Damsels in Distress: Oil Exposure Modifies Behavior and Olfaction in Bicolor Damselfish (Stegastes partitus).

In fishes, olfactory cues evoke behavioral responses that are crucial to survival; however, the receptors, olfactory sensory neurons, are directly exposed to the environment and are susceptible to damage from aquatic contaminants. In 2010, 4.9 million barrels of crude oil were released into the northern Gulf of Mexico from the Deepwater Horizon disaster, exposing marine organisms to this environmental contaminant. We examined the ability of bicolor damselfish (Stegastes partitus), exposed to the water accommodated fraction (WAF) of crude oil, to respond to chemical alarm cue (CAC) using a two-channel flume. Control bicolor damselfish avoided CAC in the flume choice test, whereas WAF-exposed conspecifics did not. This lack of avoidance persisted following 8 days of control water conditions. We then examined the physiological response to CAC, brine shrimp rinse, bile salt, and amino acid cues using the electro-olfactogram (EOG) technique and found that WAF-exposed bicolor damselfish were less likely to detect CAC as an olfactory cue but showed no difference in EOG amplitude or duration compared to controls. These data indicate that a sublethal WAF exposure directly modifies detection and avoidance of CAC beyond the exposure period and may suggest reduced predator avoidance behavior in oil-exposed fish in the wild.

Oil sands process-affected water impairs the olfactory system of rainbow trout (Oncorhynchus mykiss).

Oil sands process-affected water (OSPW), a byproduct of the extraction of bitumen in the surface mining of oil sands, is currently stored in massive on-site tailings ponds. Determining the potential effects of OSPW on aquatic ecosystems is of main concern to oil sands companies and legislators concerned about the reclamation of mining sites. In the present study, the interaction of OSPW with the chemosensory system of rainbow trout was studied. Using an electro-olfactography (EOG) technique, a 24 h inhibition curve was established and concentrations that inhibit the olfactory system by 20% and 80% (IC20 and IC80) were estimated at 3% and 22% OSPW, respectively. To study the interaction of exposure time and concentration along with the mechanism of the toxic effects, rainbow trout were exposed to 3% and 22% OSPW for 2, 24, and 96 h. An EOG investigation of olfactory sensitivity demonstrated a positive interaction between exposure time and concentration of OSPW concentration, because an increase in either or both elevated the inhibitory effect. To investigate whether or not structural damage of the olfactory epithelium could account for the observed inhibitory effects of OSPW on fish olfaction, the ultrastructure of the olfactory epithelium of exposed fish was investigated using scanning electron microscopy (SEM) and light microscopy (LM). The SEM micrographs showed no changes in the structure of the olfactory epithelium. The light micrographs revealed an increase in the number of mucous cells in 22% OSPW. The results of the present study demonstrated that exposure to OSPW impairs the olfactory system of rainbow trout and its effects increase gradually with increasing exposure time. The present study demonstrated that structural epithelial damage did not contribute to the inhibitory effects of OSPW on the olfactory system.

Use of electro-olfactography to measure olfactory acuity in the North American bullfrog (Lithobates (Rana) catesbeiana) tadpole.

Olfaction is an important sense for aquatic organisms because it provides information about their surroundings, including nearby food, mates, and predators. Electro-olfactography (EOG) is an electrophysiological technique that measures the response of olfactory tissue to olfactory stimuli, and responses are indicative of olfactory acuity. Previous studies have used this technique on a variety of species including frogs, salamanders, daphniids and, most extensively, fish. In the present study, we introduce a novel modified EOG method for use on Lithobates (Rana) catesbeiana tadpoles. Responses to a number of olfactory stimuli including amino acids, an algal extract (Spirulina), and taurocholic acid were tested, as measured by EOG. Tadpoles exhibited consistent and reliable responses to L-alanine and Spirulina extract. Tadpoles also exhibited concentration-dependent responses to Spirulina extract. These findings indicate that tadpole EOG is a viable electrophysiology technique that can be used in future research to study olfactory physiology and impairment in tadpoles.

Effects of Seasonal Changes on the Toxic Impacts of Oil Sands Process-Affected Water on Daphnia magna.

Oil sands process-affected water (OSPW), which can be potentially toxic to aquatic biota, is a major by-product of bitumen mining in northern Alberta. The effects of environmental factors on the toxicity of OSPW are understudied. In the present study, the impacts of seasonal changes in water quality on the toxic effects of OSPW (1 and 10%) on Daphnia magna was examined. Animals were chronically exposed to OSPW under conditions that represented water quality of a cold or warm seasonal condition. At each seasonal scenario survival, growth (length and mass) and reproduction of exposed D. magna were investigated. Survival and length of D. magna were only affected by OSPW in the cold-season treatment. Exposure to OSPW reduced the mass of D. magna in both cold and warm season scenarios. Daphnia magna in the cold-season treatment did not reproduce or produce eggs during the course of the experiment. The results of the present study suggest that seasonal changes in water quality may alter the toxicity of OSPW on D. magna.

Parasites and a host's sense of smell: reduced chemosensory performance of fathead minnows (Pimephales promelas) infected with a monogenean parasite.

Parasites residing within the central nervous system of their hosts have the potential to reduce various components of host performance, but such effects are rarely evaluated. We assessed the olfactory acuity of fathead minnows (Pimephales promelas) infected experimentally with the monogenean Dactylogyrus olfactorius, the adults of which live within the host's olfactory chambers. Olfactory acuity was compared between infected and uninfected hosts by assessing electro-olfactography (EOG) neural responses to chemical stimuli that indicate the presence of food (L-alanine) or the presence of conspecifics (taurocholic acid). We also compared differences in gross morphology of the olfactory epithelium in infected and uninfected minnows. Differences in EOG responses between infected and uninfected minnows to both cue types were non-significant at 30 days post-exposure. By days 60 and 90, coincident with a two times increase in parasite intensity in the olfactory chambers, the EOG responses of infected minnows were 70-90% lower than controls. When infected fish were treated with a parasiticide (Prazipro), olfactory acuity returned to control levels by day 7 post-treatment. The observed reduction in olfactory acuity is best explained by the reduced density of cilia covering the olfactory chambers of infected fish, or by the concomitant increase in the density of mucous cells that cover the olfactory chambers. These morphological changes are likely due to the direct effects of attachment and feeding by individual worms or by indirect effects associated with host responses. Our results show that infection of a commonly occurring monogenean in fathead minnows reduces olfactory acuity. Parasite-induced interference with olfactory performance may reduce a fish's ability to detect, or respond to, chemical cues originating from food, predators, competitors or mates.

A novel apparatus for evaluating contaminant effects on feeding activity and heart rate in Daphnia spp.

Cladoceran are animals of significant importance to freshwater bodies such that changes in their populations may result in drastic shifts in the food web. Numerous studies have investigated the effects of toxicants, and the effects of chemical and physical habitat changes to these animals. Most of these studies investigated more general endpoints such as mortality, reproduction, growth, and food consumption over time, and less frequently examined molecular endpoints such as enzyme activity or gene expression. However, behavioral and physiological endpoints that link the organism and molecular level endpoints are scarce. In this study, we designed an apparatus that allows for the simultaneous investigation of three essential behavioral and physiological endpoints in Daphnia, including ventilation, food uptake rate, and heart rate. Using our apparatus, we studied the effect of cadmium (Cd), suspended particles, and food on the beating rate of thoracic limbs and the frequency of mandible rolling in Daphnia magna. We also studied the effect of temperature on the heart and thoracic limb beat frequency. The results show that both Cd and suspended particles reduce the activity of mandibles and thoracic limbs. Thoracic limb movements and heart rate increased gradually with temperature. Our toxicity tests show that changes in feeding, ventilation, and heart rate are easily detected using this method.

Dactylogyrus olfactorius n. sp. (Monogenea, Dactylogyridae) from the olfactory chamber of the fathead minnow, Pimephales promelas Rafinesque (Cyprinidae).

Dactylogyrus olfactorius n. sp. (Monogenea) is described from the olfactory chamber of the fathead minnow Pimephales promelas Rafinesque in Alberta, Canada. The new species resembles Dactylogyrus bychowskyi Mizelle, 1937, D. bifurcatus Mizelle, 1937 and D. simplexus Mizelle, 1937, all parasites of Pimephales spp. in North America, in overall size and shape of the anchors and hooks, and in having a male copulatory complex with a tapered tubular penis and bifurcate accessory piece. Diagnostically, D. olfactorius n. sp. has relatively small anchors, hooks of anchor length, and a thin, long dorsal bar and no apparent ventral bar nor 4A hooks. Scanning electron microscopy revealed the body tegument of D. olfactorius n. sp. to be microvillous and in shallow annular folds, while that of D. bifurcatus, occurring on host gills of the same fish, was avillous and in gill-like folds dorsally and ventrally. Partial 28S rDNA sequences revealed significant differences between the two species, supporting establishment of D. olfactorius n. sp. and dispelling the possibility of ecophenotypic effects of site of attachment on morphology.

Metal-Polycyclic Aromatic Hydrocarbon Mixture Toxicity in Hyalella azteca. 1. Response Surfaces and Isoboles To Measure Non-additive Mixture Toxicity and Ecological Risk.

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) occur ubiquitously in aquatic environments, yet relatively little is known regarding their potential to produce non-additive toxicity (i.e., antagonism or potentiation). A review of the lethality of metal-PAH mixtures in aquatic biota revealed that more-than-additive lethality is as common as strictly additive effects. Approaches to ecological risk assessment do not consider non-additive toxicity of metal-PAH mixtures. Forty-eight-hour water-only binary mixture toxicity experiments were conducted to determine the additive toxic nature of mixtures of Cu, Cd, V, or Ni with phenanthrene (PHE) or phenanthrenequinone (PHQ) using the aquatic amphipod Hyalella azteca. In cases where more-than-additive toxicity was observed, we calculated the possible mortality rates at Canada's environmental water quality guideline concentrations. We used a three-dimensional response surface isobole model-based approach to compare the observed co-toxicity in juvenile amphipods to predicted outcomes based on concentration addition or effects addition mixtures models. More-than-additive lethality was observed for all Cu-PHE, Cu-PHQ, and several Cd-PHE, Cd-PHQ, and Ni-PHE mixtures. Our analysis predicts Cu-PHE, Cu-PHQ, Cd-PHE, and Cd-PHQ mixtures at the Canadian Water Quality Guideline concentrations would produce 7.5%, 3.7%, 4.4% and 1.4% mortality, respectively.

Metal-Polycyclic Aromatic Hydrocarbon Mixture Toxicity in Hyalella azteca. 2. Metal Accumulation and Oxidative Stress as Interactive Co-toxic Mechanisms.

Mixtures of metals and polycyclic aromatic hydrocarbons (PAHs) are commonly found in aquatic environments. Emerging reports have identified that more-than-additive mortality is common in metal-PAH mixtures. Individual aspects of PAH toxicity suggest they may alter the accumulation of metals and enhance metal-derived reactive oxygen species (ROS). Redox-active metals (e.g., Cu and Ni) are also capable of enhancing the redox cycling of PAHs. Accordingly, we explored the mutual effects redox-active metals and PAHs have on oxidative stress, and the potential for PAHs to alter the accumulation and/or homeostasis of metals in juvenile Hyalella azteca. Amphipods were exposed to binary mixtures of Cu, Cd, Ni, or V, with either phenanthrene (PHE) or phenanthrenequinone (PHQ). Mixture of Cu with either PAH produced striking more-than-additive mortality, whereas all other mixtures amounted to strictly additive mortality following 18-h exposures. We found no evidence to suggest that interactive effects on ROS production were involved in the more-than-additive mortality of Cu-PHE and Cu-PHQ mixtures. However, PHQ increased the tissue concentration of Cu in juvenile H. azteca, providing a potential mechanism for the observed more-than-additive mortality.

Biological effects and toxicity of diluted bitumen and its constituents in freshwater systems.

Approximately 50 billion cubic meters of bitumen resides within the oil sands region of Alberta, Canada. To facilitate the transport of bitumen from where it is extracted to where it is processed, the bitumen is diluted with natural gas condensate ('dilbit'), synthetic crude from hydrocracking bitumen ('synbit'), or a mixture of both ('dilsynbit'). A primary consideration for the effects of diluted bitumen products on freshwater organisms and ecosystems is whether it will float on the water surface or sink and interact with the stream or lake sediments. Evidence from a spill near Kalamazoo, MI, in 2010 and laboratory testing demonstrate that the nature of the spill and weathering of the dilbit, synbit or dilsynbit prior to and during contact with water will dictate whether the product floats or sinks. Subsequent toxicological data on the effects of dilbit and other diluted bitumen products on freshwater organisms and ecosystems are scarce. However, the current literature indicates that dilbit or bitumen can have significant effects on a wide variety of toxicological endpoints. This review synthesizes the currently available literature concerning the fate and effects of dilbit and synbit spilled into freshwater, and the effects of bitumen and bitumen products on aquatic organisms and ecosystems. Dilbit is likely to provide ecological impacts that are similar to and extend from those that follow from exposure to lighter crude oil, but the prospect of bitumen settling after binding to suspended sediments elevates the risk for benthic impacts in streams and lakes.

Recovery of Olfactory Mediated Behaviours of Fish from Metal Contaminated Lakes.

Fish mediate many biological processes by olfaction, which can be impaired by contaminants (i.e. metals). While the olfactory recovery of fish from metal contaminated lakes if subsequently cultured in clean water has been shown at the neurophysiological level, the recovery potential of olfactory mediated behaviours remains unknown. To study behavioural recovery of fish from metal contaminated lakes, wild yellow perch (Perca flavescens) were collected from two metal-contaminated lakes (Ramsey and Hannah lakes) in the metal-mining district of Sudbury, ON, Canada and cultured in clean water from a reference lake (Geneva Lake) for another 24 h. Olfactory mediated behaviours of the test organisms were tested using avoidance responses to conspecific skin extract. While olfactory mediated behaviours of fish from Ramsey Lake (low contamination) recovered after 24 h in clean water, recovery could not be observed in fish from Hannah Lake (high contamination). These results demonstrate that the recovery of behavioural deficits of fish from metal contaminated lakes is depending on the habitats' metal concentration.

Smelling salt: calcium as an odourant for fathead minnows.

Calcium plays an essential role in olfactory sensory neuron function. Studies with fish have indicated that in addition to being involved in olfactory signalling, calcium is itself an odourant. In this study we used fathead minnows (Pimephales promelas) and employed two different techniques; electro-olfactography (EOG), a neurophysiological technique that measures olfactory acuity at the olfactory epithelium, and a behavioural choice assay using a trough maze. The results demonstrate that calcium and a known odourant L-arginine are cross-adaptive, that calcium induces an EOG response in a concentration-dependent manner, and that calcium induces a strong avoidance behaviour. The behavioural avoidance was also demonstrated to be olfactory-dependent. Taken together, the results demonstrate that calcium is a potent odourant for fathead minnows. Being able to smell calcium may represent an ability to sense and avoid areas with significant changes in ionic strength, thereby avoiding physiological stress.

Chemosensory mediated behaviors and gene transcription profiles in wild yellow perch (Perca flavescens) from metal contaminated lakes.

The olfactory system of fish is sensitive to the toxic effects of low concentrations of contaminants. To investigate the effects of long-term metal exposure on olfaction in wild yellow perch (Perca flavescens), fish from one clean (Geneva Lake) and two metal-contaminated lakes (Ramsey and Hannah lakes) were collected in and around the metal-mining district of Sudbury, ON. Two different techniques were used to measure the effects of exposure to environmental contamination: (i) behavioral responses were recorded in response to conspecific skin extract and (ii) gene transcription differences in olfactory rosettes were characterized using a novel, 1000-candidate gene yellow perch microarray. Behavioral assays performed on fish from the clean lake demonstrated avoidance of a conspecific skin extract, while fish from metal contaminated lakes showed no avoidance response. A total of 109 out of the 1000 genes were differentially transcribed among the lakes. Most of the differentially transcribed genes were between the two metal contaminated lakes relative to either of the contaminated lakes and the reference lake. No genes were differentially expressed between Geneva Lake (clean) and Hannah Lake (metal contaminated). These results demonstrated that even though the different populations of fish from both Hannah and Ramey lakes were affected at the behavioral level, the impairment of olfaction was not measurable using gene transcriptional changes in olfactory rosettes.

Olfactory recovery of wild yellow perch from metal contaminated lakes.

Fish depend on their sense of smell for a wide range of vital life processes including finding food, avoiding predators and reproduction. Various contaminants, including metals, can disrupt recognition of chemical information in fish at very low concentrations. Numerous studies have investigated metal effects on fish olfaction under controlled laboratory conditions. However, few have measured olfactory acuity using wild fish in source water. In this study, we used electro-olfactography (EOG) to measure the olfactory acuity of wild yellow perch (Perca flavescens) from a clean lake (Geneva Lake) and two metal contaminated lakes (Ramsey and Hannah lakes) from Sudbury, ON, in their own lake water or in water from the other lakes. The results showed that fish from the clean lake had a greater olfactory acuity than those from metal contaminated lakes when fish were tested in their own lake water. However, when fish from the clean lake were held for 24h in water from each of the two contaminated lakes their olfactory acuity was diminished. On the other hand, fish from the contaminated lakes held for 24h in clean lake water showed a significant olfactory recovery relative to that measured in their native lake water. These results show that although fish from a clean lake demonstrated impaired olfaction after only 24h in metal-contaminated water, fish from metal contaminated lakes showed a rapid olfactory recovery when exposed to clean water for only hours.

Effects of continuous copper exposure and calcium on the olfactory response of fathead minnows.

The current gill-based Biotic Ligand Model (gbBLM) is an acute-toxicity model used to predict site-specific safe copper (Cu) concentrations. Recent effort to develop a chronic BLM has focused on the olfactory epithelium. To further this effort, the current study looked at the effect of varying Cu concentration and exposure duration on Cu-induced olfactory dysfunction, and whether calcium (Ca) protected against Cu-induced impairment as it does at the gill. Fathead minnows (Pimephales promelas) were treated with five Cu concentrations for varying exposure durations in hard and soft water. A neurophysiological technique, electro-olfactography (EOG), was employed to determine the level of olfactory dysfunction. At the low, ecologically relevant Cu concentrations tested there was significant inhibition of EOG function; however, over time there was at least a partial recovery of olfactory function, despite the continuous Cu exposure. Calcium did not appear to protect against Cu-induced olfactory dysfunction; and even alone, Ca appeared to interfere with the olfactory response to the amino acid L-arginine. Safe copper concentrations as predicted by the gbBLM, chemosensory-based BLMs, the USEPA BLM, and hardness-adjustment equations based on the exposure waters were not entirely protective against olfactory dysfunction.

Electroantennogram measurement of the olfactory response of Daphnia spp. and its impairment by waterborne copper.

In this study an electroantennogram (EAG) method was developed for use on live daphniids. The EAG response of Daphnia magna and Daphnia pulex to a variety of amino acids was measured. The strongest response measured was elicited by L-arginine and was shown to induce a concentration-dependent response indicating the response is olfactory in nature. Subsequent exposures of D. magna to a low, ecologically-relevant concentration of copper (7.5 µg/L) showed a disruption in EAG function. This study utilizes the development of an EAG method for measuring olfactory acuity of live daphniids and demonstrates that at ecologically-relevant concentrations, the olfactory dysfunction caused by copper can be detected. The EAG technique is a useful tool for investigating the olfactory response of daphniids to odourants at the cellular level and detecting the effects of toxicants on the olfactory acuity of daphniids.

Interaction of the Olfactory System of Rainbow Trout (Oncorhynchus mykiss) with Diltiazem.

Diltiazem is ubiquitously prescribed and has been reported in many effluents and freshwater bodies. Being a calcium channel blocker, diltiazem could disrupt the function of the sensory and central nervous systems. In the present study, using electro-olfactography (EOG), we investigated the interaction of diltiazem with the olfactory sensory neurons (OSNs) of rainbow trout by looking into the detection threshold and effects of immediate (~5 min) and acute (24 h) exposure to diltiazem at 6.6, 66, and 660 µg/L. We also studied the accumulation of the drug in fish plasma and whole body. Brief exposure to diltiazem impaired the OSN response to a chemosensory stimulus in a concentration-dependent manner at 6.6 µg/L and higher, whereas OSNs exposed for 24 h only displayed an impairment at 660 µg/L. Chemical analysis showed that the accumulation of diltiazem in fish plasma and body correlated with the EOG response because it was 10 times higher in the group that displayed a significant impairment (660 µg/L) compared to the other 2 groups (6.6, 66 µg/L). This correlation suggests that the impact of diltiazem on OSNs might partially be through the accumulated molecules in the fish bloodstream. Fish did not detect diltiazem as a sensory stimulus even at concentrations as high as 660 µg/L; thus, fish could potentially swim toward or fail to escape harmful concentrations of diltiazem. Environ Toxicol Chem 2022;41:554-550. © 2020 SETAC.

Emerging investigator series: use of behavioural endpoints in the regulation of chemicals.

Interest in behavioural ecotoxicology is growing, partly due to technological and computational advances in recording behaviours but also because of improvements of detection capacity facilitating reporting effects at environmentally relevant concentrations. The peer-reviewed literature now contains studies investigating the effects of chemicals, including pesticides and pharmaceuticals, on migration, dispersal, aggression, sociability, reproduction, feeding and anti-predator behaviours in vertebrates and invertebrates. To understand how behavioural studies could be used in regulatory decision-making we: (1) assessed the legal obstacles to using behavioural endpoints in EU chemicals regulation; (2) analysed the known cases of use of behavioural endpoints in EU chemicals regulation; and (3) provided examples of behavioural endpoints of relevance for population level effects. We conclude that the only legal obstacle to the use of behavioural endpoints in EU chemicals regulation is whether an endpoint is considered to be relevant at the population level or not. We also conclude that ecotoxicity studies investigating behavioural endpoints are occasionally used in the EU chemicals regulation, and underscore that behavioural endpoints can be relevant at the population level. To improve the current use of behavioural studies in regulatory decision-making contribution from all relevant stakeholders is required. We have the following recommendations: (1) researchers should conduct robust, well-designed and transparent studies that emphasize the relevance of the study for regulation of chemicals; (2) editors and scientific journals should promote detailed, reliable and clearly reported studies; (3) regulatory agencies and the chemical industry need to embrace new behavioural endpoints of relevance at the population level.

An epithelium is not just an epithelium: Effects of Na, Cl, and pH on olfaction and/or copper-induced olfactory deficits.

While the toxic effects of several substances on fish olfaction are well known, studies on how water chemistry affects contaminant-induced olfactory toxicity are rare. In the present study, the effect of water pH or Na concentration on fish olfactory response and Cu-induced olfactory toxicity was investigated. Also, the effects of two sodium salts, NaCl and NaNO3, on olfaction were studied. Juvenile rainbow trout were exposed to 6 and 32 µg/L Cu, each under five different conditions (pH 9, pH 6.5, 20 or 40 mg/L sodium added, or culture water), for 10 days before characterizing fish olfactory response using electro-olfactography (EOG). The results demonstrated that reducing the pH to 6.5 or adding 20 or 40 mg/L Na impairs the fish response to a standard olfactory cue. None of the water treatments were protective against, or synergic with, the toxic effect of Cu on the olfactory system. Of the two Na salts, NaCl caused significantly higher impairment than NaNO3. The results of the present study demonstrate that water quality modifies contaminant-induced olfactory toxicity, but differently than what is known for other tissues (i.e. gill).

The effects of diltiazem on growth, reproduction, energy reserves, and calcium-dependent physiology in Daphnia magna.

With the growth of both the pharmaceutical industry and the human population and longevity, more drugs are used and processed each day. Inevitably, these pharmaceuticals enter wastewater through human excretion and improper disposal of leftovers. One such medication, diltiazem, a calcium channel blocker, is of importance due to its widespread consumption, and prevalence in aquatic environments. To study the sub-lethal effects of diltiazem on aquatic animals, we investigated its impacts no feeding behaviour, heart rate, respiration, growth, and reproduction of a bioindicator species, Daphnia magna. When exposed to environmentally relevant concentrations, D. magna increased their heart rate by 12% and oxygen consumption by 48%. However, exposure did not have any effects on thoracic limb movement frequency or peristalsis (i.e. feeding behaviour). Individuals exposed to diltiazem for a longer duration (16 days) showed a 44% decrease in lipid reserves and produced between 17 and 28% fewer neonates which were 10-12% larger. Our study demonstrated that exposure to diltiazem creates an energy imbalance in D. magna which could, in the long run, influence their populations.