Christopher J. Kennedy

Olfactory toxicity in fishes

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.

Handling stress does not affect the expression of hepatic heat shock protein 70 and conjugation enzymes in rainbow trout treated with β-naphthoflavone

A response in heat shock protein 70 (hsp 70) expression in the beta-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 for 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.

Larvicidal and oviposition-altering activity of monoterpenoids, trans-anithole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae).

BACKGROUND 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 (LC(50) 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 beta-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 behaviors.

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)

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 microg/L; low, 9.6 +/- 2.5 microg/L; medium, 40.7 +/- 6.9 microg/L; and high, 120.2 +/- 11.4 microg/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 WSE The present study clearly shows that hydrocarbon exposure can reduce the swimming ability of fish and their ability to recover from exhaustive exercise.

A biologically based toxicokinetic model for pyrene in rainbow trout

A biologically based toxicokinetic model was developed to stimulate the metabolic disposition of pyrene in trout with an average body weight of 450 g and dosed with a single bolus injection of the chemical (10 mg/kg). The model consists of a membrane-limited muscle compartment and six flow-limited compartments including the gills, liver, gut, kidney, carcass, and blood. The compartments are represented by mass balance equations including terms for the binding of pyrene to tissue and blood proteins, biotransformation, penetration rate into the muscle, blood flow rate, tissue mass, etc. The model also provides for nonsaturable and saturable clearances of pyrene by the liver and kidney. Michaelis-Menten constants for pyrene metabolism (Km, Vmax) were determined from in vitro experiments using isolated liver cells. Renal clearance of pyrene was very close to the glomerulus filtration rate of trout. Solution of the system of equations yielded the time courses of pyrene concentration in the tissues. Predicted concentrations of pyrene in the gills, liver, gut, kidney, muscle, and blood were consistent with experimental observations for at least 6 days. The model was validated by comparing the model predicted and experimental results of trout weighing 285 g and dosed with a single intraarterial dose (3 mg/kg) of pyrene. The predicted pyrene concentrations also were in adequate agreement with the empirical data.

Toxicity of atrazine and nonylphenol in juvenile rainbow trout (Oncorhynchus mykiss): effects on general health, disease susceptibility and gene expression.

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 μg/L and 555 μg/L) or NP (2.3 μg/L or 18 μg/L) for 4d. 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 μg/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 μg/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.

CHANGES IN JUVENILE COHO SALMON ELECTRO-OLFACTOGRAM DURING AND AFTER SHORT-TERM EXPOSURE TO CURRENT-USE PESTICIDES

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 microg/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 microg/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 microg/L; higher concentrations caused decreases to occur more rapidly. Excluding IPBC and glyphosate, all EOG 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-microg/L range.

The effects of stress on the association between hsp70 and the glucocorticoid receptor in rainbow trout

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.

The relationship between cholinesterase inhibition and two types of swimming performance in chlorpyrifos‐exposed coho salmon (Oncorhynchus kisutch)

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 microg/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 microg/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(deltav), 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(deltav) values of 3.44 +/- 0.09 and 3.71 +/- 0.13 body lengths/s, respectively), and performance was significantly reduced after 20 and 40 microg/L exposures in both groups (ramp-U(crit) values: 86.4 and 83.6 %, respectively, of control; U(deltav) 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(deltav) test (11.3 +/- 0.6 vs 8.6 +/- 0.5 mmol/L), indicating a greater anaerobic effort. This increase was exaggerated after 10 microg/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.

Immunotoxicological effects of a sub-chronic exposure to selected current-use pesticides in rainbow trout (Oncorhynchus mykiss)

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 microg/L and 2 microg/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 or 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 on 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.