonid microarray (cGRASP, 32K version 1). While the low ATZ and low NP treatments had no measurable effects on the physiological endpoints measured, fish exposed to the high ATZ concentration (555 mu g/L) exhibited significantly elevated plasma cortisol, a decrease in SSI, and decreased lymphocytes and increased monocytes in peripheral blood, with suppression of early immune system processes apparent at the molecular level. In contrast, fish exposed to the high NP concentration (18 mu g/L) showed physiological (e.g. significantly elevated LSI) and gene expression changes (e.g. induction of vitellogenin) consistent with estrogenic effects, as well as decreased lymphocytes in the peripheral blood and more limited alterations in immune system related pathways in the liver transcriptome. Fish exposed to high ATZ or NP concentrations incurred higher mortality than control fish following a disease challenge with Listonella anguillarum, while fish exposed to the lower concentrations were unaffected. Microarray analysis of the liver transcriptome revealed a total of 211 unique, annotated differentially regulated genes (DRGs) following high ATZ exposure and 299 DRGs following high NP exposure. Functional (enrichment) analysis revealed effects on immune system function, metabolism, oxygen homeostasis, cell cycle, DNA damage, and other processes affected by ATZ or NP exposure. Overall, this study provides evidence at multiple levels of biological organization that both ATZ and NP are immunotoxic at sub-lethal concentrations and highlights the potential risk posed by these chemicals to wild fish populations. (C) 2012 Published by Elsevier B.V. DOI
52. Waliwitiya, R; Nicholson, RA; Kennedy, CJ; Lowenberger, CA. (2012) The Synergistic Effects of Insecticidal Essential Oils and Piperonyl Butoxide on Biotransformational Enzyme Activities in Aedes aegypti (Diptera: Culicidae).Journal of Medical Entomology 49: 614-623 The Synergistic Effects of Insecticidal Essential Oils and Piperonyl Butoxide on Biotransformational Enzyme Activities in Aedes aegypti (Diptera: Culicidae)
ethoxyresorufin O-deethylase; glutathione S-transferase; esterase; essential oil; Ae. Aegypti
The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and beta-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil + PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5-25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 +/- 1.8% over controls. The essential oils alone reduced GST activity by 3-20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58-76% and reduced GST activity by 3-85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti. DOI
51. Tierney, KB; Williams, JL; Gledhill, M; Sekela, MA; Kennedy, CJ. (2011) ENVIRONMENTAL CONCENTRATIONS OF AGRICULTURAL-USE PESTICIDE MIXTURES EVOKE PRIMARY AND SECONDARY STRESS RESPONSES IN RAINBOW TROUT.Environmental Toxicology and Chemistry 30: 2602-2607 ENVIRONMENTAL CONCENTRATIONS OF AGRICULTURAL-USE PESTICIDE MIXTURES EVOKE PRIMARY AND SECONDARY STRESS RESPONSES IN RAINBOW TROUT
Agrichemicals; Pesticides; Salmon; Stress response; Cortisol
The present study sought to determine whether environmentally realistic mixtures of agriculturally important pesticides are stressful to fish. Juvenile rainbow trout were exposed for 96 h to concentrations of a pesticide mixture found in a waterway that is the focus of salmon restoration efforts (Nicomekl River, BC, Canada). This mixture contained organochlorine, organophosphorus, phenylurea, and triazine classes of pesticides. Fish given a realistic mixture exposure (total concentration, 1.01 mu g/L) had increased plasma cortisol concentration, packed red cell volume, hematocrit (Hct), as well as decreased white cell volume, leukocrit (Lct). Similar changes in Hct and Lct were apparent after exposure to a lower concentration (0.186 mu g/L). Interestingly, no changes in plasma cortisol concentration, Hct, or Lct were noted after exposure to a higher concentration (13.9 mu g/L). This suggests that the exposure likely impaired the mechanisms enabling the stress response. Across all exposures, plasma glucose concentration was related to plasma cortisol concentration, not to pesticide mixture concentration. This suggests that a secondary stress response may be more related to variability in individual primary stress response than to differences in pesticide exposure concentrations. In summary, the present study indicates that salmon living in agrichemical-contaminated waterways may be experiencing stress, and this may pose a threat to their survival. Environ. Toxicol. Chem. 2011;30:2602-2607. (C) 2011 SETAC DOI
50. Blanc, AM; Holland, LG; Rice, SD; Kennedy, CJ. (2010) Anthropogenically sourced low concentration PAHS: In situ bioavailability to juvenile Pacific salmon.Ecotoxicology and Environmental Safety 73: 849-857 Anthropogenically sourced low concentration PAHS: In situ bioavailability to juvenile Pacific salmon
PAHs; CYP1A; Salmon; Gill; Biomarker; EROD
Gill 7-ethoxyresorufin O-deethylase (EROD) activity of juvenile Chinook salmon caged in Auke Lake, AK was used as a biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. Biomarker measurements in conjunction with a comprehensive sampling program that included grab water and sediment samples, and passive sampling devices were used to determine PAH concentrations, source(s), bioavailability, and resulting biological response. PAHs were detected at all lake locations except the reference site upstream of anthropogenic activity. Water samples were the best predictor of a biological response and EROD activity correlated to corresponding parts per trillion water pyrene concentrations (r(2)=0.9662; p=0.0004). Sediment samples yielded the clearest indication of PAN sources and amalgamated contaminant magnitude, and passive samplers served as accumulators of retrospective aqueous conditions. Results suggest that salmon stocks are being exposed to chronic low-concentrations of anthropogenically sourced PAHs during sensitive life-stages, which may be in part a contributor to their declining numbers. (C) 2010 Elsevier Inc. All rights reserved. DOI
49. Tierney, KB; Baldwin, DH; Hara, TJ; Ross, PS; Scholz, NL; Kennedy, CJ. (2010) Olfactory toxicity in fishes.Aquatic Toxicology 96: 2-26 Olfactory toxicity in fishes
Olfaction; Fish; Contaminants; Metals; Neurotoxicity; Behavior
Olfaction conveys critical environmental information to fishes, enabling activities such as mating, locating food, discriminating kin, avoiding predators and homing. All of these behaviors can be impaired or lost as a result of exposure to toxic contaminants in surface waters. Historically, teleost olfaction studies have focused on behavioral responses to anthropogenic contaminants (e.g., avoidance). More recently, there has been a shift towards understanding the underlying mechanisms and functional significance of contaminant-mediated changes in fish olfaction. This includes a consideration of how contaminants affect the olfactory nervous system and, by extension, the downstream physiological and behavioral processes that together comprise a normal response to naturally occurring stimuli (e.g., reproductive priming or releasing pheromones). Numerous studies spanning several species have shown that ecologically relevant exposures to common pollutants such as metals and pesticides can interfere with fish olfaction and disrupt life history processes that determine individual survival and reproductive success. This represents one of the pathways by which toxic chemicals in aquatic habitats may increasingly contribute to the decline and at-risk status of many commercially and ecologically important fish stocks. Despite our emerging understanding of the threats that pollution poses for chemical communication in aquatic communities, many research challenges remain. These include: (1) the determination of specific mechanisms of toxicity in the fish olfactory sensory epithelium; (2) an understanding of the impacts of complex chemical mixtures: (3) the capacity to assess olfactory toxicity in fish in situ; (4) the impacts of toxins on olfactory-mediated behaviors that are still poorly understood for many fish species; and (5) the connections between sublethal effects on individual fish and the long-term viability of wild populations. This review summarizes and integrates studies on fish olfaction-contaminant interactions, including metrics ranging from the molecular to the behavioral, and highlights directions for future research. (C) 2009 Elsevier B.V. All rights reserved. DOI
48. Gourley, ME; Kennedy, CJ. (2009) Energy allocations to xenobiotic transport and biotransformation reactions in rainbow trout (Oncorhynchus mykiss) during energy intake restriction.Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 150: 270-278 Energy allocations to xenobiotic transport and biotransformation reactions in rainbow trout (Oncorhynchus mykiss) during energy intake restriction
ISOLATED LIVER-CELLS; P-GLYCOPROTEIN; OXIDATIVE STRESS; METABOLIZING ENZYMES; FOOD-DEPRIVATION; OPSANUS-BETA; ANTIOXIDANT DEFENSES; CULTURED-HEPATOCYTES; SALMO-GAIRDNERI; GULF TOADFISH
Limited energy intake may result in the down-regulation of cellular defense mechanisms, or if maintained, result in trade-offs with other physiological systems. To examine this, juvenile rainbow trout (Oncorhynchus mykiss) were fed full-rations (1.17% body weight [BwJ]/day), half-rations (0.59% [BW]/day), or fasted for 9 weeks followed by refeeding at full-rations. BW and liver somatic index (LSI), P-glycoprotein (P-gp), ethoxyresorufin-o-deethylase (EROD), and glutathione-S-transferase (GST) activities were measured to determine if they are maintained under limited resources. P-gp and EROD activities were maintained at baseline values in ration-restricted (P-gp: 119 +/- 29 pg R123/min/mg cells, EROD: 0.58 +/- 0.17 nmol/min/mg protein) and fasted fish (P-gp: 120 +/- 14 pg R123/min/mg cells, EROD: 0.47 +/- 0.14 nmol/min/mg protein), suggesting they may be prioritized systems during fasting. GST activity was attenuated within 6 weeks of fasting (34% decrease from control), but recoverable to baseline values after refeeding. Changes in BW and LSI of calorie-restricted (BW: 16% decrease from control: LSI: 33% decrease from baseline value) and fasted trout (BW: 38% decrease from control; LSI: 44% decrease from baseline value) suggest that resources were mobilized from body stores partly to support these systems. Condition indices and defense activities in groups also varied over time, suggesting that environmental temperature may modulate these parameters. (C) 2009 Elsevier Inc. All rights reserved. DOI
47. Hildebrand, JL; Bains, OS; Lee, DSH; Kennedy, CJ. (2009) Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss) hepatocytes.Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 149: 65-72 Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss) hepatocytes
Adenylate energy charge; Energetics; Hepatocytes; Oncorhynchus mykiss; Multixenobiotic resistance; P-glycoprotein; Doxorubicin MULTIDRUG-RESISTANCE; ATP HYDROLYSIS; IMMUNOHISTOCHEMICAL DETECTION; AQUATIC ORGANISMS; RAT-LIVER; GLYCOPROTEIN; CHARGE; METABOLISM; FISH; ADRIAMYCIN
An assessment of energetic costs associated with P-glycoprotein (P-gp)-mediated xenobiotic efflux is important in understanding the energy budgets, tradeoffs, and fitness of organisms inhabiting contaminated environments. Here, a functional characterization and determination of the energetic costs associated with doxorubicin (DOX) efflux was examined in isolated hepatocytes of rainbow trout. The accumulation and efflux of DOX were both concentration dependent. The efflux of DOX over a 3 h incubation period resulted in a significant decrease in intracellular ATP concentrations (maximum decrease 25%) compared to control baseline levels, while significant increases in concentrations of ADP (max. 26%), AMP (max. 36%) and inorganic phosphate (max. 11%). were observed. In addition, significant reductions in the adenylate energy charge QAEC]: max 11%), and phosphorylation potential ([PP]: max. 53%) were shown in cells incubated with DOX compared to control cells. Inhibition of DOX efflux (max. 61%) by the non-competitive P-gp inhibitor tariquidar (XR9576), demonstrated that changes in ATP, ADP, AMP, inorganic phosphate concentrations, AEC and PP in DOX-exposed hepatocytes were mainly due to P-gp activity. Overall, these results indicate that the exposure of trout hepatocytes to DOX increases energetic and metabolic costs that are associated specifically with P-gp efflux activity. (C) 2008 Elsevier Inc. All rights reserved. DOI
46. Shelley, LK; Balfry, SK; Ross, PS; Kennedy, CJ. (2009) Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss).Aquatic Toxicology 92: 95-103 Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss)
FRESH-WATER FISH; LOWER FRASER VALLEY; BRITISH-COLUMBIA; FUNDULUS-HETEROCLITUS; DISEASE RESISTANCE; VIBRIO-ANGUILLARUM; IMMUNE FUNCTIONS; HOST-RESISTANCE; CHINOOK SALMON; PENTACHLOROPHENOL
Many current-use pesticides (CUPs) are found at increasing concentrations in aquatic environments, yet relatively little is known about their effects on the immune system of fish. We examined the in vivo effects of three pesticides (chlorothalonil, cypermethrin and pentachlorophenol) on the immune system of juvenile rainbow trout (Oncorhynchus mykiss) by assessing a suite of innate immune function tests, as well as a host resistance test using Listonella anguillarum. Increased activity of phagocytic leukocytes, as evidenced using flow cytometry, was observed following 28-day exposures to pentachlorophenol (1 mu g/L and 2 mu g/L), but not for cypermethrin or chlorothalonil, although a trend of increasing activity was noted for chlorothalonil. NO recovery was observed during a 14-day post-exposure chlorothalonil experiment, as evidenced by continued elevation of respiratory burst and percent phagocytic cells at the lowest exposure concentrations (100 ng/L and 200 ng/L), suggesting a prolonged CUP-induced impact on the immune system. No effects of any pesticide on body weights, direct lethality, serum lysozyme OF relative leukocyte differential were observed, suggesting that modulation of the cellular responses of the innate immune system represents a sensitive sub-lethal endpoint for these three pesticides. However, a lack of detectable effects of these CUPs oil host resistance to L. anguillarum in our study may reflect a dose-response range that did not elicit an effect on those immune responses responsible for control and clearance of this particular pathogen. Additional research may provide more insight into the immunomodulatory effects of these and other CUPs, and the implications for host resistance against a variety of bacterial, viral and macroparasitic pathogens. (C) 2009 Elsevier B.V. All rights reserved. DOI
45. Tierney, K.B. and C.J. Kennedy. (2009) Background Toxicology.Edited by P.J. Walsh, S. Smith, L. Fleming, H. Solo-Gabriele and W. Gerwick. Academic Press. pp. 101-120.Background Toxicology.
44. Tierney, KB; Patterson, DA; Kennedy, CJ. (2009) The influence of maternal condition on offspring performance in sockeye salmon Oncorhynchus nerka.Journal of Fish Biology 75: 1244-1257 The influence of maternal condition on offspring performance in sockeye salmon Oncorhynchus nerka
SIZE-SELECTIVE MORTALITY; JUVENILE RAINBOW-TROUT; EGG CORTISOL CONTENT; SWIMMING PERFORMANCE; PLASMA-CORTISOL; COHO SALMON; BRITISH-COLUMBIA; CHINOOK SALMON; LIFE-HISTORY; FISH HEALTH
Eggs were taken from adult sockeye salmon Oncorhynchus nerka that had reached their journey's end in spawn-ready and moribund condition, and fertilized by healthy males. Egg number, size, hatching success and offspring growth did not differ with maternal condition, which suggests the absence of any persisting physiological maternal effects. Differences were noted in the swimming behaviour and physiology of the offspring at parr stage. In a 30 min schooling test conducted using groups of five in a flume, parr from moribund females were more likely to fatigue, were not as tightly schooled, and had a diminished startle response, both in the per cent responding and the burst distance. In individual, confined swimming tests conducted within a tube, post-exercise plasma lactate concentration, which is an indicator of white muscle use, was greater for parr from moribund adult females. The moribund females also had elevated lactate following exercise (their migration), which suggests heritable differences may exist in muscle use. This study shows that juvenile O. nerka artificially propagated from females exhausted by their return migration can exhibit swimming performance differences, indicating that maternal condition may need to be considered in breeding programmes. DOI
43. Waliwitiya, R., Kennedy, C., and Lowenberger, C. (2009) Larvicidal and antiovipositional activity of monoterpenoids and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae).Pest Management Science 65: 241-248. Larvicidal and antiovipositional activity of monoterpenoids and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae)
Aedes aegypti, acute toxicity, essential oils, larvicides, monoterpenoids, oviposition, piperonyl butoxide
Aedes aegypti L. is the major vector of dengue fever and dengue hemorrhagic fever. In an effort to find effective tools for control programs to reduce mosquito populations, the authors assessed the acute toxicities of 14 monoterpenoids, trans-anithole and the essential oil of rosemary against different larval stages of Ae. aegypti. The potential for piperonyl butoxide (PBO) to act as a synergist for these compounds to increase larvicidal activity was also examined, and the oviposition response of gravid Ae. aegypti females to substrates containing these compounds was evaluated in behavioral bioassays.
RESULTS: Pulegone, thymol, eugenol, trans-anithole, rosemary oil and citronellal showed high larvicidal activity against all larval stages of Ae. aegypti (LC50 values 10.3-40.8 mg L-1). The addition of PBO significantly increased the larvicidal activity of all test compounds (3-250-fold). Eugenol, citronellal, thymol, pulegone, rosemary oil and cymene showed oviposition deterrent and/or repellent activities, while the presence of borneol, camphor and -pinene increased the number of eggs laid in test containers.
CONCLUSIONS: This study quantified the lethal and sublethal effects of several phytochemical compounds against all larval stages of Aedes aegypti, providing information that ultimately may have potential in mosquito control programs through acute toxicity and/or the ability to alter reproductive behaviorsWebsite DOI
42.Kennedy, CJ; Farrell, AP. (2008) Immunological alterations in juvenile Pacific herring, Clupea pallasi, exposed to aqueous hydrocarbons derived from crude oil.Environmental Pollution 153: 638-648 Immunological alterations in juvenile Pacific herring, Clupea pallasi, exposed to aqueous hydrocarbons derived from crude oil
fish; oil; immunology; disease; toxicity
The effects of acute and subchronic aqueous hydrocarbon exposures in the ppb range (0.2-127 mu g/L total PAH) on the immune system in Pacific herring (Clupea pallasi) were examined through specific immunocompetency assays and a host resistance model using Listonella anguillarum. Short-term hydrocarbon exposure at the highest concentration significantly enhanced respiratory burst activity (RBA) in macrophages and decreased plasma lysozyme concentrations, however, subchronic exposure (4-57 d) reduced RBA. Fish in the high exposure group were also less susceptible to the pathogen L. anguillarum following acute hydrocarbon exposure; however, this group was the most susceptible following subchronic exposures. These results are explained by a measured transient physiological stress response and long-term effects on ionoregulation. This study illustrates that hydrocarbon-elicited effects are dynamic and that toxic outcomes with respect to the teleost immune system depend on chemical concentrations and composition, exposure durations and the specific pathogen challenge. (C) 2007 Elsevier Ltd. All rights reserved. DOI
41.Kennedy, CJ; Tierney, KB. (2008) Energy intake affects the biotransformation rate, scope for induction, and metabolite profile of benzo[a]pyrene in rainbow trout.Aquatic Toxicology 90: 172-181 Energy intake affects the biotransformation rate, scope for induction, and metabolite profile of benzo[a]pyrene in rainbow trout
Benzo[a]pyrene; Rainbow trout; Biotransformation; Diet; Fasting; Hepatocytes
The metabolic conversion of benzo[a]pyrene (B[a]P) by rainbow trout (Oncorhynchus mykiss) hepatocytes was not significantly different between any group of fed fish (fed one of three isoenergetic diets that varied in protein and lipid content at full satiation levels or half rations), however at 12 weeks, fasted fish exhibited significantly reduced B[a]P biotransformation rates (by 58%). Alterations in metabolite profiles were also seen: fasted fish produced significantly more Phase I metabolites, higher levels of both glucuronide and sulphate conjugates, and lower levels of presumptive glutathione conjugates, compared to fed fish. When fish were fasted, higher proportions of phenols were produced, with lower proportions of quinones, triols and tetrols. Inducing metabolism (using beta-naphthoflavone) increased metabolic scope for B[a]P by 2-fold, regardless of each diet's baseline metabolic rate. However, the balance between Phase I and 11 reactions was altered with induction and fasting: higher proportions of Phase I metabolites were found, with lower glutathione conjugates and higher proportions of triols/tetrols. Fasting-mediated reductions in glutathione conjugation, and increased induction of oxidation vs. conjugating enzymes, can explain altered metabolite profiles. These results suggest that in contaminated habitats, where pollution-induced reductions in food quantity or quality are combined with the presence of toxic compounds and inducers, detoxification rates can be diminished. (C) 2008 Elsevier B.V. All rights reserved. DOI
40. Lizardo-Daudt, HM; Bains, OS; Singh, CR; Kennedy, CJ. (2008) Cadmium chloride-induced disruption of testicular steroidogenesis in rainbow trout, Oncorhynchus mykiss.Archives of Environmental Contamination and Toxicology 55: 103-110 Cadmium chloride-induced disruption of testicular steroidogenesis in rainbow trout, Oncorhynchus mykiss
Cadmium (Cd) is a known endocrine disruptor with the ability to affect the production of hormones involved in the regulation of reproductive processes. In the present study, the effects of CdCl2 on unstimulated and stimulated testicular steroidogenesis were examined with the intention of furthering the understanding of the potential site(s) of action in the signaling pathway for 11-KT synthesis in teleosts. In short-term (2-h) exposures, CdCl2 stimulated 11-KT production (29% and 28% over controls) in minced testicular tissues at concentrations of 10 and 100 mu M, respectively. However, 11-KT production was significantly lower than in controls (54%, 62%, and 54%) when tissues were incubated for 18 h with 1, 10, and 100 mu M Cd. Incubation of testicular tissues with 100 IU/ml human chorionic gonadotropin (hCG) and 5 mM dibutyryl-cAMP (dbcAMP), which activate rate-limiting steps in steroid synthesis, or 1.3 mu M 25-hydroxycholesterol (25-OHC), which augments production, resulted in significant increases in steroidogenesis over controls. hCG-stimulated steroidogenesis was reduced to 54% and 62% that of stimulated controls when tissues were incubated with CdCl2 at 1 and 10 mu M, respectively. 11-KT production in dbcAMP-stimulated and 25-OHC-augmented tissues was not affected by Cd exposure. The results of this study indicate that one site of action of Cd in the signaling steroidogenic pathway is located prior to cAMP formation. This impairment could be overcome when higher concentrations of Cd were used in hCG-stimulated cells, suggesting the presence of a stimulatory site at, or following, hCG receptor binding. DOI
39. Lizardo-Daudt, HM; Kennedy, C. (2008) Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages.Journal of Fish Biology 73: 702-718 Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages
cadmium; development; early life stages; endocrine disruption; trout
The sub-chronic (28-56 days) effects of exposure to low concentrations of cadmium (Cd; 0.05, 0.25, 0.50 and 2.50 mu g 1(-1)) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0.05 and 0.25 mu g 1(-1) Cd), however, delayed hatching was seen in the 2.50 mu g 1(-1) Cd group, with > 90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2.50 mu g l(-1) Cd were 13.9 +/- 0.8% shorter in total length (L-T) and weighed 22.4 +/- 3.5% (mean +/- S.E.) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0.05, 0.25 and 0.50 mu g 1(-1) Cd, respectively. These results suggest that environmentally realistic concentrations (in the mu g 1(-1) range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish. (C) 2008 The Authors Journal compilation (C) 2008 The Fisheries Society of the British Isles. DOI
38. Rudolph, BL; Andreller, I; Kennedy, CJ. (2008) Reproductive success, early life stage development, and survival of westslope cutthroat trout (Oncorhynchus clarki lewisi) exposed to elevated selenium in an area of active coal mining.Environmental Science & Technology 42: 3109-3114 Reproductive success, early life stage development, and survival of westslope cutthroat trout (Oncorhynchus clarki lewisi) exposed to elevated selenium in an area of active coal mining
The effects of accumulated Se on the reproductive success and larval development of cutthroat trout (Oncorhynchus clarki lewisi) collected from a site of active coal mining in British Columbia were assessed. Eggs from 12 fish from an exposed site (Clode Pond) and 16 from a reference site (O'Rourke Lake) were field-collected and reared in the laboratory. Egg Se concentrations ranged from 12.3 to 16.7 and 11.8 to 140.0 mu g/g dry weight (dw) from fish collected at the reference and exposed sites, respectively. Other studies, including those with this species, have not shown Se to affect egg viability; however, in the present study, eggs with Se concentrations > 86.3 mu g/g dw were not successfully fertilized or were nonviable at fertilization, while eggs with concentrations > 46.8 and < 75.4 mu g/g dw were fertilized (96% reached the eyed stage) but did not produce viable fry. A significant positive relationship between egg Se concentration and alevin mortality was observed. Deformities were analyzed in surviving fry which developed from eggs with Se concentrations between 11.8 and 20.6 mu g/g dw. No relationship between Se concentration in eggs and deformities or edema was found in this range, suggesting that the no-effect threshold for deformity is > 20.6 mu g/g dw. The present data, in conjunction with the data from several other studies in temperate fish, suggest that current Se thresholds are conservative for cold-water fish. DOI
37. Tierney, KB; Sampson, JL; Ross, PS; Sekela, MA; Kennedy, CJ. (2008) Salmon olfaction is impaired by an environmentally realistic pesticide mixture.Environmental Science & Technology 42: 4996-5001 Salmon olfaction is impaired by an environmentally realistic pesticide mixture
Many of the salmon-producing waterways of the world contain pesticides known to harm olfactory sensory neurons (OSNs) that are critically important throughout the salmon life cycle. The ability of OSNs to retain functionality after exposure to complex pesticide mixtures remains unknown. Here we show that a 96-h exposure to an environmentally realistic concentration of a mixture made from the ten most frequently occurring pesticides in British Columbia's Nicomekl River reduced the OSN responses of rainbow trout to a behaviorally relevant odorant. Odor-evoked responses were not altered by exposure to one fifth of the realistic concentration, and this may have been due an upregulation in detoxification enzymes, since glutathione-S-transferase activity reached a maximum (>32% above control) at this concentration. Mixture exposure did not help to prevent OSN impairment from a second, brief (5 min) exposure to a higher(20x) concentration of the mixture, suggesting longer-term, low-concentration exposures may not prevent damage from brief, high-concentration pulse exposures. This study demonstrates that environmentally observed pesticide mixtures can injure salmon olfactory tissue, and by extension, contribute to the threatened and endangered status of many salmon stocks. DOI
36. Lizardo-Daudt, HM; Bains, OS; Singh, CR; Kennedy, CJ. (2007) Biosynthetic capacity of rainbow trout (Oncorhynchus mykiss) interrenal tissue after cadmium exposure.Archives of Environmental Contamination and Toxicology 52: 90-96 Biosynthetic capacity of rainbow trout (Oncorhynchus mykiss) interrenal tissue after cadmium exposure
The disruption of endocrine system function in wildlife species, including teleosts, by contaminants such as metals is presently of major environmental concern. Recently, it has been shown that cadmium (Cd) exposure results in significant reductions in corticosteroid secretion by fish interrenal steroidogenic cells, likely through an inhibition of intracellular cortisol synthesis. In the present study, the effects of CdCl2 on unstimulated and stimulated interrenal steroidogenesis in rainbow trout were examined with the intention of furthering an understanding of the site(s) of Cd toxic action. CdCl2 alone reduced cortisol secretion in minced interrenal tissues to 59% and 55% of control values when exposed to 10 and 100 mu M, respectively. Incubation of interrenal tissues with 0.01 IU/mL adrenocorticotropic hormone (ACTH), which activates rate-limiting steps in steroid synthesis, resulted in significant stimulation of steroidogenesis in controls. However, ACTH-stimulated steroidogenesis was reduced when tissues were previously incubated with Cd. Maximal rates of unstimulated cortisol secretion were achieved by augmentation using 5 mu M 25-hydroxycholesterol (25-OHC) or 0.8 mu L/mL synthetic cholesterol [SyntheChol (R)(SC)]. Steroidogenesis augmentation by 25-OHC was significantly reduced in tissues incubated with Cd. Interestingly, cortisol secretion was significantly higher in SC-augmented tissue exposed to 1 and 10 mu M Cd when compared to augmented control tissues. The results of this study show that Cd affects both stimulated and unstimulated steroidogenesis in rainbow trout, and that one major site(s) of action of Cd in the cortisol synthesis pathway is likely prior to cytochrome P450 side chain cleavage.
35. Loveridge, AR; Bishop, CA; Elliott, JE; Kennedy, CJ. (2007) Polychlorinated biphenyls and organochlorine pesticides bioaccumulated in green frogs, Rana clamitans, from the lower fraser valley, British Columbia, Canada.Bulletin of Environmental Contamination and Toxicology 79: 315-318 Polychlorinated biphenyls and organochlorine pesticides bioaccumulated in green frogs, Rana clamitans, from the lower fraser valley, British Columbia, Canada
amphibians; pesticides; polychlorinated; biphenyls
Seven adult green frogs (Rana clamitans) were collected from three sites adjacent to intensive agriculture in the lower Fraser River valley, BC Canada. The highest mean concentrations of chemicals were pp'DDE at 0.313 mu g/g lipid wt. and Aroclor 1254/1260 at 2.12 mu g/g lipid wt. On a lipid weight basis, both pp'DDE and PCB concentrations varied by almost an order of magnitude among sites. Only ortho-substituted PCB congeners were detected. The concentrations of organochlorine pesticides and PCBs measured in these frogs from British Columbia are unlikely to elicit negative effects in frogs. DOI
34. Tierney, K; Casselman, M; Takeda, S; Farrell, T; Kennedy, C. (2007) The relationship between cholinesterase inhibition and two types of swimming performance in chlorpyrifos-exposed coho salmon (Oncorhynchus kisutch).Environmental Toxicology and Chemistry 26: 998-1004 The relationship between cholinesterase inhibition and two types of swimming performance in chlorpyrifos-exposed coho salmon (Oncorhynchus kisutch)
chlorpyrifos; acetylcholinesterase; salmon; swimming; performance
Brain acetylcholinesterase (AChE) activity was evaluated after two different swimming tests in coho salmon (Oncorhynchus kisutch; 238 +/- 5 g) given 96-h exposures to 0, 5, 10, 20, or 40 mu g/L of chlorpyrifos. Brain AChE activity decreased in a concentration-dependent manner (AChE activities were 81.8, 52.2, 37.3, and 21.3% of control for the 5, 10, 20, and 40 mu g/L exposures, respectively), whereas swimming performance was impaired after a threshold of AChE impairment was reached. Specifically, for swimming performance (U-crit measured using the established ramp-U-crit test (duration, 152 +/- 8 min), this threshold occurred with AChE activity of 68.5% +/- 18.1% of control. For a rapid acceleration test (U-Delta V, where V represents velocity; 27.6 +/- 0.8 min), this value was 52.6% +/- 15.4% of control. Both swim protocols resulted in similar maximum swim speeds (control ramp-U-crit and U-Delta V values of 3.44 +/- 0.09 and 3.71 +/- 0.13 body lengths/s, respectively), and performance was significantly reduced after 20 and 40 mu g/L exposures in both groups (ramp-U-crit values: 86.4 and 83.6%, respectively, of control; U-Delta V values: 85.2 and 77.8%, rsepectively, of control). Although both tests yielded similar swim speeds, postexercise plasma lactate concentrations were greater for the U-Delta V test (11.3 +/- 0.6 vs 8.6 +/- 0.5 mmol/L), indicating a greater anaerobic effort. This increase was exaggerated after 10 mu g/L of chlorpyrifos (14.6 +/- 1.3 mmol/L), indicating that anaerobic muscle was used to attain the same speed. Given the threshold relationship between AChE inhibition and swimming performance, coho salmon appear able to maintain integrated swimming activity despite significant impairment of an underlying neurological control mechanism.
33. Tierney, KB; Ross, PS; Kennedy, CJ. (2007) Linuron and carbaryl differentially impair baseline amino acid and bile salt olfactory responses in three salmonids.Toxicology 231: 175-187 Linuron and carbaryl differentially impair baseline amino acid and bile salt olfactory responses in three salmonids
olfaction; EOG; coho; sockeye; salmon; rainbow trout; carbaryl; linuron
For salmon, amino acid and bile salt detection form the basis for important behaviors including predator evasion and conspecific recognition, respectively. For this reason, decreases in olfactory sensory neuron responses to the amino acid i-serine and the bile salt taurocholic acid (TChA) have been used in studies as indicators of acute olfactory pesticide toxicity to environmental contaminants such as metals and pesticides. In this study, we first compare baseline responses to these two odorant classes across three salmonids, and then explore how two currently used pesticides alter these responses. We found baseline differences in electro-olfactogram (EOG) responses and their sensitivity to pesticide exposure between rainbow trout, coho and sockeye salmon. For example, rainbow trout had lower baseline EOGs than either coho or sockeye (e.g. 10(-5) NI TChA EOGs of 1.34 +/- 0.17 versus 2.57 +/- 0.46 and 2.72 +/- 0.43 mV, respectively). At 15 min after exposure to 10 mu g/L of the herbicide linuron, rainbow L-serine-evoked EOGs were 49.6% of control versus 78.5 and 69.8% for sockeye and coho, indicating rainbow were more sensitive to linuron. In contrast, at 30 min of exposure to 100 mu g/L carbaryl, L-serine-evoked EOGs of sockeye were 49.7% of control versus 60.3 and 62.3% for rainbow and coho, suggesting sockeye were more sensitive to carbaryl. In all species the L-serine-evoked EOGs did not return to baseline by 15 min after 100 mu g/L carbaryl exposure, suggesting persisting impairment of arnino acid detection. The TChA-evoked EOGs were less affected by carbaryl exposure (i.e. EOGs were 83.3, 84.9 and 66.0% of control 15 min after exposure) and not affected at all by 100 mu g/L linuron exposure. Species-specific differences in pesticide sensitivity may limit extrapolation of toxicity across salmonids while the generally greater sensitivity of amino acid olfaction may lead to selective impairment of behaviors such;IS predator evasion. (c) 2007 Published by Elsevier Ireland Ltd. DOI
32. Tierney, KB; Singh, CR; Ross, PS; Kennedy, CJ. (2007) Relating olfactory neurotoxicity to altered olfactory-mediated behaviors in rainbow trout exposed to three currently-used pesticides.Aquatic Toxicology 81: 55-64 Relating olfactory neurotoxicity to altered olfactory-mediated behaviors in rainbow trout exposed to three currently-used pesticides
rainbow trout; behavior; electro-olfactogram; amino acid; atrazine; roundup
Odor-evoked neurophysiological responses can form the basis for behavioral responses. Here we first characterized olfactory-mediated behavioral and neurophysiological responses of juvenile rainbow trout to the amino acid L-histidine, then looked at whether there were similar responses to the carbamate antisapstain IPBC and the herbicides atrazine and Roundup((R)), and lastly explored how exposures to these pesticides modified the L-histidine responses. Trout were behaviorally attracted to 10(-7) M L-histidine (as assayed in a counter-current olfactometer), but this preference behavior switched to indifference with higher histidine concentrations. Neurophysiologically, the summed electrical responses of peripheral olfactory neurons, as measured using electro-olfactogram (EOG), was 0.843 +/- 0.252 mV to 10(-7) M L-histidine. Of the pesticides, only Roundup((R)) evoked EOGs, indicating the amino acid-based pesticide may have acted as an odorant, and generated a behavioral response: it was avoided at active ingredient [AI; glyphosate isopropyl amine] concentrations >= 10 mg/l. With 30 min pesticide exposures, 10(-7) M L-histidine preference behavior was eliminated following exposure to 1 mu p/l IPBC and atrazine, and 100 mu g/l AI Roundup((R)). Similarly, 10(-7) M L-histidine-evoked EOGs were significantly reduced by exposure to 1 mu g/l IPBC, 10 mu g/l atrazine, and 100 mu g/l AI Roundup((R)). When combined together, the results demonstrate that typical preference behavior can be abolished when neurophysiological responses are reduced by > 60% of control. This asymmetry in response thresholds suggests that behavioral responses may be more sensitive toxicological endpoints than netirophysiological responses. (c) 2006 Published by Elsevier B.V. DOI
31.Kennedy, CJ; Farrell, AP. (2006) Effects of exposure to the water-soluble fraction of crude oil on the swimming performance and the metabolic and ionic recovery postexercise in pacific herring (Clupea pallasi).Environmental Toxicology and Chemistry 25: 2715-2724 Effects of exposure to the water-soluble fraction of crude oil on the swimming performance and the metabolic and ionic recovery postexercise in pacific herring (Clupea pallasi)
fish; oil; polycyclic aromatic hydrocarbons; swimming performance; exercise recovery
The swimming performance and recovery from exercise were determined in juvenile Pacific herring (Clupea pallasi) following exposure to the water-soluble fraction (WSF) of North Slope crude oil for more than eight weeks. Total polycyclic aromatic hydrocarbon concentrations (mean +/- standard error) at the beginning of exposures were as follows: control, 0.2 +/- 0.1 mu g/L; low, 9.6 +/- 2.5 mu g/L; medium, 40.7 +/- 6.9 mu g/L; and high, 120.2 +/- 11.4 mu g/L. Biological availability of hydrocarbons was confirmed by a significant induction of hepatic cytochrome P450 content and ethoxyresorufin-O-deethylase activity. Critical swimming speed (Ucrit) was significantly reduced in fish exposed to the highest concentration of WSF for 96 h (11% +/- 3.7% reduction) and at the two highest concentrations at four weeks (16% +/- 3.6% and 29% +/- 5.4% reductions) and eight weeks (11% +/- 3.8% and 40% +/- 5.7% reductions). Mortality occurred in all groups 24 h following Ucrit swim trials, with significantly higher mortalities observed in fish exposed to WSF in a concentration- and time-dependent manner (maximum mortality of 72.2% +/- 5.5% in the eight-week, high-exposure group). Burst swimming alone resulted in increased plasma cortisol, lactate, Na+, and Cl- concentrations and decreased muscle glycogen levels that returned to baseline values by 24 h. An interpretation of the effect of WSF exposure on postexercise metabolic recovery was complicated by pre-exercise alterations in several parameters. The time courses and magnitudes of several key postexercise parameters, including plasma cortisol, lactate, and muscle glycogen, were significantly altered by exposure to WSF. The present study clearly shows that hydrocarbon exposure can reduce the swimming ability of fish and their ability to recover from exhaustive exercise.
30. Tierney, KB; Ross, PS; Jarrard, HE; Delaney, KR; Kennedy, CJ. (2006) Changes in juvenile coho salmon electro-olfactogram during and after short-term exposure to current-use pesticides.Environmental Toxicology and Chemistry 25: 2809-2817 Changes in juvenile coho salmon electro-olfactogram during and after short-term exposure to current-use pesticides
salmon; pesticide; glyphosate; 3-iodo-2-propynyl-N-butyl carbamate; dichlorophenoxyacetic acid
For anadromous salmonids, olfaction is a critical sense, enabling return migration. In recent years, several pesticides have been identified that interfere with salmonid olfaction at concentrations in the mu g/L range; thus, they may pose a risk to species longevity. In the present study, we investigated the acute effects of five agricultural pesticides on juvenile coho salmon (Oncorhynchus kisutch) olfaction using the electro-olfactogram (EOG), a measure of odorant-evoked field potentials. Electro-olfactogram responses to the odorant L-serine were measured during and following a 30-min exposure of the left olfactory rosette to chlorothalonil, endosulfan, glyphosate acid, iodocarb (IPBC), trifluralin, and 2,4-dichlorophenoxyacetic acid. With the relatively insoluble pesticides endosulfan and trifluralin, decreases in EOG amplitude were only apparent at relatively high concentrations (100 and 300 mu g/L, respectively) following 20 min of exposure and were absent for chlorothalonil (1 mg/L). With the water-soluble herbicide glyphosate, significant EOG reductions occurred within 10 min of exposure to 1 mg/L and more rapidly with higher concentrations. Recovery of EOG post-glyphosate exposure was concentration-dependent, and complete recovery was not observed with some concentrations at 60 min postexposure. Dichlorophenoxyacetic acid only affected EOG at high concentration (100 mg/L), where it eliminated EOG within 2 min of exposure. With IPBC, EOG was decreased at 25 min of exposure to 1 mu g/L; higher concentrations caused decreases to occur more rapidly. Excluding IPBC and glyphosate, all FOG reductions occurred at concentrations greater than the current Canadian water-quality guidelines and reported 96-h lethality values. Our results show that olfactory neurons can be impaired rapidly by some current-use pesticides, even at exposures in the low-mu g/L range.
29. Tierney, KB; Taylor, AL; Ross, PS; Kennedy, CJ. (2006) The alarm reaction of coho salmon parr is impaired by the carbamate fungicide IPBC.Aquatic Toxicology 79: 149-157 The alarm reaction of coho salmon parr is impaired by the carbamate fungicide IPBC
coho salmon; Oncorhynchus kisutch; olfaction; alarm response; behavior; cortisol; carbamate; pesticide; IPBC
To determine whether the carbarnate fungicide IPBC alters the olfactory-mediated behavioral and physiologic alarm responses of coho salmon parr (Oncorhynchus kisutch), groups of coho were exposed to skin extract (an alarm pheromone source) under a variety of conditions. In the 3 min following skin extract exposure, freezing behavior was significantly increased (In the 3 min following skin extract exposure, freezing behavior was significantly increased under darkness (IR lighting) but not ambient lighting (25.3 +/- 2.6% and 7.5 +/- 5.7%, respectively; Delta calculated as: [(time (s) after/time (s) before) - 1] x 100%), and so IR was used for further experiments. Physiologically, following skin extract exposure, plasma cortisol concentration was increased at 0.5 h (58.1 +/- 14.6 ng/ml versus 4.32 +/- 1.31 ng/ml, exposed versus control), hematocrit (Hct) was increased at 2 h (50.4 +/- 1.0% versus 41.7 +/- 1.6%), and leucocrit (Lct) was decreased at 0.5 and 2 h (0.534 +/- 0.114 and 0.13 +/- 0.01% versus 1.23 +/- 0.20%). After 0.5 h exposures to 0, 1, 10 and 100 mu g/l IPBC and skin extract, the time spent dashing (> 5 cm/s) increased significantly (323 +/- 118%) in the first minute after skin extract exposure, but was absent in IPBC-exposed coho. Freezing behavior increased after skin extract exposure with control and 1 mu g/l IPBC exposures (11.0 +/- 3.0% and 17.7 +/- 11.0%, respectively), but was absent after 10 mu g/l and decreased after 100 mu g/l IPBC. Physiologically, Hct and plasma lactate concentration were significantly increased above controls after 1 mu g/l IPBC exposure (Hct: 45.7 +/- 1.6% versus 34.0 +/- 1.6%, lactate: 12.8 +/- 1.2 mM versus 3.30 +/- 1.2 mM). After 10 mu g/l exposure, IPBC alone elicited a stress response similar to skin extract. However in the 100 mu g/l treatment group the stress parameters were not different from controls. These findings suggest that the behavioral and physiologic alarm responses of juvenile salmonids may be impaired by acute exposure to >= 1 mu g/l IPBC. (c) 2006 Elsevier B.V. All rights reserved.
28. Bains, OS; Kennedy, CJ. (2005) Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P-glycoprotein substrate rhodamine 123.Toxicology 214: 87-98 Alterations in respiration rate of isolated rainbow trout hepatocytes exposed to the P-glycoprotein substrate rhodamine 123
P-glycoprotein; efflux; xenobiotic transport; rhodamine 123; rainbow trout; energetic costs
Reducing intracellular xenobiotic concentration is an important defence strategy used by cells challenged with foreign chemicals. One mechanism used to achieve this goal is via the use of P-glycoproteins (P-gps), ATP-dependent transporters that mediate the removal of hydrophobic compounds from cells. The energetic costs of this mechanism are unknown, therefore, the activity and respiratory costs associated with the P-gp-mediated efflux of rhodamine 123 (R123) was measured in isolated rainbow trout hepatocytes. The accumulation of R123 was rapid and concentration-dependent. Initial accumulation rates were 1.79 +/- 0.41, 7.29 +/- 1.06 and 15.30 +/- 1.74 ng R123/min/10(6) cells when exposed to 1, 5 and 10 mu M R123, respectively. Efflux was measured in cells 'pre-loaded' with R123 at each concentration, resulting in initial efflux rates of 0.77 +/- 0.12, 2.02 +/- 0.35 and 3.51 +/- 0.84 ng R123/min/10(6) cells, respectively. The baseline oxygen consumption rate of hepatocytes was 33.21 +/- 1.09 ng O-2/min/10(6) cells. Respiration rates were significantly higher in cells exposed to 5 and 10 mu M R123 (39.08 +/- 0.80 and 41.72 +/- 0.61 ng O-2/min/106 cells), representing increases over basal rates of 18.5 and 25.7%, respectively. Measurements of isolated mitochondrial respiration established that changes in hepatocyte oxygen consumption were not through the direct effects of R123 on mitochondria. The P-gp inhibitor, XR9576 significantly inhibited R123 efflux from cells with a concomitant return of respiration rates to baseline values. This study demonstrates that increased P-gp transport of xenobiotics can significantly raise cellular respiration rates and may result in higher energy costs for organisms living in P-gp-substrate contaminated environments. (c) 2005 Published by Elsevier Ireland Ltd.
27.Kennedy, CJ; Farrell, AP. (2005) Ion homeostasis and interrenal stress responses in juvenile Pacific herring, Clupea pallasi, exposed to the water-soluble fraction of crude oil.Journal of Experimental Marine Biology and Ecology 323: 43-56 Ion homeostasis and interrenal stress responses in juvenile Pacific herring, Clupea pallasi, exposed to the water-soluble fraction of crude oil
Clupea pallasi; cortisol; fish; ions; oil; osmoregulation; pacific herring; stress; toxicity
Juvenile Pacific herring, Clupea pallasi, were exposed both acutely (96 h) and chronically (9 weeks) to three concentrations of the water-soluble fraction (WSF) of North Slope crude oil. Mean ( +/- S.E.) total PAH (TPAH) concentrations at the beginning of the acute exposure experiment were: 9.7 +/- 6.5, 37.9 +/- 8.6 and 99.3 +/- 5.6 mu g/L. TPAH concentrations declined with time and the composition of the WSF shifted toward larger and more substituted PAHs. Significant induction of hepatic cytochrome P450 content, ethoxyresorufm O-deethylase and glutathione-S-transferase activities in WSF-exposed fish indicated that hydrocarbons were biologically available to herring. Significant but temporary, elevations in plasma cortisol (4.9-fold and 8.5-fold increase over controls in the 40 and 100 mu g/L groups, respectively), lactate (2.2-fold and 3. 1 -fold over controls in the 40 and 100 mu g/L groups) and glucose (1.3-fold, 1.4-fold and 1.6-fold over controls in the 10, 40 and 100 mu g/L groups) occurred in fish exposed acutely to WSF. All values returned to baseline levels by 96 h. Similar responses were seen with the first of several sequential WSF pulses in the chronic exposure study. Subsequent WSF pulses resulted in muted cortisol responses and fewer significant elevations in both plasma lactate and glucose concentrations. Hematocrit, leucocrit, hemoglobin concentration and liver glycogen content were not affected by acute or chronic WSF exposure. Plasma [Cl-], [Na+] and [K+] were significantly higher in the 100 mu g/L WSF-exposed group by 96 h compared to control fish, and continued to be elevated through the entire chronic exposure period. Unlike the measured stress parameters, ionoregulatory dysfunction was not modulated by WSF pulses. The results of this study suggest that chronic exposure to WSF affects at least two important physiological systems in herring: the ability of fish to maintain ion homeostasis and the interrenally-mediated organismal stress response. (c) 2005 Elsevier B.V. All rights reserved.
26. Bains, OS; Kennedy, CJ. (2004) Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss.Aquatic Toxicology 67: 217-226 Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss
pyrene; fish; rainbow trout; metabolism; detoxification; energy; costs
The respiratory costs of pyrene exposure and biotransformation were examined in isolated hepatocytes of adult rainbow trout, Oncorhynchus mykiss. Baseline oxygen consumption rates measured at an acclimation temperature of 7.5 degreesC and during an acute temperature increase to 15 degreesC were 10.1 +/- 0.1 and 22.6 +/- 10.4 ng O-2/min/mg cells, respectively. Hepatocytes exposed to pyrene at 1, 5 and 10 mug/ml exhibited concentration-dependent increases in oxygen consumption. Respiration rates of cells exposed to these concentrations at their acclimation temperature were 12.5 +/- 10.1, 14.7 +/- 0.1 and 17.1 =/- 0.2 ng O-2/min/mg cells, respectively. Exposure of cells to pyrene at 15 degreesC also elevated oxygen consumption to a maximum of 34.4 +/- 0.3 ng O-2/min/mg cells, however, the relationship with pyrene concentration was biphasic. The major metabolite identified through a series of solvent extractions, acid hydrolysis, and synchronous fluorometric spectroscopy was conjugated 1-hydroxypyrene. At 7.5 degreesC, increased pyrene metabolism correlated with increased hepatocyte respiration rates. At 15 degreesC, however, pyrene metabolism reached a maximum at 5 mug/ml, suggesting saturation of detoxification enzymes, which correlated with maximum respiration rates at this concentration. Measures of respiration by isolated mitochondria indicated that changes in hepatocyte oxygen consumption were not through direct effects of pyrene on mitochondria. This study indicates that significant respiratory costs may be accrued by teleost hepatocytes actively metabolizing and secreting xenobiotic compounds. (C) 2004 Elsevier B.V. All rights reserved.
25. Farrell, AP; Kennedy, CJ; Kolok, A. (2004) Effects of wastewater from an oil-sand-refining operation on survival, hematology, gill histology, and swimming of fathead minnows.Canadian Journal of Zoology-Revue Canadienne de Zoologie 82: 1519-1527 Effects of wastewater from an oil-sand-refining operation on survival, hematology, gill histology, and swimming of fathead minnows
This study examined the effects of various types of wastewater produced in oil-sand-refining on the survival, hematology, gill morphology, and swimming of caged fathead minnows (Pimephales promelas Rafinesque, 1820). At the reference site, all fish survived a 28-day exposure with unchanged hematocrit, leucocrit, and gill histology. In contrast, all fish did not survive a 28-day period in any of the wastewaters tested and, in some cases, they had all died within 96 h. In addition, the hematology or gill morphology of fish that had survived shorter exposure durations was found to be significantly altered; the changes included a significant decrease in lymphocytes and significant gill cellular hyperplasia and hypertrophy. The present data suggest that water remediation will be needed before the process wastewater from oil-sand-refining can support fish populations.
24. Jarrard, HE; Delaney, KR; Kennedy, CJ. (2004) Impacts of carbamate pesticides on olfactory neurophysiology and cholinesterase activity in coho salmon (Oncorhynchus kisutch).Aquatic Toxicology 69: 133-148 Impacts of carbamate pesticides on olfactory neurophysiology and cholinesterase activity in coho salmon (Oncorhynchus kisutch)
carbofuran; IPBC; mancozeb; electro-olfactogram; AChE
Many freshwater aquatic environments in the Pacific Northwest of North America contain neurotoxic pesticides, an issue of concern given the use of many of these habitats by Pacific salmon (Oncorhynchus sp.). Pesticides such as carbamates are known to affect fundamental physiological systems (such as the enzyme acetylcholinesterase (AChE)), and have been shown to affect salmonid olfactory-mediated behaviors. A neurophysiological measure of olfactory function, the electro-olfactogram (EOG), was used in this study to examine the impacts of acute localized exposure to three carbamates (the insecticide carbofuran, the antisapstain IPBC, and the fungicide mancozeb) on olfactory function in the coho salmon (Oncorhynchus kisutch). We also examine the potential for these pesticides to alter AChE levels in the,primary olfactory system and brain with brief exposures (30 min to only the olfactory rosette (OR)). In results, we find that the EOG in coho salmon is highly sensitive to brief localized exposures of two of these three carbamate pesticides. The effective nominal concentration required to cause a 50% reduction in EOG amplitude (EC50) for carbofuran was 10.4 mug/l and for IPBC was 1.28 mug/l. For mancozeb, the EC50 was higher at 2.05 mg/l. All three carbamates also affected AChE activity levels in the OR and brain (BR): carbofuran exposure at 200 mug/l significantly inhibited AChE activity in the OR, and both IPBC and mancozeb significantly increased AChE activity in BR at multiple concentrations with acute localized exposure. These carbamate effects highlight the sensitivity of salmon olfactory neurophysiology to pesticides acting not only potentially via AChE-inhibition, but also by other currently unknown modes of action. (C) 2004 Elsevier B.V. All rights reserved.
23.Kennedy, CJ; Higgs, D; Tierney, K. (2004) Influence of diet and ration level on benzo[a]pyrene metabolism and excretion in rainbow trout (Oncorhynchus mykiss).Archives of Environmental Contamination and Toxicology 47: 379-386 Influence of diet and ration level on benzo[a]pyrene metabolism and excretion in rainbow trout (Oncorhynchus mykiss)
Juvenile rainbow trout (Oncorhynchus mykiss) were fasted or fed one of three isoenergetic diets varying in protein and lipid content at full satiation levels or half rations for up to 9 weeks. At 3, 6, and 9 weeks, fish in each treatment group were dosed intraperitoneally with 10 mg tritiated benzo[a]pyrene [H-3]-B[a]P/kg (B[a]P) to examine the effects of diet composition and energy intake on xenobiotic biotransformation and excretion. The percent dose eliminated during the experiment did not differ among fish receiving the different diet compositions or rations (range 73% to 84%). However, it was significantly decreased (to 53%) in the group that was fasted for 9 weeks. Examination of fish fasted for 6 and 9 weeks showed a significant increase in the proportion of phase I metabolites and a concomitant decrease in the proportion of phase II metabolites found in bile compared with all other groups. Also, fish that were fasted for 9 weeks produced proportionately less 9,10-dihydroxy-benzo[a]pyrene-trans-9,10-diol, more 3-hydroxybenzo[a]pyrene and 9-hydroxybenzo[a]pyrene. and more glucuronic acid conjugates compared with all other groups. Thus, dietary protein and lipid concentration did not appear to affect either the rate of B[a]P metabolism or its excretion: however, prolonged fasting resulted in a shift in metabolite profiles and decreased excretion.
22. Morrow, MD; Higgs, D; Kennedy, CJ. (2004) The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss.Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 137: 143-154 The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss
biotransformation; diets; enzymes; fish; ethoxyresorufin O-deethylase; rainbow trout; ration; stress
Juvenile rainbow trout (Oncorhynchus mykiss) were fed one of three isoenergetic diets varying in protein (35-55%) and lipid content (8-18%), at full satiation levels or half rations for 6 weeks in order to investigate the effects of diet on baseline stress parameters and biotransformation enzyme activity. Growth was greatest in fish fed to satiation on a low protein and high lipid diet. Stress parameters, including plasma lactate, glucose and cortisol concentrations were not significantly affected by dietary treatment or ration. Basal biotransformation enzymes, including glutathione S-transferase (GST) and ethoxyresorufin O-deethylase (EROD) activity, were also unaffected by dietary treatment. Fish exposed to the biotransformation enzyme inducer beta-naphthoflavone did not exhibit an alteration in stress indicators or GST activity; however. EROD activity was increased (87- to 210-fold) in fish receiving all diets and rations. The results of the present study indicate that, unlike mammals, fish may be more recalcitrant to different levels of ingestion of isoenergetic diets varying in protein and lipid concentration with respect to stress responses and the maintenance of basal titers of biotransformation enzymes and their induction. (C) 2004 Elsevier Inc. All rights reserved.
21. Tierney, KB; Farrell, AP; Kennedy, CJ. (2004) The differential leucocyte landscape of four teleosts: juvenile Oncorhynchus kisutch, Clupea pallasi, Culaea inconstans and Pimephales promelas.Journal of Fish Biology 65: 906-919 The differential leucocyte landscape of four teleosts: juvenile Oncorhynchus kisutch, Clupea pallasi, Culaea inconstans and Pimephales promelas
basophil; eosinophil; leucocrit; leucocytes; monocyte; water quality
The appearance and proportions of morphologically distinct leucocytes (monocytes, thrombocytes, lymphocytes and diverse forms of granulocytes) encountered in the blood of four teleost species: coho salmon Oncorhynchus kisutch, Pacific herring Clupea pallasi, brook stickleback Culaea inconstans and fathead minnow Pimephales promelas, are presented, along with examples of how water quality influenced their relative proportions. The results clearly show that relative leucocyte number responds significantly and differently to changes in water quality. The value of using differential white cell counting as a quick and inexpensive component of assaying immune system alterations is discussed. (C) 2004 The Fisheries Society of the British Isles.
20. Tierney, KB; Stockner, E; Kennedy, CJ. (2004) Changes in immunological parameters and disease resistance in juvenile coho salmon (Oncorhynchus kisutch) in response to dehydroabietic acid exposure under varying thermal conditions.Water Quality Research Journal of Canada 39: 175-182 Changes in immunological parameters and disease resistance in juvenile coho salmon (Oncorhynchus kisutch) in response to dehydroabietic acid exposure under varying thermal conditions
dehydroabietic acid; immune system; disease; fish; salmon; temperature
This study explored the effects of a sublethal 96-h dehydroabietic acid (DHAA) exposure on aspects of the immune system of juvenile coho salmon under varying temperature conditions. Coho were exposed to DHAA concentrations below the determined LC50 value of 0.94 mg/L (95% confidence limits of 0.81 to 1.24 mg/L) for 96 h at either their acclimation temperature (8 or 18degreesC), or during an acute warm-shock (8 to 18degreesC) or cold-shock (18 to 8degreesC). Acclimation temperature alone significantly affected hematocrit (Hct), neutrophil respiratory burst activity (RBA) and leucocyte proportions. With temperature-shock, leucocrit (Lct), RBA and leucocyte proportions were altered. All parameters were affected by DHAA exposure, but not always in a dose-dependent manner. Across groups, DHAA caused Act, lysozyme, thrombocyte, neutrophil and monocyte proportions to increase, and Lct, RBA and lymphocyte proportions to decrease. DHAA-temperature interactions resulted in the exacerbation of DHAA-induced effects. Exposure temperature had the most significant effect on the susceptibility of coho to Aeromonas salmonicida; fish were more susceptible at cold temperatures and when subjected to a temperature-shock compared to their respective controls. DHAA exposure modulated the response of temperature-shocked fish to this pathogen.
19. Basu, N; Kennedy, CJ; Iwama, GK. (2003) The effects of stress on the association between hsp70 and the glucocorticoid receptor in rainbow trout.Comparative Biochemistry and Physiology A-Molecular & Integrative Physiology 134: 655-663 The effects of stress on the association between hsp70 and the glucocorticoid receptor in rainbow trout
fish; stress response; heat shock protein; glucocorticoid receptor; cortisol; beta-naphthoflavone
The purpose of this study was to characterize the association between hepatic heat shock protein 70 (hsp70) and the glucocorticoid receptor in rainbow trout that were exposed to heat stress, cortisol, and beta-naphthoflavone. This study is the first to document that the glucocorticoid receptor complex in rainbow trout hepatic tissues contains hsp70. Heat stress significantly increased levels of total cellular hsp70, and by discerning the association of hsp70 with the glucocorticoid receptor, we demonstrated that heat stress significantly increased the amount of hsp70 not bound to the glucocorticoid receptor, while significantly decreasing the amount of hsp70 bound to the glucocorticoid receptor. By calculating the ratio of hsp70 bound to the glucocorticoid receptor, to the total number of glucocorticoid receptors, stress (heat stress and cortisol-treatment) promoted the association of hsp70 with the glucocorticoid receptor. These findings demonstrate a functional and structural link between hsp70 and the glucocorticoid receptor in rainbow trout, and raise questions regarding the existence of a complex, interrelated stress response that spans all levels of biological organization within the whole animal. (C) 2002 Elsevier Science Inc. All rights reserved.
18.Kennedy, CJ. (2003) Uptake and accumulation of mercury from dental amalgam in the common goldfish, Carassius auratus.Environmental Pollution 121: 321-326 Uptake and accumulation of mercury from dental amalgam in the common goldfish, Carassius auratus
dental amalgam; mercury; accumulation; uptake; fish
In this study, the bioavailability and accumulation of mercury from external environmental exposure to mixed, cured, milled, sieved and proportioned dental amalgam was examined in the common goldfish, Carassius auratus. Fish were exposed to dental amalgam (particle size range from <0.10 to 3.15 mm) in order to represent the particle size and distribution of that found within the typical dental office wastewater discharge stream. Experimental amalgam water loadings were 0 g/l, 0.5 g/l and 1 g/l in glass aquaria at 15 &DEG;C for 28 days. Fish tissues were sampled at 5 min and 28 days of exposure, and the liver, brain, muscle and whole body analyzed for total mercury using cold vapor atomic fluorescence spectroscopy. Mercury was found in several tissues examined and generally increased with exposure to higher amounts of dental amalgam. The highest levels were found in the whole body (17.68 +/- 5.73 μg/g) followed by the liver (0.80 +/- 0.16 μg/g) and muscle (0.47 +/- 0.16 μg/g). The lowest concentrations were seen in the brain (0.28 +/- 0.19 μg/g). Compared to controls, concentrations in the whole body, muscle and liver in fish exposed for 28 days to the highest concentration of amalgam were 200-, 233-, and 40-fold higher, respectively. This study shows that mercury from an environmental exposure to representative samples of dental amalgam typically found within the dental wastewater discharge stream is bioavailable to fish and may accumulate in internal tissues. (C) 2002 Elsevier Science Ltd. All rights reserved.
17. Maltby, JB; Albright, LJ; Kennedy, CJ; Higgs, DA. (2003) Effect of route of administration and carrier on bioavailability and kinetics of astaxanthin in Atlantic salmon Salmo salar L.Aquaculture Research 34: 829-838 Effect of route of administration and carrier on bioavailability and kinetics of astaxanthin in Atlantic salmon Salmo salar L.
absorption; astaxanthin; bioavailability; i.p. injection; kinetics
This study examined astaxanthin bioavailability and kinetics in adult Atlantic salmon Salmo salar L., following two different routes of astaxanthin administration (oral vs. intraperitoneal (i.p.) injection) using two different carriers of the pigment (gelatin vs. sesame oil). The dorsal aorta of adult Atlantic salmon (mean initial weight 950 g) was cannulated. The fish received a single dose of astaxanthin (572 mug kg(-1)) in sesame oil or (514 mug kg(-1)) in gelatin via the oral or i.p. route, Plasma was sampled regularly up to 72 h post oral administration and up to 510 h post i.p. injection. The astaxanthin concentration-time curves from plasma were best fit to a one-compartment pharmacokinetic model for each of the four treatments. The gelatin carrier resulted in higher availability of astaxanthin compared to the sesame oil carrier. The bioavailability for astaxanthin in sesame oil was only 38.7% of that in gelatin by i.p. injection, and only 53.5% of that in gelatin by oral administration. Higher availability of astaxanthin was observed when i.p. injection was used compared to oral administration. The bioavailability for astaxanthin administered orally was only 12% of that by i.p. injection in sesame oil, and only 8.7% of that by i.p. injection in gelatin.
16. Basu, N; Kennedy, CJ; Hodson, PV; Iwama, GK. (2001) Altered stress responses in rainbow trout following a dietary administration of cortisol and beta-napthoflavone.Fish Physiology and Biochemistry 25: 131-140 Altered stress responses in rainbow trout following a dietary administration of cortisol and beta-napthoflavone
fish; generalized stress response; glucose; heat shock proteins; hsp70; lactate; polycyclic aromatic hydrocarbons; toxicants
Previous studies have demonstrated that fish inhabiting polluted waterways often have an impaired stress response at the organismal level. Given the possible link between the organismal (i. e. cortisol) and cellular (i. e. heat shock proteins; hsp) stress responses, we conducted this study to examine the ability of rainbow trout to respond to a 2 h, + 14 degreesC heat stress (HS) challenge following a 28 d, sub-chronic exposure to increased concentrations of cortisol (5 mg kg(-1) b. w.), beta-napthoflavone (bnf; 50 mg kg(-1) b.w.), and a combination of both (mixture), through the diet (1.5% b. w. every 48 h). While control fish responded to the HS by significantly increasing components of their organismal (cortisol, glucose, and lactate) and cellular (hepatic hsp70 protein) stress responses 6 and 24 h post HS, cortisol-, bnf-, and mixture-fed fish had impaired stress responses at both levels of organization. Additionally, hepatic hsp70 levels were significantly reduced 6 h post HS in cortisol- fed fish. While bnf- fed fish had significantly higher EROD activity, cortisol potentiated EROD activity in the mixture-fed fish. Similarly, plasma cortisol concentrations in the mixture-fed fish were significantly lower relative to cortisol-fed fish. These data are the first to indicate that sub-chronically stressed fish can have impaired stress responses at both the organismal and cellular levels. These findings raise questions regarding: (a) the universal and simple applicability of biological indicators of stress in fish; (b) the possible functional relationship between these two levels of stress responses; and (c) the importance of hsps in the generalized stress response of the whole organism.
15. Farrell, AP; Kennedy, C; Cheng, WN; Lemke, MA. (2001) Acute toxicity of monochloramine to juvenile chinook salmon (Oncorhynchus tshawytscha Walbaum) and Ceriodaphnia dubia.Water Quality Research Journal of Canada 36: 133-149 Acute toxicity of monochloramine to juvenile chinook salmon (Oncorhynchus tshawytscha Walbaum) and Ceriodaphnia dubia
chloramines; monochloramine; chinook salmon; Ceriodaphnia; LC50; TL50
A comprehensive data set for the acute toxicity of monochloramine to juvenile chinook salmon, Oncorhynchus tshawytscha, and the aquatic invertebrate Ceriodaphnia dubia are presented. For exposures up to 10 days, the equation LC50 = 7244t-(0.4525) (where LC50 = monochloramine concentration in mg/L for 50% lethality and t = time in minutes) can predict the LC50 value for monochloramine in juvenile chinook salmon and accounts for 94.4% of the variability in the experimental data. Predictions of acute toxicity were less reliable for concentrations in the range 0.5 mg/L to 0.8 mg/L since the LT50 value varied substantially in this range. Post-exposure mortality occurred only with exposures of >0.67 mg/L monochloramine. Feeding fish immediately before and during acute monochloramine exposure created a large chloramine demand in test chambers. In all tests where fish were fed, measured monochloramine concentrations were below detectable levels (<10 mg/L) and no fish mortality occurred. Monochloramine toxicity to the freshwater invertebrate Ceriodaphnia dubia was studied with time-to-lethality tests at 26 different concentrations. The equation LC50 = 61600t-(1.0748) predicted the LC50 value for chloramine in C. dubia and accounted for 94.8% of the variability in the experimental data. The mathematical equations that describe the LC50 for juvenile chinook salmon and C. dubia were used as reference lines for the evaluation of relative monochloramine toxicity Both juvenile chinook salmon and C. dubia were among the most sensitive fish and aquatic invertebrate species, respectively, C. dubia being more sensitive than juvenile chinook salmon for exposure durations >30 minutes.
14. Bendell-Young, LI; Bennett, KE; Crowe, A; Kennedy, CJ; Kermode, AR; Moore, MM; Plant, AL; Wood, A. (2000) Ecological characteristics of wetlands receiving an industrial effluent.Ecological Applications 10: 310-322 Ecological characteristics of wetlands receiving an industrial effluent
anthropogenic impacts; benthic community structure; ecosystem characteristics; fish acute lethality and stress; oil sands; wetlands
The primary objectives of this study were to evaluate the ecological characteristics of wetland ecosystems that had developed in response to oil sands effluent relative to reference wetland ecosystems and, from such an evaluation, to assess whether these wetlands were viable systems capable of integrating into the northern Canadian landscape. A secondary objective was to evaluate the use of several ecologically relevant endpoints as indicators of an ecosystem response to a known anthropogenic stress, in this case, wetlands receiving oil sands effluent. To achieve this, a suite of endpoints were compared between effluent-impacted wetlands and nonimpacted reference wetlands. Endpoints for comparison included: (1) benthic macroinvertebrate community structure, (2) chironomid density and biomass, (3) the incidence of chironomid mentum deformities, (4) the mutagenetic potential of sediment-dwelling chironomids, (5) growth and photosynthetic rare for the aquatic plant Typha latifolia (cattail), and (6) fish acute lethality and stress response as measured by changes in blood chemistry (percentage hematocrit [%hct], percentage leucocrit [%lct], and differential white blood cell count). Wetlands receiving oil sands effluent supported a low-diversity benthic community, dominated primarily by the Chironomidae and cattail. There was no evidence of mentum deformities or mutagenicity in chironomids sampled from the oil-impacted wetlands. Cattails grown in oil sands effluent and sediment demonstrated increased photosynthetic rates; however, these increased rates did not translate into increased plant growth. In contrast to the benthic community and the cattail, indigenous fish were unable to survive in wetlands containing oil sands effluent. Fish displayed altered blood chemistry (elevated %hct, depressed %lct) and ultimately death when held beyond 14 d in the oil-impacted wetlands. Of the various ecological endpoints measured, the macroinvertebrate community and changes in fish blood chemistry were the most sensitive indicators of an anthropogenic stress, demonstrating distinct differences in response between impacted and reference wetlands. To ensure that these wetlands can safely integrate into the northern Canadian landscape, future studies need to focus on their impacts at higher trophic levels indigenous to the wetland.
13.Kennedy, CJ; McDonald, LE; Loveridge, R; Strosher, MM. (2000) The effect of bioaccumulated selenium on mortalities and deformities in the eggs, larvae, and fry of a wild population of cutthroat trout (Oncorhynchus clarki lewisi).Archives of Environmental Contamination and Toxicology 39: 46-52 The effect of bioaccumulated selenium on mortalities and deformities in the eggs, larvae, and fry of a wild population of cutthroat trout (Oncorhynchus clarki lewisi)
To determine if elevated concentrations of waterborne selenium (Se), caused by coal mining, in the Elk River in southeastern British Columbia, may be causing reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory. Eggs from each female were reared separately and the percent mortalities and deformities were related to the selenium content of the eggs. Selenium concentrations in females from the exposed site were highest in the liver (36.6 +/- 22.5 mu g/g dry weight, range: 18.3 to 114), followed by the eggs (21.0 +/- 18.3 mu g/g, range: 8.7 to 81.3) and the muscle (12.5 +/- 7.7 mu g/g, range: 6.7 to 41). Despite these elevated egg Se concentrations, there was no significant effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry deformities or mortalities. Reproductive failure and embryonic terata have been reported at much lower egg Se concentrations in other fish species. The lack of any toxic response in this study may be due to an evolved tolerance to higher tissue Se concentrations in a population of fish living in a seleniferous river system.
12. Seubert, JM; Kennedy, CJ. (2000) Benzo[a]pyrene toxicokinetics in rainbow trout (Oncorhynchus mykiss) acclimated to different salinities.Archives of Environmental Contamination and Toxicology 38: 342-349 Benzo[a]pyrene toxicokinetics in rainbow trout (Oncorhynchus mykiss) acclimated to different salinities
The effects of environmental salinity on the distribution, metabolism, and elimination of benzo[a]pyrene (B[a]P) were examined in mature rainbow trout. Trout acclimated to either fresh water (0 ppt, FW) or sea water (20 ppt, SW) for 3 weeks received a single 10 mg/kg intra-arterial injection of [H-3]-benzo[a]pyrene (B[a]P) at their acclimation salinity or when subjected to an acute salinity change. Statistically significant differences in the percent body burden of B[a]P-derived radioactivity in various tissues were seen between fish in FW versus SW. Significant differences in the distribution of B[a]P and its metabolites were also noted when fish were subjected to an acute salinity change after chemical injection. Modulation of B[a]P metabolism by environmental salinity included: (1) significant differences in the proportions of Phase I metabolites in the bile of FW- (2.3%) versus SW-acclimated (14.1%) fish, and (2) alterations in the accumulations of specific metabolites (predominantly t-9, 10-dihydrodiol-B[a]P in FW fish, and 3-hydroxy-B[a]P in SW fish). The percentages of the [H-3]- B[a]P dose eliminated by 48 h was similar in FW and SW fish, but decreased in fish subjected to an acute salinity change (FW 98.8% eliminated, FW:SW 90.4%, SW 98.1%, and SW:FW 93.1%). Pharmacokinetic modeling confirmed that acute salinity changes can result in longer terminal half-lives and slower total body clearances of B[a]P.
11. Ganassin, RC; Sanders, SM; Kennedy, CJ; Joyce, EM; Bols, NC. (1999) Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus.Cell Biology and Toxicology 15: 299-309 Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus
Pacific herring; cell line; PAH toxicity; viral hemorrhagic septicemia
A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.
10. Johnston, BD; Alexander, G; Kennedy, CJ. (1999) Thermal modulation of the toxicokinetics of benzo[a]pyrene in isolated hepatocytes of sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops), and chub mackerel (Scomber japonicus).Comparative Biochemistry and Physiology C-Pharmacology Toxicology & Endocrinology 124: 157-164 Thermal modulation of the toxicokinetics of benzo[a]pyrene in isolated hepatocytes of sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops), and chub mackerel (Scomber japonicus)
benzo[a]pyrene; hepatocytes; temperature; toxicokinetics; Anoplopoma fimbria; Sebastes melanops; Scomber japonicus
Hepatocytes from sablefish (Anoplapoma fimbria), black rockfish (Sebastes melanops) and chub mackerel (Scomber japonicus) were isolated from 11 degrees C acclimated animals. The uptake, metabolism, and excretion of benzo[a]pyrene (B[a]P) in hepatocytes was measured at 6, 11 and 19 degrees C. Chub mackerel hepatocyte uptake rates were significantly lower (0.012 +/- 0.003 mu g/s per g cell) at 11 degrees C than black rockfish (0.028 +/- 0.009 mu g/s per g cell) or sablefish (0.032 +/- 0.012 mu g/s per g cell) hepatocytes at all temperatures. Hepatocytes metabolized B[a]P to phase I (1-8%) and phase II (92-99%) metabolites. Accumulation of phase II metabolites was lower in chub mackerel hepatocytes (0.016 +/- 0.004 mu g/h per g cell), than black rockfish (0.052 +/- 0.012 mu g/h per g cell), or sablefish hepatocytes (0.060 +/- 0.015 mu g/h per g cell). Phase II metabolite accumulation increased greatest with temperature in chub mackerel hepatocytes (Q(10) = 1.94 +/- 0.30), followed by sablefish (Q(10) = 1.65 +/- 0.30), and rockfish (Q(10) = 1.38 +/- 0.30). Sablefish hepatocytes had higher excretion rates of phase II metabolites (0.010 +/- 0.0023 mu g/h per g cell), than mackerel (0.0046 +/- 0.0009 mu g/h per g cell) or rockfish hepatocytes (0.0029 +/- 0.0008 mu g/h per g cell). Phase II metabolite excretion rates increased with temperature only in sablefish hepatocytes (Q(10) = 1.67 +/- 0.76). These differences in toxicokinetics may indicate distinct consequences for various species exposed to xenobiotics. (C) 1999 Elsevier Science Inc. All rights reserved.
9. Farrell, AP; Kennedy, CJ; Wood, A; Johnston, BD; Bennett, WR. (1998) Acute toxicity of a didecyldimethylammonium chloride-based wood preservative, Bardac 2280, to aquatic species.Environmental Toxicology and Chemistry 17: 1552-1557 Acute toxicity of a didecyldimethylammonium chloride-based wood preservative, Bardac 2280, to aquatic species
wood preservative; didecyldimethylammonium chloride; invertebrates; white sturgeon; salmon
The acute toxicity of the antisapstain wood preservative Bardac 2280 (principal active ingredient 80% didecyldimethylammonium chloride, DDAC) was tested with four fish and four aquatic invertebrate species. White sturgeon fry (Acipenser transmontanus) were the most sensitive fish species tested with a 24-h 50% lethal concentration (LC50) value between 1 and 10 ppb Bardac 2280. The 96-h LC50 values for the three other fish species ranged from 0.39 ppm for fathead minnows (Pimephales promelas) to 2.0 ppm for juvenile starry flounder (Platichthys stellatus). Developmental stage altered the sensitivity of coho salmon to Bardac 2280, with alevins being approximately twice as sensitive as smelts. Altering salinity up to 30 parts per thousand seawater had no significant effect on the toxicity of Bardac 2280 to coho smelts. The 48-h LC50 values for Daphnia magna, Mysidopsis bahia, Hyalella azteca, and Neomysis mercedis were 0.037 ppm, 0.039 ppm, 0.106 ppm, and 0.947 ppm, respectively. Bardac 2280 characteristically produced steep concentration-response curves for fish, i.e., a concentration range considerably less than 10-fold for a complete range of mortality, and caused no major symptoms of sublethal. stress in juvenile starry Bounder following a 24-h exposure.
8. Farrell, AP; Stockner, E; Kennedy, CJ. (1998) A study of the lethal and sublethal toxicity of polyphase P-100, an antisapstain fungicide containing 3-iodo-2-propynyl butyl carbamate (IPBC), on fish and aquatic invertebrates.Archives of Environmental Contamination and Toxicology 35: 472-478 A study of the lethal and sublethal toxicity of polyphase P-100, an antisapstain fungicide containing 3-iodo-2-propynyl butyl carbamate (IPBC), on fish and aquatic invertebrates
The acute toxicity of Polyphase P-100, an antisapstain wood preservative that contains 97% 3-iodo-2-propynyl butyl carbamate (IPBC), was determined for three species of fish (coho salmon, rainbow trout, and starry flounder) and three species of aquatic invertebrates (Daphnia magna, Hyalella azteca, and Neomysis mercedis). The 96-h LC50 values for the various fish species exposed to Polyphase P-100 ranged from 95 ppb for coho smelts (Oncorhynchus kisutch) to 370 ppm for juvenile starry flounder (Platichthys stellatus). The sensitivity of coho to Polyphase P-100 was altered by their developmental stage. Coho embryos were six to nine times more tolerant of Polyphase P-100 than coho alevins, which were twice as tolerant as coho smelts. The 48-h LC50 values for the invertebrates D. magna, H. azteca, and N. mercedis were 40 ppb, 500 ppb, and 2,920 ppb, respectively. In addition to a wider range of sensitivity to Polyphase P-100 compared with the fish species, the invertebrate species were characterized by a shallower concentration-response. In acute, 24-h sublethal tests with juvenile starry flounder and rainbow trout, there was no primary or secondary stress response (changes in hematocrit, leucocrit, hemoglobin concentration, plasma lactate concentration, and plasma cortisol concentration) at concentrations up to 50% of the 96-h LC50 value. The acute toxicity of a 1:8 mixture of Polyphase P-100 and Bardac 2280 (another antisapstain compound that contains didecyldimethylammonium chloride [DDAC] as the active ingredient) was close to additive for fish, but not for invertebrate species. The acute toxicity of the mixture was seven to eight times more than additive for H. azteca, but two to three times less than additive for D. magna. Some sublethal stress responses were revealed with the mixture that were not observed with the test chemicals alone.
7. Johnston, BD; Seubert, JM; Kennedy, CJ. (1998) Biochemical effects of didecyldimethylammonium chloride (DDAC) exposure and osmoregulatory stress on juvenile coho salmon, Oncorhynchus kisutch.Archives of Environmental Contamination and Toxicology 34: 275-279 Biochemical effects of didecyldimethylammonium chloride (DDAC) exposure and osmoregulatory stress on juvenile coho salmon, Oncorhynchus kisutch
The effects of a seawater challenge on coho salmon, Oncorhynchus kisutch, previously exposed to didecyldimethyl-ammonium chloride (DDAC) were examined, In one experiment, salmon were exposed to three sublethal concentrations of DDAC over three durations followed by a 24-h seawater challenge in a computer-controlled, intermittent-how respirometer to measure effects on several biochemical variables. After a 144-h dose, plasma cortisol, glucose, and gill Na+/K+-ATPase activity were significantly increased at a nominal DDAC concentration of 0.2 mg/L. In the second experiment, animals were exposed to five different concentrations for 24 h followed by a 24-h seawater challenge. Plasma cortisol was significantly increased at the highest exposure concentration (0.75 mg/L). Plasma Na+ was significantly elevated at exposure concentrations of 0.3, 0.5, 0.65, and 0.75 mg/L. Gill Na+/K+-ATPase activity was significantly reduced at exposure concentrations of 0.65 mg/L and 0.75 mg/L. The use of the seawater challenge to demonstrate sublethal physiological stress and impaired osmoregulatory capacity in coho salmon smelts is relevant to salmonid life history in terms of the animal's transition from freshwater to seawater during its seaward migration.
6. Wilson, JM; Vijayan, MM; Kennedy, CJ; Iwama, GK; Moon, TW. (1998) beta-naphthoflavone abolishes interrenal sensitivity to ACTH stimulation in rainbow trout.Journal of Endocrinology 157: 63-70 beta-naphthoflavone abolishes interrenal sensitivity to ACTH stimulation in rainbow trout
We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly effect either plasma turnover or tissue distribution of [H-3]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.
5. Lemke, MA; Kennedy, CJ. (1997) The uptake, distribution and metabolism of benzo[a]pyrene in coho salmon (Oncorhynchus kisutch) during the parr-smolt transformation.Environmental Toxicology and Chemistry 16: 1384-1388 The uptake, distribution and metabolism of benzo[a]pyrene in coho salmon (Oncorhynchus kisutch) during the parr-smolt transformation
benzo[a]pyrene; coho salmon; uptake; biotransformation; xenobiotics
Benzo[a]pyrene (BaP) uptake, distribution, and metabolism patterns were investigated from February to June during the transformation of freshwater coho salmon (Oncorhynchus kisutch) parr to smolts. At a BaP concentration of 5 mu g/L, uptake rates increased significantly from 0.01 +/- 0.000 mu g/g/h in February to 0.04 +/- 0.003 mu g/g/h in May and declined to 0.035 +/- 0.004 mu g/g/h in June. Following a 24-h exposure to BaP, the highest percent of body burden of BaP was found in the liver, gills, skin, and bile. The proportion of BaP in the liver and gills increased in fish from February to May and declined in June, whereas the proportion of BaP in the bile continued to rise until June when it reached a maximum of 49% of the body burden. The percent body burden of BaP in tissues such as the stomach, intestine, visceral fat, muscle, and brain did not show significant changes through the duration of the study. An analysis of bile suggests that both coho salmon parr and smolts are capable of metabolizing BaP via phase I and II biotransformation reactions to glucuronide, sulfate, and other conjugated metabolites. No significant changes occurred in the proportions of metabolite classes during the parr-smolt transformation process.
4. Seubert, JM; Kennedy, CJ. (1997) The toxicokinetics of benzo[a]pyrene in juvenile coho salmon, Oncorhynchus kisutch, during smoltification.Fish Physiology and Biochemistry 16: 437-447 The toxicokinetics of benzo[a]pyrene in juvenile coho salmon, Oncorhynchus kisutch, during smoltification
smoltification; coho; salmon; benzo[a]pyrene; biotransformation; toxicokinetics; enzymes; Oncorhynchus kisutch; pollution
The activities of Phase I and Phase II biotransformation enzymes in the livers of yearling coho salmon (Oncorhynchus kisutch), were measured biweekly from February until the release date from the hatchery in mid-June, in order to observe any alterations in baseline levels during smoltification. Peak enzyme activities occurred in February and March and then declined through to June. Total cytochrome P450 levels ranged from 0.024+/-0.009 to 0.095+/-0.010 nmol mg(-1) microsomal protein, ethoxyresorufin O-deethylase activity ranged from 2.74+/-0.75 to 9.94+/-0.85 pmol min(-1) mg(-1) microsomal protein, and glutathione S-transferase activity ranged from 0.07+/-0.01 to 0.33+/-0.01 mu mol min(-1) mg(-1) cytosolic protein during this period. Following an intraperitoneal injection of [H-3]benzo[a]pyrene (B[a]P), elimination occurred rapidly (>71% excreted into the bile within 24h) from February to June. Although the distribution of B[a]P in tissues changed through the sampling period, the highest levels of B[a]P-derived radioactivity were found in the liver, bile and fat. Analysis of the bile revealed that 55 to 63% of the radioactivity was Phase I metabolites, 16 to 24% glucuronide conjugates, 8% sulfate conjugates, 7% other conjugates and 6% aqueous-soluble metabolites. These findings suggest that the transformation from freshwater adapted coho 'parr' to 'smolts', can significantly alter biotransformation enzyme activities and the distribution and elimination of xenobiotics such as benzo[a]pyrene in these fish.
3. Vijayan, MM; Pereira, C; Forsyth, RB; Kennedy, CJ; Iwama, GK. (1997) Handling stress does not affect the expression of hepatic heat shock protein 70 and conjugation enzymes in rainbow trout treated with beta-naphthoflavone.Life Sciences 61: 117-127 Handling stress does not affect the expression of hepatic heat shock protein 70 and conjugation enzymes in rainbow trout treated with beta-naphthoflavone
trout; Oncorhynchus mykiss; heat shock protein 70; ethoxyresorufin O-deethylase; uridine-5'-diphosphoglucuronic acid transferase; gluconeogenesis; intermediary metabolism; stress; cortisol; glucose
A response in heat shock protein 70 (hsp 70) expression in the a-naphthoflavone (BNF) treated rainbow trout (Oncorhynchus mykiss) corresponded to altered metabolic status of the liver as evidenced by the lower phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase and 3-hydroxyacylcoA dehydrogenase activities. The BNF-induced increase in hsp70 levels and conjugation enzyme activities (phase I and phase II) were not modified by handling stress. Indeed handling stress did not affect either hsp 70 levels or conjugation enzyme activities in trout liver. The decrease in hepatic PEPCK activity in the BNF group may be responsible for the attenuation of the increase in liver glucose concentration after a 3 min handling stress in this species, suggesting that BNF affects liver gluconeogenic capacity in this species. Handling stress elicited a plasma cortisol and glucose response in both the sham and BNF group, however, the cortisol response with BNF was erratic compared with the sham, implying alterations in the cortisol dynamics post-stress. These results show fbr the first time that BNF affects cellular metabolic responses to stress and suggests the possibility of using hsp 70 as a biomarker for toxic effects in trout.
2. Wood, AW; Johnston, BD; Farrell, AP; Kennedy, CJ. (1996) Effects of didecyldimethylammonium chloride (DDAC) on the swimming performance, gill morphology, disease resistance, and biochemistry of rainbow trout (Oncorhynchus mykiss).Canadian Journal of Fisheries and Aquatic Sciences 53: 2424-2432 Effects of didecyldimethylammonium chloride (DDAC) on the swimming performance, gill morphology, disease resistance, and biochemistry of rainbow trout (Oncorhynchus mykiss)
The acute lethal and sublethal toxicity of didecyldimethylammonium chloride (DDAC) to juvenile rainbow trout (Oncorhynchus mykiss) was investigated. The nominal 96-h LC(50) value in a flow-through exposure system was 0.409 mg . L(-1). Investigation of the effects of acute sublethal exposures on blood chemistry, swimming performance, disease resistance, and gill morphology revealed no adverse effects after a 24-h exposure to 0.1 mg DDAC . L(-1). Primary and secondary biochemical indicators of stress, i.e., plasma cortisol, glucose, and lactate levels, were only significantly elevated after a 24-h exposure to DDAC at 0.4 mg . L(-1). However, swimming performance decreased significantly after 12- and 24-h exposures to both 0.2 and 0.4 mg . L(-1). Scanning electron microscopic analysis revealed no gross disruptions to gill epithelial surfaces as a result of toxicant exposure. A disease challenge test revealed that exposure to sublethal concentrations of DDAC for 24 h had no significant effects on the susceptibility of rainbow trout to lethal infection from Vibrio anguilarum after 14 d. The results suggest that DDAC has a very steep dose-response relationship during acute exposures. Those responsible for regulating the outflow of DDAC into fish-bearing waters should take this into account.
