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

A survey of the freshwater mussel fauna was conducted in the Powell River, Virginia, to identify critical habitat for endangered species, quantify substratum composition and coal waste deposition, and to assess population trends during the last half century. Mussels were collected as far upstream as Powell River Mile (PRM) 167.4 near Dryden, Virginia. Endangered species were collected up to PRM 144.6 at Jonesville, Virginia. The sites with greatest diversity were located furthest downstream, and there appeared to be a general decline in the number of species and diversity of mussels from downstream to upstream. Mussel densities also declined proceeding upstream, and specimens were rare above PRM 158.3 near Pennington Gap, Virginia. The highest density occurred at PRM 123.0 near the Tennessee-Virginia border, with 24 mussels/m². Collections per unit effort of sampling concurred with quadrat surveys, indicating a decline in abundance and diversity upstream. Length frequency distributions of the muckets Actinonaias pectorosa and A. ligamentina indicated an absence of smaller mussels at most sites. Sediment samples, collected in riffles at 10 sites to determine particle size distributions and the amount of coal, showed no apparent trends in waste coal from downstream to upstream; however, there were significant negative correlations between PRM location and various fractions of the substratum, indicating a longitudinal sorting of smaller size fractions. Percentages of very fine to medium sand, silt, and coal show marked increases downstream of the North Fork Powell River confluence (PRM 156.6) at Pennington Gap, Virginia. Mussel density had a slightly positive correlation with percent silt (r²=0.346, p=0.0736) but was not correlated with percent coal. Juvenile mussels of Villosa iris were placed on several types of substratum to determine differences in survival. In laboratory experiments, survival of juveniles on coal silt sometimes did not differ from that of juveniles without substratum (survival close to 100%). Survival of juveniles without substratum (93.9%), however, was significantly higher than survival (30.0-63.2%) on three sediment types from the Powell River (p< .001). In field experiments, there was a marginally significant difference in survival of juveniles between two sites (p =.070), with higher survival (47.7%) in sediments from Poteet Ford (PRM 144.6). Survival of juveniles was similar in all laboratory and field experiments. A decline in density of the mussel fauna in the Powell River over the past 15 years was apparent when compared to previous data. Contamination and siltation from coal washing facilities and abandoned mine lands are suspected of contributing to this decline. / Master of Science

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

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 federally endangered tan riffleshell (<I>Epioblasma florentina walkeri</I>) 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, <I>Etheostoma flabellare</I>), 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 (<I>Lampsilis fasciola</I>) 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

The Upper Tennessee River drainage of Virginia and Tennessee, Green River drainage of Kentucky, and Cumberland River drainage of Kentucky and Tennessee are known for their freshwater mussel species diversity. These river systems harbor dense populations of freshwater mussels that have significant impacts on surrounding ecosystems; however, due to their sedentary lifestyles, freshwater mussels are particularly susceptible to many biotic and abiotic stressors. Managers strive to preserve the fragile ecosystems that include freshwater mussels and, hence, study the life-history strategies of endangered and common freshwater mussel species. The goals of this project were to inform management decision-making based on whether Leaunio ortmanni is endemic to the Green River drainage and a species distinct from Leaunio vanuxemensis based on molecular identification, estimation of genetic diversity, and morphometric analysis, and to screen for cryptic biodiversity of populations of the Cambarunio iris species complex in the Upper Tennessee, Cumberland, and Green River drainages. I used the mitochondrial DNA (mtDNA) gene from the first subunit of the NADH dehydrogenase (ND1) as a genetic marker for species-level assessment of L. ortmanni and L. vanuxemensis from the Green and Cumberland River drainages. Additional mtDNA sequences of L. ortmanni and L. vanuxemensis were added to increase sample sizes and coverage of historical distribution. A Bayesian phylogenetic analysis of mtDNA sequences did not result in monophyletic lineages for either species based on the ND1 marker. Haplotype networks of mtDNA sequences demonstrated that haplotype sharing is occurring between the two focal taxa, and also among additional taxa, all of which previously belonged to the genus Villosa. A total of eight nuclear DNA microsatellites were successfully genotyped for the two focal taxa. The nuclear DNA microsatellites showed a strong phylogeographic signal between L. ortmanni of the Green River drainage and L. vanuxemensis of the Cumberland River drainage. An assignment-test based analysis in program STRUCTURE and a phylogenetic tree constructed using Nei's D genetic distance indicated well-differentiated populations across the two drainages. Additionally, the nuclear DNA microsatellite analysis showed a recent loss of genetic diversity across all populations, including when populations were pooled together at the sub-basin level. Further delineation of the focal taxa was assessed using geometric morphometrics and decision tree and random forest analyses. Decision tree and random forest analyses identified periostracum color, nacre color, overall shape, and sex to be distinguishing factors for field identification of L. ortmanni and L. vanuxemensis. Geometric morphometrics comparing species, shape, and drainage showed clear differentiation in shell shape between L. ortmanni and L. vanuxemensis. This study was able to delineate these two taxa, showing that L. ortmanni and L. vanuxemensis are separate species, and that L. ortmanni warrants listing under the Endangered Species Act. Management actions should focus on broodstock collection and propagation strategies to increase genetic diversity within established populations. Additionally, propagation and augmentation should look to reintroduce populations of L. ortmanni into its historical geographic range in unoccupied sections of the mainstem Green River. In addition, I assessed genetic diversity and differentiation again using ND1 and eight nuclear DNA microsatellite loci, and morphological differences among different shell forms of C. iris in the Upper Tennessee, Cumberland, and Green river basins and of the sister species C. taeniatus in the Cumberland, and Green river basins. Additional mitochondrial DNA sequences of C. iris and C. taeniatus were added to increase sample sizes and coverage of historical distribution. Mitochondrial DNA analysis demonstrated haplotype sharing between taxa, but with many unique haplotypes occurring in the upper Tennessee River basin. Nuclear DNA microsatellite loci revealed low levels of genetic diversity within populations of C. iris within the Upper Tennessee River basin, but high levels of divergence from C. iris and C. taeniatus of the Green and Cumberland River basins. The nuclear DNA analysis showed high admixture within and among sampled populations of C. iris throughout the Upper Tennessee River Basin with minimal geographic structuring among sub-basins. Further, phenotypic diversity was assessed using geometric morphometrics and decision tree and random forest analysis. Decision-tree and random forest analysis identified maximum height from the umbo to the ventral margin, periostracum color, shell width, and ray coverage to be defining characteristics for field identification of the focal taxa. Geometric morphometrics showed high overlap of shell shape for the focal taxa regardless of locality. While cryptic biodiversity was not detected in the Upper Tennessee River Basin, on a larger geographic level, cryptic biodiversity was detected using the combination of the mtDNA, nuclear DNA, and morphological data, which showed that C. taeniatus and C. iris from the Green River drainage were distinct from populations of C. iris in the upper Tennessee River basin. / Master of Science / Worldwide, freshwater mussel species diversity is greatest in North America; however, both abundance and diversity have declined in Canada, the United States, Mexico and the countries of Central America. Among rivers of North America, the Ohio River and its large tributaries, which include the Green, Cumberland, and Tennessee River drainages, have been noted for their high levels of biodiversity of freshwater mussels. Freshwater mussels contribute many services to freshwater ecosystems, including nutrient recycling and storage, structural habitat, substrate and food web modification. Dense populations of freshwater mussels have significant impacts on the surrounding ecosystem; however, due to their sedentary lifestyles, freshwater mussels are particularly susceptible to many biotic and abiotic stressors. Examples of stressors include agricultural runoff, temperature fluctuations, and dams which can alter stream conditions into more lake-like conditions and therefore affect the distributions of host fish populations. These stressors put mussels at risk for extirpation, which in turn, reduces the ecological biodiversity of these river drainages. Like other freshwater mussel species, the rainbow mussel (Cambarunio iris) of the Upper Tennessee River drainage, Mountain Creekshell (Leaunio vanuxemensis) of the Cumberland and Tennessee River drainages, and Kentucky Creekshell (Leaunio ortmanni) of the Green River drainage are in danger of decline due to anthropogenic changes in biotic and abiotic factors. Habitat degradation and loss have been of particular concern to managers. Life-history strategies for these three species are still widely unknown, and due to the overlap in their distributions, both phylogenetic and morphological analyses are needed to distinguish between species and populations to determine the best approach for management and conservation. The lack of understanding of taxonomic relationships in combination with morphologically similar characters poses a threat to conservation of these three species. The phylogenetic species concept is defined as an irreducible group whose members are descended from a common ancestor and who all possess a combination of certain defining, or derived traits, whereas the biological species concept is defined as groups or populations that are reproductively isolated from each other, meaning individuals from different groups cannot breed with one another. The results of my study have led to a better understanding of the phylogenetic relationships and species status of individuals collected from different localities of L. ortmanni and L. vanuxemensis and C. iris collected from the Green and Cumberland River drainages and the Upper Tennessee River drainage. In addition, morphological analyses were conducted to identify which traits are best for external identification of these three look-alike species, so they can be more reliably identified in the field.

published_or_final_version / Ecology and Biodiversity / Master / Master of Philosophy

A disposição de esgoto em áreas costeiras pode causar impactos para a vida marinha, alterando a biodiversidade e o equilíbrio do ecossistema chegando a causar graves doenças nos seres humanos. Neste estudo, determinou-se a concentração de um conjunto de marcadores orgânicos moleculares de introdução de esgoto no ambiente marinho: alquilbenzeno lineares (LABs) em amostras de sedimento, ostras (Crassostrea brasiliana), mexilhões (Mytella charruana e Mytella guyanensis) e peixes (Mugil curema) (Valenciennes, 1836) nos Estuário de Cananéia - Iguape e Santos - São Vicente, SP, Brasil. As concentrações de LABs (peso seco) encontradas em amostras de sedimento de Cananéia variaram de <1,0 a 80,6 ng g-1 e em Santos variaram de 6,7 a 1234,0 ng g-1. Em moluscos bivalves as concentrações detectadas em Cananéia encontravam-se na faixa de 90 a 1849 ng g-1(peso seco) e em Santos as concentrações variaram de 110 a 50495 ng g-1(peso seco). Os estudos em peixes de Cananéia apresentaram os seguintes valores médios (ng g-1 em peso seco): músculo (64,7 ± 22,6), pele (124,0 ± 60,1), fígado (164,0 ± 79,2) e rim (9,1 ± 9,9). No estuário de Santos, os tecidos de peixes apresentaram os seguintes valores médios (ng g-1 em peso seco): músculo (95,0 ± 90,1), pele (438,0 ± 437,0), fígado (274,0 ±147,0) e rim (60,7 ± 35,6). Houve diferença significativa entre as amostras analisadas nos tecidos de peixes de Cananéia e Santos evidenciando diferenças de acúmulo de LABs de acordo com o nível de antropização. A detecção de LABs nestas matrizes evidenciou a sua capacidade de acúmulo e reflete a exposição ao esgoto e portanto pode ser utilizadas no monitoramento ambiental. / The sewage disposal in coastal areas can cause impacts to marine life, altering the balance of the ecosystem and biodiversity and causing serious diseases in humans. In this study, the concentration of a set of molecular markers for the introduction of organic sewage into the marine environment: linear alkylbenzene (LAB), in samples of sediment, oysters (Crassostrea brasiliana), mussels (Mytella charruana and Mytella guyanensis) and fish (Mugil curema) (Valenciennes, 1836) from Cananéia - Iguape and Santos - São Vicente estuaries, located in Sao Paulo State, Brazil. LABs concentrations (dry weight) found in sediment samples from Cananéia ranged from <1.0 to 80.6 ng g-1 and from Santos ranged from 6.7 to 1234.0 ng g-1. In bivalve molluscs of Cananéia, the concentrations were in the range 90 to 1849 ng g-1 (dry weight) and in Santos, the concentrations ranged from 110 to 50495 ng g-1 (dry weight). Studies in fish from Cananéia showed the following average values (ng g-1 - dry weight): muscle (64.7 ± 22.6), skin (124.0 ± 60.1), liver (164.0 ± 79.2) and kidney (9.1 ± 9.9). In the Santos estuary, the fish tissue showed the following average values (ng g-1 - dry weight): muscle (95,0 ± 90.1), skin (438 ± 437), liver (274 ± 147) and kidney (60.7 ± 35.6). There were significant differences between the samples of tissues of fish from Cananéia and Santos showing differences in accumulation of LABs according to the level of anthropogenic occupation. The LABs detection in these matrices demonstrated their ability to accumulate and reflects the exposure to sewage and therefore can be used in environmental monitoring.

Environmental change is a major threat to marine ecosystems worldwide. Understanding the key biological processes and environmental factors mediating spatial and temporal species' responses to habitat alterations underpins our ability to forecast impacts on marine ecosystems under any range of scenarios. This is especially important for calcifying species, many of which have both a high climate sensitivity and disproportionately strong ecological impacts in shaping marine communities. Although geographic patterns of calcifiers' sensitivity to environmental changes are defined by interacting multiple abiotic and biotic stressors, local adaptation, and acclimation, knowledge on species' responses to disturbance is derived largely from short- and medium-term laboratory and field experiments. Therefore, little is known about the biological mechanisms and key drivers in natural environments that shape regional differences and long-term variations in species vulnerability to global changes. In this thesis, I examined natural variations in shell characteristics, both morphology and biomineralisation, under heterogeneous environmental conditions i) across large geographical scales, spanning a 30° latitudinal range (3,334 km), and ii) over historical times, using museum collections (archival specimens from 1904 to 2016 at a single location), in mussels of the genus Mytilus. The aim was to observe whether plasticity in calcareous shell morphology, production, and composition mediates spatial and temporal patterns of resistance to climate change in these critical foundation species. For the morphological analyses, the combined use of new statistical methods and multiple study systems at various geographical scales allowed the uncoupling of the contribution of development, genetic status, and environmental factors to shell morphology. I found salinity had the strongest effect on the latitudinal patterns of Mytilus shape. Temperature and food supply, however, were the main predictor of mussel shape heterogeneity. My results suggest the potential of shell shape plasticity in Mytilus as a powerful indicator of rapid environmental changes. I found decreasing shell calcification towards high latitudes. Salinity was the best predictor of regional differences in shell deposition, and its mineral and organic composition. In polar, low-salinity environments, the production of calcite and organic shell layers was increased, while aragonite deposition was enhanced under temperate, higher-salinity regimes. Interacting strong effects of decreasing salinity and increasing food availability on compositional shell plasticity predict the deposition of a thicker external organic layer (periostracum) at high latitudes under forecasted future conditions. This response potential of Mytilus shell suggests an enhanced protection of temperate mussels from predators and a strong capacity for increased resistance of polar and subpolar individuals to dissolving water conditions. Analyses of museum specimens indicated increasing shell calcification during the last century. Deposition of individual shell layers was more closely related to temporal changes in the variability of key environmental drivers than to alterations of mean habitat conditions. Calcitic layer and periostracum showed marked responses to alterations of biotic conditions, suggesting the potential of mussels to trade-off between the deposition of calcareous and organic layers as a compensatory response to strategy-specific predation pressure. These changes in biomineralisation indicated a marked resistance to environmental change over the last century in a species predicted to be vulnerable, and how locally heterogeneous environments and predation levels can have a stronger effect on Mytilus responses than global environmental trends. My work illustrates that biological mechanisms and local conditions, driving plastic responses to the spatial and temporal structure of multiple abiotic and biotic stressors, can define geographic and temporal patterns of unforeseen species resistance to global environmental change.

Compostos farmacêuticos são identificados em matrizes ambientais em ordens de grandeza de ng.L-1 a &mu;g.L-1. Dentre os fármacos, os antibióticos têm recebido atenção especial devido aos problemas que podem causar à biota aquática. O objetivo do presente estudo foi avaliar a citotoxicidade de Amoxicilina e Clavulanato de Potássio isolados e em associação para mexilhões Perna perna utilizando o ensaio do tempo de retenção do corante vermelho neutro, que avalia a estabilidade da membrana lisossômica de hemócitos dos organismos teste. A Amoxicilina causou citotoxicidade aos mexilhões nas concentrações de: CEO: 1 ng.L-1, CI25-24h: 0,44 ng.L-1, CI25-48h: 1,19 ng.L-1 e CI25-72h: 0,85 ng.L-1, o Clavulanato de Potássio foi citotóxico nas concentrações de: 10 ng.L-1 em 24h e 50 ng.L-1 e 100 ng.L-1 em 48h e 72h. Os valores de concentração inibitória foram de CI25-24h: 3,11 ng.L-1, CI25-48h: 3,45 ng.L-1 e CI25-72h: 3,43 ng.L-1. No ensaio realizado com a associação dos fármacos todas as concentrações foram citotóxicas aos mexilhões em 48h e em 72h apenas 40 ng.L-1 de Amoxicilina + 10 ng.L-1 de Clavulanato de Potássio e 200 ng.L-1 de Amoxicilina + 50 ng.L-1 de Clavulanato de Potássio. As concentrações inibitórias foram: CI25-48h: 1,67 ng.L-1 e CI25-72h: 1,36 ng.L-1 a partir dos dados de Amoxicilina e CI25-48h: 0,42 ng.L-1 e CI25-72h: 0,34 ng.L-1 a partir dos dados de Clavulanato de Potássio. / Pharmaceutical compounds are identified in environmental matrices in orders of magnitude from ng.L-1 to &mu;g.L-1. Among the drugs, antibiotics have received special attention due to the problems that can cause to aquatic biota. The aim of this study was to evaluate the cytotoxicity of Amoxicillin and Potassium Clavulanate in isolated and associated forms to marine mussels Perna perna through the neutral red retention time assay which assesses the stability of hemocytes lisossomal membrane of test organisms. Amoxicillin caused cytotoxicity to the mussels in concentrations of: OEC: 1 ng.L-1, IC25-24h: 0.44 ng.L-1, IC25- 48h: 1.19 ng.L-1 and IC25-72h: 0.85 ng.L-1, Potassium Clavulanate was cytotoxic at concentrations of 10 ng.L-1 in 24h; 50 ng.L-1 and 100 ng.L-1 at 48h and 72h. The inhibitory concentration values were IC25-24h: 3.11 ng.L-1, IC25-48h: 3.45 ng.L-1 and IC25-72h: 3.43 ng.L- 1. The test conducted with the combination of drugs all concentrations were cytotoxic to mussels in 48h and 72h only 40 ng.L-1 Amoxicillin + 10 ng.L-1 and Potassium Clavulanate 200 ng.L-1 Amoxicillin + 50 ng.L-1 Potassium Clavulanate. The inhibitory concentrations were IC25-48h: 1.67ng.L-1 and IC25-72h: 1.36 ng.L-1 from the data of Amoxicillin and IC25- 48h: 0.42 ng.L-1 and IC25-72h: 0.34 ng.L-1 from the Potassium Clavulanate data.