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.

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.

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.

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).

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.

Challenges and future prospects for developing Ca and Mg water quality guidelines: a meta-analysis.

Anthropogenic activities have the potential to increase water hardness (Ca + Mg) in receiving waters to toxic concentrations, and thus, water quality guidelines (WQG) for Ca and Mg are warranted. However, Ca can modify Mg toxicity in Ca-poor water and additional interactions with other major ions (Na+, K+, HCO3-/CO32-, SO42- and Cl-) may occur, potentially obscuring the water hardness-effect relationship. In a meta-analysis of toxicological studies, we: (i) evaluate the performance of three WQG derivation methods, and (ii) determine the influence of several variables (acute/chronic data, anions, Ca:Mg ratios, non-geographically relevant species) on the models. We find that the most sensitive species- or species sensitivity distribution (SSD)-based WQG derivation methods greatly overestimate water hardness toxicity, particularly if non-resident species are included. Broad-scale implementation of most sensitive species- or SSD-based WQG is impractical because water hardness varies beyond and within the regional scale. Anion type does not affect water hardness toxicity across species, but the Ca : Mg ratio is toxicologically relevant, underscoring the importance of considering ion ratios when developing major ion WQG. Although data supporting formal water hardness WQG are unavailable, we suggest using a two-component background condition approach that supports simultaneous management of water hardness and Ca : Mg ratio, and WQG that are applicable beyond the regional scale.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

Nickel toxicity in wood frog tadpoles: Bioaccumulation and sublethal effects on body condition, food consumption, activity, and chemosensory function.

Nickel (Ni) concentrations in aquatic ecosystems can be amplified by anthropogenic activities including resource extraction. Compared with fish and invertebrates, knowledge of Ni toxicity in amphibians is limited, especially for northern species. We examined the effect of Ni on wood frog (Lithobates sylvaticus) tadpoles, the species with the widest and most northern distribution of any anuran in North America. Wood frog tadpoles were exposed to a Ni concentration gradient (0.02-5.5 mg/L of Ni at 164 mg/L as CaCO3 water hardness) for 8 d and examined for lethality, Ni bioaccumulation, and several sublethal endpoints including body condition, food consumption, activity, and chemosensory function. Nickel induced a sublethal effect on body condition (8-d 10 and 20% effect concentrations [EC10 and EC20] of 1.07 ± 0.38 and 2.44 ± 0.51 mg/L of Ni ± standard error [SE], respectively) but not on food consumption, activity, or chemosensory function. Nickel accumulation in tadpole tissues was positively related to an increase in aqueous Ni concentration but was not lethal. Both the acute and chronic US Environmental Protection Agency water quality guideline concentrations for Ni (0.71 and 0.08 mg/L at 164 mg/L as CaCO3 water hardness, respectively) were protective against lethal and sublethal effects in wood frog tadpoles. In the present study, wood frog tadpoles were protected by current water quality guidelines for Ni and are likely not as useful as other taxa for environmental effects monitoring for this particular metal. Environ Toxicol Chem 2018;37:2458-2466. © 2018 SETAC.

Fish can smell trace metals at environmentally relevant concentrations in freshwater.

The objective of the present study was to investigate the ability of the olfactory system of rainbow trout (Oncorhynchus mykiss) to detect three trace metals, cadmium (Cd), copper (Cu), and nickel (Ni), using electro-olfactography (EOG). The olfactory response to all three metals was measured at either 10-6 M or at a concentration established by Alberta Environment and Parks (AEP) as the criterion for the protection of aquatic life. Results of the present study demonstrated that the olfactory system of rainbow trout can detect all three metals (i.e. Cd, Cu, and Ni) in water at environmentally relevant concentrations. These results provide physiological evidence for a role of the olfactory system in fish behavioural responses (as shown in previous studies) when they encounter metal contaminated waters.

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.

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 gemfibrozil on the growth, reproduction, and energy stores of Daphnia magna in the presence of varying food concentrations.

Gemfibrozil, a common lipid regulator, enters aquatic environments through treated municipal wastewater effluent that fails to remove it completely from effluent streams. When exposed to gemfibrozil concentrations of 50, 500, 5,000, and 50,000 ng L-1, Daphnia magna showed increased lipid reserves by 14-21% (significant at 500 ng L-1), increased length by 9-13% (significant at 50 ng L-1), increased mass by 6-13% (significant at 50 ng L-1) and increased neonate production by 57-74% (significant at 50 ng L-1). Gemfibrozil-exposed Daphnia held under conditions where food availability was low, grew and reproduced as well as those in the control. Taken together, these results suggest that gemfibrozil exposure within environmentally relevant concentration ranges is not toxic to Daphnia magna but has the potential to be beneficial to the species under these conditions.

Effects of oil sands process-affected water on the respiratory and circulatory system of Daphnia magna Straus, 1820.

Millions of cubic meters of oil sands process-affected water (OSPW), the major by-product of oil sand surface mining, is currently stored in tailings ponds. The present study investigated the effects of OSPW on the respiratory and circulatory system of Daphnia magna Straus 1820. The effect of OSPW on the activity (i.e. total movement and active time) of D. magna was also studied, as it has been shown to interact with the respiratory and circulatory system. Daphniids were exposed to both 1 and 10% OSPW for acute (1-day) and chronic (10-day) exposure periods. At the end of the exposures, daphniid oxygen (O2) consumption, heart rate, hemoglobin (Hb) content and activity were investigated. In response to chronic exposure to 10% OSPW, O2 consumption of D. magna increased, while the hemoglobin content and activity were reduced in both 1 and 10% OSPW. None of the OSPW treatments changed the heart rate of the test organisms. The results of the present study suggest that in response to increasing metabolic rate caused by OSPW exposure, D. magna conserve their energy by reducing their activity and probably by recycling macromolecules (i.e. hemoglobin).

Interactive toxicity of Ni, Zn, Cu, and Cd on Daphnia magna at lethal and sub-lethal concentrations.

The toxicity of metal mixtures is currently of particular interest among aquatic toxicologists. To provide insight into whether the interaction of multiple metals is similar at different biological levels, the survival and feeding behavior of Daphnia magna were studied following exposure to four metals (Cd, Cu, Ni, Zn) and their binary and quaternary combinations. In terms of survival, Zn-Cu and Cu-Cd mixtures produced more-than-additive mortality, while Ni-Cd mixtures resulted in less-than-additive mortality. Regarding behavior, Zn-Cu and Zn-Cd mixtures produced a more-than-additive reduction in feeding rate. Four (i.e. Zn-Cu, Cu-Cd, Ni-Cd, and Zn-Cd) out of six binary mixtures in the present study interacted differently at the survival and behavioral levels, strengthening the emphasis on carefully selecting the toxicological endpoint when addressing metal mixture toxicity. The results of the present study demonstrated that metals are toxic to feeding behavior of D. magna at much lower concentrations (i.e. 27-63 times lower) compared to survival, suggesting that applying sub-lethal endpoints are required for producing protective regulations.

Investigating the chronic effects of oil sands process-affected water on growth and fitness of Daphnia magna Straus 1820.

The increasing amount of stored oil sands process-affected water (OSPW), a primary by-product of oil sands mining, is an environmental concern. In the present study, we investigated the chronic effects of OSPW on growth, reproduction, and macronutrient content in Daphnia magna. To do so, we exposed D. magna to 1 and 10% OSPW (a mixture of three OSPW samples provided by major oil sands mining operators in northern Alberta) for ten days. We measured the number of the neonates produced daily in each group throughout the exposure. At the end of the exposure, we measured the mass and length of the exposed daphniids and neonates. We also measured the carbohydrate, lipid, and protein content of exposed daphniids. In the 10% OSPW group, we observed a significant reduction in all of the measured endpoints except for body length and carbohydrate and protein content of exposed daphniids. In the 1% OSPW group, on the other hand, we found a reduction only in lipid content of exposed daphniids as compared to the control group. The results of the present study demonstrated that chronic exposure to 10% OSPW affects growth and fitness of D. magna, probably due to a reduction in energy intake that causes daphniids to deplete their energy reserves.

Oil sands process-affected water impairs feeding by Daphnia magna.

Growth in extraction of bitumen from oil sands has raised concerns about influences of this industry on surrounding environments. Water clearance rate (a surrogate of feeding rate by Daphnia magna) in water containing D. magna exposed to oil sands process-affected water (OSPW) and its principal components, dissolved component (DC) and suspended particulate matter (SPM), was reduced to 72, 29, and 59% of controls, respectively. This study also examined several possible mechanisms for the observed changes algal cell density (i.e., feeding rate). There was no change in the digestive enzymes trypsin or amylase when D. magna were exposed to DC or SPM; however, exposure to total OSPW reduced trypsin activity. Mandible rolling or post-abdominal rejections, which are indicators of feeding and palatability of food, were not affected by any exposures to OSPW. Beating of thoracic limbs, which provides water flow toward the feeding groove, was reduced by exposure to SPM or total OSPW. Peristaltic activity was reduced by exposure to DC, which then might result in reduced digestion time in D. magna exposed to DC, SPM or whole OSPW. All treatments caused an increase in numbers of intact algae cells in the hindgut and excreted material. These results suggest that both DC and SPM affect feeding of D. magna by impairing actions of the digestive system, but most probably not by reducing rates of ingestion.

Rainbow trout (Oncorhynchus mykiss) detection, avoidance, and chemosensory effects of oil sands process-affected water.

Oil sands process-affected water (OSPW) - a byproduct of the oil sands industry in Northern Alberta, Canada - is currently stored in on-site tailings ponds. The goal of the present study was to investigate the interaction of OSPW with the olfactory system and olfactory-mediated behaviours of fish upon the first encounter with OSPW. The response of rainbow trout (Oncorhynchus mykiss) to different concentrations (0.1, 1, and 10%) of OSPW was studied using a choice maze and electro-olfactography (EOG), respectively. The results of the present study showed that rainbow trout are capable of detecting and avoiding OSPW at a concentration as low as 0.1%. Exposure to 1% OSPW impaired (i.e. reduced sensitivity) the olfactory response of rainbow trout to alarm and food cues within 5 min or less. The results of the present study demonstrated that fish could detect and avoid minute concentrations of OSPW. However, if fish were exposed to OSPW-contaminated water and unable to escape, their olfaction would be impaired.

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.

Rapid changes in water hardness and alkalinity: Calcite formation is lethal to Daphnia magna.

There is growing concern that freshwater ecosystems may be negatively affected by ever-increasing anthropogenic inputs of extremely hard, highly alkaline effluent containing large quantities of Ca(2+), Mg(2+), CO3(2-), and HCO3(-) ions. In this study, the toxicity of rapid and extreme shifts in water hardness (38-600mg/L as CaCO3) and alkalinity (30-420mg/L as CaCO3) to Daphnia magna was tested, both independently and in combination. Within these ranges, where no precipitation event occurred, shifts in water hardness and/or alkalinity were not toxic to D. magna. In contrast, 98-100% of D. magna died within 96h after exposure to 600mg/L as CaCO3 water hardness and 420mg/L as CaCO3 alkalinity (LT50 of 60h with a 95% CI of 54.2-66.0h). In this treatment, a CaCO3 (calcite) precipitate formed in the water column which was ingested by and thoroughly coated the D. magna. Calcite collected from a mining impacted stream contained embedded organisms, suggesting field streams may also experience similar conditions and possibly increased mortality as observed in the lab tests. Although further investigation is required to determine the exact fate of aquatic organisms exposed to rapid calcite precipitation in the field, we caution that negative effects may occur more quickly or at lower concentrations of water hardness and alkalinity in which we observed effects in D. magna, because some species, such as aquatic insects, are more sensitive than cladocerans to changes in ionic strength. Our results provide evidence that both calcite precipitation and the major ion balance of waters should be managed in industrially affected ecosystems and we support the development of a hardness+alkalinity guideline for the protection of aquatic life.