Toxicity of Chromium (VI) to Two Mussels and an Amphipod in Water-Only Exposures With or Without a Co-stressor of Elevated Temperature, Zinc, or Nitrate.

The objectives of the present study were to develop methods for propagating western pearlshell (Margaritifera falcata) for laboratory toxicity testing and evaluate acute and chronic toxicity of chromium VI [Cr(VI)] to the pearlshell and a commonly tested mussel (fatmucket, Lampsilis siliquoidea at 20 °C or in association with a co-stressor of elevated temperature (27 °C), zinc (50 µg Zn/L), or nitrate (35 mg NO3/L). A commonly tested invertebrate (amphipod, Hyalella azteca) also was tested in chronic exposures. Newly transformed pearlshell (~1 week old) were successfully cultured and tested in acute 96 h Cr exposures (control survival 100%). However, the grow-out of juveniles in culture for chronic toxicity testing was less successful and chronic 28-day Cr toxicity tests started with 4 month-old pearlshell failed due to low control survival (39-68%). Acute median effect concentration (EC50) for the pearlshell (919 µg Cr/L) and fatmucket (456 µg Cr/L) tested at 20 °C without a co-stressor decreased by a factor of > 2 at elevated temperature but did not decrease at elevated Zn or elevated NO3. Chronic 28-day Cr tests were completed successfully with the fatmucket and amphipod (control survival 83-98%). Chronic maximum acceptable toxicant concentration (MATC) for fatmucket at 20 °C (26 µg Cr/L) decreased by a factor of 2 at elevated temperature or NO3 but did not decrease at elevated Zn. However, chronic MATC for amphipod at 20 °C (13 µg Cr/L) did not decrease at elevated temperature, Zn, or NO3. Acute EC50s for both mussels tested with or without a co-stressor were above the final acute value used to derive United States Environmental Protection Agency acute water quality criterion (WQC) for Cr(VI); however, chronic MATCs for fatmucket at elevated temperature or NO3 and chronic MATCs for the amphipod at 20 °C with or without elevated Zn or NO3 were about equal to the chronic WQC. The results indicate that (1) the elevated temperature increased the acute Cr toxicity to both mussel species, (2) fatmucket was acutely more sensitive to Cr than the pearlshell, (3) elevated temperature or NO3 increased chronic Cr toxicity to fatmucket, and (4) acute WQC are protective of tested mussels with or without a co-stressor; however, the chronic WQC might not protect fatmucket at elevated temperature or NO3 and might not protect the amphipod at 20 °C with or without elevated Zn or NO3.

Metabolomic, behavioral, and reproductive effects of the aromatase inhibitor fadrozole hydrochloride on the unionid mussel Lampsilis fasciola.

Androgen-induced masculinization of female aquatic biota poses concerns for natural population stability. This research evaluated the effects of a twelve day exposure of fadrozole hydrochloride on the metabolism and reproductive status of the unionid mussel Lampsilis fasciola. Although this compound is not considered to be widespread in the aquatic environment, it was selected as a model aromatase (enzyme that converts testosterone to estradiol) inhibitor. Adult mussels were exposed to a control and 3 concentrations of fadrozole (2µg/L, 20µg/L, and 50µg/L), and samples of gill tissue were taken on days 4 and 12 for metabolomics analysis. Gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Daily observed mussel behavior included female mantle display, foot protrusion, siphoning, and larval (glochidia) releases. Glochidia mortality was significantly higher in the 20µg/L treatment. Fewer conglutinate (packets of glochidia) releases were observed in the 50µg/L treatment, and mortality was highly correlated to release numbers. Foot protrusion was significantly higher in females in nearly all treatments, including the control, during the first 4days of observations. However, this sex difference was observed only in the 50µg/L treatment during the last 8days. Generally, metabolites were significantly altered in female gill tissue in the 2µg/L treatment whereas males were mostly affected only at the highest (50µg/L) treatment. Both sexes also revealed significant reductions in fadrozole-induced metabolic effects in gill tissue sampled after 12days compared to tissue sampled after 4days, indicating time-dependent mechanisms of disruptions in metabolic pathways and homeostatic processes to compensate for such disruptions.

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.

Influence of pH on the acute toxicity of ammonia to juvenile freshwater mussels (fatmucket, Lampsilis siliquoidea).

The objective of the present study was to evaluate the influence of pH on the toxicity of ammonia to juvenile freshwater mussels. Acute 96-h ammonia toxicity tests were conducted with 10-d-old juvenile mussels (fatmucket, Lampsilis siliquoidea) at five pH levels ranging from 6.5 to 9.0 in flow-through diluter systems at 20 degrees C. Acute 48-h tests with amphipods (Hyalella azteca) and 96-h tests with oligochaetes (Lumbriculus variegatus) were conducted concurrently under the same test conditions to determine the sensitivity of mussels relative to these two commonly tested benthic invertebrate species. During the exposure, pH levels were maintained within 0.1 of a pH unit and ammonia concentrations were relatively constant through time (coefficient of variation for ammonia concentrations ranged from 2 to 30% with a median value of 7.9%). The median effective concentrations (EC50s) of total ammonia nitrogen (N) for mussels were at least two to six times lower than the EC50s for amphipods and oligochaetes, and the EC50s for mussels decreased with increasing pH and ranged from 88 mg N/L at pH 6.6 to 0.96 mg N/L at pH 9.0. The EC50s for mussels were at or below the final acute values used to derive the U.S. Environmental Protection Agency's acute water quality criterion (WQC). However, the quantitative relationship between pH and ammonia toxicity to juvenile mussels was similar to the average relationship for other taxa reported in the WQC. These results indicate that including mussel toxicity data in a revision to the WQC would lower the acute criterion but not change the WQC mathematical representation of the relative effect of pH on ammonia toxicity.

Ontogeny of juvenile freshwater pearl mussels, Margaritifera margaritifera (Bivalvia: Margaritiferidae).

The gills of juvenile freshwater bivalves undergo a complex morphogenesis that may correlate with changes in feeding ecology, but ontogenic studies on juvenile mussels are rare. Scanning electron microscopy was used to examine the ultrastructure and ontogeny of 117 juvenile freshwater pearl mussels (Margaritifera margaritifera) ranging in age from 1-44 months and length from 0.49-8.90 mm. Three stages of gill development are described. In Stage 1 (5-9 inner demibranch filaments), only unreflected inner demibranch filaments were present. In Stage 2 (9-17 inner demibranch filaments), inner demibranch filaments began to reflect when shell length exceeded 1.13 mm, at 13-16 months old. Reflection began in medial filaments and then proceeded anterior and posterior. In Stage 3 (28-94 inner demibranch filaments), outer demibranch filaments began developing at shell length > 3.1 mm and about 34 months of age. The oral groove on the inner demibranch was first observed in 34 month old specimens > 2.66 mm but was never observed on the outer demibranch. Shell length (R2 = 0.99) was a better predictor of developmental stage compared to age (R2 = 0.84). The full suite of gill ciliation was present on filaments in all stages. Interfilamentary distance averaged 31.3 µm and did not change with age (4-44 months) or with size (0.75-8.9 mm). Distance between laterofrontal cirri couplets averaged 1.54 µm and did not change significantly with size or age. Labial palp primordia were present in even the youngest individuals but ciliature became more diverse in more developed individuals. Information presented here is valuable to captive rearing programmes as it provides insight in to when juveniles may be particularly vulnerable to stressors due to specific ontogenic changes. The data are compared with two other recent studies of Margaritifera development.

Acute and chronic toxicity of technical-grade pesticides to glochidia and juveniles of freshwater mussels (Unionidae).

Chemical contaminants are among many potential factors involved in the decline of freshwater mussel populations in North America, and the effects of pesticides on early life stages of unionid mussels are largely unknown. The objective of this study was to determine the toxicity of technical-grade current-use pesticides to glochidia and juvenile life stages of freshwater mussels. We performed acute toxicity tests with glochidia (five species) and juveniles (two species) exposed to a suite of current-use pesticides including herbicides (atrazine and pendimethalin), insecticides (fipronil and permethrin), and a reference toxicant (NaCl). Because of limited availability of test organisms, not all species were tested with all pesticides. Toxicity tests with fungicides (chlorothalonil, propiconazole, and pyraclostrobin) were performed with one species (Lampsilis siliquoidea). Lampsilis siliquoidea glochidia and juveniles were highly sensitive to the fungicides tested but the technical-grade herbicides and insecticides, at concentrations approaching water solubility, were not acutely toxic to this or the other unionid species. In a 21-d chronic test with four-month-old juvenile L. siliquoidea, the 21-d median effective concentration (EC50) with atrazine was 4.3 mg/L and in atrazine treatments >or=3.8 mg/L mussel growth was significantly less than controls. The relatively high sensitivity of L. siliquoidea to chlorothalonil, propiconazole, and pyraclostrobin is similar to that reported for other aquatic organisms commonly used for toxicity testing. The relative risk associated with acute exposure of early life stages of mussels to technical-grade atrazine, pendimethalin, fipronil, and permethrin is likely low; however, survival and growth results with juvenile L. siliquoidea indicate that chronic exposure to high concentrations (>/=3.8 mg/L) of atrazine may have the potential to impact mussel populations and warrants further investigation.

Causes of Rapid Carrion Beetle (Coleoptera: Silphidae) Death in Flooded Pitfall Traps, Response to Soil Flooding, Immersion Tolerance, and Swimming Behavior.

Terrestrial insects in water can often delay or escape drowning by floating and swimming. However, we observed that flooding of pitfall traps baited with rotting carrion results in high overnight mortality of captured beetles and reasoned that this risk may be enhanced by microbial respiration. By assessing carrion beetle (Coleoptera: Silphidae) response to flooding, tolerance to immersion, and swimming behavior, we offer insights to this cause of death and beetle behavioral physiology. Response of buried Nicrophorus orbicollis Say to soil flooding resulted in beetles moving to the soil surface. The lethal time to 50% mortality (LT50 (immersion); mean ± 95% CI) for Nicrophorus investigator Zetterstedt, Nicrophorus marginatus F., Necrodes surinamensis F., and Thanatophilus lapponicus Herbst was 14.8 ± 2.3, 9.0 ± 3.3, 3.2 ± 1.1, and 12.1 ± 2.5 h, respectively. Swimming behavior and survival time of N. investigator was tested using yeast:sucrose (Y:S) solutions to create a eutrophic, severely hypoxic aqueous environment. LT50 (swimming) for N. investigator was 7.5 ± 1.4, 6.0 ± 1.7, and 4.2 ± 1.2 h for the low, medium, and high Y:S solutions, respectively, and >24.0 h in control treatments. Nicrophorus investigator survived nearly twice as long when completely immersed in deoxygenated water, as might occur in flooded burrows, than when swimming on the surface. We document for the first time, the rapid induction of hypoxic coma and death for a terrestrial insect from enhanced microbial activity and CO2 production of an aqueous environment, as well as suggestions on trapping protocols related to the federally endangered Nicrophorus americanus Olivier.

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.

Acute sensitivity of a broad range of freshwater mussels to chemicals with different modes of toxic action.

Freshwater mussels, one of the most imperiled groups of animals in the world, are generally underrepresented in toxicity databases used for the development of ambient water quality criteria and other environmental guidance values. Acute 96-h toxicity tests were conducted to evaluate the sensitivity of 5 species of juvenile mussels from 2 families and 4 tribes to 10 chemicals (ammonia, metals, major ions, and organic compounds) and to screen 10 additional chemicals (mainly organic compounds) with a commonly tested mussel species, fatmucket (Lampsilis siliquoidea). In the multi-species study, median effect concentrations (EC50s) among the 5 species differed by a factor of ≤2 for chloride, potassium, sulfate, and zinc; a factor of ≤5 for ammonia, chromium, copper, and nickel; and factors of 6 and 12 for metolachlor and alachlor, respectively, indicating that mussels representing different families or tribes had similar sensitivity to most of the tested chemicals, regardless of modes of action. There was a strong linear relationship between EC50s for fatmucket and the other 4 mussel species across the 10 chemicals (r2 = 0.97, slope close to 1.0), indicating that fatmucket was similar to other mussel species; thus, this commonly tested species can be a good surrogate for protecting other mussels in acute exposures. The sensitivity of juvenile fatmucket among different populations or cultured from larvae of wild adults and captive-cultured adults was also similar in acute exposures to copper or chloride, indicating captive-cultured adult mussels can reliably be used to reproduce juveniles for toxicity testing. In compiled databases for all freshwater species, 1 or more mussel species were among the 4 most sensitive species for alachlor, ammonia, chloride, potassium, sulfate, copper, nickel, and zinc; therefore, the development of water quality criteria and other environmental guidance values for these chemicals should reflect the sensitivity of mussels. In contrast, the EC50s of fatmucket tested in the single-species study were in the high percentiles (>75th) of species sensitivity distributions for 6 of 7 organic chemicals, indicating mussels might be relatively insensitive to organic chemicals in acute exposures. Environ Toxicol Chem 2017;36:786-796. Published 2016 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Cross-resistance of largemouth bass to glochidia of unionid mussels.

We tested whether host fish that acquired resistance to glochidia of one mussel species were cross-resistant to glochidia of other species. Largemouth bass (Micropterus salmoides) were primed with 4-5 successive infections of glochidia of Lampsilis reeveiana. The percentage of attached glochidia that survived and transformed to the juvenile stage (transformation success) was compared between primed fish and naïve controls. Transformation success of L. reeveiana, Lampsilis abrupta, Villosa iris, and Utterbackia imbecillis was significantly lower on primed fish (37.8%, 43.5%, 67.0%, and 13.2%, respectively) than on control fish (89.0%, 89.7%, 90.0%, and 22.2% respectively). Immunoblotting was used to analyze the binding of serum antibodies from primed fish with glochidia proteins. Antibodies bound to glochidia proteins of similar molecular weight from L. reeveiana and L. abrupta. Bound proteins of V. iris differed in molecular weight from those of the Lampsilis species. There was no binding to specific glochidia proteins of U. imbecillis or Strophitus undulatus. Our results indicate that host-acquired resistance can extend across mussel genera and subfamilies and might involve both specific and nonspecific mechanisms. Understanding the specificity of acquired resistance of hosts to glochidia could enhance understanding of the evolutionary and ecological relationships between mussels and their host fishes.

Evaluation of the short term 12 hour toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) to multiple life stages of Venustaconcha ellipsiformis and Epioblasma triquetra and its host fish (Percina caprodes).

The present study evaluated the risk of 12-h exposures of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) to multiple life stages of the federally endangered snuffbox (Epioblasma triquetra) and its primary host fish the common logperch (Percina caprodes) as well as a surrogate to the snuffbox, the ellipse (Venustaconcha ellipsiformis). Life stages examined included free glochidia, 1-wk juveniles, and adults of the ellipse; free glochidia, glochidia on host fish, and 1-wk juveniles of the snuffbox; and adult logperch. Larval sea lampreys were also tested alongside adult ellipse and logperch for direct comparison. Survival exceeded 82% among all life stages in both mussel species at levels up to 1.8 times what would be applied during treatments, suggesting that routine sea lamprey control operations would not adversely affect mussels. However, substantial mortality of adult logperch was observed at TFM concentrations typically applied to streams, and loss of host fish could adversely affect snuffbox reproduction. In addition, TFM had no significant effect on the number of glochidia that metamorphosed on adult logperch. Although the snuffbox is not likely to be acutely affected from sea lamprey control operations, mitigation efforts to minimize impacts to the host fish should be considered.

Metabolomic, behavioral, and reproductive effects of the synthetic estrogen 17 α-ethinylestradiol on the unionid mussel Lampsilis fasciola.

The endocrine disrupting effects of estrogenic compounds in surface waters on fish, such as feminization of males and altered sex ratios, may also occur in aquatic invertebrates. However, the underlying mechanisms of action and toxicity, especially in native freshwater mussels (Order Unionoida), remain undefined. This study evaluated the effects of a 12-day exposure of 17 α-ethinylestradiol (EE2), a synthetic estrogen in oral contraceptives commonly found in surface waters, on the behavior, condition, metabolism, and reproductive status of Lampsilis fasciola. Adult mussels of both sexes were exposed to a control and two concentrations of EE2 (0 ng/L, 5 ng/L considered to be environmentally relevant, and 1,000 ng/L designed to provide a positive metabolic response), and samples of gill tissue were taken on days 4 and 12; gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Observations of mussel behavior (mantle display, siphoning, and foot movement) were made daily, and condition of conglutinates (packets of eggs and/or glochidia) released by females was examined. No significant effects of EE2 on glochidia mortality, conglutinate condition, female marsupial gill condition, or mussel foot extension were observed. However, exposure to both concentrations of EE2 significantly reduced male siphoning and mantle display behavior of females. Metabolomics analyses identified 207 known biochemicals in mussel gill tissue and showed that environmentally relevant EE2 concentrations led to decreases in glycogen metabolism end products, glucose, and several essential fatty acids in females after 12 days, indicating reductions in energy reserves that could otherwise be used for growth or reproduction. Moreover, males and females showed significant alterations in metabolites involved in signal transduction, immune response, and neuromodulation. Most of these changes were apparent at 1,000 ng/L EE2, but similar metabolites and pathways were also affected at 5 ng/L EE2. Components of the extracellular matrix of gill tissue were also altered. These results demonstrate the utility of metabolomics when used in conjunction with traditional physiological and behavioral toxicity test endpoints and establish the usefulness of this approach in determining possible underlying toxicological mechanisms of EE2 in exposed freshwater mussels.

Assessment of toxicity test endpoints for freshwater mussel larvae (glochidia).

The objectives of the present study were to determine if the viability of freshwater mussel larvae (glochidia) is an ecologically relevant endpoint for toxicity tests and to define the appropriate duration of those tests. The authors assessed 1) how viability (the shell closure response to sodium chloride) compares with infectivity (ability to attach to a host fish and successfully metamorphose to the juvenile stage), and 2) the decline of viability and infectivity over time after glochidia were released from female mussels. Glochidia of 7 mussel species were isolated from females, placed in water, and subsampled daily for 2 d to 5 d. Viability, when ≥90%, was generally a good predictor of infectivity; however, when viability was <90%, infectivity was often disproportionately low, especially for glochidia collected near the end of the brooding period. Viability and infectivity declined more rapidly in natural water and sediment compared to reconstituted water. Following 24-h exposure to a toxicant (sodium chloride or copper), infectivity of the viable glochidia did not differ among concentrations of toxicants. The results indicate that viability is a valid proxy for infectivity and an ecologically relevant endpoint for standard toxicity tests with freshwater mussels for any test duration with control viability >90%.

Fluoxetine alters adult freshwater mussel behavior and larval metamorphosis.

We used acute and partial-lifecycle tests to examine the effects of the pharmaceutical fluoxetine on freshwater mussels (Unionida). In acute tests lasting 24-48 h, we determined median effective concentrations (EC50s) for fluoxetine with larval (glochidia viability) and juvenile (survival) life-stages of fatmucket (Lampsilis siliquoidea) and black sandshell (Ligumia recta). In a 28-d behavioral test we exposed brooding adult female wavy-rayed lampmussels (Lampsilis fasciola) to 0.37 and 29.3 µg/L fluoxetine to determine effects on adult behavior (foot protrusion, mantle lure display and glochidia parturition). We also assessed the effects of 24-h exposure of 1 and 100 µg/L fluoxetine on glochidia viability duration and metamorphosis success for the wavy-rayed lampmussel. Fluoxetine EC50s ranged from 62 µg/L for juveniles (96 h) to 293 µg/L for glochidia (24 h). In adults, statistically significant increases were observed in foot protrusion at 0.37 and 29.3 µg/L fluoxetine and lure display rates at 29.3 µg/L; glochidia parturition was not significantly affected at any test concentration. Twenty-four hour exposure of glochidia to fluoxetine did not affect viability duration, but likelihood of metamorphosis to the juvenile stage significantly increased with 1 and 100 µg/L treatments. Our results demonstrated effects of fluoxetine to unionid mussels at concentrations less than previously reported and approaching concentrations measured in surface waters.

Partial life-cycle and acute toxicity of perfluoroalkyl acids to freshwater mussels.

Freshwater mussels are among the most sensitive aquatic organisms to many contaminants and have complex life-cycles that include several distinct life stages with unique contaminant exposure pathways. Standard acute (24-96 h) and chronic (28 d) toxicity tests with free larva (glochidia) and juvenile mussels are effective at generating data on contaminant effects at two discrete life stages but do not incorporate effects on brooded glochidia. We developed a novel partial life-cycle assay that incorporates exposures to brooding adult female mussels and used this method in combination with acute toxicity tests to assess adverse effects of perfluoroctanesulfonic acid (PFOS) and perfluoroctanoic acid (PFOA) on freshwater mussels. Fatmucket (Lampsilis siliquoidea) were exposed to PFOS at two life stages: brooding glochidia (in marsupia) for 36 d and free glochidia in water for 24 h. In standard acute tests with glochidia (24-48 h exposures) and juveniles (48-96 h exposures) of fatmucket and black sandshell (Ligumia recta), glochidia were 8 to 25 times more sensitive than juveniles. Perfluoroctanesulfonic acid significantly reduced the duration of glochidia viability and reduced probability of metamorphosis at concentrations 3,000 times lower than the most sensitive acute endpoint (24-h EC50). The partial life-cycle test is adaptable to a variety of endpoints and research objectives and is useful for identifying adverse effects at contaminant concentrations below those required for an acute lethal response.

Evaluation of influence of sediment on the sensitivity of a unionid mussel (Lampsilis siliquoidea) to ammonia in 28-day water exposures.

A draft update of the U.S. Environmental Protection Agency ambient water quality criteria (AWQC) for ammonia substantially lowers the ammonia AWQC, primarily due to the inclusion of toxicity data for freshwater mussels. However, most of the mussel data used in the updated AWQC were generated from water-only exposures and limited information is available on the potential influence of the presence of a substrate on the response of mussels in laboratory toxicity tests. Our recent study demonstrated that the acute sensitivity of mussels to ammonia was not influenced by the presence of substrate in 4-d laboratory toxicity tests. The objective of the current study was to determine the sensitivity of mussels to ammonia in chronic 28-d water exposures with the sediment present (sediment treatment) or absent (water-only treatment). The chronic toxicity test was conducted starting with two-month-old juvenile mussels (fatmucket, Lampsilis siliquoidea) in a flow-through diluter system, which maintained consistent pH (≈8.3) and six concentrations of total ammonia nitrogen (N) in overlying water and in sediment pore water. The chronic value (ChV, geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) was 0.36 mg N/L for survival or biomass in the water-only treatment, and was 0.66 mg N/L for survival and 0.20 mg N/L for biomass in the sediment treatment. The 20% effect concentration (EC20) for survival was 0.63 mg N/L in the water-only treatment and was 0.86 mg N/L in the sediment treatment (with overlapping 95% confidence intervals; no EC20 for biomass was estimated because the data did not meet the conditions for any logistic regression analysis). The similar ChVs or EC20s between the water-only treatment and the sediment treatment indicate that the presence of sediment did not substantially influence the sensitivity of juvenile mussels to ammonia in the 28-d chronic laboratory water exposures.

An evaluation of the influence of substrate on the response of juvenile freshwater mussels (fatmucket, Lampsilis siliquoidea) in acute water exposures to ammonia.

Acute 96-h ammonia toxicity to three-month-old juvenile mussels (Lampsilis siliquoidea) was evaluated in four treatments (water-only, water-only with feeding, water and soil, and water and sand) using an exposure unit designed to maintain consistent pH and ammonia concentrations in overlying water and in pore water surrounding the substrates. Median effect concentrations (EC50s) for total ammonia nitrogen in the four treatments ranged from 5.6 to 7.7 mg/L and median lethal concentrations (LC50s) ranged from 7.0 to 11 mg/L at a mean pH of 8.4. Similar EC50s or LC50s with overlapping 95% confidence intervals among treatments indicated no influence of substrate on the response of mussels in acute exposures to ammonia.

Persistence of host response against glochidia larvae in Micropterus salmoides.

Host fish acquire resistance to the parasitic larvae (glochidia) of freshwater mussels (Unionidae). Glochidia metamorphose into juvenile mussels while encysted on host fish. We investigated the duration of acquired resistance of largemouth bass, Micropterus salmoides (Lacepède, 1802) to glochidia of the broken rays mussel, Lampsilis reeveiana (Call, 1887). Fish received three successive priming infections with glochidia to induce an immune response. Primed fish were held at 22-23 degrees C and were challenged (re-infected) at intervals after priming. Metamorphosis success was quantified as the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive. Metamorphosis success at 3, 7, and 12 months after priming was significantly lower on primed fish (26%, 40%, and 68% respectively) than on control fish (85%, 93%, and 92% respectively). A second group of largemouth bass was similarly primed and blood was extracted. Immunoblotting was used to detect host serum antibodies to L. reeveiana glochidia proteins. Serum antibodies were evident in primed fish, but not in naive control fish. Acquired resistance of host fish potentially affects natural reproduction and artificial propagation of unionids, many of which are of conservation concern.