Homogeneity at nuclear microsatellite loci masks mitochondrial haplotype diversity in the endangered fanshell pearlymussel (Cyprogenia stegaria).

We report on multiple patterns of differentiation and connectivity in the fanshell pearlymussel (Cyprogenia stegaria), based on different markers. Knowledge of genetic variation and genetic connectivity among remaining populations of this federally endangered species is needed to initiate implementation of the species recovery plan. We collected tissue samples from 96 specimens from the Green, Rolling Fork, and Licking Rivers, tributaries to the Ohio River, and the Clinch River, a tributary to the Tennessee River, providing broad coverage of the current distributional range of the species. Results from 7 nuclear DNA microsatellite markers suggested minimal population-level differentiation, whereas a mitochondrial DNA (mtDNA) marker (ND1) exhibited significant differentiation between C. stegaria in the Clinch River and the Ohio River populations. The ND1 data also confirm the existence of 2 distinct mtDNA lineages in the genus that transcends species boundaries. Further analyses suggest that the disproportionally strong signal from 2 very divergent ND1 lineages possibly masks finer-grained structure in the Ohio River population, based on one of the mtDNA lineages only. We recommend further sampling to confirm the absence of one lineage from the upper Clinch River drainage and suggest that provisional management guidelines should limit reciprocal exchanges among C. stegaria populations from the Clinch River and those in the Ohio River system.

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.

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.

Chlorine toxicity to early life stages of freshwater mussels (Bivalvia: Unionidae).

Chlorine (Cl) is a highly toxic, widely used halogen disinfectant that is present in point-source pollution discharges from wastewater treatment plants and industrial facilities. The U.S. Environmental Protection Agency freshwater criteria for Cl are 19 microg total residual Cl (TRC)/L as a maximum 1-h average concentration and 11 microg TRC/L as a maximum 4-d average; however, toxicological data for unionids were not used in these calculations. To address this void in the data, we conducted acute tests with glochidia from several species and 21-d bioassays with three-month-old Epioblasma capsaeformis and three-, six-, and 12-month-old Villosa iris juveniles. The 24-h lethal concentration 50 values for glochidia were between 70 and 220 [Lg TRC/L, which are 2.5 to 37 times higher than those reported in other studies for cladocerans. Significant declines in growth and survivorship were observed in the 21-d test with E. capsaeformis at 20 microg TRC/L. Lowest-observed-adverse-effects concentrations in bioassays with juvenile V. iris were higher (30-60 microg TRC/L) but showed a significant trend of declining toxicity with increased age. Although endpoints were above water quality criteria, the long life spans of unionids and potential implications of chronic exposure to endangered juvenile mussels still warrant concern.

Acute and chronic toxicity of mercury to early life stages of the rainbow mussel, Villosa iris (Bivalvia: Unionidae).

Mercury (Hg) contamination is receiving increased attention globally because of human health and environmental concerns. Few laboratory studies have examined the toxicity of Hg on early life stages of freshwater mussels, despite evidence that glochidia and juvenile life stages are more sensitive to contaminants than adults. Three bioassays (72-h acute glochidia, 96-h acute juvenile, and 21-d chronic juvenile toxicity tests) were conducted by exposing Villosa iris to mercuric chloride salt (HgCl2). Glochidia were more sensitive to acute exposure than were juvenile mussels, as 24-, 48-, and 72-h median lethal concentration values (LC50) for glochidia were >107, 39, and 14 microg Hg/L, respectively. The 24-, 48-, 72-, and 96-h values for juveniles were 162, 135, 114, and 99 microg Hg/L, respectively. In the chronic test, juveniles exposed to Hg treatments > or = 8 microg/L grew significantly less than did control organisms. The substantial difference in juvenile test endpoints emphasizes the importance of assessing chronic exposure and sublethal effects. Overall, our study supports the use of glochidia as a surrogate life stage for juveniles in acute toxicity tests. However, as glochidia may be used only in short-term tests, it is imperative that an integrated approach be taken when assessing risk to freshwater mussels, as their unique life history is atypical of standard test organisms. Therefore, we strongly advocate the use of both glochidia and juvenile life stages for risk assessment.

Freshwater mussel shells as environmental chronicles: geochemical and taphonomic signatures of mercury-related extirpations in the North Fork Holston River, Virginia.

This study utilized freshwater mussel shells to assess mercury (Hg) contamination in the North Fork Holston River that extirpated (caused local extinctions of) a diverse mussel fauna. Shells (n = 366) were collected from five sites situated upstream (two sites), just below (one site), and downstream (two sites) of the town of Saltville, Virginia, where Hg was used to produce chlorine and caustic soda from 1950 to 1972. Shell samples were used to test the (1) utility of geochemical signatures of shells for assessing the spatial variation in Hg levels in the river relative to the contamination source and (2) value of taphonomy (post-mortem shell alteration) for distinguishing sites that differ in extirpation histories. Geochemical signatures of 40 shells, analyzed using atomic absorption spectroscopy, indicated a strong longitudinal pattern. All shells from the two upstream sites had low Hg concentrations (<5-31 microg/kg), shells directly below Saltville had variable, but dramatically higher concentrations (23-4637 microg/kg), and shells from the two downstream sites displayed intermediate Hg levels (<5-115 microg/kg) that declined with distance from Saltville. Two pre-industrial shells, collected at Saltville in 1917, yielded very low Hg estimates (5-6 microg/kg). Hg signatures were consistent among mussel species, suggesting that Hg concentrations were invariant to species type; most likely, highly variable Hg levels, both across sites and through time, overwhelmed any interspecific differences in Hg acquisition. Also, a notable postmortem incorporation of Hg in mussel shells seemed unlikely, as the Hg content was not correlated with shell taphonomy (r= 0.18; p = 0.28). The taphonomic analysis (n = 366) showed that the degree of shell alteration reliably distinguished sites with different extirpation histories. At Saltville, where live mussels have been absent for at least 30 years, shells were most heavily altered and fragmented. Conversely, fresh-looking shells abounded upstream, where reproducing mussel populations are still present. In summary, relic shells offered valuable spatio-temporal data on Hg concentrations in a polluted ecosystem, and shell taphonomic signatures discriminated sites with different extirpation histories. The shell-based strategies exemplified here do not require sampling live specimens and may augment more standard strategies applied to environmental monitoring. The approach should prove especially useful in areas with unknown extirpation and pollution histories.

Sensitivity of juvenile freshwater mussels (Lampsilis fasciola, Villosa iris) to total and un-ionized ammonia.

This study evaluated the sensitivity of juveniles of two freshwater unionid mussel species (Villosa iris [Lea] and Lampsilis fasciola [Rafinesquel) to un-ionized and total ammonia. Five concentrations of ammonium chloride were tested using 96-h static-renewal toxicity tests at 12 and 20 degrees C. Based on their respective mean 96-h lethal concentration to 50% (LC50s), V. iris (0.11 mg/ L NH3-N) was more sensitive than L. fasciola (0.26 mg/L NH3-N). At 96 h, significant differences in sensitivity to un-ionized ammonia between the two temperatures were not observed for either species. Comparison of LC50s reported for other aquatic organisms to the 96-h LC50s calculated for juvenile L. fasciola and V. iris shows these two mussel species to be among the most sensitive to un-ionized ammonia. Based on reported levels of un-ionized ammonia in the aquatic environment from anthropogenic sources, un-ionized ammonia may be an important limiting toxicological factor to freshwater mussel populations.