In aquatic environments, organisms such as freshwater mussels are likely exposed to complex contaminant mixtures related to industrial, agricultural, and urban activities. With growing interest in understanding the risk that chemical mixtures pose to mussels, this investigation focused on the effects of various waterborne contaminants (ammonia, chloride, copper, and potassium) and selected binary mixtures of these chemicals following a fixed-ratio design to Villosa iris glochidia and juvenile Lampsilis fasciola. In individual exposures, 48-h EC50 values were determined for V. iris glochidia exposed to ammonia chloride (7.4 [95% confidence interval (CI) 6.6–8.2] mg N/L), ammonia sulfate (8.4 [7.6–9.1] mg N/L), copper sulfate (14.2 [12.9–15.4] μg Cu2+/L), potassium chloride (12.8 [11.9–13.7] mg K+/L), potassium sulfate (10.1 [8.9–11.2] mg K+/L), and sodium chloride (480.5 [435.5–525.5] mg Cl−/L). The 7-d LC50 values for juvenile L. fasciola were determined for potassium sulfate (45.0 [18.8–71.2] mg K+/L), and sodium chloride (1738.2 [1418.6–2057.8] mg Cl−/L). In Ontario these waterborne contaminants have been reported to co-occur, with concentrations exceeding the EC10 for both life stages at some locations. Data from binary mixture exposures for V. iris glochidia (chloride-ammonia, chloride-copper, and copper-ammonia) and juvenile L. fasciola (chloride-potassium) were analyzed using a regression-based, dose-response mixture analysis modeling framework. Results from the mixture analysis were used to determine if an additive model for mixture toxicity [concentration addition (CA) or independent action (IA)] best described the toxicity of each mixture and if deviation towards dose-ratio (DR) or dose-level (DL) synergism/antagonism (S/A) occurred. For all glochidia binary mixture exposures, CA was the best fit model with DL deviation reported for the chloride-copper mixture and DR deviation reported for the copper-ammonia mixture. Using the model deviation ratio (MDR), the observed toxicity in all three glochidia mixture exposures were adequately described by both CA (mean = 0.71) and IA (mean = 0.97) whereas the juvenile mixture exposure was only adequately described by CA (mean = 0.64; IA mean = 0.05).

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.

Neonicotinoid insecticides can be transported from agricultural fields, where they are used as foliar sprays or seed treatments, to surface waters by surface or sub-surface runoff. Few studies have investigated the toxicity of neonicotinoid or the related butenolide insecticides to freshwater mollusk species. The current study examined the effect of neonicotinoid and butenolide exposures to the early-life stages of the ramshorn snail, Planorbella pilsbryi, and the wavy-rayed lampmussel, Lampsilis fasciola. Juvenile P. pilsbryi were exposed to imidacloprid, clothianidin, or thiamethoxam for 7 or 28 d and mortality, growth, and biomass production were measured. The viability of larval (glochidia) L. fasciola was monitored during a 48 h exposure to six neonicotinoids (imidacloprid, thiamethoxam, clothianidin, acetamiprid, thiacloprid, or dinotefuran), or a butenolide (flupyradifurone). The 7-d LC50s of P. pilsbryi for imidacloprid, clothianidin, and thiamethoxam were ≥4000 μg/L and the 28-d LC50s were ≥182 μg/L. Growth and biomass production were considerably more sensitive endpoints than mortality with EC50s ranging from 33.2 to 122.0 μg/L. The 48-h LC50s for the viability of glochidia were ≥456 μg/L for all seven insecticides tested. Our data indicate that neonicotinoid and butenolide insecticides pose less of a hazard with respect to mortality of the two species of mollusk compared to the potential hazard to other non-target aquatic insects.

Substituted phenylamines (SPAs) are incorporated into a variety of consumer products (e.g., polymers, lubricants) in order to increase the lifespan of the products by acting as a primary antioxidant. Based on their physicochemical properties, if SPAs were to enter the aquatic environment, they would likely partition into sediment. No studies to date have investigated the effect of sediment-associated SPAs on aquatic organisms. The current study examined the effect of four SPAs (diphenylamine (DPA); N-phenyl-1-napthylamine (PNA); N-(1,3-dimethylbutyl)-N’-phenyl-1,4-phenylenediamine (DPPDA); 4,4’-methylene-bis[N-sec-butylaniline] (MBA)) on three different life stages of the freshwater mussel, Lampsilis siliquoidea. The viability of larvae (glochidia) of L. siliquoidea and Lampsilis fasciola was assessed after 48 h of exposure to SPAs in water. The 48-h EC50s for glochidia viability of L. siliquoidea were 5951, 606, 439, and 258 μg/L for DPA, PNA, DPPDA, and MBA, respectively, and 7946, 591, 137, and 47 μg/L, respectively, for L. fasciola. Juvenile (7–15 months) and adult L. siliquoidea were exposed to sediment-associated SPAs for 28 d. LC50s for juvenile mussels were 18, 55, 62, and 109 μg/g dry weight (dw) of sediment for DPA, PNA, DPPDA, and MBA, respectively. Adult mussels were exposed to sub-lethal concentrations of sediment-associated SPAs in order to investigate reactive oxygen species (ROS), lipid peroxidation and total glutathione in the gill, gonad, and digestive gland tissue, and viability and DNA damage in hemocytes. No significant concentration-dependent trend in any of these biochemical and cellular endpoints relative to the concentration of sediment-associated SPAs was observed in any tissues. Investigations into the concentration of SPAs in the aquatic environment are required before a conclusion can be made on whether these compounds pose a hazard to the different life stages of freshwater mussels.

Chloride concentrations in surface waters have increased significantly, a rise attributed to road salt use. In Canada, this may be a concern for endangered freshwater mussels, many with ranges limited to southern Ontario, Canada’s most road-dense region. The acute toxicity of NaCl was determined for glochidia, the mussel’s larval stage. The 24h EC50s of four (including two Canadian endangered) species ranged from 113–1430mgClL⁻¹ (reconstituted water, 100mg CaCO₃L⁻¹). To determine how mussels would respond to a chloride pulse, natural river water (hardness 278–322mg CaCO₃L⁻¹) was augmented with salt. Lampsilis fasciola glochidia were significantly less sensitive to salt in natural water (EC50s 1265–1559mg Cl L⁻¹) than in reconstituted water (EC50 285mgL⁻¹). Chloride data from mussel habitats revealed chloride reaches levels acutely toxic to glochidia (1300mgL⁻¹). The increased salinization of freshwater could negatively impact freshwater mussels, including numerous species at risk.

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.