Browsing by Author "W. Gregory Cope, Committee Chair"

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Assessing DNA Damage in Hemocytes of the Freshwater Mussel Elliptio complanata with the Comet Assay
(2007-12-21) Prochazka, Sharon Tencza; W. Gregory Cope, Committee Chair; Heather M. Cheshire, Committee Member; Leslie Recio, Committee Member; David B. Buchwalter, Committee Member
The single cell gel electrophoresis or comet assay is widely used to detect DNA damage following exposure to genotoxic compounds with cells isolated from tissue or circulatory fluids from various organisms. The cell sampling method can often lead to the fatality of the test organism, depending on the type of tissue sampled. The objective of this study was to assess genotoxicity with the comet assay from in vitro and in vivo studies using a non-lethal hemolymph sampling method on Elliptio complanata, a native freshwater mussel found abundantly in North Carolina. The hemolymph was withdrawn from the anterior adductor muscle sinus and exposed in vitro to various concentrations (previously tested for cell viability) of selected pesticide formulations, Aatrex 4L herbicide (atrazine), Roundup herbicide (glyphosate), Lorsban 4E insecticide (chlorpyrifos), and Thionex 3EC insecticide (endosulfan), technical grade copper sulfate and a mixture of 46 different PAHs for 4-hours at 4°C. After exposure, the hemocytes were isolated and the comet assay was performed on the treated cells, control cells, and positive control cells exposed to 160 uM hydrogen peroxide (H2O2). For quality assurance purposes, CometAssay Control Cells™ were analyzed throughout each comet assay procedure in the laboratory to ensure the validity of results. The levels of DNA damage measured were expressed as % tail DNA and olive tail moment (OTM). Of all the compounds and concentrations tested during the in vitro exposures, excluding the positive H2O2 controls, only the mixture of PAHs yielded statistically significant (P < 0.05) levels of DNA damage compared to the controls for both DNA damage parameters. The DNA damage was observed at 50 and 100 mg⁄L PAHs, with % tail DNA of 40.70 % and 38.55 %, and OTM of 12.41 and 11.03, respectively. The remaining test compounds yielded no detectable genotoxic effects, regardless of the DNA damage parameter, under the specified concentrations and test conditions. Because genotoxicity was detected during the in vitro exposure with PAHs, an in vivo exposure with PAHs was performed to assess the predictive capabilities of the in vitro test. The in vitro PAH exposure produced a much greater genotoxic response with both parameters then was detected in vivo, in which only the positive control yielded statistically significant levels of DNA damage. Thus, under the conditions tested in this study, in vitro exposures with freshwater mussel hemolymph were unable to predict a similar in vivo response, based on the presented data. Nonetheless, a method with a high degree of accuracy was demonstrated during this study with consistent and repeatable levels of DNA damage measured in the CometAssay Control cells™ and positive control treatments. Therefore, we are confident that if the chemicals tested had been genotoxic under the conditions tested, the effects would have been detected. This research investigated the use of a non-lethal genotoxicity screening tool using freshwater mussel hemolymph. Further testing and evaluation is needed before this tool could be widely implemented. Moreover, there is need for a better understanding of freshwater mussel hemolymph and the functions and capabilities of hemocytes in the defense of genotoxic compounds.
Bioturbation as a Novel Method to Characterize the Toxicity of Aquatic Sediment.
(2005-05-29) Cho, Eun-ah; W. Gregory Cope, Committee Chair
Bioturbation, the biological process through which many species of infaunal benthic invertebrates suspend bottom sediments into the water column through their burrowing, feeding, respiratory, and locomotor activities, may be a sub-lethal endpoint that can be exploited to assess the toxicity of aquatic sediments. Therefore, we developed a novel test method that used bioturbation (BioTurbTox test) generated by the activities of second in-star Chironomus tentans larvae as the toxicity endpoint (Chapter 2). To validate this method, copper (Cu) and fluoranthene were individually spiked into relatively uncontaminated aquatic sediment to assess changes in bioturbation and mobilization of the chemicals into the overlying water. Turbidity production responded to the chemicals in the sediment in a concentration-dependent manner and was an excellent indicator of sediment toxicity. Moreover, substantial concentrations of Cu were released into the overlying water from the Cu-spiked sediment, whereas little fluoranthene was mobilized into the overlying water from the fluoranthene-spiked sediment. Sediment samples were then collected from the field and used to evaluate the similarity of response of the BioTurbTox test to other more standardized toxicity tests. In the summer of 2003, sediment samples were collected at six sites in the Neuse River of North Carolina tested for toxicity, and analyzed for chemical contaminants (Chapter 3). Atrazine was the most frequently detected current-use pesticide and pyrene and fluoranthene were measured at relatively high concentrations from the Neuse River sites. Concentrations of fluoranthene were correlated with results from the Ceriodaphnia dubia porewater and BioTurbTox tests. We concluded that the new BioTurbTox test was useful as a rapid screening method for sediment toxicity information, but required normalization to the clay content or to the total organic carbon content of field collected sediments. In Chapter 4, the toxicity of environmental pharmaceuticals and personal care products (PPCPs) were evaluated with the BioTurbTox and C. dubia reproductive tests. Fluoxetine and bisphenol A significantly affected bioturbation caused by C. tentans, especially at high concentrations (1-2 mg/L), and the turbidity change induced by caffeine, fluoxetine, and bisphenol A showed a concentration-response relation. Triclosan affected reproduction of C. dubia at relatively low concentrations (IC50: 85.4 μg/L). However, most of the tested PPCPs were not acutely toxic at environmentally relevant concentrations, but were relatively toxic at high concentrations. In Chapter 5, two sediment-spiking methods (extract mixing vs. whole sediment dilution methods) were compared with the BioTurbTox test and a gradient response was observed from both methods. Based on the similarity of the toxic response, we determined that either of the spiking methods was appropriate for estimating the toxicity of aquatic sediments in screening level assessments. The overall conclusion from this research was that the newly developed BioTurbTox test shows promise as a tool to assess the toxicity and mobilization of contaminants from aquatic sediments.
Sensitivity of Early Life Stages of Freshwater Mussels to a Range of Common and Extreme Water Temperatures
(2008-12-05) Pandolfo, Tamara Jane; Thomas Kwak, Committee Member; W. Gregory Cope, Committee Chair; David Buchwalter, Committee Member
Freshwater mussels fulfill an essential role in benthic aquatic communities, but are also one of the most rapidly declining faunal groups in North America. Rising water temperatures, caused by global climate change, industrial discharges, drought, or land development, can further challenge impaired unionid communities. The aim of this study was to determine the upper thermal tolerances of the early life stages, glochidia and juveniles, of freshwater mussels. Glochidia of eight species of mussels were tested: Lampsilis siliquoidea, Potamilus alatus, Ligumia recta, Ellipsaria lineolata, Lasmigona complanata, Megalonaias nervosa, Alasmidonta varicosa, and Villosa delumbis. Seven of these species were also used in juvenile tests. Survival trends were monitored as mussels held at three acclimation temperatures, 17Â°C, 22Â°C, and 27Â°C, were exposed to a range of common and extreme water temperatures (20 - 42Â°C) in standard acute laboratory tests. The average median lethal temperature (ET50) in 24-h tests with glochidia was 33.7Â°C, ranging from 29.1 to 37.5Â°C. The mean ET50 in 96-h juvenile tests was 34.8Â°C, and ranged from 32.9 to 36.7Â°C. As an indicator of sublethal thermal stress, heart rate patterns for seven species of juvenile freshwater mussels were assessed visually through direct observation. Species differences were observed; L. recta and V. delumbis displayed significant changes in heart rate associated with increasing temperature at all three acclimation temperatures. Thermal stress is almost certainly not the only stressor affecting freshwater mussels. Metals, such as copper, are a common source of toxicant exposure in aquatic environments. The toxic effects of copper on the early life stages of freshwater mussels have been well-studied, and freshwater mussels are more sensitive to copper than most aquatic organisms. The effect of a sublethal copper concentration on the upper thermal tolerance of three species, Lampsilis siliquoidea, Potamilus alatus, and Ligumia recta, of juvenile freshwater mussels was determined. ET50s were calculated in the absence and presence of copper, and they ranged from 32.9ÂºC to 36.7ÂºC with a mean of 34.8ÂºC. Based on 95% confidence interval overlap, there were no differences among ET50s caused by acclimation temperature, species, or presence of copper. However, survival trends showed evidence of interactive effects between copper and temperature for all three species, suggesting this is an area that warrants further study. In freshwater systems, the larval life stage, glochidia, of Unionoida mussel species must develop as obligate parasites on host fish gills or fins before transforming into the juvenile life stage and dropping to the sediment to perpetuate their life cycle. Because of the relationship between mussels and their often specific host fish species, freshwater mussels are not only potentially affected by their own variable thermal tolerance limits, but also by the thermal tolerances of their fish hosts. Thermal tolerance data for eight species of freshwater mussels and their host fish was compiled and compared in order to determine whether the community structure of these systems is at risk from rising environmental temperatures; relationships were complicated with mussels being both more and less thermally sensitive than certain host fish species. Freshwater mussels are a valuable part of benthic aquatic ecosystems, and this study has shown that thermal stress can negatively impact these animals; therefore it is necessary to keep rivers and streams thermally acceptable to protect these threatened fauna.