Sulfate sensitivity of early life stages of freshwater mussels Unio crassus and Margaritifera margaritifera.

Sulfate is increasingly found in elevated concentrations in freshwater ecosystems due to anthropogenic activities. Chronic exposure to sulfate has been reported to cause sublethal effects on freshwater invertebrates. Previous sulfate toxicity tests have mostly been conducted in hard or moderately hard waters, and research on species inhabiting soft water is needed, given that freshwater organisms face heightened sensitivity to toxicants in water of lower hardness. In the present study, we examined sulfate sensitivity of two endangered freshwater mussel species, Unio crassus, and Margaritifera margaritifera. Glochidia and juveniles of both species were subjected to acute and/or chronic sulfate exposures in soft water to compare sulfate sensitivity across age groups, and effective concentrations (EC)/lethal concentrations (LC) values were estimated. Mussels were individually exposed to allow relatively larger numbers of replicates per treatment. Chronic sulfate exposure significantly reduced growth, foot movement, and relative water content (RWC) in juvenile mussels of M. margaritifera. Mussels at younger stages were not necessarily more sensitive to sulfate. In the acute tests, LC50 of glochidia of M. margaritifera and U. crassus was 1301 and 857 mg/L, respectively. Chronic LC10 was 843 mg/L for 3-week-old U. crassus juveniles, 1051 mg/L for 7-week-old M. margaritifera juveniles, and 683 mg/L for 2-year-old M. margaritifera juveniles. True chronic Lowest Effective Concentration for 7-week-old M. margaritifera may be within the 95% interval of EC10 based on RWC (EC10 = 446 mg/L, 95%CI = 265-626 mg/L). Our study contributed to the understanding of sulfate toxicity to endangered freshwater mussel species in soft water.

THE EFFECTS OF GLOCHIDIA INFECTION ON THE METABOLIC RATE AND HYPOXIA TOLERANCE OF BLUEGILL LEPOMIS MACROCHIRUS AND LARGEMOUTH BASS MICROPTERUS SALMOIDES.

Gill parasites can negatively affect hosts by altering behavior or causing adverse effects to host physiology. Most unionid mussel larvae (glochidia) are obligate parasites requiring fish hosts, but much of the literature concerning how these parasites affect their hosts has been limited to only a few study species of salmonids and mussels. Here, we test the effects of natural glochidia infection levels on resting metabolic rate and hypoxia tolerance of bluegill Lepomis macrochirus and largemouth bass Micropterus salmoides using glochidia of the southern fatmucket mussel Lampsilis straminea, a warm-water unionid. We quantified oxygen uptake, hypoxia tolerance, and ability to regulate metabolic rate via respiration rate, critical dissolved oxygen values (DOcrit), and a regulation index (RI), respectively, to compare the effects of glochidia infection between infected and uninfected fish over 11 wk after host inoculation. Hosts were infected with glochidia at levels similar to those seen in wild, naturally occurring infections. At these levels, we observed no effects of glochidia infection on metabolic rate, DOcrit, or RI of infected versus control fish of either species over our 11-wk experiment. Glochidia infection on fish gills at levels expected under field conditions may not always induce an organismal-level (i.e., individual organism) response or cause host respiratory stress. Preventing respiratory stress from infection would be beneficial for both host and parasite, given that glochidia survival and dispersal depend on host survival.

Effects of parasitic freshwater mussels on their host fishes: a review.

Freshwater mussels in the order Unionida are highly adapted to parasitize fish for the primary purpose of dispersal. The parasitic larval stage affixes itself to the gills or fins of the host where it becomes encysted in the tissue, eventually excysting to develop into a free-living adult. Research on the parasitic interactions between unionids and their host fishes has garnered attention recently due to the increase in worldwide preservation efforts surrounding this highly endangered and ecologically significant order. With the exception of heavy infestation events, these mussels cause minor effects to their hosts, typically only observable effect in combination with other stressors. Moreover, the range of effect intensities on the host varies greatly with the species involved in the interaction, an effect that may arise from different evolutionary strategies between long- and short-infesting mussels; a distinction not typically made in conservation practices. Lower growth and reduced osmotic potential in infested hosts are commonly observed and correlated with infestation load. These effects are typically also associated with increases in metabolic rate and behaviour indicative of stress. Host fish seem to compensate for this through a combination of rapid wound healing in the parasitized areas and higher ventilation rates. The findings are heavily biased towards Margaritifera margaritifera, a unique mussel not well suited for cross-species generalizations. Furthermore, the small body of molecular and genetic studies should be expanded as many conclusions are drawn from studies on the ultimate effects of glochidiosis rather than proximate studies on the underlying mechanisms.

First Immunodetection of Sensory and Nervous Systems of Parasitic Larvae (Glochidia) of Freshwater Bivalve Nodularia douglasiae.

Most freshwater mussels have an unusual life cycle that requires host fish species for larval (glochidia) development and dispersal. Glochidia have a unique morphological structure that adapts to parasitic lifestyles and survival. The morphology of the glochidial shells of most Unionoidea, a group of freshwater bivalve mollusks, has been studied in detail using light and scanning electron microscopy. This study summarizes our data on the glochidia shell morphology of the Asian mussel Nodularia douglasiae from two localities in the Primorsky Territory, the Russian Far East. In contrast to the shell morphology of glochidia, little is known about the neurodevelopment of the Unionoidea. Herein, we first demonstrate that the structures of the sensory, muscle, and nervous systems of the glochidia larvae of N. douglasiae differ dramatically from those of the comparable larval systems of marine bivalve species, as revealed through alpha-acetylated tubulin, serotonin (5-HT), and FMRFamide antibodies as well as phalloidin for detection of F-actin and whole-mount confocal microscopy. We found that the glochidia sensory system included four pairs of tubulin-lir multicilia hair cells. Non-ciliar tubulin-lir cells synthesize the neuropeptide FMRFamide and are identified as afferent neurons collecting information from peripheral tubulin-lir hair sensory cells to nervous regulators. The glochidia's muscular system was represented by a smooth adductor, retractors, and minor muscle bundles associated with the shell and visceral organs. The 5-HT-lir larval system is arranged most simply and consists of two immunopositive neurons innervating the adductor. The FMRFamide-lir system is more complicated and consists of several neuronal centers comprising neuronal bodies and their neurites in different areas of the larva. The FMRFamide-lir neurons are closely associated with sensory hair cells, and others, together with 5-HT-lir neurons, may be involved in the anlagen of adult ganglia. Thus, the nervous system of N. douglasiae glochidia is drastically different from other mollusks and lophotrochozoans because of the absence of an apical organ and the location and composition of FMRFamide and 5-HT cells. Morphological, molecular, and behavioral investigations of Unionoidea taxa need to be further conducted to investigate the parasite-host relationship, nerve-dependent regulation of parasite behavior, and evolution of mollusks.

Higher mortality of the less suitable brown trout host compared to the principal Atlantic salmon host when infested with freshwater pearl mussel (Margaritifera margaritifera) glochidia.

The freshwater pearl mussel (Margaritifera margaritifera) is a highly host-specific parasite, with an obligate parasitic stage on salmonid fish. Atlantic salmon (Salmo salar) and brown trout (Salmo trutta f. trutta and Salmo trutta f. fario) are the only hosts in their European distribution. Some M. margaritifera populations exclusively infest either Atlantic salmon or brown trout, while others infest both hosts with one salmonid species typically being the principal host and the other a less suitable host. Glochidial abundance, prevalence and growth are often used as parameters to measure host suitability, with the most suitable host species displaying the highest parameters. However, it is not known if the degree of host specialisation will negatively influence host fitness (virulence) among different host species. In this study we examined the hypothesis that glochidial infestation would result in differential virulence in two salmonid host species and that lower virulence would be observed on the most suitable host. Atlantic salmon and brown trout were infested with glochidia from two M. margaritifera populations that use Atlantic salmon as their principal host, and the difference in host mortality among infested and control (sham infested) fish was examined. Higher mortality was observed in infested brown trout (the less suitable host) groups, compared to the other test groups. Genetic assignment was used to identify offspring from individual mother mussels. We found that glochidia from individual mothers can infest both the salmonid hosts; however, some mothers displayed a bias towards either salmon or trout. We believe that the differences in host-dependent virulence and the host bias displayed by individual mothers were a result of genotype × genotype interactions between the glochidia and their hosts, indicating that there is an underlying genetic component for this parasite-host interaction.

Growth patterns of the pan-European freshwater mussel, Anodonta anatina (Linnaeus, 1758) (Bivalvia: Unionidae), vary with sex and mortality in populations.

Post-maturation growth leading to indeterminate growth patterns is widespread in nature. However, its adaptive value is unclear. Life history theory suggests this allocation strategy may be favored by temporal pulses in the intensity of mortality and/or the capacity to produce new tissues.Addressing the origin of indeterminate growth and the variability of growth patterns, we studied the growth of duck mussels, Anodonta anatina, a pan-European unionid, in 18 Polish lakes. For each population, the sex, size, and age of collected mussels were measured to estimate Bertalanffy's growth curve parameters. We integrated information on A. anatina mortality rates, lake trophy, biofouling by zebra mussels, Dreissena polymorpha, and the prevalence of parasitic trematode larvae to identify selective conditions in lakes.We found two sources of mortality in A. anatina populations, pertaining to adverse effects of zebra mussel biofouling and trophy state on mussel survival. Additionally, populations with heavier biofouling presented a smaller abundance of parasites, indicative of a relationship between filtering intensity and contraction of water-borne trematode larvae by filtering A. anatina.Consistently for each sex, populations with a greater trophy-related mortality were characterized in A. anatina by a smaller asymptotic size Lmax , indicative of a life history response to mortality risk involving early maturation at a smaller body size. In all populations, females featured higher mortality and larger asymptotic size versus males.Our findings support a theoretical view that adaptive responses to selection involve adjustments in the lifetime resource allocation patterns. These adjustments should be considered drivers of the origin of indeterminate growth strategy in species taking parental care by offspring brooding in body cavities.

In situ and low-cost monitoring of particles falling from freshwater animals: from microplastics to parasites.

A simple and low-cost method of monitoring and collecting particulate matter detaching from (or interacting with) aquatic animals is described using a novel device based on an airlift pump principle applied to floating cages. The efficiency of the technique in particle collection is demonstrated using polyethylene microspheres interacting with a cyprinid fish (Carassius carassius) and a temporarily parasitic stage (glochidia) of an endangered freshwater mussel (Margaritifera margaritifera) dropping from experimentally infested host fish (Salmo trutta). The technique enables the monitoring of temporal dynamics of particle detachment and their continuous collection both in the laboratory and in situ, allowing the experimental animals to be kept under natural water quality regimes and reducing the need for handling and transport. The technique can improve the representativeness of current experimental methods used in the fields of environmental parasitology, animal feeding ecology and microplastic pathway studies in aquatic environments. In particular, it makes it accessible to study the physiological compatibility of glochidia and their hosts, which is an essential but understudied autecological feature in mussel conservation programs worldwide. Field placement of the technique can also aid in outreach programs with pay-offs in the increase of scientific literacy of citizens concerning neglected issues such as the importance of fish hosts for the conservation of freshwater mussels.

Cumulative effects of cadmium and natural stressors (temperature and parasite infection) on molecular and biochemical responses of juvenile rainbow trout.

The simultaneous presence of natural and anthropogenic stressors in aquatic ecosystems can challenge the identification of factors causing decline in fish populations. These stressors include chemical mixtures and natural abiotic and biotic factors such as water temperature and parasitism. Effects of cumulative stressors may vary from antagonism to synergism at the organismal or population levels and may not be predicted from exposure to individual stressors. This study aimed to evaluate the combined effects of chronic exposure to cadmium (Cd) and elevated water temperature (23 °C) or parasite infection in juvenile rainbow trout (Oncorhynchus mykiss) using a multi-level biological approach, including RNA-sequencing. Fish were exposed to diet-borne Cd (6 µg Cd/g wet feed), individually and in combination with thermal (23 °C) or parasitic stressors, for 28 days. The parasite challenge consisted of a single exposure to glochidia (larvae) of the freshwater mussel (Strophitus undulatus), which encysts in fish gills, fins and skin. Results indicated lower fish length, weight, and relative growth rate in fish exposed to a higher water temperature (23 °C). Body condition and hepatosomatic index of trout were, however, higher in the 23 °C temperature treatment compared to the control fish kept at 15 °C. Exposure to thermal stress or parasitism did not influence tissue Cd bioaccumulation. More than 700 genes were differentially transcribed in fish exposed to the individual thermal stress treatment. However, neither Cd exposure nor parasite infection affected the number of differentially transcribed genes, compared to controls. The highest number of differentially transcribed genes (969 genes) was observed in trout exposed to combined Cd and high temperature stressors; these genes were mainly related to stress response, protein folding, calcium metabolism, bone growth, energy metabolism, and immune system; functions overlapped with responses found in fish solely exposed to higher water temperature. Only 40 genes were differentially transcribed when fish were exposed to Cd and glochidia and were related to the immune system, apoptosis process, energy metabolism and malignant tumor. These results suggest that dietary Cd may exacerbate the temperature stress and, to a lesser extent, parasitic infection stress on trout transcriptomic responses. Changes in the concentrations of liver ethoxyresorufin-o-deethylase, heat shock protein 70 and thiobarbituric acid reactive substances coupled to changes in the activities of cellular glutathione S-transferase and glucose-6-phosphate dehydrogenase were also observed at the cellular level. This study may help understand effects of freshwater fish exposure to cumulative stressors in a changing environment.

Stress-related gene transcription in fish exposed to parasitic larvae of two freshwater mussels with divergent infection strategies.

Freshwater unionoid mussels have a unique life cycle involving a temporary parasitic phase. Their larvae (glochidia) attach to the gills or fins of fish hosts where they remain encysted until metamorphosis into free-living juveniles. The physiological response of fish during the critical period of glochidial attachment is not well understood, but recent work suggests that glochidia retention and survival is enhanced in stressed and cortisol-injected hosts. In this study, the early changes induced by glochidiosis were investigated for the first time at the transcriptional level. In 2 separate experiments, juvenile yellow perch Perca flavescens were inoculated with glochidia of Elliptio complanata (a host generalist) and Lampsilis radiata (a host specialist) following a standardized procedure. The transcriptional levels of 5 genes involved in the fish response to stress were assessed in the host liver and gills 24 h post-infection using quantitative real-time PCR. The number of encysted glochidia did not significantly differ between fish inoculated with E. complanata and L. radiata. Both species induced a 3-fold increase of 70 kDa heat-shock protein gene (hsp70) transcription in host liver. However, only E. complanata influenced the transcription of cortisol-regulated genes, notably glucocorticoid receptor DNA-binding factor 1 (grlf1). This gene, known to modulate tissue responsiveness to cortisol, was downregulated in infected fish compared to controls. Our findings suggest that different glochidia species interact with their fish host in distinct ways. Additional studies are required to address this hypothesis and further investigate the significance of the observed host transcriptional responses.

Host-parasite interaction as a toxicity test endpoint using asymmetrical exposures.

Interspecific relationships frequently determine the effect a pollutant can have on an organism, and this is especially true in closely interacting species such as hosts and parasites. The high spatial and temporal variability of contaminant concentrations combined with the movement of aquatic biota can further influence the consequences that are associated with contamination. We used a full factorial design for the exposed and unexposed partners of the relationship between the parasitic larvae (glochidia) of the European freshwater mussel (Anodonta anatina) and its host fish (Squalius cephalus) to identify the sources of variation in the sublethal endpoints of species interaction (the intensity of parasite attachment, the spatial position of glochidia on the host body, and encapsulation success). We used the water-borne human pharmaceutical compounds methamphetamine (a central nervous system stimulant) and tramadol (an opioid) at environmentally relevant concentrations (˜ 6.7 and 3.8 nmol L-1 of methamphetamine and tramadol, respectively) as a proxy for contaminant exposure because these compounds are emerging aquatic stressors that are known for high spatial and temporal variability in their detected concentration levels. The relationship between the bivalve and the fish species was influenced by the preceding contact with both methamphetamine and tramadol, but this effect was highly asymmetric. Our experimental design enabled us to identify the specific changes in the relationship outcome that are elicited by the exposure of individual partners, such as the significant increase in glochidia infection success rate from 59.6 ± 3.9% to 78.7 ± 2.8% (means ± s.e.) that was associated with host exposure to methamphetamine. Additionally, the significant interaction effect of the exposure was demonstrated by the lowered proportion of glochidia attached to gills after the coexposure of both partners to tramadol. The impact of pharmaceuticals on wild aquatic host-parasite relationships provides an example of the risks that are associated with the unintentional discharge of biologically active compounds into freshwater habitats. Given the increasing evidence showing the ecological impact of waste pharmaceuticals, the use of multitrophic interaction endpoints after joint and unilateral exposures provides an important step towards the realistic risk assessment of these compounds.

Host (Salmo trutta) age influences resistance to infestation by freshwater pearl mussel (Margaritifera margaritifera) glochidia.

The freshwater pearl mussel (Margaritifera margaritifera) is an endangered bivalve with an obligate parasitic stage on salmonids. Host suitability studies have shown that glochidial growth and load vary significantly between host strains as well as among individuals of a suitable strain. Variation in host suitability has been linked to environmental conditions, host age and/or size, genetic composition of the host and parasite, or a combination of these factors. In our study, we wanted to investigate if brown trout (Salmo trutta) displayed an age-dependent response to glochidial infestation. We hypothesised that 1+ naive brown trout hosts tolerate glochidial infestation better than 0+ hosts. In order to test our hypothesis, we infested 0+ and 1+ hatchery reared brown trout with glochidia from closely related mothers and kept them under common garden conditions. This allowed us to observe a pure age dependent host response to infestation, as we eliminated the confounding effect of genotype-specific host interactions. We analysed the interaction between glochidial load and host condition, weight and length, and observed a significant age-dependent relationship. Glochidial load was negatively correlated to host condition in 0+ fish hosts and positively correlated in 1+ hosts. These contradictory findings can be explained by a change in host response strategy, from resistance in young to a higher tolerance in older fish. In addition, we also examined the relationship between glochidial load and haematocrit values in the 1+ hosts and observed that haematocrit values were significantly higher in heavily infested hosts. Our results have important conservation implications for the management of wild pearl mussel populations, as well as for captive breeding programmes.

Potential impacts of the invasive species Corbicula fluminea on the survival of glochidia.

Freshwater mussels (Bivalvia, Unionida) are one of the most imperilled faunal groups globally, being the introduction of invasive species a possible major mechanism of threat. The Asian clam Corbicula fluminea is a problematic invasive species in aquatic ecosystems and can impair the survival of parasitic larvae (glochidia) of native freshwater mussels. However, this possible mechanism of threat remains speculative and to date very few studies addressed quantitatively this issue. In order to cover this gap, we have performed a series of manipulative laboratory studies to assess how distinct densities of C. fluminea can affect the survival of glochidia after 6, 12, 24 and 48 h of exposure, using larvae of the native freshwater mussel Anodonta anatina. Our results suggest an increase in mortality of A. anatina glochidia with an increase in density of C. fluminea. Two main mechanisms may possibly explain our results: 1) the high filtration capacity of C. fluminea that can contribute to the mortality of glochidia due to the mechanical damage of their fragile shells when passing by siphons and/or digestive tract of C. fluminea and 2) the high excretion capacity of C. fluminea that can lead to mortality of glochidia due to increase in ammonia concentration. Mortality of glochidia was also time dependent with higher values registered after 48 h. This work is one of the first showing the influence of C. fluminea density on the survival of glochidia, being filtration (and consequent passage in the digestive tract) and biodeposition the main potential mechanisms explaining overall mortality. These results also suggest that sites with high densities of C. fluminea may be highly detrimental for the conservation of freshwater mussels, potentially impairing the survival of glochidia and negatively affecting the recruitment of juveniles.

Acute toxicity of sodium chloride and potassium chloride to a unionid mussel (Lampsilis siliquoidea) in water exposures.

Freshwater mussels (order Unionoida) are one of the most imperiled groups of animals in the world. However, many ambient water quality criteria and other environmental guideline values do not include data for freshwater mussels, in part because mussel toxicity test methods are comparatively new and data may not have been available when criteria and guidelines were derived. The objectives of the present study were to evaluate the acute toxicity of sodium chloride (NaCl) and potassium chloride (KCl) to larvae (glochidia) and/or juveniles of a unionid mussel (fatmucket, Lampsilis siliquoidea) and to determine the potential influences of water hardness (50, 100, 200, and 300 mg/L as CaCO3 ) and other major ions (Ca, K, SO4 , or HCO3 ) on the acute toxicity of NaCl to the mussels. From the KCl test, the 50% effect concentration (EC50) for fatmucket glochidia was 30 mg K/L, similar to or slightly lower than the EC50s for juvenile fatmucket (37-46 mg K/L) tested previously in our laboratory. From the NaCl tests, the EC50s for glochidia increased from 441 to 1597 mg Cl/L and the EC50s for juvenile mussels increased from 911 to 3092 mg Cl/L with increasing water hardness from 50 to 300 mg/L. Increasing K from 0.4 to 1.9 mg/L, SO4 from 13 to 40 mg/L, or HCO3 from 44 to 200 mg/L in the 50 mg/L hardness water did not substantially change the NaCl EC50s for juvenile mussels, whereas increasing Ca from 9.9 to 42 mg/L increased the EC50s by a factor of 2. The overall results indicate that glochidia were equally or more sensitive to NaCl and KCl compared with juvenile mussels and that the increased water hardness ameliorated the acute toxicity of NaCl to glochidia and juveniles. These responses rank fatmucket among the most acutely sensitive freshwater organisms to NaCl and KCl. Environ Toxicol Chem 2018;37:3041-3049. © 2018 SETAC. This article is a US government work and, as such, is in thepublic domain in the United States of America.

Tetrahymena glochidiophila n. sp., a new species of Tetrahymena (Ciliophora) that causes mortality to glochidia larvae of freshwater mussels (Bivalvia).

A ciliate protozoan was discovered whose presence coincided with a rapid decrease in the viability (i.e. ability to close valves) of glochidia of the freshwater mussel Lampsilis siliquoidea. Microscopic examination showed it to be a histophagous tetrahymenine ciliate. Small subunit (SSU) rRNA and cytochrome c oxidase subunit 1 (cox1) barcode sequences from cultured cells showed that it belongs to the same new species isolated from water samples as a free-living ciliate. Phylogenetic analyses place this new ciliate in the same clade with the macrostome species Tetrahymena paravorax, and we propose the name T. glochidiophila n. sp. for this new species. The phylogeny provides further support for the hypothesis that histophagy was a life history trait of the ancestor of Tetrahymena.

Copper uptake, patterns of bioaccumulation, and effects in glochidia (larvae) of the freshwater mussel (Lampsilis cardium).

The early life stages of freshwater mussels are particularly sensitive to copper (Cu) contamination. We measured the acute toxicity, bioaccumulation, and sublethal effects of Cu in glochidia. In addition, we used radiolabeled Cu (64 Cu) to examine the time-dependent kinetics of uptake over 24 h. Uptake of 64 Cu by live and dead glochidia exposed to 0.11 µmol/L exhibited similar hyperbolic patterns over the first 40 min, indicating an adsorptive phase independent of larval metabolism. Thereafter, uptake was linear with time, with a 10-fold lower bioaccumulation rate in live than in dead animals, representing a close to steady state of Cu regulation. In contrast, dead glochidia exhibited a progressively increasing uptake, possibly attributable to the fact that metal-binding sites become more accessible. Mortality was strongly correlated with bioaccumulation (48 h); live glochidia exposed to Cu concentrations >0.27 µmol/L lost their regulatory ability and accumulated Cu to an even greater extent than dead animals. Exposure to Cu induced significant decreases in whole-body Na+ and Mg2+ concentrations; increases in reactive oxygen species concentration, lipid peroxidation, and protein carbonylation; and a decrease in antioxidant capacity against peroxyl radicals. Overall, these results clarify the patterns of Cu uptake and regulation, emphasize the importance of distinguishing between live and dead larvae, and indicate that toxicity is associated with Cu bioaccumulation, involving both ionoregulatory disturbance and oxidative stress. Environ Toxicol Chem 2018;37:1092-1103. © 2017 SETAC.

Acute toxicity of chlorpyrifos and carbosulfan to glochidia of the freshwater mussel Hyriopsis bialata Simpson, 1900.

The acute toxicity of carbosulfan and chlorpyrifos in formulated pesticides to glochidia (larvae) of the freshwater mussel (Hyriopsis bialata Simpson, 1900) was evaluated under static conditions in moderately hard dechlorinated tap water. Measured pesticide concentrations were 26 to 34% lower than nominal concentrations; therefore, all results are expressed in terms of measured active ingredient. Carbosulfan was relatively non-toxic to the mussel larvae with median effective concentrations (EC50) of carbosulfan at 24 and 48 h greater than 0.10 mg/L. The EC50s of chlorpyrifos at 24 and 48 h were 0.083 and 0.078 mg/L, respectively (measured concentrations). The 48-h EC50 of a combined exposure to a mixture of chlorpyrifos and carbosulfan at a constant ratio of 2.9:1 was 0.0142:0.049 mg CP:CB/L. In a separate experiment, the effect of water hardness on carbosulfan, chlorpyrifos, or a combined exposure was assessed using glochidia exposed to either soft, moderately hard, or hard reconstituted water. There was no effect of water hardness on the survival of glochidia after 24- or 48-h exposure to carbosulfan. The chlorpyrifos 48-h EC50s in soft water, moderately hard water, and hard water were 0.18, 0.11, and 0.16 mg/L, respectively. The data indicate that the lowest water hardness resulted in the highest survival of glochidia, whereas an increase to moderate water hardness resulted in significantly decreased survival of glochidia (F = 15.5, P < 0.05). The EC50s of a combined exposure at 48 h in soft water, moderately hard water, and hard water were 0.124:0.044, 0.132:0.047, and 0.064:0.022 mg CP:CB/L, respectively. The data indicate that the combined toxicity was lowest at low and moderate water hardness, whereas an increase to high water hardness resulted in a significantly decreased survival of glochidia. After 48 h, the toxicity of the combined chlorpyrifos and carbosulfan exposure in soft and hard water was greater than that of chlorpyrifos alone.

Assessing the toxicity and risk of salt-impacted winter road runoff to the early life stages of freshwater mussels in the Canadian province of Ontario.

In temperate urbanized areas where road salting is used for winter road maintenance, the level of chloride in surface waters has been increasing. While a number of studies have shown that the early-life stages of freshwater mussels are particularly sensitive to salt; few studies have examined the toxicity of salt-impacted winter road runoff to the early-life stages of freshwater mussels to confirm that chloride is the driver of toxicity in this mixture. This study examines the acute toxicity of field-collected winter road runoff to the glochidia of wavy-rayed lampmussels (Lampsilis fasciola) (48 h exposure) and newly released juvenile fatmucket mussels (Lampsilis siliquoidea) (<1 week old; 96 h exposure) under different water hardness. The chronic toxicity (28 d) to older juvenile L. siliquoidea (7-12 months old) was also investigated. The 48-h EC50 and 96-h LC50 for L. fasciola glochidia and L. siliquoidea juveniles exposed to different dilutions of road run-off created with moderately hard synthetic water (∼80 mg CaCO3/L) were 1177 (95% confidence interval (CI): 1011-1344 mg Cl-/L) and 2276 mg Cl-/L (95% CI: 1698-2854 mg Cl-/L), respectively. These effect concentrations correspond with the toxicity of chloride reported in other studies, indicating that chloride is likely the driver of toxicity in salt-impacted road-runoff, with other contaminants (e.g., metals, polycyclic aromatic hydrocarbons) playing a de minimis role. Toxicity data from the current study and literature and concentrations of chloride in the surface waters of Ontario were used to conduct a probabilistic risk assessment of chloride to early-life stage freshwater mussels. The assessment indicated that chronic exposure to elevated chloride levels could pose a risk to freshwater mussels; further investigation is warranted to ensure that the most sensitive organisms are protected.

Encystment of parasitic freshwater pearl mussel (Margaritifera margaritifera) larvae coincides with increased metabolic rate and haematocrit in juvenile brown trout (Salmo trutta).

Gill parasites on fish are likely to negatively influence their host by inhibiting respiration, oxygen transport capacity and overall fitness. The glochidia larvae of the endangered freshwater pearl mussel (FPM, Margaritifera margaritifera (Linnaeus, 1758)) are obligate parasites on the gills of juvenile salmonid fish. We investigated the effects of FPM glochidia encystment on the metabolism and haematology of brown trout (Salmo trutta Linnaeus, 1758). Specifically, we measured whole-animal oxygen uptake rates at rest and following an exhaustive exercise protocol using intermittent flow-through respirometry, as well as haematocrit, in infested and uninfested trout. Glochidia encystment significantly affected whole-animal metabolic rate, as infested trout exhibited higher standard and maximum metabolic rates. Furthermore, glochidia-infested trout also had elevated levels of haematocrit. The combination of an increased metabolism and haematocrit in infested fish indicates that glochidia encystment has a physiological effect on the trout, perhaps as a compensatory response to the potential respiratory stress caused by the glochidia. When relating glochidia load to metabolism and haematocrit, fish with low numbers of encysted glochidia were the ones with particularly elevated metabolism and haematocrit. Standard metabolic rate decreased with substantial glochidia loads towards levels similar to those of uninfested fish. This suggests that initial effects visible at low levels of encystment may be countered by additional physiological effects at high loads, e.g. potential changes in energy utilization, and also that high numbers of glochidia may restrict oxygen uptake by the gills.

Extending the toxicity-testing paradigm for freshwater mussels: Assessing chronic reproductive effects of the synthetic estrogen 17α-ethinylestradiol on the unionid mussel Elliptio complanata.

Surface water concentrations of the synthetic estrogen 17α-ethinylestradiol (EE2) as low as 1ng/L can cause adverse reproductive effects in fish under acute and chronic exposure conditions, whereas higher concentrations (> 5ng/L) in acute studies are necessary to elicit adverse effects in freshwater mussels. Prolonged chronic exposures of freshwater mussels to EE2 remain un-evaluated. An extended duration testing paradigm was used to examine reproductive and biochemical (carbohydrate, lipid, protein) effects of EE2 on the unionid mussel, Elliptio complanata, throughout its reproductive cycle. Mussels were exposed to a control and EE2 concentrations (5 and 50ng/L) in six discrete and sequential 28 d tests, and in one discrete and simultaneous 180 d test, from February through August. Foot protrusion and siphoning behavior were recorded daily, along with conglutinate releases and larval (glochidia) mortality. Gonad, hemolymph, and gonad fluid samples were taken for biochemical and vitellogenin-like protein (Vtg) analysis post-exposure. Female mussels released eggs and conglutinates during the months of April to June, indicating sexual maturation during this time. Conglutinates released in the 5ng/L treatment in 28 d exposures contained fewer glochidia and more eggs, and increased concentrations of Vtg in hemolymph were observed from April to August in the 5ng/L treatment during the 180 d exposure. Results indicate that the 180 d test approach, concurrent with the sequence of 28 d tests, enabled a more robust evaluation of mussel behavior and physiology than would have been possible with a single short-term (28 d) test.