Christopher W. Theodorakis

Size segregation and the effects of oddity on predation risk in minnow schools

Abstract Size segregation was determined in bluntnose minnow, Pimephales notatus, fathead minnow, P. promelas, and stroneroller minnow, Campostoma anomalum, schools by measuring distances between similar- and dissimilar-sized neighbours. Size segregation was compared among species and between schools with and without a predator present. In all cases, there was strong evidence for size segregation. In addition, schools of predominately large fish (25 large, five small fish) and predominately small fish (25 small, five large) were used to determine if odd-sized fish were eaten by large mouth bass, Micropterus salmoides, more often than other school members. In all cases, the odd-sized fish were eatenmore often than expected.

Preliminary assessment of perchlorate in ecological receptors at the Longhorn Army Ammunition Plant (LHAAP), Karnack, Texas.

There have been increasing human health and ecological concerns about ionic perchlorate (ClO4) since it was detected in drinking water sources in 1997. Perchlorate is known to affect thyroid function, causing subsequent hormone disruption and potential perturbations of metabolic activities. According to current estimates, perchlorate is found in the surface or groundwater of 14 states, including Texas. Longhorn Army Ammunition Plant, located in east central Texas, was a facility historically associated with perchlorate-containing propellants and rocket motors. Subsequently, perchlorate contamination in ground and surface waters at the facility has been reported. Soil, sediment, water, vegetation, and animal tissue samples were collected from several locations within the plant for a preliminary site assessment of perchlorate contamination. Perchlorate concentrations ranged from 555–5,557,000 ppb in vegetation, 811–2038 ppb in aquatic insects, below detection limits (ND) to 207 ppb in fish, ND-580 ppb in frogs, and ND–2328 ppb in mammals.Consistent with our hypothesis, aquatic organisms inhabiting perchlorate-contaminated surface water bodies contained detectable concentrations of perchlorate. Additionally, terrestrial organisms were exposed through pathways not necessarily related to contaminated surface waters. Therefore, these data demonstrate that aquatic and terrestrial species are exposed to perchlorate in the environment. To our knowledge, this represents the first incidence of perchlorate exposure among wild animals reported in the scientific literature.

Integration of genotoxic and population genetic endpoints in biomonitoring and risk assessment.

Genetic ecotoxicology is a multifaceted discipline that examines the effects of xenobiotic compounds on the structure and function of DNA. This paper discusses the role of genetic ecotoxicology in environmental biomonitoring and risk assessment. Genetic ecotoxicology may include somatic effects (e.g., DNA damage) or population genetic effects (changes in genetic diversity or gene frequencies). Traditionally, genetic ecotoxicology studies have focused on either one of these sub-disciplines, but integration of these two approaches would be advantageous for three reasons. First, at the population level, concordant responses between changes in population genetic structure and elevated levels of DNA damage may provide evidence that the population genetic changes are influenced by exposure to genotoxic chemicals. Second, if the frequencies of alleles or other genetic markers differ between genotoxicant-contaminated and reference populations, associations between relative amount of DNA damage and genotype may provide evidence that these changes are due to genotoxicant-induced selection. Third, genetic analysis of gene flow may provide insight into patterns of dispersal that could obscure differences between contaminated and reference populations. In order to demonstrate the application of these ideas, three lines of research are summarized herein. The first is a series of studies that focus on radionuclide-contaminated populations of mosquitofish (Gambusia). This research identified RAPD markers that may be indicative of genetic adaptation to radionuclide stress. Relative amounts of DNA damage among genotypes presented evidence that these markers may be indicators of relative radioresistance. The second study examined DNA damage and population genetic structure in radionuclide-contaminated kangaroo rat (Dipodomys) populations. It was found that between-population differences in genetic diversity paralleled those for DNA damage and relative levels of contamination. Also, population genetic analysis indicated that there was dispersal between contaminated and reference populations, and that between-population differences in the amount of DNA damage could not be detected until this dispersal was taken into account. In the third study, populations of redbreast sunfish (Lepomis auritis) from streams contaminated with complex mixtures of industrial chemicals were examined. It was found that the genetic distances between populations within the contaminated stream corresponded with the relative magnitude of molecular and community-level effects. It was concluded that genetic ecotoxicology could make significant contributions to the fields of environmental biomonitoring and ecological risk assessment, and that integration of genotoxicology and population genetic studies would be a definite advantage toward this end.

Acute effects of Fe2O3, TiO2, ZnO and CuO nanomaterials on Xenopus laevis

Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO(2), Fe(2)O(3), and CuO nanomaterials (20-100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1,000 mg L(-1) for TiO(2), Fe(2)O(3), CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L(-1) (ZnO). An EC(50) of 10.3 mg L(-1) ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L(-1). The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.

Sequential expression of biomarkers in Bluegill Sunfish exposed to contaminated sediment.

The temporal expression of various biological rsponses was determined in Bluegill SunfishLepomis macrochirus exposed under controlled laboratory conditions to sediment containing high concentrations of polynuclear aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. Liver, gill, blood, kidney, brain, spleen and intestine were removed from Sunfish sampled at 1, 2, 4, 8, 16, and 40 weeks post-exposure. Biomarker data were recorded for specific proteins, enzymatic activities, DNA integrity, and histopathology. Biomarkers in the laboratory exposed fish were similar to those of indigenous Sunfish sampled from the site of origin of the contaminated sediment. Several patterns of development of biomarkers over time were also evident. For example, the responses of certain biomarkers are not time-dependent (i.e., intestine and gill ATPase activities) while that of others, such as brain ATPase activity, liver cytochrome P450 and NADPH content, stress proteins, chromatin proteins and DNA strand breaks, fluctuate over time. Still other biomarkers, such as EROD activity, zinc protoporphyrin content of the blood, and DNA adducts, showed marked increases over time. Such patterns need to be considered when comparing laboratory and field results and deciding which biomarkers to use for biomonitoring programs. Implications for natural selection and population/community level responses are also discussed.

Genetic ecotoxicology II: population genetic structure in mosquitofish exposed in situ to radionuclides

In 1977, approximately 250 mosquitofish (Gambusia affinis) from a relatively uncontaminated site (Crystal Springs) were transplanted into a small pond on the Department of Energy Oak Ridge Reservation which is heavily contaminated with radionuclides (Pond 3513). Starting in 1992, DNA polymorphism was evaluated using the RAPD (Randomly Amplified Polymorphic DNA) and allozyme genotype techniques to determine if genetic differentiation had occurred between the two populations. Fish from a second radionuclide-contaminated population (White Oak Lake) and another unrelated non-contaminated population (Wolf Creek) were also examined. For the RAPD analyes, 15 RAPD primers (from a total of 40) were found to produce polymorphic banding patterns in at least two of the four populations and subsequently were used to produce a total of 142 bands. Data generated by these RAPD primers indicated an increased genetic diversity in radionuclide-contaminated sites relative to reference sites. Furthermore, the patterns from six RAPD primers produced a higher average number of bands when using DNA from radionuclide- contaminated populations than from non-contaminated, and for three RAPD primers the average number of bands from radionuclide- contaminated populations was lower. In addition, 17 bands occurred at a higher frequency in the radionuclide-contaminated compared to the non-contaminated populations. For the allozyme analyses, it was found that there was a higher percentage of polymorphism and heterozygosity in the radionuclide-contaminated relative to non-contaminated sites. These findings contribute to our understanding of the evolutionary effects of contaminant exposure as well as to the development of population-level biomarkers

Genetic ecotoxicology I: DNA integrity and reproduction in mosquitofish exposed in situ to radionuclides

Female mosquitofish (Gambusia affinis) were collected from two sites located on the US Department of Energys Oak Ridge Reservation that are contaminated with 137Cs, 90Sr, other radionuclides and chemical genotoxicants. Fish from non- radionuclide contaminated environments located off the reservation were also collected. DNA, extracted from liver tissue and blood cells, was examined by gel electrophoresis for structural damage in the form of strand breakage. In general, the level of DNA strand breaks was elevated in fish from radionuclide-contaminated sites with observed differences in the number and type of strand breaks between liver tissue and blood cells. The number of malformed embryos was higher in fish at the contaminated sites, and varied with season. Fecundity was negatively correlated with the level of double strand breaks in the DNA of fish from one contaminated site. Females with broods that included malformed embryos had more DNA strand breakage than those that did not; and furthermore, a threshold effect was observed between the occurrence of malformed embryos and the presence of double strand breaks in the DNA of the mother. These findings have implications for both ecological risk assessment and evolutionary ecology

Relationship between genotoxicity, mutagenicity, and fish community structure in a contaminated stream

Genotoxic responses (chromosomal damage, DNA strandbreakage) of redbreast sunfish (Lepomis auritis)populations exposed to industrial effluent andmutagenicity of the associated sediments weredetermined in order to compare them to changes incommunity structure. Data were collected from areference stream and East Fork Poplar Creek (EFPC), afirst-order stream which originates on the grounds ofthe Department of Energy Y-12 Plant at Oak Ridge, TN. This stream is contaminated with mercury, PCBs, andnumerous other compounds. Previous studies have shownthat sediment contaminant concentrations, as well asphysiological biomarker responses of the local fishpopulations, are highest at the headwaters of EFPC anddecrease with increasing distance from the DOEfacility as contaminant loading decreases. Chromosomal damage was measured by flow cytometry – asreflected by variation in cellular DNA content – andstrand breakage was determined by agarose gelelectrophoresis using blood as the source of DNA. Mutagenicity was determined by theSalmonella/microsome assay using organic solventextracts of sediment surface samples. Community levelresponses included community diversity and percentpollution-tolerant species. Biomarker responses andmutagenicity were found to be highest at theheadwaters of EFPC, and tended to decrease withincreasing distance from the effluent. In general,biomarker responses appeared to be correlated withmutagenicity of the sediment, and both of theserelated to fish community disturbance and level ofstream contamination. Because responses at severallevels of biological organization show similarpatterns of downstream effects, this suggests thatthere may be a causal relationship betweencontamination and biological effects.

New trends in biological monitoring: application of biomarkers to genetic ecotoxicology.

Environmental pollution is a complex issue because of the diversity of anthropogenic agents, both chemical and physical, that have been detected and catalogued. The consequences to biota from exposure to genotoxic agents present an additional problem because of the potential for these agents to produce adverse change at the cellular and organismal levels. Organismal responses at the genetic level to exposure to environmental genotoxicants have been well documented. Past studies in genetic toxicology at the Oak Ridge National Laboratory have focused on structural damage to the DNA of environmental species that may occur after exposure to genotoxic agents and the use of this information to document exposure and to monitor remediation. Current studies in genetic ecotoxicology are attempting to characterize the biological mechanisms at the gene level that regulate and limit the response of an individual organism to genotoxic factors in their environment. An elucidation of the molecular mechanisms involved with these responses, as well as an assessment of the changes that may occur to the genetic material, will provide an understanding of the potential for deleterious consequences at higher levels of biological organization. Moreover, modern procedures of molecular biology offer the hope that alterations and changes to genetic material can be readily detected.

Effects of ZnO nanomaterials on Xenopus laevis growth and development

The objectives of this study were to quantify uptake and developmental effects of zinc oxide nanomaterials (nano-ZnO) on Xenopus laevis throughout the metomormosis process. To accomplish this, X. laevis were exposed to aqueous suspensions of 40-100 nm nano-ZnO beginning in-ovo and proceeding through metamorphosis. Nanomaterials were dispersed via sonication methods into reconstituted moderately hard water test solutions. A flow-through system was utilized to decrease the likelihood of depletion in ZnO concentration. Exposure to 2 mg/L nano-ZnO significantly increased mortality incidence to 40% and negatively affected metamorphosis of X. laevis. Tadpoles exposed to 2 mg/L nano-ZnO developed slower as indicated by tadpoles with an average stage of 56 at the conclusion of the study which was significantly lower than the control tadpole stages. No tadpoles exposed to 2 mg/L of nano-ZnO completed metamorphosis by the conclusion of the study. Tadpoles exposed to 0.125 mg/L nano-ZnO experienced faster development along with larger body measurements indicating that low dose exposure to nano-ZnO can stimulate growth and metamorphosis of X. laevis.