Lethal and Sub-lethal Implications of Sodium Chloride Exposure for Adult Unionid Mussel Species: Eurynia dilatata and Lasmigona costata.

The elevated use of salt as a de-icing agent on roads in Canada is causing an increase in the chloride concentration of freshwater ecosystems. Freshwater Unionid mussels are a group of organisms that are sensitive to increases in chloride levels. Unionids have greater diversity in North America than anywhere else on Earth, but they are also one of the most imperiled groups of organisms. This underscores the importance of understanding the effect that increasing salt exposure has on these threatened species. There are more data on the acute toxicity of chloride to Unionids than on chronic toxicity. This study investigated the effect of chronic sodium chloride exposure on the survival and filtering activity of two Unionid species (Eurynia dilatata, and Lasmigona costata) and assessed the effect on the metabolome in L. costata hemolymph. The concentration causing mortality after 28 days of exposure was similar for E. dilatata (1893 mg Cl-/L) and L. costata (1903 mg Cl-/L). Significant changes in the metabolome of the L. costata hemolymph were observed for mussels exposed to non-lethal concentrations. For example, several phosphatidylethanolamines, several hydroxyeicosatetraenoic acids, pyropheophorbide-a, and alpha-linolenic acid were significantly upregulated in the hemolymph of mussels exposed to 1000 mg Cl-/L for 28 days. While no mortality occurred in the treatment, elevated metabolites in the hemolymph are an indicator of stress.

Effects of municipal wastewater effluents on the digestive gland microbiome of wild freshwater mussels (Lasmigona costata).

Gut microbial communities are vital for maintaining host health, and are sensitive to diet, environment, and chemical exposures. Wastewater treatment plants (WWTPs) release effluents containing antimicrobials, pharmaceuticals, and other contaminants that may negatively affect the gut microbiome of downstream organisms. This study investigated changes in the diversity and composition of the digestive gland microbiome of flutedshell mussels (Lasmigona costata) from upstream and downstream of two large (service >100,000) WWTPs. Mussel digestive gland microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16 S rRNA gene. Bacterial alpha diversity decreased at sites downstream of the second WWTP and these sites were dissimilar in beta diversity from sites upstream and downstream of the first upstream WWTP. The microbiomes of mussels collected downstream of the first WWTP had increased relative abundances of Actinobacteria, Bacteroidetes, and Firmicutes, with a decrease in Cyanobacteria, compared to upstream mussels. Meanwhile, those collected downstream of the second WWTP increased in Proteobacteria and decreased in Actinobacteria, Bacteroidetes, and Tenericutes. Increased Proteobacteria has been linked to adverse effects in mammals, but their functions in mussels is currently unknown. Finally, effluent-derived bacteria were found in the microbiome of mussels downstream of both WWTPs but not in those from upstream. Overall, results show that the digestive gland microbiome of mussels collected upstream and downstream of WWTPs differed, which has implications for altered host health and the transport of WWTP-derived bacteria through aquatic ecosystems.

Salt-Laden Winter Runoff and Freshwater Mussels; Assessing the Effect on Early Life Stages in the Laboratory and Wild Mussel Populations in Receiving Waters.

The widespread use of road salt for winter road maintenance has led to an increase in the salinity of surface water in many seasonally cold areas. Freshwater mussels have a heightened sensitivity to salt, which is a concern, because many Canadian mussel species at risk have ranges limited to southern Ontario, Canada's most road-dense region. This study examined the effect of winter road runoff on freshwater mussels. The impact of two bridges that span mussel habitat in the Thames River watershed (Ontario, Canada), the second most species-rich watershed for mussels in Canada, were studied. During a winter melt event, bridge runoff, as well as creek surface water surrounding the bridges were collected. Chloride concentrations in samples from bridge deck and tile drains varied (99-8250 mg/L). In general, survival of Lampsilis fasciola glochidia exposed to those samples reflected chloride levels (e.g. 84% at 99 mg/L; 0% at 8250 mg/L), although potassium (60 mg/L) may have at least contributed to toxicity in one sample. Serial dilution exposures with the two most toxic runoff samples revealed 48-h glochidia EC50s of 44% (McGregor Creek Tile Drain) and 26% (Baptiste Creek Deck Drain). During the melt event, the chloride concentrations in creek surface waters downstream of the bridges ranged from 69 to 179 mg Cl-/L; effects on glochidia (viability 77-91%) exposed to those waters was minimal. There were no live mussels surrounding one bridge (Baptiste Creek), likely due to poor habitat. At the other targeted bridge (McGregor Creek), fewer mussels were found close (< 100 m up- or downstream) to the bridge than further (> 200 m) away. However, other contributing factors, including agriculture, were present at both study areas.

Acute and chronic toxicity of neonicotinoid and butenolide insecticides to the freshwater amphipod, Hyalella azteca.

Neonicotinoids are the most widely used insecticides in the world. They are preferentially toxic to insects while displaying a low toxicity toward vertebrates, and this selective toxicity has resulted in the rapid and ubiquitous use of these compounds. However, neonicotinoids have been detected in agricultural surface waters and are known to cause adverse effects in non-target aquatic organisms. A wide range of toxicity has been reported for aquatic crustaceans, but most of the studies focus on the acute effects of imidacloprid, and few data are available regarding chronic effects of other neonicotinoids or neonicotinoid replacements (e.g., butenolides). The objective of this study was to assess the acute and chronic toxicity of six neonicotinoids (imidacloprid, thiamethoxam, acetamiprid, clothianidin, thiacloprid, and dinotefuran) and one butenolide (flupyradifurone) to the freshwater amphipod Hyalella azteca. Chronic (28-d), water-only, static-renewal tests were conducted. Survival was assessed weekly, and growth was measured at the end of the exposure. Effects of neonicotinoids varied depending on the compound. Acute (7-d) LC50s were 4.0, 4.7, 60, 68, 230, and 290 µg/L for clothianidin, acetamiprid, dinotefuran, thiacloprid, imidacloprid, and thiamethoxam, respectively. Chronic (28-d) survival and growth were reduced at similar concentrations to acute (7-d) survival for thiamethoxam, acetamiprid, clothianidin, and dinotefuran. However, chronic survival and growth of amphipods exposed to imidacloprid and thiacloprid were reduced at lower concentrations than acute survival, with respective 28-d LC50s of 90 and 44 µg/L, and EC50s of 4 and 3 µg/L. Flupyradifurone was intermediate in toxicity compared to the neonicotinoids: 7-d LC50, 28-d LC50, and 28-d EC50 were 26, 20, and 16 µg/L, respectively. The concentrations of imidacloprid and clothianidin reported for North American surface waters fall within the effect ranges observed in this study, indicating the potential for these compounds to cause adverse effects to indigenous populations of H. azteca.

Investigation of clearance rate as an endpoint in toxicity testing with freshwater mussels (Unionidae).

The implementation of ecologically relevant sub-lethal endpoints in toxicity testing with freshwater mussels can provide valuable information during risk assessment, especially since these organisms are often exposed to low levels of contaminants. This study examined how to optimize quantifying the filtering capacity or clearance rate (CR) of mussels after exposure to a reference toxicant, sodium chloride (NaCl). CR was defined as the number of algal cells an individual mussel can remove from the overlying water by filtration over time and was determined using spectrophotometric absorbance and direct microscopic examination. Optimization included consideration of the following factors: concentration of algae mixture at test initiation, duration of CR assay, and statistical power. Experimental vessels contained either juvenile (ten, ~ 4 months old) or adult (one, ~ 2.5 years old) Lampsilis siliquoidea. To detect a 10% change in filtering capacity, the optimized adult CR assay was run for 48 h with 2.7 × 107 cells/mL of algae added at test initiation and a minimum of 6 replicates per treatment. The optimized juvenile mussel CR assay was run for 48 h with 1.77 × 107 cells/mL of algae added at test initiation; however, 13 replicates would be required to detect a 10% change to satisfy each method. To reduce the number of juvenile mussels used in testing, a minimum of 4 replicates per treatment was recommended to detect a 25% change in CR. After exposure to a reference toxicant (NaCl), EC50s from the optimized CR assay were compared to two other mussel toxicity endpoints: survival and burial (ability of mussels to bury in clean sand). CR by direct microscopic examination was slightly more sensitive than survival and burial in juveniles and only slightly more sensitive than survival in adults. No significant differences (p > 0.05) were detected between the EC/LC50 values determined from CR and the less labour-intensive survival and burial endpoints. The present study suggests the CR for juvenile and adult L. siliquoidea remained largely unaffected in mussels that survived a 7-day NaCl exposure.

Effects of sodium chloride exposure on ion regulation in larvae (glochidia) of the freshwater mussel Lampsilis fasciola.

The salinization of freshwater can have negative effects on ecosystem health, with heightened effects in salt-sensitive biota such as glochidia, the larvae of freshwater mussels. However, the toxicological mechanism underlying this sensitivity is unknown. Therefore, Lampsilis fasciola glochidia were exposed to NaCl (nominally 0.25 and 1.0 g/L) prepared in reconstituted moderately-hard water (control), as well as to a dilution of that water (1:4) with ultrapure reference water (diluted control). Unidirectional Na(+) influx (measured with (22)Na) was evaluated after 1, 3 and 48 h of exposure. In addition, unidirectional Cl(-) influx (measured with (36)Cl), whole-body ion (Cl(-) and Na(+)) concentrations, and glochidia viability (measured as the ability to close valves) were assessed after 48 h of exposure. Significantly reduced glochidia viability (56%) was observed after exposure to 1.0 g/L NaCl. Na(+) influx was significantly higher in glochidia exposed to both 0.25 and 1.0 g/L NaCl for 1h than in those kept under control conditions. After 3 and 48 h of exposure, differences in Na(+) influx rate between salt-exposed and control glochidia were generally reduced, indicating that larvae may be able to, at least temporarily, recover their ability to regulate Na(+) influx when exposed to elevated NaCl concentration. Compared to the moderately-hard water control, whole-body Na(+) and Cl(-) concentrations were relatively unchanged in glochidia exposed to 0.25 g/L NaCl, but were significantly elevated in glochidia exposed to 1.0 g/L NaCl and the diluted control. While Na(+) influx rate had recovered to the control level after 48 h of exposure to 1.0 g/L NaCl, Cl(-) influx rate remained elevated, being ~7-fold higher than the Na(+) influx rate. These findings suggest that the loss of viability observed when glochidia were exposed to a high NaCl concentration (1.0 g/L) could be caused by ionoregulatory disturbances mainly associated with an elevated Cl(-) influx.

Evidence of oxidative stress in wild freshwater mussels (Lasmigona costata) exposed to urban-derived contaminants.

The physiological effect of complex mixtures of anthropogenic contaminants on aquatic organisms is not well understood. This study employed a suite of sub-cellular biomarkers and general health measurements to assess the effect of urban-derived contaminants on wild freshwater mussels. Adult Lasmigona costata were collected from four sites in the Grand River (ON, Canada) that receive incremental amounts of municipal wastewater effluents and road runoff. Biomarkers of metal exposure, oxidative stress, and general health were examined in the gills of wild mussels. Concentrations of nine metals as well as the metal-binding protein, metallothionein (MT), were significantly higher (p<0.05) in mussels living downstream of the urban area. For example the concentrations of Pb, Cr and Zn were five-fold, and Ag more than 20 fold higher in mussels collected downstream of 11 municipal wastewater treatment plants and four cities compared to levels in upstream mussels. Downstream mussels showed evidence of oxidative stress, such that lipid peroxidation (LPO) (as thiobarbiturate reactive substances) was significantly elevated and the antioxidant capacity against peroxyl radicals (ACAP) was significantly decreased (p<0.01) in downstream mussels compared to upstream mussels. Regarding general health indicators, although gill lipid concentrations were similar across sites, protein concentration was significantly (p<0.001) higher in mussels collected from the upstream reference site compared to all downstream sites. The trends observed indicate that there are physiological effects in mussels associated with chronic exposure to complex urban inputs and that some biomarkers respond to municipal wastewater effluent and road runoff exposure in a cumulative manner. The observed oxidative stress response (ACAP) along with the elevation in MT, suggest that even though the defense mechanisms in the chronically exposed mussels have been activated, there is still an excess of reactive oxygen species that result in oxidative damage. The physiological effects of exposure reported in this study correspond with previously reported whole-organism impacts and declines in freshwater mussel populations in the urban-impacted region of this watershed.

Responses of biomarkers in wild freshwater mussels chronically exposed to complex contaminant mixtures.

Subcellular biochemical biomarkers are valuable early warning indicators of environmental contaminant effects. Thus, the present study evaluated several biomarkers and the relationships among them in wild freshwater mussels (Lasmigona costata) from a gradient of metal exposure and differential levels of other urban-related influences in the Grand River (ON, Canada). The biomarkers examined are related to metal exposure [gill ion and metal concentrations (Na, K, Ca, Mg, Cd, Cu, Ni, Pb and Zn)], oxidative status [reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), antioxidant capacity (ACAP)], sulfhydryl (SH) metabolism [glutathione (GSH), protein sulfhydryl groups (SH protein), glutathione S-transferase (GST), glutathione reductase (GR)], and lipid peroxidation. Gill metal concentration increased proportionally to waterborne metal concentration and disturbances in osmotic and divalent cations (Ca and Mg) concentrations were observed. This suggests that the observed effects are associated with metal exposure, although simultaneous relationships with other contaminants are also possible. Oxidative status biomarkers (ROS, SOD, CAT and ACAP) were more sensitive to urban-influences than gill metal concentration. In contrast, biomarkers involving SH metabolism (GSH, SH protein, total SH, GR and GST) were more correlated with gill metal concentration. Oxidative damage occurred when both metal and urban-related influences were high. Mechanistically, the way of dealing with oxidative stress changed when mussels were exposed to high levels of contaminants. The reduction in ROS content, SOD and CAT activity, and ACAP accompanying the stimulation of detoxification metabolism via SH (GSH and SH protein contents, GST and GR activities) and their association with gill metal concentration are discussed.

Microparticles in Wild and Caged Biota, Sediments, and Water Relative to Large Municipal Wastewater Treatment Plant Discharges.

Anthropogenically modified microparticles including microplastics are present in municipal wastewater treatment plant (WWTP) effluents; however, it is unclear whether biotic exposures are elevated downstream of these outfalls. In the fall of 2019, the present study examined whether microparticle levels in resident fish, environmental samples, and caged organisms were elevated near the Waterloo and Kitchener WWTP outfalls along the Grand River, Ontario, Canada. Wild rainbow darters (Etheostoma caeruleum) were collected from a total of 10 sites upstream and downstream of both WWTPs, along with surface water and sediment samples to assess spatial patterns over an approximately 70-km river stretch. Amphipods (Hyalella azteca), fluted-shell mussels (Lasmigona costata), and rainbow trout (Oncorhynchus mykiss) were also caged upstream and downstream of one WWTP for 14 or 28 days. Whole amphipods, fish digestive tracts, and mussel tissues (hemolymph, digestive glands, gills) were digested with potassium hydroxide, whereas environmental samples were processed using filtration and density separation. Visual identification, measurement, and chemical confirmation (subset only) of microparticles were completed. Elevated abiotic microparticles were found at several upstream reference sites as well as at one or both wastewater-impacted sites. Microparticles in amphipods, all mussel tissues, and wild fish did not show patterns indicative of increased exposures downstream of effluent discharges. In contrast, elevated microparticle counts were found in trout caged directly downstream of the outfall. Across all samples, cellulose fibers (mainly blue and clear colors) were the most common. Overall, results suggest little influence of WWTP effluents on microparticles in biota but rather a ubiquitous presence across most sites that indicates the importance of other point and nonpoint sources to this system. Environ Toxicol Chem 2024;43:1047-1061. © 2024 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Review and Development of Best Practices for Toxicity Tests with Dreissenid Mussels.

Since their introduction to North America in the 1980s, research to develop effective control tools for invasive mussels (Dreissena polymorpha and D. rostriformis bugensis) has been ongoing across various research institutions using a range of testing methods. Inconsistencies in experimental methods and reporting present challenges for comparing data, repeating experiments, and applying results. The Invasive Mussel Collaborative established the Toxicity Testing Work Group (TTWG) in 2019 to identify "best practices" and guide development of a standard framework for dreissenid mussel toxicity testing protocols. We reviewed the literature related to laboratory-based dreissenid mussel toxicity tests and determined the degree to which standard guidelines have been used and their applicability to dreissenid mussel testing. We extracted detailed methodology from 99 studies from the peer-reviewed and gray literature and conducted a separate analysis for studies using presettlement and postsettlement mussels. We identified specific components of methods and approaches that could be refined or standardized for dreissenid mussels. These components included species identification, collection methods, size/age class distinction, maintenance practices, testing criteria, sample size, response measures, reporting parameters, exposure methods, and mortality criteria. We consulted experts in the field of aquatic toxicology and dreissenid mussel biology on our proposed. The final recommendations contained in the present review are based on published standard guidelines, methods reported in the published and gray literature, and the expertise of TTWG members and an external panel. In addition, our review identifies research needs for dreissenid mussel testing including improved methods for early-life stage testing, comparative data on life stages and between dreissenid mussel species, inclusion of a reference toxicant, and additional testing of nontarget species (i.e., other aquatic organisms). Environ Toxicol Chem 2023;42:1649-1666. © 2023 His Majesty the King in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

In vitro assessment of the genotoxicity and immunotoxicity of treated and untreated municipal effluents and receiving waters in freshwater organisms.

Municipal wastewater effluent is one of the largest sources of pollution entering surface waters in the Laurentian Great Lakes. Exposure to wastewater effluent has been associated with impaired immune systems and induction of genotoxicity to aquatic animals. Due to habitat degradation and environmental pollution linked to industrial development and population growth, several regions of the Great Lakes have been designated Areas of Concern (AOCs). In this study, we assessed the effect of extracts of sewage influent, (treated) effluent and receiving surface waters from the Hamilton Harbour AOC and the Toronto and Region AOC (Ontario, Canada) on the phagocytic immune response of rainbow trout (Oncorhynchus mykiss) kidney leukocytes and the genotoxicity (DNA strand breaks) of these extracts on freshwater mussel (Eurynia dilatata) hemocytes. We identified and quantified numerous chemicals present in the various samples extracted for exposure. In freshwater mussels, extracts from Hamilton Harbour AOC induced DNA damage with the most frequency (12 out of 28 samples) regardless of sample type, reflecting past and present industrial activities. In contrast, extracts from Toronto and Region AOC induced DNA damage infrequently (2 out of 32 (summer) and 5 out of 32 (fall) samples, respectively) and from different WWTPs at different times. None of the extracts induced any significant effect on phagocytosis of rainbow trout kidney leukocytes. The present study indicates that despite overall improvements to effluent quality, treatment of influent by WWTPs may not result in a corresponding improvement of the genotoxicity of effluents. In vitro bioassays are useful and cost-effective rapid-screening tools for preliminary assessments of contamination of aquatic ecosystems.

Freshwater mussels in an impacted watershed: Influences of pollution from point and non-point sources.

The Grand River watershed in a densely populated region of Ontario supports one of the richest assemblages of freshwater mussels in Canada. However, water quality in this watershed is influenced by urban development, agriculture, and industry. Mussel populations and water chemistry in the lower Grand River and the Boston Creek tributary were evaluated to determine whether point sources of pollution such as discharges of domestic wastewater and industrial effluent, and non-point sources of pollution are affecting mussel distribution and population structure. Semi-quantitative population surveys conducted at 9 study sites identified 20 mussel species, including 3 Species at Risk. Mussel abundance (34-160 mussels/search hour) and species richness indicated that mussel populations in the lower Grand River watershed are continuing to recover from historical lows reported in the 1970s. However, changes in populations at some sites were consistent with altered water chemistry. Most notable was that the three most abundant mussel species in the Boston Creek tributary downstream of a gypsum plant discharge were significantly smaller in length than those upstream of this site. The water chemistry in this habitat was characterized by elevated conductivity (∼2000 µS/cm) and calcium (∼500 mg/L), as well as concentrations of sulfate (∼1000 mg/L) that can be toxic to freshwater mussels. In the Grand River downstream of the confluence with Boston Creek, there tended to be (p > 0.05) fewer mussels (mean 34 ± 20/search h) compared to upstream (mean 67 ± 15/search h) and this corresponded to altered water chemistry, including elevated sulfate (239 mg/L) downstream of the confluence relative to upstream (58 mg/L). These data indicate that chronic exposures to high levels of major ions is likely driving changes to mussel population structure. In addition, the discharges of wash water from a gypsum plant may be impacting sensitive biota in the main stem Grand River well beyond the immediate tributary receiving environment.

The acute toxicity of bitumen-influenced groundwaters from the oil sands region to aquatic organisms.

The extraction of surface mined bitumen from oil sands deposits in northern Alberta, Canada produces large quantities of liquid tailings waste, termed oil sands process-affected water (OSPW), which are stored in large tailings ponds. OSPW-derived chemicals from several tailings ponds migrating past containment structures and through groundwater systems pose a concern for surface water contamination. The present study investigated the toxicity of groundwater from near-field sites adjacent to a tailings pond with OPSW influence and far-field sites with only natural oil sands bitumen influence. The acute toxicity of unfractionated groundwater and isolated organic fractions was assessed using a suite of aquatic organisms (Pimephales promelas, Oryzias latipes, Daphnia magna, Hyalella azteca, Lampsilis spp., Ceriodaphnia dubia, Hexagenia spp., and Vibrio fischeri). Assessment of unfractionated groundwater demonstrated toxicity towards all invertebrates in at least one far-field sample, with both near-field and far-field samples with bitumen influence toxic towards P. promelas, while no toxicity was observed for O. latipes. When assessing the unfractionated groundwater and isolated organic fractions from near-field and far-field groundwater sites, P. promelas and H. azteca were the most sensitive to organic components, while D. magna and L. cardium were most sensitive to the inorganic components. Groundwater containing appreciable amounts of dissolved organics exhibited similar toxicities to sensitive species regardless of an OSPW or natural bitumen source. The lack of a clear distinction in relative acute toxicities between near-field and far-field samples indicates that the water-soluble chemicals associated with bitumen are acutely toxic to several aquatic organisms. This result, combined with the similarities in chemical profiles between bitumen-influenced groundwater originating from OSPW and/or natural sources, suggests that the industrial bitumen extraction processes corresponding to the tailings pond in this study are not contributing unique toxic substances to groundwater, relative to natural bitumen compounds present in groundwater flow systems.

Method Development for a Short-Term 7-Day Toxicity Test with Unionid Mussels.

The US Environmental Protection Agency's short-term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short-term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field-collected effluent in 7-day exposures. Both mussel species were equally or more sensitive (more than two-fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7-day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7-day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%-52% length increase) when the 1- or 2-week-old mussels were fed 2 ml twice daily and the 3-week-old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314-520 mg Cl/L) among the three age groups, indicating that an age range of 1- to 3-week-old mussels can be used for a 7-day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7-day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392-3409. © 2021 SETAC.

Toxicity of the pharmaceuticals finasteride and melengestrol acetate to benthic invertebrates.

The toxicity of endocrinologically active pharmaceuticals finasteride (FIN) and melengestrol acetate (MGA) was assessed in freshwater mussels, including acute (48 h) aqueous tests with glochidia from Lampsilis siliquoidea, sub-chronic (14 days) sediment tests with gravid female Lampsilis fasciola, and chronic (28 days) sediment tests with juvenile L. siliquoidea, and in chronic (42 days) sediment tests with the amphipod Hyalella azteca and the mayfly Hexagenia spp. Finasteride was not toxic in acute aqueous tests with L. siliquoidea glochidia (up to 23 mg/L), whereas significant toxicity to survival and burial ability was detected in chronic sediment tests with juvenile L. siliquoidea (chronic value (ChV, the geometric mean of LOEC and NOEC) = 58 mg/kg (1 mg/L)). Amphipods (survival, growth, reproduction, and sex ratio) and mayflies (growth) were similarly sensitive (ChV = 58 mg/kg (1 mg/L)). Melengestrol acetate was acutely toxic to L. siliquoidea glochidia at 4 mg/L in aqueous tests; in sediment tests, mayflies were the most sensitive species, with significant growth effects observed at 37 mg/kg (0.25 mg/L) (ChV = 21 mg/kg (0.1 mg/L)). Exposure to sublethal concentrations of FIN and MGA had no effect on the (luring and filtering) behaviour of gravid L. fasciola, or the viability of their brooding glochidia. Based on the limited number of measured environmental concentrations of both chemicals, and their projected concentrations, no direct effects are expected by these compounds individually on the invertebrates tested. However, organisms are exposed to contaminant mixtures in the aquatic environment, and thus, the effects of FIN and MGA as components of these mixtures require further investigation.

Investigating a potential mechanism of Cd resistance in Chironomus riparius larvae using kinetic analysis of calcium and cadmium uptake.

The uptake kinetics of waterborne Ca and Cd, both independently and in combination, were examined in C. riparius larvae, which are extremely Cd tolerant. Larvae exposed to Ca (100-2500 micromol L(-1)), exhibited classic Michaelis-Menten saturation kinetics for Ca influx, measured using (45)Ca as a radio-tracer. The maximum rate of Ca influx (J(max)(Ca)) was 0.39 micromol g(-1)h(-1), and the Ca concentration where the carrier reached half saturation (K(M)(Ca)) was 289 micromol L(-1). Cd influx was measured using (109)Cd as a radio-tracer in larvae exposed to Cd (0-1400 micromol L(-1)) while the Ca concentration was set to the K(M)(Ca). This revealed a J(max)(Cd) (2.26 micromol g(-1)h(-1)) which was nearly 6-fold higher that of Ca. This unusually high capacity for Cd uptake is in accordance with the huge tissue Cd burdens that chironomid larvae are able to accumulate during high level exposures. The apparent K(M)(Cd) (1133 micromol Cd L(-1)), when recalculated to account for the background Ca level, was still high (567 micromol Cd L(-1)), suggesting that this organism has a low affinity for Cd relative to most aquatic animals, indeed lower or comparable to its affinity for Ca. In consequence, even well above environmentally relevant Cd exposures, C. riparius does not accumulate Cd at the expense of Ca, thereby avoiding internal hypocalcaemia, in contrast to most other organisms which are much more sensitive to Cd. However, Ca influx was significantly reduced when 1200 micromol Cd L(-1) was added to Ca exposures (96-2410 micromol L(-1)). Michaelis-Menten analysis revealed a similar J(max)(Ca) in Cd-exposed and control larvae (i.e. exposed only to Ca), but that the apparent K(M)(Ca) was many-fold higher in larvae which were simultaneously exposed to Ca and Cd. Conversely, increasing Ca concentrations (96-2410 micromol L(-1)) progressively inhibited Cd uptake from a Cd exposure concentration (1200 micromol L(-1)), providing additional support for a common transport system. These results suggest that the interaction of Cd and Ca in C. riparius is one of simple competitive interaction, and that the unusual Cd transport kinetics (low affinity, high capacity) relative to fairly standard Ca transport kinetics help explain the unusual tolerance that this organism has to acute Cd exposure.

Sensitivity of the glochidia (larvae) of freshwater mussels to copper: assessing the effect of water hardness and dissolved organic carbon on the sensitivity of endangered species.

The assessment of the potential impact of waterborne contaminants on imperilled freshwater mussels is needed. Acute copper toxicity was assessed in a standardized soft water (hardness 40-48 mg CaCO(3)equivalents L(-1)) using the larvae (glochidia) from three common and six (Canadian) endangered mussel species. The resulting 24h EC50s ranged from 7 to 36 microg Cu L(-1), with the EC50s of two endangered species <10 microg Cu L(-1). Acute copper sensitivity was also determined in Ptychobranchus fasciolaris, a species that employs conglutinates (packets of glochidia) in its reproductive strategy. Conglutinates were found to provide significant protection from acute copper exposure as the EC50 of the encased glochidia was more than four-fold higher than freed glochidia (72.6 microg Cu L(-1) vs. 16.3 microg Cu L(-1)). The glochidia from two endangered species, Epioblasma triquetra and Lampsilis fasciola, were used to examine the influence of water hardness and dissolved organic carbon (DOC) on copper sensitivity. Exposures in moderately-hard water (165 mg CaCO(3) L(-1)) demonstrated that an increase in water hardness resulted in a significant reduction in copper sensitivity. For example, in L. fasciola the 24 h EC50s were 17.6 (14.2-22.6) microg Cu L(-1) and 50.4 (43.5-60.0) microg Cu L(-1) in soft water and moderately-hard water, respectively. The addition of DOC (as Aldrich Humic Acid) also resulted in a significant decrease in Cu sensitivity, such that a 10-fold increase in the EC50 of E. triquetra was observed when the reconstituted soft water was augmented with 1.6 mg DOC L(-1). To determine if current water quality regulations for copper would protect all glochidia, the USEPA's Biotic Ligand Model (BLM) was used to derive water quality criteria for these exposures. While BLM-derived criteria for the soft water exposures indicate that protection would be marginal for the sensitive endangered species, the criteria derived for the DOC exposures suggest that the natural complexity of most natural waters in Southern Ontario (Canada) will provide glochidia with protection from acute copper exposure.

The effect of extreme waterborne cadmium exposure on the internal concentrations of cadmium, calcium, and sodium in Chironomus riparius larvae.

Chironomus riparius larvae (3rd-4th instar) were extremely resistant to waterborne Cd with 48h LC50s of 331 mg Cd/L in soft water (10 mg/L Ca CO(3)) and 1106 mg Cd/L in moderately hard (140 mg CaCO(3)/L) water. Unexposed larvae had whole body Ca and Na concentrations of 11.2(0.3) and 84.5(3.0) micromol/g, respectively. The larvae exposed through acute toxicity tests accumulated massive amounts of Cd, reaching >50 micromol/g in larvae exposed to 437 mg Cd/L, though burdens were lower at higher exposure concentrations. These Cd burdens were approximately fivefold greater than whole-body Ca concentrations. Cd exposure also had a significant negative effect on internal Ca: whole-body Ca declined by over 70% in larvae exposed to Cd above the LC50 concentration. The effect of Cd exposure on whole-body Na was much less dramatic as levels dropped by 10-28% in the acutely exposed larvae. Time series exposures (up to 72h) across a range of Cd concentrations (0.1-865 mg/L) revealed that internal Ca dropped within the first hour of exposure regardless of the concentration of Cd. In all but the highest (865 mg Cd/L) exposure, internal Ca eventually recovered to the control level. Cd resistance in C. riparius may lie in its ability to maintain internal Ca balance even when exposed to extreme (>100 mg/L) levels of Cd, coupled with remarkable capacities for storage-detoxification and excretion of Cd.

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.