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The Powell River is located in southwestern Virginia and northeastern Tennessee, USA and supports a diverse freshwater mussel assemblage of 29 extant species. Throughout the river major ion and trace element concentrations have increased over the last several decades due to extensive surface coal-mining in the headwaters in Virginia. As watershed area affected by mining has increased, mussel populations have declined, especially in Virginia where populations have been severely reduced or extirpated. The upper watershed now has been extensively mined for coal, causing widespread effects on water and sediment quality. To investigate how mining may be affecting mussel populations, I first conducted a laboratory bio-assay to assess the effects of elevated major ions and the trace element nickel (Ni) on growth and survival of juvenile mussels, including one common species (Villosa iris) and one endangered species (Epioblasma capsaeformis). No significant differences in overall survival between treatments and control were observed for either species over a 70 day test period. Total growth was not significantly different between treatments and control for either species. However, overall growth varied significantly (p=0.009) between species, with V. iris (2.49 mm) exhibiting greater growth compared to E. capsaeformis (1.97 mm). Results suggest that major ion chronic toxicity alone or in combination with Ni at or below my test concentration is not a likely source of toxicity to juvenile mussels in the Powell River. Secondly, I conducted a field study in the Powell River using two cohorts of juveniles of Villosa iris to assess the effects of trace elements and PAH contamination related to mining on mussel survival and growth. Specific conductance was elevated throughout the Powell River, where site means ranged from 450 to 900 µS/cm. While mortality was high at all eight sites it was not significantly different among these sites (p>0.28); however, growth of juvenile mussels was significantly higher (p

The Clinch River (CR) and North Fork Holston River (NFHR) contain some of the most diverse freshwater mussel assemblages in the United States; however, both rivers are experiencing declines in mussel populations. The first component of this study used histological evaluations and water quality data to determine whether mussels were negatively impacted in the CR zone of decline (ZD) and to inform future management of freshwater mussels in the river. In the 91 kilometer (km) section from Carbo, Virginia (CRKM 431) downstream to Speers Ferry, Virginia (CRKM 340), referred to as the ZD, mussel density decreased >90% from 1979 to 2014 at key sites such as Semones Island (CRKM 378.3) and Pendleton Island (CRKM 364.2). Laboratory propagated mussels were placed in cages in the river for one year from June 2012 to May 2013 at four sites within the ZD and four sites in reaches where mussel populations remain stable or are increasing, a zone of stability (ZS). The survival, growth and histological results indicated that there are continuing impacts to mussels in the ZD. Research investigating impacts to the ZD and methods to improve water quality in this zone are needed. The laboratory component of this study examined sublethal effects of potassium (K⁺), chloride (Cl⁻), and un-ionized ammonia (NH₃-N) on mussel tissues at concentrations relevant to those found in the NFHR. Historical industrial activities at Saltville, Virginia, as well as continued pollution of the NFHR from chemical waste ponds at this location, are believed to be significant contributors to mussel declines. Contaminant seepages from the waste ponds that include Cl⁻, K⁺, and NH₃-N have been shown to be toxic to adult and juvenile mussels. A three-month laboratory study was conducted to assess impacts to organ tissues (gills, digestive glands, kidneys, and gonads) of adult Villosa iris exposed to environmentally relevant concentrations of K⁻ (4 and 8 mg/L), Cl⁻ (230 and 705 mg/L), and NH₃-N (0.014 and 0.15 mg/L) using histological evaluations. No detectable differences were observed among the histological endpoints from mussels held in treatments and control (p>0.05). The study design was modified and repeated using increased concentrations of K⁺ (8, 16, and 32 mg/L) and Cl⁻ (705, 1410, and 2820 mg/L) for a two-month exposure period. Due to issues with maintaining NH3-N in mussel holding chambers, the second study did not the second study did not include NH₃-N exposures. Control mussels in both studies had a higher abundance of lipofuscin in kidneys and degraded cytoplasm in the digestive gland diverticula compared to baseline mussels, indicating that captivity influenced mussel tissues. Future studies are needed to more thoroughly address these captivity effects. Both survival and histological data in the second test showed a significant negative effect of the increased concentrations of Cl⁻ and K⁻, which were representative of those found at some sites in the NFHR downstream of Saltville, Virginia. Master of Science

Select freshwater mussels in the genera Fusconaia, Pleurobema, and Pleuronaia were collected primarily in the upper Tennessee River basin from 2012 to 2014 for phylogenetic and morphological assessments. Freshwater mussels in these genera are similar in appearance, hence the need for phylogenetic verification and morphological assessment. Phylogenetic analyses of the mitochondrial gene ND1 and the nuclear gene ITS1 revealed three unrecognized, phylogenetically distinct species. These species were separated from their closest congener by 2.85%, 3.17%, and 6.32% based on pairwise genetic distances of ND1. Gaps created from aligning ITS1 sequences were coded as fifth characters, which phylogenetically separated most closely related species. Analyses of ND1 agreed with previous literature on the phylogenetic distinctiveness of Pleuronaia species, with the exception of the DNA sequences of P. gibberum, which grouped outside this genus based on the analyses conducted in this study. Morphological variation was recorded for eight of the species to include quantitative and qualitative characters as well as geometric morphometric analyses. Three decision trees were created from quantitative and qualitative characters using classification and regression tree analyses. The best-performing tree used quantitative and qualitative characters describing shell-only scenarios and obtained 80.6% correct classification on terminal nodes. Canonical variates analysis on geometric morphometric shell data revealed large morphological overlap between species. Goodall's F-tests between pairs of species revealed significant differences (a=0.05) between all but one species pairs; however, examination of landmarks on shells concluded large overlap of landmarks between species pairs. Lack of morphologically distinct characters to readily identify these phylogenetically distinct species indicates large morphological overlap among these species. Biologists need to be cognizant that morphologically cryptic species may exist in systems often explored. Three dichotomous keys were created from classification trees to identify select individuals in the genera Fusconaia, Pleurobema, and Pleuronaia; two of these keys, one for shells and one for live mussels were tested by participants with varying mussel identification skills to represent novices and experts. Both keys used continuous (quantitative) and categorical variables to guide participants to identifications. Novices, who had no prior mussel identification experience, correctly identified mussels with a 50% accuracy using the shell key and with a 51% accuracy using the live key. Experts, who had at least three years of experience identifying mussels, correctly identified mussels with a 58% accuracy using the shell key and with a 68% accuracy using the live key; however one expert noted that they did not use the live key to correctly identify one mussel. Morphological overlap of variables between mussels likely resulted in failure to consistently identify mussels correctly. Important management decisions and project implementations require accurate assessment of species' localities and populations. Incorrect species identification could hinder species' recovery efforts or prevent projects that otherwise could have continued if species are misidentified. If a mussel collection is thought to be a new record or could affect a project, I recommend that molecular genetic identifications be used to verify the species identity. Master of Science

From 2006-2011, four population reintroduction techniques were applied to three sites within a reach of the upper Clinch River in Virginia designated suitable for population restoration of the federally endangered oyster mussel (Epioblasma capsaeformis). These techniques were: 1) translocation of adults (Site 1), 2) release of laboratory-propagated sub-adults (Site 1), 3) release of 8-week old laboratory-propagated juveniles (Site 2), and 4) release of stream-side infested host fishes (Site 3). Demographic data were collected in 2011 and 2012 by systematic quadrat and capture-mark-recapture sampling to assess reintroduction success, evaluate reintroduction techniques, and compare survey approaches for monitoring freshwater mussels. Estimates of abundance and density of translocated adults ranged from 450-577 individuals and 0.09-0.11/m2 in 2011, and 371-645 individuals and 0.07-0.13/m2 in 2012. Estimates of abundance and density of laboratory-propagated sub-adults ranged from 1,678-1,943 individuals and 0.33-0.38/m2 in 2011, and 1,389-1,700 individuals and 0.27-0.33/m2 in 2012. Additionally, three recruits were collected at Site 1. No E. capsaeformis were collected at Sites 2 and 3. Capture-mark-recapture sampling produced similar mean point estimates as systematic quadrat sampling, but with typically more precision. My results indicated that the release of larger individuals (>10 mm) is the most effective technique for restoring populations of E. capsaeformis, and that systematic quadrat and capture-mark-recapture sampling have useful applications in population monitoring that are dependent on project objectives. Systematic quadrat sampling is recommended when the objective is to simply estimate and detect trends in population size for species of moderate to larger densities (>0.2/m2). Capture-mark-recapture sampling should be used when objectives include assessing a reintroduced population of endangered species or at low density, obtaining precise estimates of population demographic parameters, or estimating population size for established species of low to moderate density (0.1-0.2/m2). The ability to grow endangered juveniles to larger sizes in captivity requires improving grow-out culture methods of laboratory-propagated individuals. A laboratory experiment was conducted to investigate the effects of temperature (20-28 C) on growth and survival of laboratory-propagated juveniles of the Cumberlandian combshell (Epioblasma brevidens), E. capsaeformis, and the wavyrayed lampmussel (Lampsilis fasciola) in captivity. Results indicated that 26 C is the optimum temperature to maximize growth of laboratory-propagated juveniles in small water-recirculating aquaculture systems. Growing endangered juveniles to larger sizes will improve survival in captivity and after release into the wild. As a result, hatcheries can reduce the time that juveniles spend in captivity and thus increase their overall production and enhance the likelihood of success of mussel population recovery efforts by federal and state agencies, and other partners. Master of Science

Propagation and culture has been accepted as an approved aquaculture method for resource managers to enhance and recover freshwater mussel populations. Although juveniles can be produced readily for many mussel species, achieving high growth and survival in the laboratory remains difficult. The goal of my project was to improve growth and survival of juvenile mussels by comparing diets, algae concentration, and culture systems. The first objective determined a suitable diet for feeding juvenile northern riffleshell, Epioblasma torulosa rangiana, a species listed as federally endangered. Three algal diets were evaluated to determine differences in growth and survival of juveniles of E. t. rangiana. After 60 d, mean survival on Phytofeast, Shellfish Diet, and WSSNFH mix were 75.1 (95% CI: 72.2 to 78.0), 78.9 (95% CI: 74.5 to 83.2), and 85.0 (95% CI: 80.6 to 89.3) percent, respectively. WSSNFH mix had the highest survival which was significantly different from Phytofeast (p=0.01). Mean shell lengths were 2.37 mm (95% CI: 2.27 to 2.47), 2.62 mm (95% CI: 2.52 to 2.72), and 3.11 mm (95% CI: 3.01 to 3.22), respectively. Juvenile length on all three diet treatments was significantly different from each other (p5 mm. Mean incremental length of juveniles of E. t. rangiana at 60 d in Pans, Buckets, and Upwellers was 1.19 mm (95% CI: 0.746 to 1.62), 1.05 mm (95% CI: 0.608 to 1.49), and 2.07 mm (95% CI: 1.63 to 2.51), respectively. Incremental lengths were significantly higher in the Upwellers (p=0.03). The mean lengths for Bucket and Pan systems were not significantly different from each other (p=0.54). Percent survival of juveniles for the Pans, Buckets, and Upwellers were 91.7 (95% CI: 87.4 to 96.0), 90.0 (95% CI: 80.6 to 99.4), and 100 (95% CI: 100 to 100), respectively. Survival in the Upwellers was significantly higher than in the Buckets (p=0.018). Survival of juveniles in the Pan system and Upwellers were not significantly different from each other (p=0.05). Mean growth for A. ligamentina was 1.96 mm (95% CI: 1.03 to 2.9), 0.88 mm (95% CI: 0.048 to 1.80), and 2.46 mm (95% CI: 1.537 to 3.38), respectively (p=0.07). Mean percent survival of juveniles of Actinonaias ligamentina in the Pans, Buckets, and Upwellers were 100 (95% CI: 100 to 100), 86.7 (95% CI: 74.0 to 99.4), and 100 (95% CI: 100 to 100), respectively. Survival of A. ligamentina in the Upwellers was significantly higher than in Buckets (p

Qualitative and quantitative sampling was conducted along a 165 km reach of the river from PRKM 269.4 near Dryden, VA to PRKM 104.8 near Harrogate, TN. Twenty-nine species were observed throughout the entire river, and the highest diversity of 23 species was collected at PRKM 152.6. Mussel abundances (mussels/person-h and mussels/m2) ranged from 0.33 to 21.98 mussels/person-h and 0.00 to 2.24 mussels/m2. Recent recruitment (individuals < 40 mm, depending on the species) was observed for 15 species, including the endangered Epioblasma brevidens, Lemiox rimosus, and Quadrula intermedia. The greatest number of species (6) with evidence of recent recruitment also was found at PRKM 152.6. Data from the quantitative survey were used to simulate several sampling protocols that could be used to develop a long-term monitoring program for the Powell River. Five sites, PRKMs 197.9, 171.4, 159.6, 152.6, and 129.4, were selected for long-term monitoring because of high mussel densities and species richness. Six sampling protocols were simulated using the statistical program MONITOR to determine which protocols, if any, could monitor statistically significant changes in mussel abundance at rates + 10%. Each of the simulated sampling protocols lasted between 15 and 30 y, and employed quantitative sampling at 3 to 5 y intervals. None of the sampling protocols simulated during this study were able to detect declines in mussel abundance < 10%. Two sampling programs were able to detect increases in mussel abundance > 6% when the level of significance was > 0.10, and four sampling programs were able to detect a density increase of > 8% when the level of significance was 0.05. Despite the inability to monitor declines in mussel abundances, a long-term monitoring program is needed for the Powell River. Because qualitative sampling has been repeatedly shown to document species presence more effectively than quantitative sampling, it should be used to monitor changes in species presence and distribution. Quantitative sampling should be employed to monitor juvenile recruitment and changes in size-class structure of populations. Quantitative sampling also should be conducted to monitor overall mussel abundance at sites. Despite the inability to statistically detect changes in mussel density in the Powell River, quantitative sampling can provide valuable information, and the data collected can be used to qualitatively monitor changes in total density at sites. Both species share a similar distribution in the Powell River. Eighteen specimens of Quadrula sparsa were collected between PRKM 230.9 and 152.6, and 68 individuals of Q. intermedia were collected between PRKM 230.9 and 129.4. The highest density of each species was collected at PRKM 152.6, and recent recruitment was observed at PRKMs 152.6 and 153.4. Fresh-dead and relic shells of both species were thin-sectioned to determine individual growth rate and life span. These species complete the majority of their growth during the first 10 y of life, and likely live for a total of 40 to 50 y. One gravid female of Q. intermedia was collected during this study, but no gravid females of Q. sparsa were observed. Master of Science

Genetic variation was examined in two endangered mussel species, Epioblasma brevidens and E. capsaeformis, and a common species Lampsilis fasciola, in the Clinch River, TN, by screening mitochondrial DNA (mtDNA) sequences and nuclear DNA microsatellites. These species use fish hosts with varying dispersal capabilities, ranging from low, moderate, and high, respectively. Patterns of mtDNA polymorphism exhibited different trends for long-term population sizes for each species during the Holocene (~10,000 ya to present); namely, E. brevidens has declined over time, E. capsaeformis has remained stable, and L. fasciola has expanded. Long-term effective population size (Ne) was smallest in E. brevidens, intermediate in E. capsaeformis, and highest in L. fasciola. Moderately diverged mtDNA lineages, perhaps indicative of secondary contact, were observed in E. brevidens and E. capsaeformis. High levels of gene flow (Nm) were estimated among demes of L. fasciola using traditional F-statistics and likelihood estimates of Nm, whereas such metrics were lower in E. brevidens and E. capsaeformis. Data are consistent with population dynamics and life history traits of each species and their fish hosts. Age, shell growth, and population demography of Epioblasma brevidens, E. capsaeformis, and Lampsilis fasciola were studied from 2004-2007 in a 32-km reach of the Clinch River, TN. Observed maximum age and length of E. brevidens was 28 y and 71.5 mm for males and 11 y and 56.6 mm for females; of E. capsaeformis, 12 y and 54.6 mm for males and 9 y and 48.6 mm for females; and of L. fasciola, 45 y and 91.3 mm for males and 13 y and 62.6 mm for females. For all three species, observed maximum age and length was greater among males than females. Estimated population size in this river reach was approximately 43,000 individuals for E. brevidens, 579,000 individuals for E. capsaeformis, and 30,000 individuals for L. fasciola. Mean recruitment y-1 of 1 y-old E. brevidens ranged from 7.1% to 20%, of E. capsaeformis from 4.0% to 32.4%, and of L. fasciola from 5.8% to 25.6%. Population growth rate y-1 was 24.9% for E. brevidens, 34.6% for E. capsaeformis, and -22.4% for L. fasciola. Mortality rates of females were higher than for males of E. capsaeformis and L. fasciola, but not E. brevidens. Juvenile mussels were collected but temporally and spatially variable in occurrence, and a significant component of the age-class structure of all three species. Recruitment was very high during 2006-2007 for E. capsaeformis and other species, likely due to low river discharges in the spring-summer of 2005-2007. Surplus individuals of E. brevidens and E. capsaeformis are currently available to conduct translocations for restoration purposes. Population modeling of Epioblasma brevidens and E. capsaeformis in the Clinch River was conducted to determine suitable harvest levels for translocation of sub-adults and adults, and to determine quantitative criteria for evaluating performance and recovery of extant and reintroduced populations. For both species, the recommended annual harvest was

Epioblasma capsaeformis, commonly named the oyster mussel, once occupied thousands of miles of stream reaches, but has now been reduced in range to small, isolated populations in a few river reaches. Due to this significant decline in population numbers, a study was conducted to develop a diet for propagating this endangered species under captive conditions. Oyster mussel juveniles were collected from several sites on the Clinch River and sacrificed for gut content and biochemical composition analyses in summer. Feces and pseudofeces from live river-collected juveniles were examined seasonally for algae, detritus, and bacteria to qualitatively determine diet of specimens. Two feeding trials also were conducted in this study to evaluate effect of diet (commercial and non-commercial diets), on growth and survival of oyster mussel juveniles. From examination of gut contents, fecal and pseudofecal samples, it was apparent that algae and a significant amount of detritus (~90%) composed wild juvenile diets. E. capsaeformis juveniles (1-3 y of age) could have fed on particles up to 20 µm in size and seemed they were mostly ingesting particles within the 1.5-12 µm size range. Protein content of sacrificed juveniles ranged from 313 to 884 mg/g and was highly variable. Glycogen content ranged from 49-171 mg/g. Caloric content of four juveniles ranged from 2,935.10 to 4,287.94 cal/g, providing a preliminary baseline range for future energetic studies on freshwater mussels. Growth was significantly higher in those juveniles fed the triple concentration algae-mix (62,076 cells/ml) than all other diets tested in trial 1. Results of both feeding trials indicated that survival of juvenile oyster mussels was enhanced when fed an algal diet supplemented by bioflocs. Master of Science

Mercury contamination of the North Fork Holston River below Saltville, Virginia has nearly extirpated most mussel populations. Because natural recovery of these populations has not occurred, this research combined field and laboratory assessments to determine the extent of ecological impairment in the river. In situ 60-day Asian clam (Corbicula fluminea) growth studies in 2005 showed a positive correlation (p=0.03) between low clam growth and sediment mercury levels. Because of severe low flow conditions of the NFHR in late 2005 conductivity dissipation from a point source brine discharge downstream rarely reached background level (~345 µS/cm) and was observed as high as 690 µS/cm 640 m below the discharge site. In addition, conductivity doubled in the river section adjacent to the remediated Ponds 5 and 6 (rm 81.6 and 80.4). Such low flow conditions (mean flow < 50 ft3/sec) occur in the NFHR approximately every five years. This low flow situation also evidenced a thick white flocculent or floc observed to accumulate at the base of the two remediated ponds. Analysis of the flocculent determined it to be high in aluminum (1.9-38 mg/L) and iron (2.0-51.0 mg/L), well above US Environmental Protection Agency Water Quality Criteria limits (0.0087 and 1.0 mg/L, respectively); riverine sediments collected below the accumulated floc also had high levels of calcium (240,000-380,000 mg/kg) and mercury(0.62-1.7 mg/kg). Acute tests with juveniles of Villosa iris and

In 1998, a tanker truck overturned on U.S. Route 460 in Tazewell County, Virginia, releasing approximately 5110 L of Octocure 554-revised, a rubber accelerant, into an unnamed tributary approximately 162 m from its confluence with the Clinch River. The spill killed nearly all aquatic life in the river, including three species of federally endangered mussels, for 11 km downstream. The restoration plan developed after the spill included community outreach to complement the physical, on-site restoration effort. Although numerous studies have been made of the aquatic resources in the upper Clinch River, Tazewell County, Virginia, there had been no prior assessment of the local residents' knowledge, attitudes, and opinions about the natural resources in this biodiversity hotspot. I surveyed residents of Tazewell County, Virginia to assess their baseline knowledge about the upper Clinch River watershed, endangered mussels, aquatic conservation, and water quality issues. The survey response rate was 42%. Survey respondents were aware that mussels are present in the Clinch River watershed, but were not aware of their imperiled status or the ecosystem services that mussels perform. On average, respondents expressed stronger moralistic, naturalistic, and ecologistic than negativistic and utilitarian attitude orientations toward endangered freshwater mussels. Respondents considered water quality important and expressed support for mussel restoration. However, respondents indicated that the presence of mussel populations might negatively impact economic development of the county. Local community leaders and conservation educators, when interviewed to explore their opinions on conservation of aquatic resources, placed high value upon the Clinch River's water quality. They also have sought information regarding water quality and the Clinch River from local organizations and agencies and/or have developed a relationship with them as a result of their long-term presence in the community. The majority of respondents believed that human impacts contribute to species decline, but human impacts may not be the primary contributor to such decline. Respondents indicated that governmental and nongovernmental conservation agencies and organizations can assist localities by providing additional funding opportunities, seminars, and training sessions. Tazewell County conservation educators focus broadly on water resources of the county. Conservation educators wised that adults and teenagers displayed greater interest in conservation issues and that local governments were involved more actively in conservation. Overwhelmingly, educators believed that forming and fostering partnerships is the most effective way to inform audiences about conservation. Barriers to conservation education faced by educators include lack of funding, audience apathy, and/or audience negativity. Nearly all respondents indicated that balance between conservation and development currently exists or that achieving such a balance should be a goal of the local government. Survey and interview results were used to develop specific outreach recommendations to generate community support for mussel restoration and aquatic conservation in Tazewell County. Recommendations for the Certus Spill Outreach Plan include: frame mussel outreach messages in a broader aquatic ecosystem context in order to emphasize the human connection to aquatic ecosystems, communicate positive messages about conservation with decision-makers, regularly communicate positive messages about conservation in the local press, and partner with the local school system and agencies that have established positive images in the towns and county. Master of Science

The Big South Fork National River and Recreation Area (NRRA), located in Tennessee and Kentucky, has prepared a management plan to include restoration of its mussel fauna to historic levels. Restoration activities include propagation of juvenile mussels and relocation of adults to suitable sites in the Big South Fork of the Cumberland River (BSF) and its tributaries. This study was conducted to identify host fish for Cumberland elktoe (Alasmidonta atropurpurea) and Cumberland bean (Villosa trabalis), to determine suitable juvenile culture conditions for Epioblasma brevidens and V. trabalis, and to locate sites important to future mussel restoration efforts in the NRRA. Host fish identifications and propagation techniques were determined for two of the endangered species in the NRRA, Cumberland elktoe (Alasmidonta atropurpurea) and Cumberland bean (Villosa trabalis). Of seven host species tested, banded sculpin (Cottus carolinae) was the most suitable host fish for propagation of A. atropurpurea. Of five host species tested, fantail darters (Etheostoma flabellare) were the most suitable host fish for propagation of V. trabalis. Culture techniques to raise juvenile mussels in captivity were evaluated, using newly metamorphosed juveniles of V. trabalis and E. brevidens in recirculating systems. No differences in juvenile growth or survival were detected among substrates used (fine sediment, coarse sand, and a mixture of the two). Recirculating system design seemed to affect juvenile growth and survival; however, variable condition of juveniles also seemed to affect results, making it difficult to determine effects from trial treatments. Finally, an assessment of potential sites in the NRRA for restoration activities was conducted using spatial analysis in a geographic information system (GIS) and several measures of conservation value. Mussel restoration sites were assessed for potential threats from adjacent land uses that may negatively affect mussels, including coal mines, oil and gas wells, transportation corridors, agriculture and urban development. Sites were also evaluated on their current conservation value to designate which sites are most important to long-term maintenance of mussel fauna. Several sites were identified that contain relatively few land-use threats, and are appropriate for mussel restoration activities, including Big Island, Station Camp Creek, and Parchcorn Creek sites on the mainstem BSF, as well as sites on Clear Fork and North White Oak Creek. Many of these sites also have high conservation values. Other sites had relatively high land-use threats that need to be addressed before restoration activities take place. Such sites include Leatherwood Ford, Rough Shoals Branch, Blue Heron, and Yamacraw on the mainstem BSF. The dominant threat to most sites came from transportation corridors, whereas some sites in southern and eastern portions of the watershed also were threatened by coal mines, and oil and gas wells. Master of Science

The river drainages originating in the Cumberland region of Virginia, Tennessee and Kentucky are home to some of the last surviving and most diverse assemblages of native freshwater mussels. This region of the country also has historically and continues to be a major source of coal for the United States. Numerous experiments were carried out in an attempt to determine what ecotoxicological effects these activities have had on mussels as well as what has been done to correct some of the most severe cases of environmental pollution due to historical coal mining operations. Analysis of interstitial water (IW), sediment and in situ toxicity testing and chemical analyses showed that the most likely cause for mussel declines was elevated metal concentrations (Al, Cu, Fe, Pb) found in IW. Ecotoxicological assessments of the two streams (Black and Ely Creeks) most impacted by acid mine drainage (AMD) in the state of Virginia were carried out to determine their potential for future degradation of the Powell River watershed into which they drain. The Powell River is a major system still inhabited by native mussels. Sophisticated wetland systems built at Ely Creek have significantly improved the ecological health of Ely Creek, decreasing the pollution into the Powell River. Reclamation and wetland construction at Black Creek have had a positive impact but active coal mining and un-remediated AMD are still negatively affecting this system. After the watershed has been fully reclaimed the discharge from Black Creek will likely be improved. Master of Science

The Clinch River, Virginia is known for high aquatic biodiversity, particularly Unionidae which are declining at remarkable rates. Studies conducted on the mainstem have only addressed effects of point-source stressors (various toxic spills and effluents from the Clinch River Plant (CRP), Carbo, Virginia) that have been introduced into the Clinch River. It is hypothesized that the tributaries of the Clinch River deliver a variety of stressors to the mainstem, which may affect the diverse fauna. The aquatic health of 19 upper Clinch River tributaries, Virginia, was assessed via ecotoxicological ratings that indicated the least healthy catchments were associated with mining activity (Dumps, Russell and Coal Creeks). Tributaries were categorized by land use and mining streams were significantly different from agricultural and forested streams (F = 9.63, p80) were Big Stony, Copper, Indian, Stock, Little River and Cove Creeks. Master of Science

Three spined, mussel species occur in the United States along the Atlantic slope; James spinymussel (Pleurobema collina), Tar spinymussel (Elliptio steinstansana), and Altamaha spinymussel (E. spinosa). The James spinymussel was listed as endangered in 1988, and was until recently considered to be endemic to the James River basin (Clarke and Neves 1984; USFWS 1990). Biologists with the North Carolina Department of Transportation (NCDOT) discovered spinymussel populations in the Dan and Mayo rivers in NC in 2000 and 2001, respectively. The U.S. Fish & Wildlife Service (USFWS) tentatively identified this species as Pleurobema collina. My project proposed by the Virginia Cooperative Fish and Wildlife Research Unit to the USFWS and the Virginia Transportation Research Council, determined where P. collina resides in VA and what the extent of its range is within the state. An informal preliminary survey design for P. collina was used during the summer of 2002 and simple random sampling was deployed in 2003-2004 surveys to provide a good basis for comparison to gauge the efficiency of the informal sampling design. In 2002, a total of 116 person-hours were spent surveying 39 localities on the Mayo, Dan, and Smith rivers. A total of 96 P. collina was observed in the South Fork of the Mayo River, Patrick and Henry counties, VA. A documented range of 24 rkm was established in the South Fork Mayo River. During the summers of 2003 and 2004, a total of 228 person-hours were spent surveying 38 equal-area river reaches (10, 000 m2) on the mainstems of the Dan, Smith, South Mayo, and Banister rivers. No specimens of P. collina (live or relic shells) were detected. A simple random sampling approach was designed to be easy, relatively quick and cost effective, applicable to most rivers, and to provide actual numbers for comparison. Negative results were only reported after 6 person-hours of searching within each randomly selected, equal-area river reach had been expended. P. collina was declared absent from the VA random sites surveyed in 2003-2004 with a confidence of ~90%. A genetic characterization of four extant populations of P. collina was conducted to assess its taxonomic affinity and to resolve conservation issues related to recovery planning and management actions. The populations were examined for phenotypic variation, and were characterized phylogenetically using DNA sequences. A comprehensive analysis was performed for both separate and combined mitochondrial (357 bp of cytochrome-b, 916 bp of ND-1) and nuclear (502 bp of ITS-1) DNA sequences. Based on comprehensive molecular, morphological, and life history data, populations of P. collina sampled from the Dan River sub-drainage do not warrant separate species designation from P. collina sampled from the James River drainage. Master of Science

Due to the increasing need to provide refugia for freshwater mussels impacted by anthropogenic activities and exotic species, facilities should be identified and protocols developed for holding mussels in captivity. White Sulphur Springs National Fish Hatchery (WSSNFH), White Sulphur Springs, WV, has held freshwater mussels for nearly eight years, and has the potential to become an important refugium and propagation facility for conservation of mussels in the Ohio River Basin and elsewhere. The goal of this study was to determine the feasibility of holding adult freshwater mussels in long-term captivity at WSSNFH by evaluating survival, energy reserves, and gametogenesis of captive mussels in a recirculating pond system. I relocated three mussel species in the summer of 2001 and 10 mussel species in the summer of 2002 to a recirculating pond system (reservoir and raceway) at the hatchery. Water quality parameters of pH, alkalinity, hardness, temperature, and dissolved oxygen; and algal concentrations were measured periodically from summer 2001 to summer 2003. Annual survival rates of 10 species were estimated (August 2002 to August 2003) using the program MARK. Glycogen, protein, and lipid concentrations in mantle tissue of three captive species (Actinonaias ligamentina, Cyclonaias tuberculata, and Tritogonia verrucosa) were compared to those of wild mussels in the New River. Gametogenic activity and synchrony in A. ligamentina and C. tuberculata were compared between captive and wild mussels. Water quality parameters, with the exception of temperature, were within desirable ranges for most of the study. Temperatures of > 28° C were observed for several days during summers 2002 and 2003. Algal concentrations averaged 1903 cells ml-1 in the raceway (range: 300 to 4658 cells ml-1), which is comparable to algal concentrations reported for nearby rivers. The overall survival rate for 10 freshwater mussel species held in the raceway for one year was 77%. Villosa vanuxemensis had the highest survival rate (96%), and Lampsilis cardium had the lowest survival rate (31%). Although there were fluctuations in glycogen, protein, and lipid levels over 2 yr, there were no overall differences in energy substrates between captive and wild mussels at the end of the study. Captivity did not appear to have a negative affect on gametogenesis. Captive C. tuberculata spawned within the expected time frame between January and June, but slightly earlier than their wild counterparts in the New River. Due to the infestation of the gonads of both captive and wild A. ligamentina by digenean trematodes, little gametogenesis was observed. However, captive holding did not appear to have an effect on trematode infestation rates. From these results, I conclude that captive holding conditions in the recirculating pond system at WSSNFH were adequate for long-term holding of a wide range of mussel taxa. I recommend that WSSNFH continue to be used as an adult holding facility. Further research should be conducted to determine food and habitat preferences of freshwater mussel species in captivity so that optimal holding conditions can be provided for each species. Master of Science

To determine the effects of different algal diets on freshwater mussels, tissues of Elliptio complanata were sampled for physiological, somatic, and gametogenic condition from August 1999 to May 2000. Treatments included mussels fed Scenedesmus quadricauda (S), Neochloris oleoabundans (N), a no feed treatment (NF), and a reference group of mussels from the Nottoway River (NR), Virginia. The levels of protein and glucose differed among treatments (p0.17). Production of ripe and developing gametes differed significantly among treatments (p=0.001), but stage of gamete development did not (p=0.70). Lipid levels and muscle fiber areas of treatment groups differed significantly (p

The use of glass racks and suspended pocket nets for holding freshwater mussels collected from the Ohio River and relocated to lined-ponds was studied over 3 years. Survival of mussels in ponds was 73 % after 1 y, 44 % after 2 y, and 5 % after 3 y. The glycogen levels of mussels in ponds for ly were significantly greater than that of mussels in ponds after 2.5 y and 3 y, indicating a chronic decline in body condition in mussels. Despite the presence of a diverse and dense assemblage of algae and organic detritus in the ponds, the stomachs examined at 3 y were empty and the bodies were emaciated. In the laboratory, I determined the amount of algae cleared by the rainbow mussel, Villosa iris (Lea, 1829) fed different algal rations, and estimated the algae concentration needed to maintain mussels in captivity. Filtration rate in the first feeding hour was highest in ration B (1.0 mg dry wt L⁻¹) and lowest in ration C (3.4 mg dry wt L⁻¹). After 1 h, filtration rates declined in ration B but increased in rations C and A. V.iris likely achieved gut satiation in the first hour using maximum filtration (712.5 mL h⁻¹g⁻¹) and then decreased filtration (259 mL h⁻¹g⁻¹) thereby regulating ingestion rate during the following 2 h. I estimate, therefore, that K V. iris daily maintenance requirement for carbon is 8.2 mg C (1.2 x 10⁹ cells of N. oleoabundans) or ca. 2.4% of dry body weight. Assimilation efficiencies (AE) and carbon budgets also were established for the rainbow mussel, Villosa iris (Lea 1829), using radio-labeled cultures of Neochloris oleoabundans (Chantanachat and Bold 1962) at three cell concentrations. Approximately 70% of the ingested carbon was assimilated (assimilation efficiency) by V. iris fed 5 x 10⁵ cells mL⁻¹) (3.4 mg dry weight L⁻¹). At 5 x 10⁴ cells' mL⁻¹) (0.34 mg dry weight L⁻¹), AE was 47.5 %. At 5 X 10³ cells mL⁻¹) (0.034 mg dry weight L⁻¹), AE was 40%. V. iris had the greatest amount of energy available for growth, reproduction, and body condition in captivity at 3.4 mg dry weight L⁻¹). The gross composition (protein, carbohydrate (CHO), and lipid) of four algae (Bracteacoccus grandis, Neochloris oleoabundans, and Scenedesmus quadricauda, and Phaeodactylum tricomutum) was examined at four different phases of growth. The CHO content (% algal dry wt) increased with growth phase (age of the algal culture) with the exception of B. grandis. N. oleoabundans and P. tricomutum contained the greatest CHO content (33.07 ± 6.89% and 39.37 ± 3.07%, respectively) at late stationary phase. The total lipid content increased with growth phase for N. oleoabundans and P. tricomutum. Lipid content of B. grandis decreased with age, and S. quadricauda showed no difference in lipid content (% algal dry wt) between growth phase. N. oleoabundans' lipid content (31.85 ± 9.4%) was greater than all other species. Generally, there was no effect of phase on the sterol content, with the exception of the sterol content of S. quadricauda increased with growth phase. The mean sterol content of the four algae ranged 1.0 % ± 0.4 to 1.8 ± 1.8 of the total lipid dry wt; maximum sterol % of lipid was 5 % for Scenedesmus and 4.4% for B. grandis. There was no effect of growth phase or species on the protein content (% of algal dry wt). The protein content ranged 60.6 ± 17.1 to 70.3 ± 9.5 % of algal dry wt. Ph. D.

Recent efforts to restore depressed or extirpated populations of freshwater mussels have focused on artificial propagation as an effective and practical conservation strategy. Although artificially cultured juveniles have been produced and released to the wild, no study has investigated the best time of year or the best developmental stage to release these juveniles. Several experiments were conducted to gain knowledge of appropriate culture and release techniques. Juvenile wavyrayed lampmussels (Lampsilis fasciola) were artificially propagated in the laboratory and subsequently release into a fish hatchery raceway during June, September and March. Juveniles released in June experienced a gradual decline in survival rate, with 50% survival after 72 days and stable survival thereafter until 200 days. Juveniles released in September and March experienced high mortality within the first month, and were unsuccessful in surviving the cold water conditions typical of those seasons. Temperature was strongly associated with growth; thus, juveniles released in June exhibited considerably greater growth than those released in September and March. Survival was positively correlated with shell length in the first 32 days post-metamorphosis. Fall and spring survival values, shell length-frequency data, and a significant increase in overwinter mean shell length (p = 0.045) suggest that overwinter survival is size-dependent. Survival rates of juveniles released in June exceed those of previous culture studies reported in the literature. From these results, I suggest that the best time for release of propagated juvenile freshwater mussels should be at the beginning of the growing season in late spring, when water temperatures exceed 15 degrees C (plus/minus 1°C). Offspring of four, gravid female L. fasciola were tested to evaluate the extent of variability in growth and survival among brood stock. Comparison of growth and survival of progeny showed significantly lower performance of only one female (p < 0.05). Although results indicate variability in growth and survival among broods, it is not known whether heritabilities or physiological fitness caused this variability. Infested fish (IF), newly metamorphosed juveniles (NMJ) and juveniles cultured for 1 month (CJ) were released to a fish hatchery raceway in order to determine the most appropriate developmental stage to release juveniles. Significant differences were recorded among the three release methods, with CJ attaining the greatest growth (2.47 mm plus/minus 0.02), NMJ with the next best growth (1.86 mm plus/minus 0.02), and IF exhibiting the least growth (1.34 mm plus/minus 0.02) (p < 0.0001). Survival among release methods was not statistically different because of high variability within each release method. High mortality from predacious fish was presumably the cause of this variability. With minimal predation, L. fasciola experienced 82.2% (plus/minus 3.6) survival at 90 days. Survival of hatchery-reared juveniles was comparably higher than laboratory-reared juveniles, suggesting that culturing freshwater mussels in a hatchery raceway is a preferable alternative to laboratory culture. An experiment was conducted to compare growth of confined and unconfined juvenile L. fasciola released to a fish hatchery raceway. After 72 days, juveniles confined to small open dishes within the raceway (1.04 mm plus/minus 0.08) exhibited significantly less growth than juveniles released to the raceway that were not held in containers (2.15 mm plus/minus 0.07) (p < 0.0001). Results of additional comparisons with compiled growth data suggest that juvenile growth is retarded when cultured in small dishes. Master of Science

The federally endangered tan riffleshell (Epioblasma florentina walkeri) is restricted to only one known reproducing population, in Indian Creek, Tazewell County, Virginia. Attempts to recover this species by augmenting relic populations throughout its historic range are aided through knowledge of its population biology and requirements in culture environments. Infestations of host fish (fantail darters, Etheostoma flabellare), obtained from four river drainages, with tan riffleshell glochidia showed that significantly more juveniles transformed per fish from infestations on fantail darters from Indian Creek (mean = 59.22 ± 10.01) than on fantail darters from the Roanoke River (mean = 9.45 ± 10.64) (p = 0.024). Number of juveniles from fantail darters collected from Elk Garden and the South Fork Holston River were not significantly different from those of either Indian Creek fish or Roanoke River fish. These results support the hypothesis that mussel-host fish relationships are likely mediated by fish immune responses. Furthermore, this study suggests that this compatibility has resulted from coadaptation between the tan riffleshell and fantail darter populations in Indian Creek. The tan riffleshell population in Indian Creek was estimated to be 1078 adults (95% CI= 760 - 1853), using Schumacher's modification of Schnabel's maximum likelihood estimator. The sex ratio and size distribution of males and females were approximately equal. Specimen ages, determined from thin-sections of shells, showed that mussels aged by external annuli on shells likely underestimates the true ages of individuals. Appropriate culture conditions for this species were examined using juveniles of the wavyrayed lampmussel (Lampsilis fasciola) as a surrogate. In the first experiment, juvenile growth and survival was compared between four substratum types (fine sediment, < 120μm; fine sand, 500 μm-800 μm; coarse sand, 1000 μm-1400 μm; and mixed sediment, < 1400 μm) and two light treatments in open versus covered recirculating troughs (2.8 m). Juveniles in fine sediment substratum and covered troughs fared poorest, with 7% survival and growth to only 0.86 mm in length after 16 wk. Juveniles in mixed sediment and open troughs fared best, with 26% survival and growth to 1.09 mm after 16 wk. Additionally, juveniles in fine sand in covered troughs had significantly higher survival (23.1%) than juveniles in fine sediment (p = 0.04), and juveniles in fine sand survived consistently better between light treatments than in the other substrata. There were no significant differences among the other treatments. A second experiment was performed to determine whether juveniles were responding directly to the presence of light or whether only the increased autochthonous production improved growth and survival. One-half of each of three 2.8 m troughs were covered with 50% shade cloth, while the other sides were left open to ambient light. Additionally, the best and worst sediments from the first experiment (fine sand and fine sediment) were used again to verify the results from the previous experiment. In this case, juveniles in both sides of the troughs grew equally well, but juveniles in the open sides had significantly poorer survival (open mean: 1.78%, sd = 5.01; covered mean: 7.4%, sd = 5.01) (p = 0.046). Fine sediment yielded significantly higher growth of juveniles than fine sand (p = 0.009), with shell lengths of 2.63 mm (sd = 0.075) in fine sediment and 1.94 mm (sd = 0.102) in fine sand. The differences in survival and growth between the two experiments were attributed to differential numbers of chironomids and platyhelminths, which are predators of young juveniles. Additionally, the fine sediment was more tightly packed in the first experiment than in the second, which may have restricted movement and subsequently reduced survival. Light alone likely did not affect juvenile survival and growth; rather, it was seemingly the greater abundance of aufwuchs available as food. This hypothesis was corroborated by a juvenile behavior experiment, which showed that juveniles did not act differently when in tanks not exposed to light versus those open to ambient light. Master of Science

American Electric Power's (AEP) coal-fired Clinch River Plant, a power-generating facility in Carbo, Russell County, Virginia (USA), has impaired Clinch River biota through toxic spills in 1967 and 1970, and effluent copper (Cu) concentrations that were reported to have exceeded water quality criteria from 1985-1989. These impacts have provided impetus for many research projects addressing the absence of bivalves, including federally protected species of native mussels (Unionoidea), from sites influenced by CRP effluent. Modifications in CRP effluent during 1987 and 1993 drastically reduced Cu levels and warranted the present study, which assessed long-term biological recovery in Clinch River biota near the CRP. In 2000-2001, surveys of benthic macroinvertebrate communities and instantaneous measures of effluent toxicity did not foretell significant reductions in survivorship and growth of field-caged Asian clams (Corbicula fluminea) at sites downstream of the CRP. More importantly, these results indicated renewed toxicity in CRP effluent. Additional transplant studies using two enclosure types were conducted to isolate effects attributable to CRP effluent from the potentially confounding effects of substrate variability among study sites. While it was found that mean growth of clams was greatest in the enclosure that minimized substrate variability (p=0.0157), both enclosure types clearly distinguished significant impairment of survivorship and growth at sites downstream of the CRP discharge, and strengthened the association between impairment and CRP effluent. An intensive field investigation was undertaken to determine whether impairment observed in transplant studies extended to resident bivalves. During 2001-2002, densities and age structures of C. fluminea and distributions of mussels suggested that impairment indeed extended to resident bivalves for a distance of 0.5 to 0.6 km downstream of the CRP discharge. Impairment of bivalves was less evident below (1) a fly ash landfill and (2) coal mining activities and low-volume leachate from a bottom ash settling pond. With respect to long-term recovery, modifications in CRP effluent treatment have reduced Cu concentrations from an average of 436 mg/L in 1985-1989 to 13 mg/L in 1991-2002. Subsequently, Cu body burdens of Asian clams (Corbicula fluminea) transplanted within CRP influence have decreased from 442% of levels accumulated at reference sites in 1986, to 163% of these levels in 2002. The reduction in effluent Cu largely explains recovery of most benthic macroinvertebrate community parameters (e.g., richness, diversity) at influenced sites from levels that were typically less than 70% of reference levels, to levels that frequently range from 80 to greater than 100% of reference levels. Nevertheless, bivalves remain impaired downstream of the CRP; survivorship and growth of C. fluminea transplanted to CRP-influenced sites have typically been less than 40 and 20% of reference values, respectively. Furthermore, C. fluminea has seldom been encountered within CRP influence for nearly two decades. Likewise, native mussels remain absent within CRP influence, but recent surveys suggest their downstream distributions are more proximate to the CRP discharge than has been reported previously. A preliminary assessment of factors potentially contributing to toxicity revealed that (1) water reclaimed from settling basins for discharge with CRP effluent significantly impaired fecundity of ceriodaphnids at concentrations of 50%, (2) LC50 values for industrial treatment chemicals were misrepresented on Material Safety Data Sheets and consequently, were subject to misapplication by operators, (3) Cu concentrations of 96 mg/L significantly impaired growth of Asian clams in artificial stream testing, and (4) effluent Al exceeded acute and chronic water quality criteria, suggesting this ion should receive further consideration in future studies. Master of Science

Understanding the respiratory physiology and energy metabolism is important for establishing the dissolved oxygen (DO) requirements of freshwater mussels, determining the metabolite(s) indicative of environmental stress, and interpreting environmental conditions based on physiological indicators of mussels. Three studies were undertaken to address these questions. The first study was conducted with seven mussel species collected from reservoir and riverine habitats. The two objectives were to determine the diurnal patterns of valve gaping of freshwater mussels from different habitats, and to monitor heart rate changes of a mussel species that exhibited the diurnal gaping. The results showed that night gaping is evident for the mussels collected from lentic areas, but not for those collected from lotic areas. The heart rate of Pyganodon grandis increased when they gaped. The second study was conducted with nine species of freshwater mussels from different habitats. The three objectives were to determine the patterns (i.e., regulator and conformer) of oxygen consumption (OC) rate under declining DO, evaluate the effects of temperature on ability to regulate OC under declining DO, and finally to use this information to predict DO criteria for maintaining freshwater mussels in captivity. The results showed that mussels living in the habitats subjected to low DO have a better ability to regulate the OC and were more tolerant to hypoxia. The third study assessed three mussel species from different habitats with different abilities to regulate OC under low DO. The two objectives in this study were to identify the energetic metabolite changes under different DO levels and air exposure for the three species, and to determine the appropriate tissue(s) and metabolite(s) to use for estimating the stress in mussels. The results showed that different biochemical strategies were used by Villosa iris, Elliptio complanata, and Pyganodon grandis. Villosa iris had the lowest anaerobic capacity. Elliptio complanata had a more efficient anaerobic metabolism, and P. grandis reduced energy metabolism under low DO and air exposure. posterior adductor muscle, gill and mantle were good tissues for evaluating hypoxic stress. The mantle tissue had the highest glycogen store and was the best tissue for non-lethal study of physiological condition. Ph. D.

There is a great deal of concern about the declining freshwater mussel fauna of North America. Although deteriorating water quality and habitat degradation may account for much of the decline, it has been suggested that the exotic Asian clam, Corbicula fluminea, may be having an effect on native unionids. Negative impacts may result directly from competition or indirectly, because of Corbicula population crashes that release ammonia and reduce dissolved oxygen in the sediment. Laboratory tests were conducted to determine the relative sensitivity of native mussel and Asian clam life stages to unionized ammonia, and mussel glochidia were the most sensitive (24-hr LC50 of 0.11 mg/L NH₃-N). Juvenile and adult mussels were similarly sensitivity, with average 96-hr LC50's of 0.49 and 0.52 mg/L NH₃-N, respectively. Adult C. fluminea were the least sensitive, having an average LC50 of 0.80 mg/L NH₃-N. The EPA standard test organism, Ceriodaphnia dubia, had one of the lowest LC50's (0.07 mg/L NH₃-N) of the five species, and the fathead minnow, Pimephales promelas, had the highest (1.18 mg/L). The differing sensitivities of the various life stages are important when trying to determine the impact of an Asian clam dieoff. If a dieoff occurs at a time of year when the more sensitive life stages, such as glochidia are present, then the impact on mussel recruitment may be greater. Two miniature artificial stream tests were used to determine the effect of clam density on dieoff rate, ammonia production and dissolved oxygen levels. Only clams at the highest density of 10,000/m2 experienced 100% mortality. Unionized ammonia levels exceeded 4.0 mg/L, and dissolved oxygen levels dropped below 1.0 mg/L during the dieoff. The amount of unionized ammonia produced was twofold greater than the concentration that produced an LC50 in adult C. fluminea and ~40 times greater than the LC50 for V. iris glochidia. Factors thought to have contributed to the C. fluminea dieoff were flow rate, low dissolved oxygen levels, temperature and perhaps ammonia. A complete dieoff did not occur until flow was stopped and dissolved oxygen concentrations began to drop. One-hundred percent mortality occurred in 38 days for the first test, and 21 days in the second test. Higher water temperatures in the first test (26° C) compared to the second test (average = 21.7°C) are thought to have resulted in the faster dieoff. Master of Science

Four life stages of freshwater mussels were tested for their sensitivity to copper and a metal-containing effluent. This permitted an assessment of the variability in sensitivity among the life stages in order to identify those stages most threatened in the wild from copper exposure. Glochidia, while held within the marsupia of the adult, the released or isolated glochidia, the encysted glochidia, and the gravid mussel were tested. Little effect on glochidia was detected following 30- day artificial stream exposures of gravid adults to 19.1 ug Cu/L and an effluent containing an average of 23.9 ug Cu/L. Isolated glochidia were killed by copper concentrations ranging from 20-80 ug/L in 24-hour exposures, with sensitivity increasing with hardness and temperature. Encysted glochidia were resistant to exposures up to 400 ug Cu/L. No significant effect on metamorphosis to the stage was detected. This is likely due to the encapsulation of the glochidium by the host fish. Juvenile mussels reduced their activity during 24-hour exposures to copper concentrations as low as 17 to 24 ug/L and concentrations of 30 to 42 ug/L caused mortality. Juvenile mussels and glochidia within the marsupia are probably the two most sensitive stages in the life cycle of the freshwater mussel. Copper pollution will have its greatest impact in the summer, during periods of high water temperature and low flow. Master of Science