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.

Evaluation of Chronic Effects of Potassium Chloride and Nickel on Survival, Growth, and Reproduction of a Unionid Mussel (Lampsilis siliquoidea).

The ASTM International standard test method for freshwater mussels (E2455-13) recommends 4-week toxicity testing with juveniles to evaluate chronic effects on survival and growth. However, concerns remain that the method may not adequately address the sensitivity of mussels to longer term exposures (>4 weeks), particularly in relation to potential reproductive impairments. No standard method directly evaluates toxicant effects on mussel reproduction. The objectives of the present study were to (1) evaluate toxicity endpoints related to reproduction in fatmucket (Lampsilis siliquoidea) using two common reference toxicants, potassium chloride (KCl) and nickel (Ni); (2) evaluate the survival and growth of juvenile fatmucket in standard 4-week and longer term (12-week) KCl and Ni tests following a method refined from the standard method; and (3) compare the sensitivity of the reproductive endpoints with the endpoints obtained from the juvenile mussel tests. Reproductive toxicity tests were conducted by first exposing female fatmucket brooding mature larvae (glochidia) to five test concentrations of KCl and Ni for 6 weeks. Subsamples of the glochidia were then removed from the adults to determine three reproductive endpoints: (1) the viability of brooded glochidia; (2) the viability of free glochidia in a 24-h exposure to the same toxicant concentrations as their mother; and (3) the success of glochidia parasitism on host fish. Mean viability of brooded glochidia was significantly reduced in the high KCl concentration (26 mg K/L) relative to the control, with a 20% effect concentration (EC20) of 14 mg K/L, but there were no significant differences between the control and any Ni treatment (EC20 > 95 µg Ni/L). The EC20s for viability of free glochidia after the additional 24-h exposure and parasitism success were similar to the EC20s of brooded glochidia. The EC20s based on the most sensitive biomass endpoint in the 4-week juvenile tests were 15 mg K/L and 91 µg Ni/L, similar to or greater than the EC20s from the reproductive KCl and Ni tests, respectively. When exposure duration in the juvenile tests was extended from 4 to 12 weeks, the EC20s decreased by more than 50% in the KCl test but by only 8% in the Ni test. Overall, these results indicate that a standard 4-week test with juvenile mussels can prove effective for estimating effects in chronic exposures with different life stages although a longer term 12-week exposure with juvenile mussels may reveal higher sensitivity of mussels to some toxicants, such as KCl. Environ Toxicol Chem 2024;43:1097-1111. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

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.

Long-term radiographic appearance of a bioabsorbable biocomposite tibial tuberosity advancement cage implant.

OBJECTIVE: To report the radiographic appearance of a bioabsorbable biocomposite tibial tuberosity advancement cage at least 1 year after implantation. Design Retrospective case series. METHODS: Medical records (February 2014-March 2015) of dogs receiving a biocomposite tibial tuberosity advancement cage were reviewed. Cases were selected if they had undergone surgery at least 1 year before the selection, no additional surgeries were performed, and no known surgical site infection had occurred. Medical record information assessed included signalment, body weight (kg), affected stifle joint (left or right), date of original surgery and the size of biocomposite cage used (9 or 12 mm). Radiographs were evaluated by two blinded radiologists who calculated percentages of osteolucency present in five zones around the cage and assigned a numerical score based on these calculations. Variables were evaluated statistically for effect on lucency percentage and numerical score. RESULTS: Fifty dogs were included. Zone 5 (caudoproximal area) was found to have the lowest lucency percentage and score and zone 3 (distal area) had the highest lucency percentage and score. Twelve-millimetre cages were significantly associated with a higher lucency numerical score than 9 mm cages. CONCLUSION: A biocomposite tibial tuberosity advancement cage was found to have variable amounts of radiographically apparent osseous integration at least 1 year after implantation.

Prevention of mucosal Escherichia coli infection by FimH-adhesin-based systemic vaccination.

Virtually all uropathogenic strains of Escherichia coli, the primary cause of cystitis, assemble adhesive surface organelles called type 1 pili that contain the FimH adhesin. Sera from animals vaccinated with candidate FimH vaccines inhibited uropathogenic E. coli from binding to human bladder cells in vitro. Immunization with FimH reduced in vivo colonization of the bladder mucosa by more than 99 percent in a murine cystitis model, and immunoglobulin G to FimH was detected in urinary samples from protected mice. Furthermore, passive systemic administration of immune sera to FimH also resulted in reduced bladder colonization by uropathogenic E. coli. This approach may represent a means of preventing recurrent and acute infections of the urogenital mucosa.

Evaluation of acute copper toxicity to juvenile freshwater mussels (fatmucket, Lampsilis siliquoidea) in natural and reconstituted waters.

The influence of dissolved organic carbon (DOC) and water composition on the toxicity of copper to juvenile freshwater mussels (fatmucket, Lampsilis siliquoidea) were evaluated in natural and reconstituted waters. Acute 96-h copper toxicity tests werec onducted at four nominal DOC concentrations (0, 2.5, 5, and 10 mg/L as carbon [C]) in dilutions of natural waters and in American Society for Testing and Materials (ASTM) reconstituted hard water. Toxicity tests also were conducted in ASTM soft, moderately hard, hard, and very hard reconstituted waters (nominal hardness 45-300 mg/L as CaCO3). Three natural surface waters (9.5-11 mg/L DOC) were diluted to obtain a series of DOC concentrations with diluted well water, and an extract of natural organic matter and commercial humic acid was mixed with ASTM hard water to prepare a series of DOC concentrations for toxicity testing. Median effective concentrations (EC50s) for dissolved copper varied >40-fold (9.9 to >396 gg Cu/L) over all 21 treatments in various DOC waters. Within a particular type of DOC water, EC50s increased 5- to 12-fold across DOC concentrations of 0.3 to up to 11 mg C/L. However, EC50s increased by only a factor of 1.4 (21-30 gg Cu/L) in the four ASTM waters with wide range of water hardness (52-300 mg CaCO3/L). Predictions from the biotic ligand model (BLM) for copper explained nearly 90% of the variability in EC50s. Nearly 70% of BLM-normalized EC50s for fatmucket tested in natural waters were below the final acute value used to derive the U.S. Environmental Protection Agency acute water quality criterion for copper, indicating that the criterion might not be protective of fatmucket and perhaps other mussel species.

Pharmacokinetics of an immediate and extended release oral morphine formulation utilizing the spheroidal oral drug absorption system in dogs.

This study investigated the pharmacokinetics of a human-labeled oral morphine formulation consisting of both immediate and extended release components in dogs. In a randomized design, 14 dogs were administered either 1 or 2 mg/kg morphine orally. Blood samples were collected up to 24 h post drug administration. Plasma concentrations of morphine were measured using high-pressure liquid chromatography with electrochemical coulometric detection. For both groups, maximal concentration occurred at 3 h post drug administration followed by a gradual decrease in morphine concentration over 24 h. There was substantial variability in morphine concentrations among dogs. The higher dose group produced a greater exposure (higher area-under-the-curve), higher peak concentration, longer half-life and a shorter time to peak concentration (t(max)). The specific oral morphine formulation used in this study produced sustained plasma morphine concentrations over 24 h compared with previous intravenous dosing and immediate-release oral morphine studies. However, the low morphine plasma concentrations and high variability produced from this formulation, suggest that the clinical application of this formulation at the doses evaluated in this study are limited.

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.

Congenital dysfibrinogenemia: fibrinogen Detroit.

A 17 yr old female with a congenital bleeding disorder was found to suffer from dysfibrinogenemia. Whole blood and plasma coagulation times were delayed and thrombelastograms were grossly abnormal. Clottability of plasma fibrinogen by addition of thrombin was not demonstrated during the 30 min test period. Fibrinogen was revealed by turbidometric and immunologic techniques. Other coagulation factors were present in normal amounts and prothrombin activation was normal. Patient's plasma inhibited thrombin clotting times of normal plasma and purified normal fibrinogen. Fibrinolysis was not detected. The plasma fibrinogen migrated normally on paper and cellulose acetate electrophoresis, but on immunoelectrophoresis it displayed a faster mobility than normal fibrinogen. On immunodiffusion the antigenic determinants were similar to those of normal fibrinogen. The patient's fibrinogen-antifibrinogen precipitins required longer to appear and the resultant precipitin was broader and hazier than those elicited with normal fibrinogen. These findings suggest the presence of two discrete populations of fibrinogen molecules. Investigation of the family of the patient suggested that the defect has an autosomal dominant pattern of heredity. Immunologic comparisons of our patient's plasma and of her relatives with plasma of patients with "Fibrinogen Baltimore" and "Fibrinogen Cleveland" revealed certain differences in immunoelectrophoretic mobility as well as in immunodiffusion. In keeping with the nomenclatures of abnormal fibrinogens in the literature, we propose the term "Fibrinogen Detroit" for this fibrinogen.Physicochemical properties of "Fibrinogen Detroit" were investigated also and compared with those of normal fibrinogen. Purified normal fibrinogen (clottability 96.7%) and "Fibrinogen Detroit" revealed homogeneity when studied by ultracentrifugation and immunoelectrophoresis. Native and cleaved "Fibrinogen Detroit" had the same sedimentation constants and molecular weights as the normal. In fresh samples. 3 moles of free SH groups/mole of fibrinogen were titrated in both. Determination of the amino acid composition revealed a decreased content of lysine, glucosamine, and galactosamine in abnormal fibrinogen. Total carbohydrates, protein-bound hexoses, sialic acid, and hexosamine were decreased in the abnormal fibrinogen. In an investigation with Doctors Blombäck a specific molecular defect was revealed in the N-terminal disulfide knot of the alpha (A) chain in which the arginine at the 19th position was replaced by serine. It is believed that the substitution of a strongly basic amino acid with a neutral hydroxy acid may result in considerable conformational changes in the N-terminal disulfide knot of fibrinogen which might affect the "active site" for polymerization. The lower carbohydrate content observed in "Fibrinogen Detroit" may have been the result of a change in primary and tertiary structure of the protein.

Chaperone-assisted pilus assembly and bacterial attachment.

Bacterial pili assembled by the chaperone-usher pathway can mediate microbial attachment, an early step in the establishment of an infection, by binding specifically to sugars present in host tissues. Recent work has begun to reveal the structural basis both of chaperone function in the biogenesis of these pili and of bacterial attachment.

Acute and chronic toxicity of glyphosate compounds to glochidia and juveniles of Lampsilis siliquoidea (Unionidae).

Native freshwater mussels (family Unionidae) are among the most imperiled faunal groups in the world. Factors contributing to the decline of mussel populations likely include pesticides and other aquatic contaminants; however, there is a paucity of data regarding the toxicity of even the most globally distributed pesticides, including glyphosate, to mussels. Therefore, the toxicity of several forms of glyphosate, its formulations, and a surfactant (MON 0818) used in several glyphosate formulations was determined for early life stages of Lampsilis siliquoidea, a native freshwater mussel. Acute and chronic toxicity tests were performed with a newly established American Society of Testing and Materials (ASTM) standard guide for conducting toxicity tests with freshwater mussels. Roundup, its active ingredient, the technical-grade isopropylamine (IPA) salt of glyphosate, IPA alone, and MON 0818 (the surfactant in Roundup formulations) were each acutely toxic to L. siliquoidea glochidia. MON 0818 was most toxic of the compounds tested and the 48-h median effective concentration (0.5 mg/L) for L. siliquoidea glochidia is the lowest reported for any aquatic organism tested to date. Juvenile L. siliquoidea were also acutely sensitive to MON 0818, Roundup, glyphosate IPA salt, and IPA alone. Technical-grade glyphosate and Aqua Star were not acutely toxic to glochidia or juveniles. Ranking of relative chronic toxicity of the glyphosate-related compounds to juvenile mussels was similar to the ranking of relative acute toxicity to juveniles. Growth data from chronic tests was largely inconclusive. In summary, these results indicate that L. siliquoidea, a representative of the nearly 300 freshwater mussel taxa in North America, is among the most sensitive aquatic organisms tested to date with glyphosate-based chemicals and the surfactant MON 0818.

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.

Acute and chronic toxicity of pesticide formulations (atrazine, chlorpyrifos, and permethrin) to glochidia and juveniles of Lampsilis siliquoidea.

Freshwater mussels are among the most imperiled faunal groups in North America; approximately 67% of the nearly 300 native freshwater mussel species (family Unionidae) are listed as endangered, threatened, or of special concern. Despite evidence that glochidia and juvenile life stages are highly sensitive to some chemical contaminants, the effects of pesticides on early life stages of unionid mussels are largely unknown. In the United States, pesticide registration is based on toxicity data of the active ingredient, not formulations as they are sold and applied. Some pesticide formulations, however, are more toxic than their active ingredient (technical-grade pesticide) alone because of the presence of surfactants, adjuvants, or other ingredients in the formulation. The objective of the present study was to compare the toxicity of active ingredients of several current-use pesticides (atrazine, chlorpyrifos, and permethrin) to the toxicity of pesticide formulations to glochidia and juvenile life stages of a freshwater mussel (Lampsilis siliquoidea). The atrazine formulation (Aatrex) was more toxic than technical-grade atrazine in chronic tests with juvenile L. siliquoidea. For other pesticides, acute and chronic toxicity of technical-grade pesticides were similar to the toxicity of pesticide formulations. Median effective concentrations for chlorpyrifos were 0.43 mg/L for glochidia at 48 h, 0.25 mg/L for juveniles at 96 h, and 0.06 mg/L for juveniles at 21 d. Atrazine and permethrin as well as their formulations did not cause significant acute toxicity in glochidia or juveniles at exposure concentrations approaching water-solubility limits. Additional research is needed on other pesticides with different modes of action, on the role of different routes of exposure, and with other species of unionid mussels to evaluate similarities of toxic response.

Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae).

The objective of the present study was to determine acute toxicity of copper, ammonia, or chlorine to larval (glochidia) and juvenile mussels using the recently published American Society for Testing and Materials (ASTM) Standard guide for conducting laboratory toxicity tests with freshwater mussels. Toxicity tests were conducted with glochidia (24- to 48-h exposures) and juveniles (96-h exposures) of up to 11 mussel species in reconstituted ASTM hard water using copper, ammonia, or chlorine as a toxicant. Copper and ammonia tests also were conducted with five commonly tested species, including cladocerans (Daphnia magna and Ceriodaphnia dubia; 48-h exposures), amphipod (Hyalella azteca; 48-h exposures), rainbow trout (Oncorhynchus mykiss; 96-h exposures), and fathead minnow (Pimephales promelas; 96-h exposures). Median effective concentrations (EC50s) for commonly tested species were >58 microg Cu/L (except 15 microg Cu/L for C. dubia) and >13 mg total ammonia N/L, whereas the EC50s for mussels in most cases were <45 microg Cu/L or <12 mg N/L and were often at or below the final acute values (FAVs) used to derive the U.S. Environmental Protection Agency 1996 acute water quality criterion (WQC) for copper and 1999 acute WQC for ammonia. However, the chlorine EC50s for mussels generally were >40 microg/L and above the FAV in the WQC for chlorine. The results indicate that the early life stages of mussels generally were more sensitive to copper and ammonia than other organisms and that, including mussel toxicity data in a revision to the WQC, would lower the WQC for copper or ammonia. Furthermore, including additional mussel data in 2007 WQC for copper based on biotic ligand model would further lower the WQC.

Chronic toxicity of copper and ammonia to juvenile freshwater mussels (Unionidae).

The objectives of the present study were to develop methods for conducting chronic toxicity tests with juvenile mussels under flow-through conditions and to determine the chronic toxicity of copper and ammonia to juvenile mussels using these methods. In two feeding tests, two-month-old fatmucket (Lampsilis siliquoidea) and rainbow mussel (Villosa iris) were fed various live algae or nonviable algal mixture for 28 d. The algal mixture was the best food resulting in high survival (>or=90%) and growth. Multiple copper and ammonia toxicity tests were conducted for 28 d starting with two-month-old mussels. Six toxicity tests using the algal mixture were successfully completed with a control survival of 88 to 100%. Among copper tests with rainbow mussel, fatmucket, and oyster mussel (Epioblasma capsaeformis), chronic value ([ChV], geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) ranged from 8.5 to 9.8 microg Cu/L for survival and from 4.6 to 8.5 microg Cu/L for growth. Among ammonia tests with rainbow mussel, fatmucket, and wavy-rayed lampmussel (L. fasciola), the ChV ranged from 0.37 to 1.2 mg total ammonia N/L for survival and from 0.37 to 0.67 mg N/L for growth. These ChVs were below the U.S. Environmental Protection Agency 1996 chronic water quality criterion (WQC) for copper (15 microg/L; hardness 170 mg/L) and 1999 WQC for total ammonia (1.26 mg N/L; pH 8.2 and 20 degrees C). Results indicate that toxicity tests with two-month-old mussels can be conducted for 28 d with >80% control survival; growth was frequently a more sensitive endpoint compared to survival; and the 1996 chronic WQC for copper and the 1999 chronic WQC for total ammonia might not be adequately protective of the mussel species tested. However, a recently revised 2007 chronic WQC for copper based on the biotic ligand model may be more protective in the water tested.

Intra- and interlaboratory variability in acute toxicity tests with glochidia and juveniles of freshwater mussels (Unionidae).

The present study evaluated the performance and variability in acute toxicity tests with glochidia and newly transformed juvenile mussels using the standard methods outlined in American Society for Testing and Materials (ASTM). Multiple 48-h toxicity tests with glochidia and 96-h tests with juvenile mussels were conducted within a single laboratory and among five laboratories. All tests met the test acceptability requirements (e.g., >or=90% control survival). Intralaboratory tests were conducted over two consecutive mussel-spawning seasons with mucket (Actinonaias ligamentina) or fatmucket (Lampsilis siliquoidea) using copper, ammonia, or chlorine as a toxicant. For the glochidia of both species, the variability of intralaboratory median effective concentrations (EC50s) for the three toxicants, expressed as the coefficient of variation (CV), ranged from 14 to 27% in 24-h exposures and from 13 to 36% in 48-h exposures. The intralaboratory CV of copper EC50s for juvenile fatmucket was 24% in 48-h exposures and 13% in 96-h exposures. Interlaboratory tests were conducted with fatmucket glochidia and juveniles by five laboratories using copper as a toxicant. The interlaboratory CV of copper EC50s for glochidia was 13% in 24-h exposures and 24% in 48-h exposures, and the interlaboratory CV for juveniles was 22% in 48-h exposures and 42% in 96-h exposures. The high completion success and the overall low variability in test results indicate that the test methods have acceptable precision and can be performed routinely.

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.