Draft submitted 29 June 2005 · 2018-06-18 · Westinghouse Savannah River Company and the South Carolina Department of Natural Resources, published “Fishes of the Middle Savannah

ANNUAL TECHNICAL PROGRESS REPORT

OF ECOLOGICAL RESEARCH

Draft submitted 29 June 2005Final submitted 19 July 2005

Supported under Cooperative AgreementDE-FC09-96SR18546

between

The University of Georgiaand the

U.S. Department of Energyfor

The University of Georgia fiscal year endingJune 30, 2005

Paul M. Bertsch, Director

Prepared by Laura Janecek

Savannah River Ecology LaboratoryDrawer E

Aiken, SC 29802PH (803) 725-2472 FAX 725-3309 E-mail: [email protected]

www.uga.edu/srel/

SAVANNAH RIVER

ECOLOGY LABORATORY

This report is provided for information only and is not to be considered formallypublished literature. We request that no citations be made of information contained

herein without the express consent of the investigator.

Savannah River Ecology Laboratory FY2005 Annual Technical Progress Report

Table of Contents

SREL FY2005 Overview .............................................................................................................................................. 1

Special Accomplishments of SREL Personnel ............................................................................................................. 5

An Overview of Research Themes ............................................................................................................................... 7

Characterization ......................................................................................................................................................... 8Question 1: What are the roles of microorganisms in biogeochemical processes? ......................................... 8Question 2: How do coupled biological and geochemical processes interact? .............................................. 10Question 3: What processes control chemical speciation? ............................................................................. 12Question 4: How are bioavailability, bioconcentration, and biomagnification related? ............................. 20Question 5: What types of statistical models best describe contaminant distributions? ............................. 22Question 6: Can sentinel species be used as surrogates to determine environmental health? .................... 24Question 7: What techniques permit studies of bacterial community structure and function?.................. 26Question 8: How do seasonal variations influence contaminant speciation?.............................................. 27Question 9: How can amphibian conservation plans protect against rare events? ...................................... 28

Ecological Risks and Effects ..................................................................................................................................... 30Question 1: How does contaminant speciation influence bioavailability? .................................................. 30Question 2: How do contaminant effects integrate over different levels of biological organization? ......... 31Question 3: How do variation in contamination, remediation practices, and ecology alter risk? .............. 34Question 4: What are the potential interactions from exposures to mixed contaminants? ........................ 37Question 5: What are the risks from low-dose, chronic exposures to radiation? ......................................... 37Question 6: What are the most cost-effective biomarkers for impacts to biota? .......................................... 37Question 7: What are the mechanisms of trophic and maternal transfer of contaminants? ....................... 38Question 8: What are the risks associated with indirect selection for antibiotic resistance? ...................... 39Question 9: What statistical techniques provide the most power when estimating dose or exposure? ....... 41

Remediation and Restoration ................................................................................................................................... 42Question 1: How can solute transport be incorporated in models of contaminant migration? .................. 42Question 2: What are the hydrogeochemical processes that determine contaminant migration? ............... 42Question 3: What hydrogeochemical factors control permeable reactive barrier function? ........................ 43Question 4: What are the mechanisms by which amendments immobilize contaminants? ........................ 44Question 5: Can plant genes involved in remediation of groundwater contaminants be identified? ......... 46Question 6: How do microbes affect metal speciation transformations? ..................................................... 46Question 7: What traits of native plants determine their use in remediation and restoration? .................. 47Question 8: Can natural processes be directed or accelerated for restoration? ............................................ 48

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Research Support Programs .................................................................................................................................... 51Environmental Health and Safety Program .................................................................................................... 52Distance Learning Program ............................................................................................................................ 52Quality Assurance Program ............................................................................................................................. 53Research Data Archives ................................................................................................................................... 53SREL Undergraduate and Graduate Education Program ................................................................................ 53Environmental Outreach Program.................................................................................................................. 56DOE Research Set-Aside Areas ......................................................................................................................... 58

Externally Funded Grants ......................................................................................................................................... 62

Publications ............................................................................................................................................................. 69

SREL Organizational Chart ....................................................................................................................................... 75

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The Savannah River Ecology Laboratory (SREL) is aresearch unit of The University of Georgia (UGA) and hasbeen conducting ecological research on the Savannah RiverSite (SRS) near Aiken, South Carolina for almost 54 years.The overall mission of the Laboratory is to acquire andcommunicate knowledge of ecological processes andprinciples. SREL conducts fundamental and appliedecological research, as well as education and outreachprograms, under a Cooperative Agreement with the U.S.Department of Energy (DOE).

The Laboratory’s research mission during the 2005 fiscalyear was fulfilled with the publication of 102 journal articlesand book chapters by faculty, technical staff, students, andvisiting scientists. Two books were also published with SRELpersonnel as authors. An additional 43 journal articles havebeen submitted or are in press. Other noteworthy eventstook place as faculty members, staff, and graduate studentsreceived awards. These are described in the section titledSpecial Accomplishments of SREL Personnel on page 5.

Notable scientific accomplishments during the past yearincluded work in the areas of environmental remediation,radiation ecology, and environmental microbiology.� SREL researchers, in collaboration with others from

Clark Atlanta University, WSRC, and the U.S. ForestService-Savannah River, developed and implementedan automated vadose zone monitoring system tomeasure soil moisture and soil matric potential, andto collect lysimeter samples. A telemetry componentof the system allows for real-time monitoring of vadosezone conditions in the field from a remote location.Also, the sampling frequency and data retrieval intervalcan be adjusted without visits to the field site. Thesystem is currently being tested at the Mixed WasteManagement Facility on the SRS.

� In a paper published in the journal RadiationResearch, Dr. Thomas Hinton and colleaguesexamined whether dose-rate limits established for

FY2005 OverviewSavannah River Ecology Laboratory

humans also protect other organisms in theenvironment, as is currently assumed. Theyemphasized there is a need to understand howradiation effects are integrated across different levelsof biological organization, but to gain suchunderstanding better collaboration is needed betweenthe traditionally separate disciplines of radiationbiology and radiation ecology.

� Dr. Paul Bertsch and colleagues received a $239,000grant from the Department of Energy Office of ScienceEMSP program to study the link between chemicalspeciation and bioavailability of uranium and nickelin contaminated sediments. This study, to be conductedin the Tims Branch/Steed Pond system of the SavannahRiver Site, will provide valuable information on thescientific basis for natural attenuation of similarlycontaminated areas.

� SREL researchers received a $500,000 grant from theNational Oceanic and Atmospheric Administration(NOAA) to study the role of metal contamination inthe proliferation of antibiotic resistance in coastalwater-borne pathogens. The three-year award buildsupon novel field and laboratory research done at theSRS by Drs. J Vaun McArthur and RamunasStepanauskas that suggested microbes resistant tometal contamination may also be resistant toantibiotics.

During the past year several faculty had accomplishmentsworthy of special note.� SREL director Dr. Paul Bertsch received the Soil

Science Research Award, a career achievement awardfrom the 11,000-member Soil Science Society ofAmerica, the largest professional soil scienceorganization in the world.

� Dr. Andrew Neal was senior author of a publicationthat was in the top 5% of most-downloaded papers ofthe 350 published in Geochimica et CosmochimicaActa in the past year. The paper, “Surface structure


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effects on direct reduction of iron oxides byShewanella oneidensis,” was done in collaborationwith other scientists from Pacific Northwest NationalLaboratory, Idaho National Laboratory, Montana StateUniversity and the U.S. Naval Research Laboratory.

� Dr. Christopher Romanek was recognized for his highlycited research in the geosciences by ISI EssentialScience Indicators. Dr. Romanek’s record includes19 papers cited a total of 870 times to date in the fieldof Geosciences.

During the past year SREL hosted two workshops ofnational significance. In November, 2004, a workshop on“Inventory and Monitoring of Amphibians and Reptiles”was held at the SREL Conference Center. Co-sponsored bySREL, U.S. Forest Service, and Partners for Amphibian andReptile Conservation (PARC), this workshop broughttogether Forest Service personnel from across the countryto learn herpetofaunal inventory and monitoringtechniques that could be implemented on national forestlands. Over 30 participants took part in this four-dayworkshop.

In March, 2005, a workshop entitled “Partners along theFall Line II: Nurturing Ecoregional Partnerships forPreserving Regional Resources and Sharing Data, ScientificFindings, and Technical Approaches” was held at the SRELConference Center. Sponsored by the Department ofDefense SERDP Ecosystem Management Program, the goalsof this workshop were to share ecosystem managementapproaches, address specific opportunities, and nurtureecosystem planning partnerships. Participants representingseveral state and federal governmental and non-governmental agencies attended this three-day workshop.

2005 saw the retirement of one senior SREL facultymember. Dr. Domy Adriano retired after a 30-year careerat SREL, where he concentrated his research mainly ontrace elements in the environment. Among otherachievements, he published over 200 scientific articles andbook chapters and wrote two highly regarded books ontrace elements. Dr. Adriano, who retired in April, continuesto collaborate on research projects with colleagues at SREL,in the U.S., and abroad.

SREL personnel produced two books during the past year.Researcher Dean Fletcher, with co-authors fromWestinghouse Savannah River Company and the SouthCarolina Department of Natural Resources, published“Fishes of the Middle Savannah River Basin, With Emphasison the Savannah River Site” in March 2005. This 480-pagebook features over 200 color photographs of fish species

and habitats taken bySREL researcherDavid Scott. SRELSenior Ecologist Dr.Whit Gibbons and co-author Dr. MikeDorcas published “Snakes of the Southeast” in May, 2005.This volume features over 300 color photographs anddistribution maps of the 52 snake species found in thesoutheastern U.S.

The Savannah River Ecology Laboratory’s primary fundingsource is a Cooperative Agreement between the U.S.Department of Energy and The University of GeorgiaResearch Foundation that currently covers a five-yearperiod from July 1, 2001 through June 30, 2006. Theestimated total cost of this agreement since its inceptionin 1996 is over $94 million, with DOE contributing about$89 million and the University of Georgia about $4 million.SREL’s total operating budgets from DOE and other federalsources in FY02, FY03, FY04, and FY05 were $10.2, $10.2,$9.11, and $8.7 million, respectively. In FY05 SRELreceived about $7.75M from DOE Office of Science (DOE-SC). The FY06 budget from DOE-SC was projected to be


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$0, based upon the President’s budget request; SREL hadrequested $7.978M for FY06 from DOE-SC. However, as aresult of discussions with DOE-EM, DOE-SC, NNSA, andUGA, SREL expects the FY06 budget to be $4.3M. SRELalso receives almost $798,000 per year from The Universityof Georgia. During FY05 SREL received $115.6K from DOEand WSRC for specific SRS-related tasks.

Researchers at SREL currently have funding totaling about$2.242M from 33 grants received during FY05, in additionto funds provided by DOE-SR. Sources of grant awardsrange from private foundations such as the National Fishand Wildlife Foundation to federal agencies such as theU.S. Environmental Protection Agency, the National ScienceFoundation, and the Department of Defense (DoD).Important grants awarded this year included $115K fromthe National Institutes of Health to Dr. Travis Glenn; $239Kfrom the DOE-SC Environmental Management ScienceProgram (EMSP) to Dr. Paul Bertsch; and $534K fromThe National Oceanic and Atmospheric Administration toDrs. Ramunas Stephanauskas and J Vaun McArthur.

As of 30 June 2005 SREL had a permanent staff of about145 people, nearly all of whom are employees of TheUniversity of Georgia. The staff includes 21 researchscientists, six of whom are co-staffed through tenure-trackpositions in various departments at The University ofGeorgia and one who is co-staffed through a tenure-trackposition in the School of Public Health of the University ofSouth Carolina. There are another 10 Ph.D.s inpostdoctoral appointments. Research technicians (44),clerical and other support personnel (45), and graduatestudents (25) comprise the remaining staff categories.

In addition to holding faculty positions in 12 departmentsat the University of Georgia, various SREL faculty haveadjunct status at 18 other colleges and universities. Faculty,staff, and students also are active in providing outreachand service to the scientific community. Representativesfrom the laboratory hold more than 60 editorial orcommittee positions in national groups and organizationsand also serve on several UGA academic and administrativecommittees. Over 100 lectures, scientific presentations,and posters were presented during the past year at scientificmeetings, colleges, and universities, including minority

institutions.

In FY05 SREL spent about $100K to replace some existingequipment, including a microwave digester, ultra coldfreezer and thermal cyclers, and to purchase a portableX-ray fluorescence analyzer. SREL also purchased areplacement DNA sequencer for its molecular ecologylaboratory at a cost of about $180K.

Participants in the SREL Education Program during FY05came from schools located throughout the United Statesand included 18 undergraduate students and 37 graduatestudents. The graduate students came from 7 differentuniversities in the U.S. and abroad, emphasizing thenational and international stature of the SREL program. Inthe past year 4 graduate students from SREL earned MastersDegrees and 4 earned Doctor of Philosophy Degrees. ANational Science Foundation grant from the ResearchExperiences for Undergraduates Program for a proposaltitled “The Impact of Energy Technologies on NaturalEnvironmental Systems” continued to provide funding forthe undergraduate program at SREL.

The SREL Outreach Program reaches a different audiencein its efforts to communicate scientific awareness to thegeneral public. During the past year, SREL scheduled 247talks, 14 tours, 19 exhibits, and 62 workshops, reaching atotal of 64,761 people. Topics of these presentationsincluded biodiversity, the process of science, animaladaptations, plants and wetlands, environmental scienceand chemistry, local ecosystems and conservation,classification, and careers in ecology and research.

The SREL Distance Learning Program continued to focusits efforts on programming related to the Laboratory’s coreprograms in ecology and environmental science. SREL, incooperation with and with funding from the UGA Collegeof Pharmacy, has offered a multidisciplinary Master’sDegree in Environmental Toxicology via the GeorgiaStatewide Academic and Medical Systems (GSAMS)network. This is the first advanced degree offered by UGAthrough any distance learning site in Georgia or SouthCarolina. Eight students have completed all requiredcoursework and are working on the research componentof the degree and four students have graduated from the


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program. In an effort to expand its audience, the SRELOutreach Program presented 5 Ecotalks via distancelearning to elementary, middle, and high schools in bothSouth Carolina and Georgia. Many Georgia schools havenot been able to maintain compatibility with GSAMS andthe Georgia audiences are declining.

The Conference Center has continued to see wide use, bothby SREL personnel and the local community. The facilitywas used to host a total of 67 scientific meetings andenvironmental education programs for students, teachers,and the general public this past year, and almost 2,000people visited the facility.

Representatives of the Laboratory also serve local andstatewide communities by organizing a canned goods drivein November, hosting an auction to benefit the SouthCarolina Chapter of The Nature Conservancy in December,managing a recycling program, participating generouslyin the UGA Campaign for Charities, and participating inthe regional Heart Walk to benefit the American HeartAssociation.


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Special Accomplishmentsof SREL Personnel

Dr. Andrew Neal authored a very popular scientific paperin the journal Geochimica et Cosmochimica Acta, aninternational publication devoted to geochemical issues.Andrew’s paper was in the top 25 most-downloaded papersfor the period of October 2003 to September 2004. Thepaper covered the groundbreaking work he and hiscolleagues are pursuing on the environmentally importantprocess of bacterial iron reduction.

Dr. Paul Bertsch received the Soil Science ResearchAward, a career achievement award from the Soil ScienceSociety of America that recognizes outstanding researchcontributions in soil science. Paul also received the D.W.Brooks Award for Excellence in Research, from the Collegeof Agriculture and Environmental Sciences at The Universityof Georgia.

Graduate student Meredith Wright, a student of Dr. JVaun McArthur, was awarded a 2004 EnvironmentalProtection Agency (EPA) STAR Fellowship, valued at over$37,000. Her study is titled “The Transfer and Transportof Antibiotic Resistance in Heavy-Metal ContaminatedStreams.

Dr. Christopher Romanek was recognized as a top tenresearcher in the field of “Mars Science” for the years1993-2004 by ISI Essential Science Indicator (http://www.esi-topics.com/mars/index.html).

Dr. Paul Bertsch was appointed to serve on two NationalAcademy of Sciences committees: the U.S. NationalCommittee for Soil Science and the Committee on EarthResources. Paul was also appointed to a National Academyof Sciences Steering Committee for a Workshop onFrontiers in Soil Science Research, by Executive Directorof the Policy and Global Affairs Division of the NationalResearch Council, 2005.

Dr. Domy Adriano was appointed Honorary Presidentof the International Society of Trace ElementBiogeochemistry, Vienna, Austria (Autumn 2004).

Dr. Tracy Punshon received the 2004 Editor’s Citationfor Excellence in Manuscript Review Award from theJournal of Environmental Quality, in recognition of heroutstanding professional contributions in reviewing papersduring 2004.

Graduate student Sarah Strycharz, a student of Dr. LeeNewman, won a conference award for Best StudentPresentation for her poster “Development of a genome-wide screening method to identify gene candidates involvedin the degradation of halogenated hydrocarbons using ionchromatography” at the Phytoremediation Session of theAssociation for Environmental Health Sciences meeting inAmherst, MA.

Graduate student Chris Winne, a student of Dr. WhitGibbons, won a travel award from the South AtlanticChapter of the Society of Wetland Scientists to facilitate hisattendance at the Association of Southeastern Biologistsannual meeting in Alabama. While at the ASB annualmeeting, Chris won the Association of Southeastern BiologyStudent Research Award in Aquatic Biology for his talk on“Adaptation of a small aquatic snake (Seminatrix pygaea)to a dynamic habitat: selection on body size andreproduction after aestivating during drought.”

The first SREL Graduate Student Symposium was held inJuly, 2004; 35 students presented oral or posterpresentations. Winners in the platform session included:Jason Unrine (first place, $300), Kimberly Andrews(second place, $200), and Jacqueline Litzgus (thirdplace, $100). Poster session winners included AngelKelsey Wall (first place, $300), Tracey Tuberville(second place, $200), and Meredith Wright (third place,$100).


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Graduate student Kimberly Andrews, a student of Dr.Whit Gibbons, received the UGA Institute of Ecology’sBest Student Paper Award in the applied category. The $500case prize was awarded for the paper judged to be thebest published during the preceding year by an Institute ofEcology graduate student. Her paper was titled “How dohighways influence snake movement? Behavioral responsesto roads and vehicles;” it has been accepted for publicationin the journal Copeia.

Graduate students Gabrielle Graeter and J.D. Willsoneach received Student Travel Awards from the Society ofWetland Scientists. Both are students of Dr. Whit Gibbons.

The SREL Environmental Outreach Program was oneof two nominations from UGA for a Board of Regents ofthe University System of Georgia-Best Practicescompetition.

Dr. Nanthi Bolan, of Massey University, New Zealand,received the M.L. Leamy Award for 2004 from the NewZealand Soil Science Society for most meritoriouspublications in the last three years (2001-2003). Dr. Bolanwas a visiting scientist at SREL for several months during2004, working primarily with Dr. Domy Adriano.

Dr. Kenneth McLeod was appointed to the ScienceWorking Group of the Governor’s Task Force on “CoastalWetland Forest Conservation and Use,” for the State ofLouisiana.

Undergraduate student Tom Luhring won two awardsthis year. He was the recipient of the Institute of Ecology’sJosh Laerm Memorial Outstanding Undergraduate Award.Tom also received the Georgia Museum of Natural HistoryLaerm Memorial Award. Tom participated in the SREL REUundergraduate program in the summer of 2004; his SRELsponsors were Drs. Whit Gibbons and BetsieRothermel.


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SREL receives its DOE funding as a single operating grantto support the activities outlined in the Laboratory’sCooperative Agreement. SREL’s management and facultyare responsible for establishing the research programsand plans that implement the terms of the Agreement.

The long-term ecological studies that provide anindependent assessment of SRS activities on theenvironment and demonstrate the relative health of SRSecosystems have been central to SREL’s researchthroughout its nearly 54-year history. Discussions withDOE-SR administrators in 1999 resulted in a reaffirmationof the importance and value of long-term ecologicalstudies, while at the same time delineating SREL’s role inemerging issues related to environmental remediation andrestoration.

To detail the research to be conducted with DOE-SCfunding, SREL prepares research plans and milestones.

An Overview ofResearch Themes

Beginning with FY04, research plans have been assembledaccording to three research themes: CHARACTERIZATION,ECOLOGICAL RISK AND EFFECTS, and REMEDIATION AND

RESTORATION—which reflect how the environmentalremediation process proceeds. This is done to provide amore useful document for DOE managers and contractors,and to provide researchers with a perspective on how theymight identify and address knowledge gaps in each of thethemes through scientific research. Numerous knowledgegaps are associated with each theme and some cut acrossall three themes. For example, processes controllingbioavailability emerge as gaps across all themes; however,the specific gaps and the approaches to dealing with themwithin the three themes vary based on the level oforganization or scale at which they are addressed. Theknowledge gaps identified for each theme are notcomprehensive, but rather represent those gaps that wereaddressed in studies planned for FY05.


Characterization

Characterization is a necessary first step in determining environmental and health risks and devising remediation andrestoration strategies. Characterization has physical, chemical, and biological components, and spans molecular tolandscape scales. Although it includes a descriptive component, characterization is more than simply measuringcontaminant concentrations in biota or other media, or reporting the presence or quantity of various organisms atcontaminated locations. It includes developing an understanding of the processes that control distributions ofcontaminants, chemical speciation, and bioavailability. Characterization also includes elucidating the environmentaland ecological patterns and processes that influence distributions of organisms, biological diversity and function, health,and population processes. Characterization is necessary to construct models of how natural and engineered systemsfunction, both in the presence and absence of environmental perturbations such as anthropogenic contamination. Inother words, characterization is best viewed as an activity to better describe and understand particular habitats andsystems, both contaminant-stressed and more pristine.

Significant knowledge gaps exist that impair accurate risk assessment, limit remediation and restoration activities, andmake cost-effective management decisions difficult or impossible. Only with scientific advancements can informeddecisions be made about, for example, (1) whether there is a need to restore or remediate a given site; (2) the state towhich a site should be restored; (3) when the process has been completed, and (4) how successful and cost-effective aremediation activity has been. There are significant knowledge gaps that must be closed to achieve the types ofcharacterization necessary to meet remediation and restoration goals. Research at SREL addresses these knowledgegaps by taking advantage of unique expertise in the environmental sciences and ecology, the unparalleled field researchopportunities at the SRS, and the long-term data sets, research tools and capabilities that SREL has developed over thelast half-century.

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Report on FY05 Characterization Milestones

Question 1:What are the roles of microorganisms in biogeochemicalprocesses that affect contaminant cycling, speciation, andmobility, and how are these affected by co-contaminants?How do the microbial populations and species diversityaffect or contribute to the overall function of the ecosystem?

(A) Use of field flow fractionation to investigate uraniumadsorption by ShewanellaInvestigators: A.L. Neal and B.P. Jackson

We have continued to study the sorption of uraniumto Shewanella cells and a paper was published

recently in Analytical Chemistry (SREL reprint 2812)summarizing our results to date. More recently, wehave received samples from Dr. Alex Belieav of PacificNorthwest National Laboratory of exopolymerssecreted by Shewanella oneidensis MR-1 with a highaffinity for U. These are believed to be similar toexopolymers we observed during our FFFexperimentation. We are currently utilizing HighPerformance Size Exclusion Chromatography (HP-SEC) to assess the molecular weight of the exopolymer;recent results suggest an approximate size of 200 kDa,but we are aiming to improve our estimate byoptimizing the HP-SEC separation.


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In preliminary experiments aimed at determining theaffect of dissimilatory iron reduction upon the fate ofuranium in natural soils, we have been performingexperiments using chromate into soils from SavannahRiver site. These experiments, using Shewanellaputrefaciens strain CN-32, have focused on theinteraction between cells and naturally occurring fulvicacids in surface soils of the Orangeburg series andvadose soils from the Tobacco Road formation.Preliminary experiments indicate that autoclaving doesnot alter Cr(VI) sorption behavior to the two soils.Very limited abiotic Cr(VI) reduction was observedfor either sterile soil upon addition of 0.1 mM Cr(VI),assessed using the diphenylcarbazide method. Incontrast, rapid reduction was observed uponinoculation with CN-32. Simultaneously, Fe(II) washigher in treatments inoculated with CN-32 asdetermined using the ferrozine method. Although thevadose zone soil contained greater quantities ofextractable Fe (14.62 ± 0.65 g kg-1, compared to 2.04± 0.04 g kg-1 in the surface soil) less Fe(II) wasidentified in solution (0.25 mg L-1) compared to thesurface soil (1.6 mg L-1) following 7 days incubation.There is a significant difference between the totalorganic content of the two soils, the surface soil having13.5 mg g-1 compared to 4.2 mg g-1 in the vadose zonesoil. Amendment of the vadose zone soil with humicmaterials extracted from the surface soil effectivelyincreased Fe(II) concentrations to surface soil levels,suggesting that the iron reduction observed in thevadose zone soil could be augmented by humic acids.The presence of humic acids also had a significanteffect upon Cr(III) speciation. While in humic acidfree vadose zone soil total [Cr] (i.e. [Cr(VI) +Cr(III)]) in solution assessed by ICP-MassSpectrometry was not significantly different from[Cr(VI)], in the surface soil containing significanthumic material total [Cr] (2 mg L-1) far exceeded[Cr(VI)] (0.5 mg L-1), suggesting much of the Cr(III)was associated with humic material in the aqueousphase, not as precipitates associated with the soil itself.This has significant implications for the concept ofreduced metal species being an acceptable endpointin surface soils containing high levels of humicmaterials.

Upon completion of these experiments we aim to studythe fate of uranium under identical conditions.

(B) How do radionuclide and heavy metal inputs influencemicrobial soil populations within a riparian system?Investigators: C.L. Zhang, G.L. Mills, A.L. Neal, B.PJackson, and P.M. Bertsch, in collaboration withcolleagues at the Savannah River NationalLaboratory and the Medical University of SouthCarolina

Past DOE activities have created significantenvironmental contamination, which can have adverseeffect on all forms of life including microorganisms.On the other hand, certain groups of microorganismsare known to enhance the degradation of organicpollutants and the transformation and immobilizationof heavy metals and radionuclides. Considerableresearch has been conducted by DOE to determinethe transport and fate of contaminants in soils andgroundwater. Technologies have also been developedto enhance biodegradation of chlorinated organics andpetroleum hydrocarbons and biotransformation ofheavy metals and radionuclides at SRS. However, adeeper understanding of microbial diversity,community structure, and activity is needed to betteraccomplish DOE missions and respond to new DOEinitiatives and environmental bioremediationstrategies.

Our initial research focus was on three sites at SRS:(1) Steeds Pond, which is heavily contaminated withmetals and radionuclides; (2) Lower Tims Branch,which is less contaminated with metals andradionuclides; (3) an iron creek at D-Area, which isimpacted by the seepage of a holding pond forremediation practices. DNA was extracted from soilor sediments using a commercial kit (MoBio Co.).Primers specific for bacteria (27F/1492R) were usedfor PCR amplification of these DNA samples. The PCRproducts were screened using denaturing gradient gelelectrophoresis (DGGE). The DGGE patterns werecompared using statistical methods, which resultedin a similarity tree using the Pearson indices and theun-weighted pair group with arithmetic means


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algorithm (UPGMA).

The bands in DGGE lanes were scanned by the ImagerSystem (Alpha Imager 3400) and intensities of bandswere measured and the percentages of the bands inthe community were calculated using the software.The data were then reorganized and submitted to thedatabase for UPGMA analysis. TreeView software wasused to generate a relationship tree. The resultsshowed that about 40 bands in all of five samples canbe identified in the urea concentration region from30% to 55%. Based on the same quantity DNA andreferences controls, different bands referred todifferent bacteria species and different intensitiesreferred to different abundances of differentpopulations. Stream sediments and surface soil fromSteed Pond showed similar distribution patterns exceptthat the sediment diversity was less evenly distributedthan the surface soil. Community evenness for LowerTims Branch was to some extent short because of theminor dominances of several bands. In the ion creeksamples, the sediments and surface mats showed thelarge likelihood between their diversity patterns andcommunity structures.

Bacterial diversity is high at all three sites. Thecommunity structure at each location is significantlydifferent, suggesting a geographical variation ofbacterial diversity at the SRS. The community structureat Steeds Pond is more similar to that at the LowerTims Branch than to those at the iron creek. This mayreflect environmental differences between the soil andaquatic systems. At the iron creek, black sedimentbelow the iron mat has a higher diversity than thesurface mat. This suggests that microbial communitiesvary in the context of the changing geochemicalenvironment.

Question 2:How do coupled biological and geochemical processesinteract to determine the fate and transport of contaminantsin near-surface and surface environments?

(A) Upland land-use effects on downslope conditions

Investigators: S.J. Harper, B.S. Collins, and R.R.Sharitz

Upland land-use can alter forest composition,structure, and biogeochemistry, including movementof materials along geomorphological gradients. Forexample, forestry practices such as cutting andprescribed fire can result in greater carbon, nitrogen,and sediment movement downslope and inputs ofthese materials at the upland-wetland interface. Inturn, these inputs can influence important drivers ofbiogeochemical processes, including redoxconditions, carbon pools, and nutrient dynamics.Improved understanding of the effects of uplandprocesses on drivers of biogeochemical conditionslocated downslope will enhance efforts to remediateorganic and inorganic contaminants concentrated atthe wetland-upland interface.

In FY05 we continued field research alonggeomorphological gradients to characterize land-useeffects on soil characteristics and input of materials,principally nitrogen, at the upland-wetland interface.Over winter 2003-04, we established three sets of sitesalong the gradient from managed pine forest throughslope hardwoods to the wetland-upland interface alongTims Branch. These sites represent the followingcombinations of land use activities: (a) undisturbedupland, slope, and interface; (b) burned [2004]upland, undisturbed slope, and interface; and (c)burned [2004] upland, thinned [ca. 2000] slope, andinterface. Pre- and post-fire sampling of lysimeters,deployed in upland, slope, and upland-wetlandinterface regions in each set of sites, revealed positionalong the slope, but not land use, affected NO

3-N

concentrations, while land use, but not slope position,affected NH

4-N concentrations. NO

3-N concentrations

were highest in slope locations, while NH4-N was

marginally greater in undisturbed sites than in siteswith recently burned upland. Mass of organic layersamples was less at the upland-wetland interface thanat slope or upland positions. Further, downslope sitesleading from burned and cleared uplands had lessorganic layer mass than those leading from burnedor undisturbed uplands. These field data suggest there


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is little immediate effect of land-use activities onnitrogen loss/availability in lysimeter samples alongthe topographic gradient, but prescribed fire andclearing can affect carbon and nitrogen cycling byreducing organic layer pools available fordecomposition.

In FY05 we also developed spatially-explicit modelsusing GIS to characterize the physical conditions ofthe Tims Branch subwatershed on the SRS. Based ona 10-m elevation model and derived slope estimates,we divided the study area (3571 ha) into zonesrepresenting upland, slope, and interface areas (65,12, and 23% of total area, respectively). Model resultsindicated that these zones differed with regard todominant soil (Fuquay sand, Vaucluse-Ailey complex,and fluvaquents, respectively) and vegetation types(loblolly/upland hardwoods, open-canopy loblolly,and flood plain sweetgum forest, respectively). Despitethese physical differences, all three zones wereextensively burned in conjunction with forestmanagement during the past decade of the availablehistorical record (61, 64, and 55%, respectively).Given the prevalence of burning and its importanceto nutrient cycling within the ecosystem, further workis needed to determine effects of upland land usepractices on biogeochemical drivers at the wetland-upland interface.

(B) Effects of coal fly ash on ecosystemsInvestigators: K.W. McLeod, R.R. Sharitz, B.C.Collins, W.A. Hopkins, B.E. Taylor, and B.P. Jackson

Fly ash was released from the D-Area coal-burningpower plant into a bottomland forest from 1967 to1977 as a result of runoff from a filled settling basin.We characterized the extent (length, width, and depth)of fly ash in a 3-dimensional grid at the site. Up to a2.75 m depth of fly ash was deposited in some areasand total aerial coverage was approximately 40hectares. Twenty-four hectares held at least 30 cm offly ash. Arsenic (As) and selenium (Se) were theprimary contaminants-of-concern, with meanconcentrations in areas, with at least 30 cm of fly ash,of 7 and 4 ppm in the forest floor litter layer, 31 and 6

ppm in the fly ash, and 5 and 3 ppm in the originalsoil, respectively. Arsenic and selenium concentrationsin the ash-contaminated areas were 10 and 2 timesgreater than in control areas, respectively. Strontiumwas also elevated (7X) in the ash containing areas.This large scale deposition of the fly ash has alsosignificantly altered the topography of the forestedlandscape, with ash deposited in low spots and areaswith slow water flow rates.

Contaminant concentrations in foliage of herbaceousplants and trees were elevated in the ash plume area(generally by a factor of 2-3 for As and 2-8 for Se),but As and Se concentrations were less than 2 and 4ppm, respectively. Higher Se concentrations weregenerally found in tree than herbaceous foliage, whilehigher As concentrations were more likely found inthe herbaceous plants. Earthworms (Lumbricusrubellus) and land snails (several species) hadelevated levels of both As and Se at sampling siteslocated on the ash plume. Earthworms containedmuch higher levels of Se (up to 35 ppm) than snails.Larval amphibians contained more As than adultamphibians, but Se concentrations did not decline withmaturing life stage. Elemental concentrations weregenerally higher in Bufo terrestris (southern toad)than Rana sphenocephala (southern leopard frog).Concentrations of As, Se, and strontium were up to11-35 times higher in amphibian metamorphs fromthe ash area than from an unpolluted area.

While the plant species composition on the ash plumearea has shifted to an earlier successional stage, thecommunity characteristics such as basal area anddensity are similar to those of an adjacent non-contaminated early successional clearcut site. Thepresent vegetation is in transition from the formermature forest community to an impacted and/orrecovering community.

The herpetofaunal community is characterized by atleast 18 amphibian (14 anuran and 4 salamanderspecies) and 17 reptile (9 snake, 3 lizard, 4 turtleand 1 alligator) species. The diversity is not unusualfor this community type. Abundance and species-


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richness of soil-dwelling macroinvertebrates (mainlyearthworms and land snails) were substantially higherat control sites than at sites located on the ash plume.

Since the plant community is adjusting to the depositedash, the best management for this site might bemonitored natural attenuation, although it isrecognized that the metal contaminants will never beconverted to less harmful forms and through naturalattenuation the contaminants will simply dilute overtime into the environment. Then, it must bedemonstrated that the migrating ash contaminants arenot harming the biota, either individually or byaffecting community diversity.

Question 3:What processes control chemical speciation and mobilityof toxic metals, organic contaminants and radionuclidesin biota, soil, wetlands, ground water and surface water?What linkages exist between chemical speciation (andother properties of contaminants at the molecular level)and bioavailability, uptake and transfer of contaminantsby organisms?

(A) Use of transgenic nematodes to assay heavy metalexposureInvestigators: T.C. Glenn, C.H. Jagoe, B.P. Jackson,P.M. Bertsch, and collaborators from the IdahoNational Engineering and EnvironmentalLaboratory (INEEL) and the Universities of Georgiaand South Carolina

Work continued on the transgenic nematodes(Caenorhabditis elegans) with a metal-specificpromoter, metallothionein-2, linked to a GreenFluorescent Protein reporter (mtl-2::GFP), which wehave developed and described previously. We havecontinued development of high throughput assaysusing plate readers, and a COPAS Biosort (flowcytometer modified to gently handle large particles,such as C. elegans). During testing of the COPASBiosort, we measured fluorescence of unexposed andmetal exposed mtl-2::GFP worms. Much of theinformation below is modified from data gained during

a product demonstration and resulting report writtenby B. Wang of Union Biometrica, who set up theinstrument and conducted assays in our labs withworms we provided to him. This information will beimportant for our resulting publications, and we arelikely to invite Dr. Wang to be a co-author.

The worms clearly responded to Cd by producingincreased levels of GFP. The treated mtl-GFP wormsshowed a large variance among the individualfluorescence intensities. Indeed, examination of theseworms under a fluorescent microscope revealed thatalthough the majority of these worms were highlyfluorescent, the rest of them showed low fluorescencelevels comparable to that of the untreated ones. The“bright” and “dim” subpopulations of the treatedworms can be separated by the COPAS instrumentbased on these different fluorescence intensity levels.We are now investigating the causes for variance inGFP expression and working to reduce the variancein brightness within our mtl-2::GFP strains.

Spatial distribution and chemical speciation ofcontaminants

We also used synchrotron X-ray fluorescence to studythe spatial distribution of Pb and Cu in C. elegansinitially exposed to each contaminant in solution.Qualitatively there was a striking difference in thespatial distribution of each contaminant in thenematodes; Cu was relatively homogeneouslydistributed while Pb was highly localized in thepharynx. The analytical technique was very sensitiveto assessing exposure to these metals and in the caseof Pb, significant uptake was discernable for aqueousexposure of 2.4 µm. The highly localized nature of Pbuptake and the high concentration of Pb at this hotspotsuggested that this was a Pb solid phase. We used µ-Xray diffraction to investigate this hotspot and obtaineda diffraction pattern consistent with the Pb phosphatemineral pyromorphite. This work was presented atthe Society for Environmental Toxicology andChemistry 4th World Congress in Portland, October2004. A manuscript has been prepared, submitted andaccepted for publication in Environmental Science


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and Technology.

(B) Effects of mercury in the environmentInvestigators: C.H. Jagoe, W.A. Hopkins, C.S.Romanek, I.L. Brisbin, Jr., and A.L. Bryan Jr.

Although the form of mercury that generallyaccumulates in fish and wildlife is methyl mercury,most mercury released to the environment bycommercial and industrial sources is in inorganicforms. These inorganic species are methylated in soils,waters, and wetlands by microbial processes. Thus,better understanding of the processes that control orinfluence in situ mercury methylation, speciation, andtransport is essential to assessing and mitigatingenvironmental mercury contamination. Also, most ofthe mercury accumulating in fish and wildlifeoriginates in their diet. Therefore, documenting andunderstanding trophic transfer and trophicrelationships within food webs is critical tounderstanding and managing mercury contaminationproblems.

In FY 2005, we published a manuscript on mercuryconcentrations and speciation in amphibian larvae inwetlands, and their food items. Mercuryconcentrations in larvae from wetlands withoutobvious local pollution sources were similar to thosethat caused adverse effects in laboratory experiments.Amphibian larvae from these wetlands also act as preyitems for a variety of wildlife, so these mercuryconcentrations might indicate risks to consumerspecies. Speciation data were used to calculatebioaccumulation factors for inorganic and methylmercury in a simple food web composed of periphytonto amphibian larvae. Our results indicated thatstandard, widely used EPA models do not accuratelypredict mercury bioaccumulation in these wetlandsystems.

We published a study in FY05 exploring therelationships between tissue mercury concentrationsin benthic bivalves and dissolved mercuryconcentrations in water. This study examined therelative contributions of small streams and wetlands

to mercury in a large river (the Savannah River).Corbicula are small, introduced clams. They aresessile filter feeders, so contaminant concentrationsin their tissues are highly reflective of local conditions.Corbicula living at the mouths of small streamsdraining wetlands on the South Carolina coastal plainhad higher tissue mercury concentrations than thosefrom sites nearby in the main river channel. Methylmercury concentrations were also higher in tributarystream waters than in the main river channel. Theseresults indicate that small streams draining wetlandson the coastal plain are important sites of mercurymethylation, and suggest that such small catchmentsare of considerable importance in the biogeochemicalcycling of mercury on the SRS and, probably, in othersoutheastern systems.

In concert with other ongoing projects, we havecollected fish from SRS reservoirs and offsite locationsfor analysis of contaminants, including total tissuemercury, for several years. Otoliths from subsamplesof these fish were removed and used to measure fishage. Fish size and age data allow calculation of relativegrowth rates of fish from SRS reservoirs versus offsitelocations, and allow for examination of relationshipsbetween size, age, and tissue mercury concentrations.A manuscript describing these relationships inreservoirs on the SRS and offsite locations has beensubmitted for peer-review.

For almost 10 years, we have collected samples oftissues (blood, down, feathers) and food items fromwood storks (Mycteria americana), an endangeredspecies that nests in the southeastern U.S. This workwas partially funded by the U.S. Fish and Wildlifeservice. This one-of-a-kind, long-term data set showsthat mercury exposure and accumulation in nestlingwood storks varies with location and annual rainfallpatterns. Parent storks forage in wetlands near nestingcolonies and return prey to their nestlings. Mercuryconcentrations were consistently higher in juvenilebirds fed from freshwater wetlands than in those fedfrom estuarine or salt-water wetlands. This isconsistent with the concept that methylation potentialsare greater in freshwater systems than in estuarine


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systems. Analysis of stable carbon isotopes in tissuesprovides direct evidence for carbon sources in thediet. Nestlings from colonies where freshwater preyrepresents the only available carbon source haddifferent δ13C signatures than those from colonieswhere saltwater and estuarine prey were available. Incoastal colonies, where both fresh and saltwater preywas available, mercury concentrations in nestlingswere higher in wet years, when foraging habitatexpanded in freshwater marshes filled by rainwater.Shifts in nestling δ13C signatures between wet and dryyears were consistent with this interpretation. Amanuscript describing these results is in preparation.

Feather samples have been collected from more than10 other species of nestling wading birds (includingegrets, herons, and ibis) from coastal and inlandcolonies. This work was partially funded by externalgrants, and includes a student funded by NOAA.Samples were obtained from inland nesting colonieswhere parent birds foraged exclusively in fresh waterand from birds in coastal colonies where parentsforaged in both fresh and estuarine/salt water habitats.Analyses of samples collected in FY05 have beencompleted. Mercury concentration varies amongspecies, with those feeding at the highest trophic levels(as identified by δ15N signatures) having the highestmercury concentrations. This work interfaces withstudies of trophic relationships and contaminantconcentrations in other birds (waterfowl and eagles)as described in other sections of this report.

(C) Earthworms as indicators of uranium and nickelInvestigators: T. Punshon (CRESP), P.M. Bertsch, andB.P. Jackson

The extent to which metal and radionuclidecontaminants of soils and sediments pose anenvironmental hazard depends upon their potentialfor movement within the environment and their entryinto biota. An accurate evaluation of this hazardinevitably involves a determination of bioavailability,because it is now widely accepted that only a fractionof the total contaminant is subject to such movements.Direct measurement of bioavailability in soils and

sediments has proved a challenge to environmentalscience which has so far resisted incorporation into areadily usable test. Current approaches to measuringbioavailability involve chemical and biological tests.Chemical tests involve applying a series of increasinglyaggressive extractants and measuring the metalconcentration removed by each extraction as afunction of the total. Commonly known as a sequentialextraction scheme, it was not intended to inform onbiological responses. No single fraction orcombination of fractions represents that accessibleto biota. Although a great many scientific studies haveused the scheme to make inferences about speciationand bioavailability, the scheme is actually a measureof chemical lability. Biological tests are consideredmore appropriate measures of bioavailability becausevarious toxicity and mortality endpoints are related toa definite contaminant concentration. These testsinvolve exposure of certain test organisms to measuredamounts of contaminant in a situation which ideallyresembles a field situation. For soils and sediments,in particular, earthworms and nematodes have beenused. Our aim in carrying out these earthworm studiesis to bridge knowledge gaps between chemical andbiological tests, so that we can move toward a greaterunderstanding of bioavailability and derive moreaccurate measures for risk assessment andremediation.

The contaminant source for the Tims Branch ripariansystem has been the subject of several keybiogeochemical studies at SREL, all of which havefound a contrast in the chemical lability of the maincontaminants, Ni and U. Geochemical tests have shownhigher chemical lability for Ni than U, with the latterbeing complexed by dissolved organic matter or boundto a colloidal fraction. A survey of the chemicalcomposition of dominant flora corroborated this withthe presence of Ni and absence of U within planttissues.

Our previous work using the U.S. EnvironmentalProtection Agency’s Earthworm Subchronic Toxicitytest (EST) indicated that the substrate collected in TimsBranch was not toxic to earthworms, and yet they


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accumulated extremely high concentrations of U withintheir tissues (>200 µg g-1), contradicting the coreinferences of geochemical tests and evidence fromvegetation. Although the EST does not claim to beapplicable to U specifically, the data it provided forNi, a contaminant for which it should be applicable,were also counter to geochemical tests. Nickelbioavailability exceeded that of U by an order ofmagnitude, but it did not accumulate in earthwormtissues during the EST. Further, U accumulation wasstrongly affected by components of the artificial soilmixture prescribed in the EST methodology, inparticular the level of calcium carbonate incorporatedinto the soil. Addition of organic matter alsosuppressed earthworm U accumulation. Our studiesindicated that the composition of the artificial soil wasjust as influential—if not more so—to U accumulation(and by inference, bioavailability) than Uconcentration of the soil.

The next stage of the research involved examiningearthworms using synchrotron X-ray fluorescencespectroscopy (SXRF) available at beamline X26A ofthe National Synchrotron Light Source (BrookhavenNational Laboratory). This non-destructive techniqueshows the relative abundance and spatial distributionof metals (including U) within environmental samples,and was applied to samples of contaminant-exposedearthworm digestive tract samples (preparation ofwhich is described in the 2004 SREL Annual TechnicalProgress Report). Collection of compositional mapsfor earthworms yielded excellent results in FY04 afterthe EST was repeated using a 50/50 mix of TimsBranch soil and artificial soil, the treatment level inwhich the highest U accumulation in earthworms wasobserved. By collecting multiple elemental maps athigh resolution (i.e. low pixel: step size ratio), andcombining maps to form a composite image of theentire worm, we were able to show the extent of Uaccumulation in the earthworm gut.

Clearly, U is found only in the tissues within the coelom,and not in the exterior tissues from peritoneum toepidermis. These U-enriched tissues comprise thechloragogue cells, intestinal epithelium, intestine and,

tentatively, typhlosole. Uranium distribution isextensive within the crop and gizzard. Choragogeoustissues are of interest in this work, because they areresponsible for the formation of mineralized depositsarising from the combination of metals filtered out ofthe gut with phosphates and carbonates. This is asequestration mechanism, which protects theearthworm from metal toxicity, and may be the reasonbehind the low EST mortality but high tissue Uconcentrations. X-ray fluorescence spectra showedthat areas of the earthworm that were particularlyenriched with U were also enriched with P and Ca.

Determinations of the speciation of the U accumulatedby earthworms has been carried out both by extendedX-ray fine structure spectroscopy (EXAFS) and alsoby size-exclusion chromatography interfaced withinductively coupled plasma mass spectroscopy (SEC-ICP-MS). The oxidation state of the U within theearthworms is known to be in the U(VI) form, althoughthe binding environment of the U is still unknown. Byconducting replicated analysis in the EXAFS range ofthe XAS spectra, and comparing data to known Ustandards, we are able to determine the bindingenvironment of the U within earthworms. Sizeexclusion chromatography has been conducted onmineralized granules extracted from earthwormtissues.

The results of the studies conducted on earthwormswill be published as a three-paper series, andremaining speciation studies are nearing completion.The first paper will focus on the results of the EST, inparticular the effects of calcium carbonate and organicmatter, while the second paper will present resultsfrom the SXRF and EXAFS studies on abundance,distribution and speciation of U within the earthwormtissues. The final study will present data from size-exclusion ICP-MS of extracted earthworm granules.

(D) Whole-lake experiments on the dynamics of cesiumInvestigators: T.G. Hinton and B. Taylor, withcollaborators from Colorado State University

2005 Progress: The rich data set derived from our


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whole-lake addition of stable cesium to a 137Cs-contaminated pond on the SRS continues to revealthe interesting dynamics of the contaminant. Thisresearch is important because whole-lake experimentsprovide a realistic experimental scale, but are rareand difficult to conduct. Additionally, our data fill avoid in the SRS data base by providing information onthe first 250 days following contamination by Cs. Twomanuscripts were produced from this research inFY05.

The first manuscript (Pinder III, J.E., et al.. 2005.The influence of a whole-lake addition of stablecesium on the remobilization of aged 137Cs in acontaminated reservoir. J. Environ. Radioactivity80:225-243; SREL reprint 2818) quantifies the releaseof 137Cs from the sediments of the lake following theaddition of stable Cs to the water column. Desorptionof 137Cs from sediments is an important process thatprolongs the bioavailability of the contaminant,enhances the probability of subsequent uptake of thecontaminant by biota, and directly relates to thedetermination of human and ecological risks.European research following the Chernobyl accidentexamined the remediation potential of adding stablepotassium to 137Cs contaminated lakes, but no studieshave considered the addition of stable Cs. Wedocumented a 3-fold increase in the dissolved 137Csinventory of the lake’s water column following theaddition of 4kg of stable Cs. Several lines of evidenceindicated that the source of the increased inventorywas a release of 137Cs from the sediments. Thedynamics within the water column were modeled witha simple steady-state model of the form: Y = 3.1 +6.1 (1 – e (-0.028 * t )); where Y predicts the inventory of137Cs in the water column of the lake and t is thenumber of days following the addition of the stableCs.

The second manuscript (Pinder III, J. E., T. G. Hintonand F. W. Whicker. In press. Foliar uptake of cesiumfrom the water column by aquatic macrophytes. J.Environ. Radioactivity) examines the impacts ofaquatic vegetation on the dynamics of Cs. Specifically,we were interested in the relative importance of foliar

uptake of Cs by aquatic plants from the water columnversus root uptake of Cs from the contaminatedsediments. Although foliar uptake has been previouslydemonstrated for some species of aquatic plants, mostmodels of the transport and fate of Cs in lakes do notcontain components and pathways that consideraquatic plants. We anticipated that this absence mightbe contributing to a lack of accuracy in someChernobyl-derived models that predict the long termdynamics of cesium in aquatic systems. We found thataquatic plants can indeed have an important influenceon Cs dynamics within a lake, and that foliar uptakewas the principal absorption mechanism for fourcommon species of aquatic plants (Braseniaschreberi, Nymphaea odorata, Myriophyllumspicatum and Utricularia inflata), but was notimportant in Typha latifolia, Alternantheraphilozeroides or Sagittaria latifolia.

Following the Chernobyl accident 137Cs concentrationsin fish were 200 to 20,000 times greater than that inthe water. Because freshwater fish are a significantpart of the European diet, this lead to 137Cs radiationexposure to humans. Our stable Cs research showed(1) declining concentrations in the water due touptake by biota and deposition onto sediments, and(2) continuing accumulation in largemouth bass(Micropterus salmoides), a common food and gamefish. Mathematical modeling was used to estimate thatratios of fish concentrations to water concentrationsat equilibrium would be approximately 70,000. Thisvalue is greater than those in European lakes. Thegreater ratio is due to the lower concentrations ofpotassium in the water of Southeastern lakes.

(E) Chemical speciation in the environmentInvestigators: B.P. Jackson, W.A. Hopkins, T.Punshon (CRESP), C.S. Romanek, and P.M. Bertsch

Knowledge of chemical speciation is imperative tounderstanding mechanisms of toxicity or tolerance tocontaminants in organisms. Plants and bacteria canproduce extracellular ligands either to enhance uptakeof an otherwise unavailable element or to limit uptakeof a potential toxicant. We have developed methods


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based on interfacing a separation procedure such asfield flow fractionation or liquid chromatography withinductively coupled plasma mass spectrometry toinvestigate trace element speciation in environmentaland biological samples in support of a number ofresearch areas at SREL:

Selenium contamination of soils and sediments

SREL studies have focused on Se contamination arisingas a result of coal combustion where Se is input intothe system in inorganic form either as selenate orselenite. However, plants readily convert inorganic Seto selenomethionine, which is bioavailable to higherorganisms and may cause toxicity at elevatedconcentrations. Additionally, numerous other lowmolecular weight organoselenium compounds havebeen identified, although little is known regarding theirchemical reactivity and toxicity. We tested a numberof different reagents for effectiveness in extracting Secompounds from bullfrogs (Rana catesbeiana)collected from a coal fly ash settling basin. Theseextracts were analyzed by size exclusionchromatography (SEC)-ICP-MS to determine themolecular weight of Se compounds in the extract. IfSEC-ICP-MS identified low molecular weight Secompounds (<1000 Da), then the extract wasanalyzed by ion chromatography (IC)-ICP-MS toidentify the compound. SEC-ICP-MS analysis usingextractants that do not degrade proteins indicated thatSe was a constituent of a high molecular weightcompound (>30,000 Da). The use of protease orchitinase as extractants, two enzymes that degradeproteins to the constituent amino-acids, liberatedpredominantly low molecular weight Se compoundsthat were identified as selenomethionine by IC-ICP-MS. Future work will focus on determining Sespeciation in the sediment and food resources ofbullfrogs and in higher trophic organisms. Resultsfrom this study will be presented at an internationalPlasma Mass Spectrometry conference in Durham,England, and a publication will be submitted to a peerreviewed journal in FY05.

This work has been completed, and was presented at

the International Plasma Mass SpectrometryConference in Durham, England, in September 2004.A manuscript has been accepted for publication inthe proceeding volume for the conference. (Jackson,B.P., et al. In Press. Selenium speciation inamphibian larvae developing in a coal fly ashsettling basin. Proceedings of the 9th InternationalConference on Plasma Source Mass Spectrometry,Durham UK, 2004).

Arsenic and selenium speciation in soils

Arsenic and selenium have several stable oxidationstates and can exist in a number of low molecularweight organic complexes. These different speciesexhibit differing reactivity, toxicity, and availability, soit is important to quantify As and Se on a species ratherthan a total element basis. Ion exchangechromatographic methods have been developed toquantify different As and Se species that can occur inenvironmental samples. This methodology was appliedto study residual As and Se in a soil profile five yearsafter application of flue gas desulfurized gypsumstabilized with coal fly ash (both coal combustionresidues). Arsenic was found to have not significantlyleached from the 0-15 cm soil profile but Se hadsignificantly leached over 5 years. Increased watersoluble Se concentrations persisted five years afterapplication. Increased As concentrations weremanifest in the ligand exchangeable fraction. The mainspecies detected were arsenate and selenite, for Asand Se respectively. Plant uptake of Se was significantlyincreased in direct relation to initial treatment, withselenium present as selenate in plant tissue extractscollected from invasive plants growing on the treatedsoils. The one-time soil amendment with coalcombustion by-products was shown to have long-termeffects on soil As and Se concentrations and increasedSe solubility and availability five years after application.The results of this study suggest that repeatedapplication of coal combustion byproducts to soilcould be detrimental due to build up of As in the soilprofile and the long-term availability of Se. The resultsof this study were presented at the InternationalConference on the Biogeochemistry of Trace Elements


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in Uppsala, Sweden, in July 2003 and will be publishedin an upcoming proceedings volume.

This work is complete, the manuscript has beensubmitted, and the proceedings volume is in press.(Punshon, T., et al. Arsenic and Selenium Speciationin Aged, FGD-Amended soil. In: Coal CombustionProducts and Environmental Issues. Sajwan, K.S.,I. Twardowska, T. Punshon, and A.K. Alva (Eds).Springer-Verlag Publishers, New York, USA).

Trace metal complexation by low molecular weightorganic acids

High concentrations of Ni and U exist in the sedimentsof Steeds Pond and Tims Branch at the SRS. Thisriparian site is the focus of a number of researchprojects at SREL studying the solid phase speciation,bioavailability, and trophic transfer of Ni and U.Previous studies have shown that Ni is bioavailable atthis site with increased concentrations detected inseveral plant species. Various low molecular weightorganic acids, notably citric acid and histadine, areknown to be important complexers of Ni in plants.We have investigated analytical techniques to identifyand quantify Ni-organic ligands in the rhizosphere andplant extracts from Steeds Pond/Tims Branch. Usinghigh performance size exclusion columns we haveseparated Ni-oxalate, -EDTA, -citrate, and -histadine,although the retention time for each species was notsolely based on molecular weight. Analysis of a plantextract from Tims Branch showed that Ni wascomplexed in a low molecular weight compound, mostlikely Ni-citrate. We are continuing to study the use ofSEC-ICP-MS for the separation of low molecular weightmetal-organic ligand complexes to characterize theeffect of the gel phase on the stability of the metal-ligand complex with the goal of using the techniquefor unambiguous speciation data of metal-ligandinteractions in biological samples.

Preliminary results of Ni and U speciation in plantextracts using these techniques were presented at the8th International Conference on the Biogeochemistryof Trace Elements, Adelaide, Australia, March 2005.

The techniques of using ion chromatography and sizeexclusion chromatography coupled to ICP-MS to studymetal interactions with low molecular weight acidsare promising and our preliminary results suggest thatfurther work in this area is warranted.

Laser Ablation ICP-MS

Laser ablation ICP-MS is being used with increasedfrequency in the environmental sciences because ofthe spatial control afforded by laser sampling, thereduced sample size requirements, and the ability tosample the solid phase directly without needing todigest the material. This technology was employed tostudy the trace element content of mussel shellscollected from unimpacted and polluted streams onthe SRS. Various standardization protocols are beingdeveloped to relate the trace element geochemistry ofshell carbonate to laboratory standards of similarmineralogy. In addition, a micro-digestion techniquewas developed to ground truth laser sampling resultswith ultra small volumes of carbonate that weremechanically removed and dissolved for conventionalICP-MS analysis. Trace element analysis of shellmaterial from the umbo to the outer shell margin willprovide a near continuous record of exposure overthe time period of shell growth. Stable carbon andoxygen isotope analyses of material collected over anadjacent area provide an independent measure of shellchronology that will be tied to historical records ofstream chemistry that reside in various SRS databases.This work will provide information on the factors thatinfluence contaminant exposure and transport influvial systems of the SRS.

Laser ablation ICP-MS was also employed to study soilparticle resuspension to understory foliage at TimsBranch. High concentrations of U were found in theleaf tissue of understory plants that were not observedin other plant samples collected from the site. We usedLA-ICP-MS to directly analyze leaf samples taken fromthe site. By optimizing the laser power used for theablation we were able to adjust the time for ablationthrough the leaf to 1 minute. During this ablationprocess elemental data were continuously collected


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by ICP-MS to create an element profile with depth.Uranium was found to be associated with the leafsurface, consistent with U being bound to sedimentparticles adhering to the leaf surface. Nickel was moreevenly distributed throughout the leaf, consistent withuptake of Ni by the plant. Washing removed 80% of Uand 32% of Ni, further substantiating the differentialpartitioning of these two elements in the understoryleaves. The results of this study were published in theJournal of Environmental Monitoring.

A number of studies using laser ablation ICP-MS havebeen on-going during the last year. Work on usingmussel shells as an indicator of contaminationexposure is continuing. Other studies have used LA-ICP-MS to map the 2-D distribution of Fe, Cu, and Znin sections of cryofrozen biological soft tissue; thepreliminary results on brain thin sections arepromising and an analytical methods paper is inpreparation. It is anticipated that these methods canbe applied to examine contaminant distribution andlocalization in exposed organisms as a complementarytechnique to PIXE, SEM, and SXRF.

Field flow Fractionation

Field flow fractionation (FFF) has similarities tochromatography in that it is an analytical separationtechnique that separates the constituents of a samplebased on their hydrodynamic radius. Flow-FFF is avery versatile technique and, depending on the carrierand cross flow rates, it can be used to separatebiomolecules or colloidal particles. By coupling flow-FFF to ICP-MS it is possible to determine the elementalconstituents of eluting particles in addition to the sizeinformation that can be gained from UV detection aftercalibration of the Fl-FFF technique. We have employedsize exclusion chromatography and Fl-FFF coupled toICP-MS to study the speciation of U in sedimentporewaters taken from Steeds Pond. Size exclusionchromatography-ICP-MS revealed that a large fractionof ‘soluble’ U was excluded from the column, whichindicates that U is associated with constituents of thesolution phase >30,000 Da. The distribution wasbimodal and in addition to the excluded fraction, U

was also bound by dissolved organic matter (DOM)of approximately 1000 Da; lower concentrations ofNi were also associated with DOM. Over 80% ofsoluble U eluted from the SEC column either in theexcluded volume or bound by DOM, hence the >0.22µm fraction that is ostensibly the dissolved fractionactually has very little ‘free’ uranyl cation in solution.The high Al signal in the excluded peak concurrentwith the U peak suggested that this phase may be aninorganic colloid. To investigate this observationfurther, Fl-FFF was used. This technique was calibratedusing sized polystyrene beads to allow accurate sizingof the colloidal particle. Fl-FFF-ICP-MS showed thatthe colloidal particle was approximately 0.1 µm andcontained Al, U, Mn, and Fe.

Flow field flow fractionation (Fl FFF) coupled to ICP-MS was used to study the binding of Ni and U to humicacid and colloidal phases in contaminated sedimentporewaters. Results of this study were presented atthe 8th International Conference on the Biogeo-chemistry of Trace Elements, Adelaide, Australia,March 2005. A manuscript of this work was prepared,submitted and published during 2005. (Jackson, B.P.et al. 2005. Characterization of colloidal andhumic-bound Ni and U in the ‘dissolved’ fractionof contaminated sediment extracts. EnvironmentalScience and Technology. 39, 2478-2485; SRELreprint 2827).

Flow FFF-ICP-MS was used as an on-line technique tostudy sorption and pH isotherms of U binding tobacteria. A manuscript of this work was prepared,submitted and published during 2005. (Jackson,B.P.,J.F. Ranville, and A.L. Neal. 2005. Application of flowfield flow fractionation-ICP-MS for the study ofuranium binding in bacterial cell suspensions.Analytical Chemistry. 77:1393-1397; SREL reprint2812).

(F) Use of Monitored Natural Attenuation for chlorinatedsolvent remediationInvestigators: L.A. Newman and faculty atUniversity of South Carolina


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Study of this system is ongoing. We have had someunexpected metabolites show up in the trunks of treesthat are being examined on the site, and have startedmore frequent monitoring of the system to determinewhat is happening. We have started sampling thedeeper aquifer to have more frequent samples to checkto chlorinated solvent metabolites. We are also doingDNA extractions, which will be analyzed by microarrayto test for the presence of solvent-degrading organismsin the soil and groundwater. This work has beenpresented by the student at the Association forEnvironmental Health Sciences annual conference(2004), and the EPA International Phytotechnologiesmeeting (2005).

Question 4:How are bioavailability, bioconcentration, and bio-magnification related, and what extrinsic and intrinsicproperties of systems control these processes?

(A) Modeling cesium dynamics in an SRS cooling pondInvestigators: B.E. Taylor and T.G. Hinton

Radioactive cesium has been an important constituentof the contaminants released accidentally from nuclearreactors. Twenty years after the releases to Pond B onthe SRS were stopped, 99% of the radioactivity wasdue to 137Cs, and 99% of the cesium was in thesediments, where it is adsorbed onto clays. However,this adsorption is reversible, particularly under anoxicconditions (ammonia competes with cesium forbinding sites on clays), and about 1% of the cesiumwas distributed among macrophytes, water, seston,and animals. A group headed by Tom Hinton and JohnPinder designed a tracer experiment to elucidatedynamics of cesium in Pond 4, another impoundmenton the SRS. Intensive sampling included sediments,water, and biota. This part of the project focused oninvertebrates. Our objectives were to measure ratesof accumulation by invertebrates and to infer pathwaysof uptake. In the tracer experiment, we monitoredlarvae of the phantom midge Chaoboruspunctipennis, a planktonic predator, and the snailHelisoma trivolvis, a littoral consumer. Before the

experiment, we sampled the plankton and benthoson a quarterly basis for a year and a half to characterizecomposition and abundances of the invertebrates. Inshallow water, molluscs, including Helisoma, werethe most abundant macroinvertebrates (median 90animals·m-2). In deep water, macroinvertebrates weresparse, and Chaoborus was the most abundantdipteran (median 4200 animals·m-2). We alsocollected common taxa of invertebrates and vegetationfor analysis of stable isotopes of carbon and nitrogento provide further information about trophic pathways.Further analysis of these data was completed in FY05.

The experiment revealed rapid uptake andaccumulation of cesium (due to regulatory issues,133Cs, which is stable, was used an analog for 137Cs).Ten days after tracer was added to the epilimnion ofthe pond in August 1999, cesium concentrations weretwo orders of magnitude higher in the snail than inwater and an order of magnitude higher in Chaoborusthan in water. Stable isotope data indicated that algaeand submerged macrophytes were likely trophicsources for Helisoma, even though emergent andfloating-leaved macrophytes constitute the bulk of thebiomass in the pond. Chaoborus were likely feedingmainly on planktonic cladocerans, which feed in turnon planktonic algae and other small particles. Throughlate fall, concentrations in both taxa changed inparallel with concentrations in the water. However, inspring and summer of the following year,concentrations in both invertebrates doubled, whilewater concentrations continued generally to decline.Clearly, the concentration of the contaminant in waterdoes not fully govern its availability to the biota.Submissions of two manuscripts from this project arescheduled for FY06.

(B) The potential of red-eared sliders to serve aschronicled indicators of contaminant exposureInvestigators: P.M. Bertsch, B.P. Jackson, T. Hinton,W. Hopkins, and collaborators from the Universityof South Dakota

With increased interest in the use of indigenousorganisms as indicators of environmental


21

contamination, it has been suggested that turtles wouldbe ideal indicators of metal contamination. They areextremely long-lived, have a wide geographicdistribution, occupy a variety of habitats, appear to berelatively tolerant to a range of pollutants, and haveshells comprised of bone (apatite), which is a well-known target organ for a number of transition andheavier elements. Furthermore, turtle shells exhibitgrowth annuli for both the bone and protein (keratin)sheath coating, within which periodic growthdeposition bands are discernible. This provides thepotential to examine historical information related tometal, metalloid, and radionuclide exposure, akin todendroanalysis. Our initial studies using spatiallyresolved synchrotron-based X-ray microanalysis havedemonstrated that turtles collected from contaminatedenvironments associated with a former nuclearmaterials processing facility and a coal combustionwaste repository have spatial heterogeneity of Ni, Se,As, and other metals and metalloids, which have beenhypothesized to be related to specific exposure events.

Controlled laboratory experiments, using a knowncontaminant dose over a standard time frame, wouldoffer valuable information supporting the use of turtlesas bioindicators. To further explore this idea, 500hatchling red-eared sliders (Trachemys scriptaelegans) were obtained in June 2003. The hatchlingswere divided evenly into twelve tanks and each tankwas assigned to one of four treatment groups. Withthe exception of the winter months (exotherm inactiveperiods), turtles have been dosed with nickel citrate(Ni

3(C

6H

5O

7)

2·XH

2O) on a bi-monthly gavaging regime

since July 2003. The nickel is administered orally usinga transfer pipette, and regurgitation events arerecorded. Measurements of individuals (carapacelength and width, plastron width and length, and mass)were taken upon initial receipt of turtles and prior toall gavaging events.

All measurements indicate notable growth of survivingturtles regardless of treatment group. Marginal bonefragments, plastron segments, and keratin scutesheaths have been sectioned from individuals in thetwo highest dosing groups, and control tanks, and

analyzed using synchrotron-based X-ray microanalysisat beamline X26-A at the National Synchrotron LightSource (Brookhaven National Laboratory, Upton, NY).Results indicate spatially variable higher nickelconcentrations in higher dosing group turtles incomparison to lower and control group individuals,and a gradient of nickel levels is apparent inaccordance with dosing. In addition, preliminary ICP-MS and HRTGA analyses have been conducted usingmarginal scute sections. Additional trials of allanalytical techniques, including synchrotron-basedmicroanalysis, ICP-MS (including laser ablation), andHRTGA, are necessary for more conclusive results.The bi-monthly gavaging regime is planned for anothergrowth season (through October 2005), at which timeindividuals will be sacrificed and additional analysesconducted. After the first round of gavaging, photoswere taken of each individual for use in future analysesof fluctuating asymmetry. In addition, DNA strandbreakage analyses will be conducted during the winterof 2005-2006. With the combined results of the abovetechniques, it will be possible to further support theuse of turtles as bioindicators through developmentof a spatial-time distribution of nickel dosing events.

Preliminary results were presented at the Joint Meetingof Ichthyologists and Herpetologists (May 2004). Theconcept of the experiment was presented, as well asevidence detailing the utility of Synchrotron-basedtechnology in this project and results indicatingdeposition patterns of contaminants in bone sections.An update will be presented at the Joint Meeting thisyear (July 2005) which will go into further detail onquantities and patterns of nickel present in accordancewith dosing levels, as well as results from HRTGA andICP-MS analyses.

(C) Processes controlling the distribution of metals in treesinhabiting contaminated environmentsInvestigators: T. Punshon (CRESP), P.M. Bertsch, B.P.Jackson, S. Harper, and collaborators from theUniversity of Chicago

This project was completed in FY04.


22

(D) Using gray foxes as indicators of contaminantdistributions and trophic level relationshipsInvestigators: I.L. Brisbin, Jr., C.S. Romanek, B.P.Jackson, and collaborators at the University of SouthDakota

During the past year a spatially explicit model wascompleted for the gray fox (Urocyoncinereoargenteus) at the SRS. This model is one ofseveral that have now been developed for variouswildlife species at the site for use in ecological riskassessment. This now allows this widely distributedand omnivorous canid to serve as a “receptor species”for studies and evaluations of the fate and effects ofsite contaminants in the higher trophic levels of naturalterrestrial food webs on the site.

Previously collected data describing the relationshipsbetween contaminant levels and stable isotope profilesfor carbon and nitrogen in gray foxes collected on vs.off of the SRS have now been analyzed. A draftmanuscript has been written on these relationshipsand is now being reviewed prior to being submittedfor publication. In addition, using historical andcurrent museum specimens, over 150 hair sampleshave been collected from gray foxes from throughouttheir range, along with comparable hair samples fromred foxes (Vulpes fulva) and coyotes (Canis latrans).These hair samples are being analyzed for stableisotopes of carbon and nitrogen to determine whetherthe trophic relationships of the two fox species havebeen altered since the time of the unprecedented rangeexpansion of the coyote into the Southeast during thepast 20 years.

Widely held opinions have suggested that the coyotemay now have become an effective trophic competitorfor these other two canid species and that the trophicrole of the gray fox at the SRS (and hence its role as areceptor species in ecological risk assessment) maybe in the process of being altered as a result of thearrival and establishment of the coyote in the naturalterrestrial ecosystems of the SRS.

(E) Patterns and extent of contamination in Americanalligators from the southeastern U.S.Investigators: I.L. Brisbin, Jr., C.H. Jagoe, T.C. Glenn,and C.S. Romanek

This work had not been projected to continue throughFY05. However an opportunity was provided to submitan invited paper for publication summarizing our 35+years of study of alligator populations on the SRS. Thispaper emphasized studies evaluating the effects ofreservoir impoundment and management upon thealligator population inhabiting the Par Pond reactorcooling reservoir. A partial reservoir drawdown in1991-1994 proved to be the major source of negativeimpacts upon the population while radionuclidecontamination of alligator meat was not of concern.

As is the case elsewhere throughout the alligator’srange in the Southeast, however, levels of mercurycontamination of alligator meat could, under somecircumstances, exceed those generally considered safefor human consumption. Two papers describing thisproblem have now been prepared and submitted forpresentation at a national environmental toxicologymeeting, and both continue to be prepared forpublication. In one of these papers, mercury levels inmeat from SRS alligators were lower than thosecollected from an area near a polluted Superfund sitein south Georgia, while lowest mercury levels werefound in meat of alligators from Louisiana and alligatorfarms.

Question 5:What types of mathematical or statistical models bestdescribe contaminant distributions and the process ofcontaminant uptake and accumulation in biota and theirhabitats? Does the selection of the best model make adifference in terms of decisions about how (or if) toremediate contaminated areas?

(A) Modeling spatial distributions of cesium in reservoirsedimentsInvestigators: S.J. Harper, T.G. Hinton, and M.D.Wilson


23

Relatively little is known about the spatial distributionof radionuclides within cooling reservoirs. Tocharacterize and predict the distribution and dynamicsof such contaminants, a spatially-explicit modelingapproach is needed. Surveys on the SRS indicate thatthe vast majority of 137Cs contamination is confined tothe sediment of former cooling reservoirs, thoughtremendous variability in concentrations is observedamong sample locations. Much of this variation islikely attributable to underlying spatial heterogeneityin sediment characteristics, as strong gradients indepth, slope, and soil texture can exist in these man-made systems. Given this variability, statistical poweranalyses indicate that it could take thousands ofsediment samples to detect significant changes inradionuclide inventories over time due to remediationefforts or natural attenuation. Thus, while the vastmajority of radionuclide contamination is containedwithin sediment, it is very difficult to quantify spatialdistributions and document subsequent declines incontamination. Advances in the field of hydroacousticsafford a technological solution to this problem.Hydroacoustic sampling provides enhanced statisticalpower to determine concentration distributions byaccounting for the spatial variability in factors knownto influence radionuclide dynamics, includingbathymetric features (depth, slope) and sedimentcharacteristics (soil texture, water content, organicmatter).

The merit of this spatial modeling approach wasdemonstrated for 137Cs in Pond B at the SRS. We usedmultiple transducer frequencies during extensivehydroacoustic surveys to provide detailed spatialinformation for bathymetric features (e.g., depth,distance from bank, and slope), lake-bottom sedimentclass types, and submerged aquatic vegetation. Basedupon these maps, point-based sediment samples werecollected across observed ranges in depth, sedimenttype, and slope. Laboratory analyses of these sampleswere conducted to quantify 137Cs concentrations,moisture content, organic content, and texture(percent sand, silt, and clay) of sediments. Significantlinear and nonlinear relationships were found, andwere especially strong among soil moisture, organic

content, and 137Cs. Extensive sampling of thesecorrelative factors via hydroacoustics allowed a full-coverage map of 137Cs to be generated. In FY05 westrengthened and validated the model developed forPond B by repeating our detailed hydroacousticsurveys and laboratory analyses. Results indicated thathydroacoustic-derived predictors of bathymetric andsediment characteristics were very reliable andconsistent among sample dates. In addition, resultsindicated significant redistribution of 137Cs in surficialsediments occurred due to focusing near the bottomof slopes. Resuspension and redistribution due toeffects of wind or other extrinsic factors was notdetected over this relatively short time period.Continuation of this research could provide greaterunderstanding of the distributions of radionuclideswithin reservoirs, and thus contribute towards betterresource management. Such models should proveuseful for documenting and predicting contaminationdeclines, predicting responses of contaminants toreservoir draw down or environmental change, andenhancing estimates of exposure risks for target fishesand wildlife species that utilize these systems.

(B) Use of non-normal statistical models to analyzeenvironmental contaminant dataInvestigators: C.H. Jagoe, M.D. Wilson, I.L. Brisbin,Jr., T.C. Glenn, and colleagues from The Universityof Georgia and the National Institutes of Health

Many statistical models assume that variables arenormally distributed. These models are powerful andwell developed, but assumptions of normality arefrequently not met in environmental sampling. In fact,frequency distributions of contaminants in organismsoften appear non-normal, and may be highly skewed.Selection of appropriate data analysis techniquesrequires knowledge of underlying distributions andtheir properties. Probability-based risk assessmentmodels are also affected by the underlying frequencydistributions of the input data. For example, widelyused modeling tools such as Crystal Ball(Decisioneering, Inc.) typically require specificationof the frequency distribution of variables forprobabilistic simulations. We have published several


24

papers describing radiocesium distributions in biota,and demonstrated that these are best fitted using non-normal models (lognormal or Weibull). This workextends this analysis to other contaminants,particularly heavy metals such as mercury. Weidentified representative data sets that included tissuecontaminant concentrations from both vertebrates andinvertebrates, from on the SRS and from other areas.Data were tested for normality and lognormality usingLillefors tests. Contaminant distributions were non-normal in all the data sets tested to date. In some cases,the data were lognormally distributed. This projectinterfaces with work on bootstrap goodness of fit testreported in the effects and risk assessment undersection 9. A publication related to this work,demonstrating a preferred method for calculatingaverage radiation dose to groups of individuals incurrently in press in the Journal of RadiologicalProtection. We anticipate that these studies will yieldadditional manuscripts for submission later this year.We will continue such goodness of fit analyses in FY06,and incorporate additional data from existing data sets.

Question 6:Can sentinel species be used as surrogates to determineenvironmental health, and can this information be used todetermine whether remediation is needed in a given area?

(A) Using sentinel species to monitor the effects ofenvironmental contaminants on biotaInvestigators: I.L. Brisbin, Jr., C.H. Jagoe, and C.S.Romanek

With the refocusing of the SREL mission to confinefield work on contaminants to several specific siteson the SRS, it has been determined that none of thepotential sentinel species that were being consideredduring the previous year of this work would beappropriate for use in such areas. Accordingly, plansto release and study sentinel populations incontaminated habitats on the SRS have now beenplaced in abeyance. However, an opportunity wasprovided to prepare an invited paper for publicationdescribing long-term studies designed to evaluate the

potential for radio-telemetered eastern box turtles(Terrapene carolina) to serve as a multiplyrecapturable free-ranging surrogate to evaluatecontaminant bioavailability in contaminated SRShabitats. Because of the large size and long time frameof the data base involved, this paper was able, webelieve for the first time ever, to estimate cohortsurvival simultaneously by two independent methods:standard mark-and-recapture analysis and a modifiedmark-and-recapture method based on long-termdocumentation of the survival and fates of turtlescarrying radio transmitters. This paper has now beencompleted and submitted to the editors of the invitedpublication.

(B) Examination of a small mammal community in thecontaminated Tims Branch watershedInvestigators: I.L. Brisbin, Jr., C.S. Romanek, C.H.Jagoe, B.P. Jackson, and collaborators at theUniversity of South Dakota

A Master’s thesis has now been completed andsuccessfully defended dealing with the relationship ofheavy metal contamination to trophic position (asrevealed by stable isotope analyses) within smallmammal communities inhabiting contaminatedhabitats on the SRS and off-site control areas. Thisthesis, The Use of Small Mammals as Indicators ofHeavy Metal Bioavailability in a ContaminatedRiparian Zone (University of Georgia, 2003), is nowin the process of being edited into a format forsubmission for publication in a peer-reviewedtoxicology journal. This process should be completeand the paper submitted by the end of summer, 2005,since an off-site collaborator from the University ofSouth Dakota is spending part of the summer at theSRS and will be working with the other authors onthis paper. This thesis research found no statisticallysignificant relationship between trophic position andlevels of metal body burdens in the small mammalssampled. This suggests that some environmental,demographic, or perhaps seasonal factor is workingto confound what would normally be expected to be apattern of increasing levels of metals contaminationat higher trophic levels.


25

(C) Analyses of contaminant levels in feral swine from theSRSInvestigators: I.L. Brisbin, Jr., C.S. Romanek, B.P.Jackson, and collaborators at the University of SouthDakota.

During the past year heavy metal analyses werecompleted on feral swine muscle samples for whichradiocesium levels had already been determined. Themajority of stable isotope analyses have now also beencompleted and the remainder should be completedwithin the next few months. At that time final dataanalyses will be undertaken and a manuscript will beprepared for publication. In the meantime a paperdescribing a spatially explicit model for the wild hogas a receptor species for ecological risk assessmentactivities at the SRS has been revised with regard toreviewers’ comments and resubmitted forconsideration for publication. In addition, an invitedpaper has been completed and submitted forpublication describing the importance of long-termdata bases in the development of management plansfor free-ranging wild hogs. This paper was presentedduring the previous year and will now appear as partof the published proceedings of an SRS-sponsoredsymposium on the ecology and management of wildhogs.

(D) Herpetofaunal diversity on the SRSInvestigators: J.W. Gibbons and collaborators

Long-term data on the herpetofaunal distribution andabundance patterns on the SRS will prove to be helpfulin addressing remediation and restoration goals. Bycontinuing to collect long-term data and then usingthose data sets in conjunction with GIS coverage ofSRS waste sites, we can develop simple models toestimate how many species occur in waste sites ofparticular size and habitat characteristics.

Long-term data have been collected at many sites onthe SRS—most noteworthy are Rainbow Bay (RB;continuous data since 1978), Ellenton Bay (EB; 20yrs of data since 1975), Ginger’s Bay (GB; yearly datasince 1986). These continuous or nearly continuous

records are supplemented by shorter duration studiesthat encompass a broader range of habitats, such assnake sightings on SRS roads. In recent years we havealso used outside funding from the National ScienceFoundation (NSF) and other agencies and foundationsto sample additional SRS locales (e.g., four Carolinabay wetlands that are part of the NSF “Land Use Effectson Amphibian Populations” study) and additionalspecies (e.g., studies of gopher tortoise relocationfunded by the Department of Defense and the U.S.Forest Service).

In FY 2005 we continued sampling efforts at RB (whichis the longest running amphibian study in the world)for all herp species and at GB and EB for select species.At RB to date more than 600,000 captures of 75 reptileand amphibian species have been recorded. Thesedata were integral to two manuscripts currently inpress (Taylor, B.E., D.E. Scott, and J.W. Gibbons.Coupling catastrophic reproductive failure andterrestrial survival: Implications for managementof pond-breeding salamanders. ConservationBiology; and Dixon, P. and J.J.K. Pechmann. AStatistical Test to Show Negligible Trend. Ecology)and one submitted to Ecology (Daszak, P., et al.Chytridiomycosis does not cause populationdeclines in long-term monitored amphibianpopulations at the Savannah River Site). Recentcapture data from EB during FY 2004 were used toprepare and submit the manuscript How ProductiveCan an Isolated Wetland Be? RemarkableAmphibian Biomass and Abundance by J. W.Gibbons et al.

Accurate assessment of distribution and abundancedata for all species of herpetofauna requires extensivecompilation and consolidation of all data sets collectedsince 1951. In FY05 we continued gathering historicaldata from SRS inventory and research projects,verifying data set records, and reformatting the dataas necessary so that individual files are compatible.We currently have completed the SRS turtle datasetthrough FY05 (beginning with captures in 1967; 12species, 35,084 captures and recaptures). Severallarge snake data sets have been reformatted and


26

merged that include 7,419 captures of 34 species from1967 through 1988. We have also added severalsmaller data sets, including ones from 1951-1955,and from 1989-2004, to bring the snake records above16,000 captures. More than one million amphibiancaptures from RB, EB, and GB are currently beingcompiled in addition to other amphibian projectsbeing obtained through direct contact with formerSREL faculty and students. The consolidated databasewill include captures of all representative SRSherpetofauna, but several rare, unusual, and potentialreceptor species have been selected for in-depth studyof demographics, habitat preferences, and distributionpatterns. These include the spotted turtle, gopher frog,tiger salamander, pine snake, and southern hognosesnake, all of which have been recognized as sensitivespecies in need of protection at the federal level. Otherspecies will be added to this list as finalized datasetsare analyzed.

Question 7:What effective techniques permit studies of bacterialcommunity structure and function without isolation? Howcan these techniques be applied to studies of contaminantdynamics or other environmental problems?

(A) Fatty acids as an indicator of microbial diversity inenvironmental samplesInvestigators: C.L. Zhang, G.L. Mills, and C.S.Romanek

Membrane phospholipids are one of the essentialcomponents of microbes that function at the interfacebetween the intracellular biochemical cycling and theexternal environments. Phospholipids are sensitive toenvironmental changes and may vary in relativeabundance and structures. These features can berecognized as biomarkers, which, along with theirisotopic composition, can provide information abouttheir chemical taxonomy, type of carbon cycling andenvironmental nutrition. The SRS has been animportant place to observe the impacts of heavy metalsand radionuclides on local ecology. This study aimedto determine environmental factors that may have

impacts on the microbial cell membrane.

Surface soil, pond sediment, stream mud, and sandsamples were collected from Beaver Pond, BeaverDam, and soil at the Lower Tims Branch on the SRS.Samples were freeze-dried before lipid extraction. Afraction of the sample was extracted by a mixture ofchloroform-methanol-H

2O for total lipids. The total

lipids were fractioned into polar, neutral, and glycollipids by organic solvents with different polarities ona silicic column. The polar fraction of lipids wasfurther methylated for gas chromatographic or gaschromatographic-mass spectroscopy analyses.Information about lipid profiles and single lipidmolecular structures was analyzed. The carbonisotopic compositions of single molecular fatty acidswere also measured.

Generally, the SRS samples have unusually highnumbers of phospholipid fatty acids (PLFA). More than50 fatty acids with different structures and carbonnumbers could be discriminated. Among them, thestrain chains C

16, C

18 are most common. Terminal

branched and unsaturated lipids were also significant.Monounsaturated C

16 and C

18 were common in all

samples, with some samples containing a smallfraction of monounsaturated C

17 chains. Iron-

reducing, sulfate-reducing, and photoautotrophicmicroorganisms were expected to contribute to thelipid pool in these samples. The carbon-isotopeprofiles indicated considerable variation between fattyacids within one sample as well as between samples.The Beaver Pond and Creek mud have lower 13C thanthe soil samples, which may indicate more extensivein situ carbon cycling in the aquatic environment thanin the soil environment.

This is an ongoing project. The phospholipid data willfinally be combined to the molecular data to allow usto have a better understanding of carbon flow in thisenvironment. Consequently the microbial metabolismof Fe and S will also be elucidated. These links areimportant in microbial remediation because the iron(oxyhydr) oxides and iron sulfur compounds areimportant carriers of radionuclides, whose trans-


27

formation requires coupling between carbon, sulfur,and iron cycling.

(B) Screening microbial communities to determine theeffects of contaminant exposure on antibioticresistanceInvestigators: JV. McArthur, T.C. Glenn, and C.H.Jagoe

Industrial contamination of aquatic systems is a majorproblem on DOE properties. Included in thiscontamination are significant amounts of heavy metalsand organic pollutants. From past research we haveshown that bacterial exposure to heavy metalcontamination at field concentrations appears to selectfor increased incidences of antibiotic resistance. Wehave sought to determine whether indirect selectioncan result in increased levels of antibiotic resistanceand to elucidate the mechanisms involved. Consideringthat <1-10% of bacteria in nature can be cultured,we have developed culture-independent methodsbased on flow cytometry that allow the screening ofcomplete microbial assemblages. Existing cultivationtechniques target only a minor fraction of naturalbacteria. Therefore it is most likely that platingexperiments fail to account for and to identify most ofbacteria resistant to antibiotics and heavy metals innatural samples. To overcome this methodologicallimitation, we have developed a combination ofcutting-edge culture-independent techniques toseparate and identify resistant and sensitive bacterialcells. We have begun a series of experimentsinvestigating the role of macroinvertebrates as “hotspots” of microbial activity to harbor and promoteantibiotic resistance. Meredith Wright, a Ph.D. studentworking on this project, has received an EPA StarFellowship to study the role of macroinvertebrates inthe spread of antibiotic resistance in bacteria. She hasfound that bacteria associated with Corbicula, anintroduced bivalve, in contaminated streams on theSRS have higher proportions of antibiotic resistancethan do Corbicula in uncontaminated streams.

Another graduate student, Ginger Humphries, is usingculture-independent techniques based on flow

cytometry to study interactions of mercury andselenium exposure in bacteria from Savannah Riverwater. In eukaryotes, small amounts of selenium canreduce or ameliorate mercury toxicity. This study willtest whether selenium has similar effects on mercurytoxicity in prokaryote communities, and whetherselenium reduces the development of antibioticresistance associated with mercury exposure. Thisproject thus links both characterization of thedevelopment of antibiotic resistance with a basictoxicological study.

(C) Evaluating the antibiotic resistance of knownwaterborne pathogens E. coli and Vibrio sp. in theenvironmentInvestigators: JV. McArthur, T.C. Glenn, and C.H.Jagoe

We sampled sediment and water from Shipyard Creek,Charleston, SC in March and June and the ACE Basinin June and isolated E. coli and vibrios using standardmethods. These cultures will be screened for antibioticresistant by the NOAA Charleston lab. DNA extractedfrom all of the isolates and from bacteria in thesediments has been amplified by PCR. Integraseprimers have been used to detect occurrence of theintegrase gene. In addition to the microbiologicalsampling we have collected samples from the sedimentand water at each site for analysis of metal content,dissolved organic carbon, antibiotic content, andnutrient analysis.

Question 8:How do seasonal variations or episodic perturbationsinfluence contaminant speciation, bioavailability andaccumulation?

(A) Bioavailability of metals in relation to wetlandhydroperiodInvestigators: C.H. Jagoe, A.L. Bryan Jr., and I.L.Brisbin Jr.

Both natural and man-made wetland systems aresubject to periodic flooding and drying. Such flooding


28

and drying can be due to seasonal variations,environmental factors such as beaver dams or erosion,or human activities such as wetland restoration or damconstruction. Intermittent flooding, whether frequentof infrequent, can influence metal speciation. Previouswork, including studies at SRS locations, suggestedthat mercury bioavailability and accumulation isrelated to hydroperiod (relative amount of time that awetland contains water) and intermittent flooding ofsoils and sediments. In preliminary laboratoryexperiments with crayfish (Procambarus clarkii),repeated flooding and drying of mesocosms increasedbioavailability of some metals. In FY05 we continuedcollection of samples of biota from Carolina bays(intermittently flooded wetlands on the coastal plain),where previous studies had demonstrated arelationship between bay hydroperiod and fishmercury concentrations. Surveys of amphibian larvaedid not indicate similar relationships. Differences inaccumulation patterns among fish and amphibiansmay reflect differences in the form of mercuryaccumulated by these organisms. This reflects theimportance of metal speciation to bioavailability. Plansto place crayfish in bays with different hydroperiodsto compare measure metal uptake over time werepostponed indefinitely in FY05 because of therealignment of SREL research activities. We developeda proposal to look at effects of temporaryimpoundments on the dynamics of contaminants in acontaminated stream. This proposal seeks to expandstudies on hydroperiod to examine the role ofimpoundments in contaminant sequestration,speciation, and mobilization. We began samplingnative biota from free-flowing and impounded sectionsof streams on SRS with historic inputs of contaminantsto gather preliminary data for the proposed activities.Results of these studies will allow evaluation ofpotential impacts of wetland remediation projects,particularly those where water levels vary annually orseasonally.

Question 9:How can conservation plans for amphibian populationsprovide insurance against rare but catastrophic events?

(A) Modeling population dynamics of an amphibianspecies to encourage sound natural resourcemanagementInvestigators: B. Taylor and J.W. Gibbons

The fundamental challenge of population ecology isto identify and quantify the factors needed for reliableunderstanding and sound management. Processescritical to the success of a population may operate atspatial and temporal scales outside the scope ofordinary field studies. The long history of ecologicalstudies at the SRS provides data that can be used toaddress such issues. Wide variation in reproductivesuccess is common among amphibians that breed inseasonal ponds, but persistence of adults can bufferagainst these fluctuations, particularly for long-livedspecies such as the marbled salamander (Ambystomaopacum). We hypothesized that the frequent episodesof catastrophic failure observed in a 24-year field studyat Rainbow Bay in South Carolina would enhance theimportance of high terrestrial survival. To assess themagnitude of these responses, we adapted an age-structured, individual-based model with density-dependent growth and survival of larvae. The modelwas based on extensive data from local field studiesand experiments. At Rainbow Bay, reproductivesuccess was poor (<1 metamorph per breedingfemale) in nearly half of the 22 years that the speciesbred; complete failure occurred in 6 of 22 years, whenpond hydroperiod was <2 mo. To estimate the density-independent component of survival in the pond stages(eggs and larvae), we fitted stock-recruitment curvesfrom the model to field data for years of goodreproduction. Initial conditions in the simulationsdescribed the colonization observed at Rainbow Bay.We measured effects of catastrophic reproductivefailure, occurring randomly with probability 0.5 peryear, by comparing simulations with catastrophicfailure and good survival in the pond stage tosimulations with equivalent average survival in thepond stage. With consistently good survival in the pond


29

stages, the simulated population required survivalprobabilities in the upland stages (juveniles andadults) near 0.5 per year to persist and near 0.8 peryear to achieve the increases observed at Rainbow Bay.Catastrophic failure created additional fluctuations inthe population, raised the thresholds of survivalrequired for persistence, and caused extinction underconditions that were otherwise favorable. The marbledsalamander at Rainbow Bay is not at great risk ofextinction due to catastrophic failure, but the riskincreases dramatically if life span is decreased orfrequency of failure is increased. Any reduction interrestrial survival will have deleterious consequencesby reducing the breeding populations at equilibrium,even if it does not jeopardize persistence. The modelfor the marbled salamander provides assessments ofrisk that can be applied to poorly-studied species withsimilar life histories, such as the endangered flatwoodssalamander (A. cingulatum).

A manuscript by Taylor, Scott, and Gibbons describingthis work is in press in Conservation Biology.


Ecological Risks and Effects

30

Report on FY05 Risks and Effects Milestones

Investigators in the Ecological Risks and Effects Research Theme at SREL conducted a wide variety of research duringFY05 that will assist DOE in making better-informed decisions about remediation and land management. This researchattempts to reduce many of the knowledge gaps currently associated with ecological risk analyses and the effects thatcontaminants have on biota. In FY05 researchers associated with this theme conducted research on:

� measuring the impact of mercury speciation on amphibians� understanding trophic transfers of contaminants in terrestrial ecosystems� integrating contaminant effects among different levels of biological organization� developing biomarkers to measure transgenerational risks from contaminant exposures� developing fish models for understanding key issues in ecological risk analyses� understanding how the spatial and temporal variation of contamination alters risks� examining potential negative impacts to biota due to indirect effects of remediation� determining levels of sensitivity among biota exposed to organic contaminants� determining the importance of fish and waterfowl as vectors for contaminant dispersal� quantifying contaminant exposures to endangered species on the SRS� determining interactive effects of mixed contaminants� examining risks from chronic low-dose rate irradiation� using stable isotopes as biomarkers of contaminant effects� measuring maternal transfers of contaminants� determining if contaminant exposure promotes antibiotic resistance in bacteria� improving the statistical analyses of ecological risk data sets

Question 1:How does contaminant speciation influence bioavailability,and how do changes in bioavailability alter contaminantdose-response and toxicity relationships?

A. Impacts of mercury speciation on amphibiansInvestigators: C.H. Jagoe and W.A. Hopkins

The possible effects of dietary mercury exposure atenvironmentally realistic concentrations on developingamphibians are poorly understood. Existing datasuggest that amphibians can accumulate mercury fromtheir diet while feeding in wetlands, but it is unclear

whether this exposure negatively impacts amphibianpopulations. We published a paper in 2005 quantifyingthe trophic transfer of inorganic and organic mercuryspecies from aufwuchs (periphyton) to amphibians.In two other papers published in FY05, we reporteddietary mercury accumulation in laboratory feedingstudies and showed that this exposure resulted ineffects on larval development and life history traitsimportant to amphibian population dynamics.Southern leopard frog (Rana sphenocephala) larvaewere exposed to experimental diets formulated tocontain mercury concentrations and species similarto those in aufwuchs from aquatic habitats


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contaminated by atmospheric deposition.Observations on mortality, incidence of malformation,and larval growth and development were made for254 days. Increased mortality, malformations, andchanges in growth and development were observedat mercury concentrations (1,500-3,300 ng Hg/g DW)similar to the highest concentrations in amphibiandiets expected from atmospheric deposition. Theresults of this study are more ecologically realistic thanresults obtained from aqueous mercury exposure inprevious studies. Our results indicate that dietarymercury exposure in habitats primarily contaminatedby atmospheric deposition has the potential to causeadverse effects in amphibian larvae. A new study wasbegun using a similar experimental design (frog larvaewere fed experimental diets with ecologically realisticmercury concentrations), to examine changes inbehavior with Hg accumulation. Mercury isneurotoxic, so changes in nervous functions affectinglocomotion, sensory perception, or memory shouldbe manifested as changes in behavior. We hypothesizethat behavioral changes represent a sensitive indicatorof mercury toxicity, and that behavioral changes mightimpact ecologically relevant activities such as locatingfood, feeding, and predator avoidance. This study willbe completed in early FY06 and results prepared forpublication.

B. Trophic transfers in contaminated terrestrial systemsInvestigators: W.A. Hopkins and B.P. Jackson

Additional research is needed to understand howcontaminant speciation affects bioavailability interrestrial organisms, particularly in relation to trophictransfer (often the most important route of exposurein terrestrial organisms). In FY04 we developedmethods for exposing terrestrial organisms to dietarytrace elements under conditions that allowquantification of trophic transfer, chemical speciation,and biological effects at multiple trophic levels. Thisresearch was completed in FY04 and a manuscriptdetailing our findings was published in FY05(Hopkins, W.A., et al. 2005. Transfer of Se from preyto predators in a simulated terrestrial chain.Environmental Pollution 134:447-456; SREL reprint

2811). Additional manuscripts are in preparation thatdescribes changes in chemical speciation that occurdepending upon the fate of transported selenium.

Question 2:How do contaminant effects integrate over different levelsof biological organization (i.e., how much molecular andcellular damage is required before effects becomesignificant to individuals, populations, and communities)?

(A) Integrating effects over different levels of biologicalorganizationsInvestigators: T.G. Hinton, T.C. Glenn, and W.A.Hopkins, with additional collaborators fromColorado State University and The University ofGeorgia

Two manuscripts were published in FY05 relative tothe linking of molecular level effects from radiologicalexposure to population-level endpoints. The firstmanuscript (Hinton, T.G., et al. 2004. Radiologicaleffects on the environment: A need to question oldparadigms and enhance collaboration amongradiation biologists and radiation ecologists; SRELreprint 2791) provides a historical perspective of thecurrent paradigm that does not explicitly protectnonhuman biota from radiation, but instead relies onthe concept that if dose limits are set to protecthumans, then the environment is automaticallyprotected as well. The paper summarizes recentinternational questioning of this paradigm and pointsout the controversial aspects of three differentframeworks being proposed to protect theenvironment from ionizing radiation, including thatproposed by DOE. We emphasize that to properlyaddress radiation protection of the environment weneed to understand how effects are integrated acrossdifferent levels of biological organization. Toaccomplish this, an enhanced collaboration is neededamong the traditionally separate disciplines ofradiation biology and radiation ecology.

The second manuscript (Hinton, T.G. and F.Bréchignac. In press. A case against biomarkers as


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they are currently used in radioecological riskanalyses: A problem of linkage. IRSN CollectionSeries, Suppl. 1, vol. 39. EDP Sciences, Paris)identifies the problem of biomarkers in ecological riskanalyses generally lacking a linkage betweenmolecular-level effects and quantifiable impactsobserved in individuals and populations. The paperstresses that from an ecological risk analysisperspective, the challenge is to evaluate whetherchanges in contaminant levels constitute a significantrisk to a population. That challenge is even moredaunting when biomarkers of damage are used as asurrogate indicator of a population effect. Althoughthe biomarker may be a very sensitive and earlywarning sign of a possible effect, the applicability ofsuch damage to ecological risk can only be assessedonce the connection has been made to changes inindividual resource allocation-based life histories thatare coupled to population dynamics.

In collaboration with colleagues at the Medical Collegeof Georgia, we conducted preliminary studies ofadaptive response (AR) with ionizing radiation (IR)as the stressor. Adaptive response is a phenomenonin which an organism’s response to a contaminant isreduced if it has been previously exposed to a primingdose. The concept has been demonstrated many timesin vitro, but rarely in vivo, and has implications toradiation workers and the dose response curves uponwhich regulations governing radiological exposuresare based. Medaka fish and SREL’s Low Dose-rateIrradiation Facility are ideally suited to examine thephenomenon.

The first step was to determine the dose response curvefor IR exposure of the medaka embryo. The main, or“challenge” dose of ionizing radiation was deliveredat the pre-early gastrula stage (medaka stage 12,approximately 10-12 hours post fertilization, hpf).Following the challenge dose, embryos are incubatedfor about 40 h to allow onset of apoptosis. At that time(medaka stage 25), organ systems are present,including the brain, heart, circulatory system, digestivetract, and kidney. Considerable morphologicdifferentiation has occurred, and spatial distribution

of apoptotic cells can be assessed relative toanatomical landmarks. Sporadic apoptosis occurs inthe absence of IR exposure as part of normaldevelopment (0 mGy). Little or no change in this lowbackground level of apoptosis was evident at 150 mGy.At the next higher dose of 500 mGy, a monolayer ofapoptotic cells became visible in the region lining theventricle of the brain, particularly toward the rostralsurface. Apoptosis was more pronounced at 1.5 Gy,encompassing the entire surface of the ventricle. Atdoses of 5 Gy and 15 Gy, apoptosis becomes evidentthroughout the head of the embryo. Somemorphologic abnormality (eye reduction) occurs at15 Gy, reflecting extensive cell death. The ventricularsurface is a region of active cell proliferation that atslightly later times in development forms the“ventricular zone”, a region of intense cellproliferation and neuronal birth. The occurrence ofradiation-induced apoptosis in this region is consistentwith the differential sensitivity of rapidly dividing cellsto radiation. The principal target of IR is DNA, andthe dose of IR needed to produce a given effect scaleslinearly with genome size. The medaka genome isabout five times smaller than the human genome andtwo to three times smaller than that of the zebrafish.The 150 mGy to 15 Gy range used here is thusequivalent to about 30 mGy to 3 Gy for humans. Toprovide context, the low end of the range, adjusted tothe equivalent human exposure and for radiationquality, is 10-fold higher than the annual naturalbackground exposure (3 mSv; 1 mGy=1 mSv for137Cs), less than the annual NRC dose limit for radiationworkers (50 mSv), and less than the dose from a singlewhole-body CT scan (50-150 mSv). The results thusillustrate a strength of the medaka embryo system,which is the presence of a measurable response atdoses that approach actual exposure scenarios.

We next examined an adaptive response evoked byexposure to priming doses of radiation, followed bylarger challenge doses. To maximize the chances ofseeing an effect, two priming doses were used, onedelivered to the parents (priming dose 1) and another,much lower dose, delivered directly to the very earlyembryos (priming dose 2). For priming dose 1, a


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breeding population was exposed to 137Cs radiation,for 6 days at 0, 4, or 40 mGy d-1 (0, 24, or 240 mGytotal dose). On day 6, newly deposited embryos werecollected and exposed briefly to priming dose 2 (0,0.15, 0.3, 1.5, or 3.0 mGy as indicated). Embryos weretransported to the Medical College of Georgia andincubated until they reached stage 12. Embryos werethen challenged with an acute 1.5 Gy dose of 137Csirradiation, incubated until stage 25 to allow for onsetof apoptosis and TUNEL-stained to reveal apoptoticcells.

Consistent with previous results, embryos that receivedthe challenge dose in the absence of priming hadapoptotic cells at the boundary of the ventricle andtelencephalon, in the hindbrain, and in the tail. Controlembryos that did not receive the challenge doseshowed only the expected background level ofapoptosis. In contrast, offspring of parents thatreceived a 240 mGy (40 mGy per day) priming dosewere resistant to the challenge dose. There was littleor no increase in apoptosis above the spontaneouslevel, and no sign of the increased apoptosis at theventricular surface that characterizes radiation-injuredembryos. The other treatment groups showedsuboptimal AR. Among embryos receiving thechallenge dose, there was less apoptosis than in theunprimed group. The primed embryos showed verylittle of the apoptosis at the ventricular-telencephalonboundary that is diagnostic of radiation injury inducedby stage 12 challenge. Embryos did, however, show asomewhat elevated level of apoptotic cells dispersedthroughout the head region. This more dispersedpattern of apoptosis was seen even in the absence ofchallenge. We speculate that elevated, but dispersed,apoptosis may reflect injury from priming dose 2.Further experiments, where parents and embryos areirradiated separately, will be required to test thishypothesis.

In summary, clear evidence for AR was seen at 240mGy, equivalent to about 50 mGy in humans. This doseis expected to produce only 0.5-1 Double StrandBreaks/cell. Suboptimal AR was seen also at a 10-foldlower dose, sufficient to induce only one Double

Strand Break per 10 or 20 cells, cumulatively over a6 d priming period. Results indicate that the medakaembryo model can be used to study and quantifyadaptive response induced at very low doses and doserates.

We also submitted three proposals in FY05 to DOE’sLow Dose Program. All three proposals used medakaas the model organism and SREL’s Low Dose-rateIrradiation Facility. The first two proposals receivedgood reviews but did not score high enough to garnerfunds. The third proposal was fully funded, and theresearch is targeted to begin in late July 2005.

(B)Development of biomarkers to measure trans-generational risks from contaminant exposureInvestigators: T.C. Glenn, T.G. Hinton, C.H. Jagoe,and I.L. Brisbin, Jr.

This fiscal year, we emphasized work in the medakafish. We have previously identified STR loci fromtraditional laboratory methods of constructingenriched libraries. We also searched Genbank for STRloci in medaka sequences. Raw and modestlyprocessed whole-genome shotgun (WGS) sequencedata of the medaka have recently become availablefrom sequencing done in Japan (e.g., http://shigen.lab.nig.ac.jp/medaka/genome/top.jsp). Wesearched the available WGS data for all possible STRDNA sequences. We have discovered 70,245 uniquesequences with at least one microsatellite repeat.Tetra- and penta-nucleotide repeats are abundant. Areasonably large number of loci were discovered withthe same core repeats at the most often studied ESTRloci in mice. Approximately one third of the primerpairs that we have designed yielded scorablepolymorphic loci (26 loci of a total of 72). About 50%of tetra and penta-nucleotide repeat loci identifiedfrom the WGS data have yielded scorable polymorphicloci. The 26 candidate loci yielded 2-11 alleles perlocus from a survey of 19 fish in the SREL breedingcolony. These were used to identify mutant alleles thatwere then used in determination of effects of low dose-rate irradiation.


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We screened the 26 candidate primer pairs amongparents and 200 offspring from control breeding pairsto identify loci with the highest mutation rates. Wehave identified 7 loci in which an average of at least1% of the offspring contain mutant (non-parental)alleles.

We have also screened the 7 loci with observedmutations in the control breeding pairs, along with 2other loci that could be easily incorporated into themulti-locus genotyping panels, in medaka that hadbeen irradiated at the Low Dose-rate IrradiationFacility (LoDIF) at SREL. All medaka in the irradiatedgroup were exposed as juveniles to 68 mGy/d from a137Cs source. They were then housed unirradiated forseveral months. For 7 days immediately prior tobreeding, the males were irradiated again. Offspringderiving from eggs obtained 30 days post-irradiation(i.e., sperm derived from irradiated spermatogonia)were collected. A total of 188 offspring from 4 breedingpairs were analyzed. Offspring from the irradiatedparents have elevated mutation frequencies relativeto unirradiated controls (p=0.03; Fisher’s Exact Test:parental vs. non-parental alleles, control vs. exposed).Similar results are obtained, and the same conclusionis drawn from using alleles or numbers of offspringwith mutations. It is clear that two of the loci (#21and #45) are responsible for most of the responseobserved. Additional experiments to determine theconsistency of this response are underway.

Although the Fisher’s Exact Test is used mostcommonly in published studies of this type, it doesnot account for differences among families. To accountfor variation among families, mutation rates in theexposed and control groups were compared usinggeneralized estimating equations to fit a Poissonregression model that accounted for multiplemeasurements on fish within several families in SAS.In this analysis, the number of non-parental alleles ineach individual was modeled as a Poisson variablewith treatment (exposed vs. control) as the mainfactor, and family nested within treatment as the sourceof repeated measurements. The mean number ofmutations for the control fish was 0.080 and this was

significantly less than the mean number of mutationsin exposed fish (0.149; p = 0.018).

Thus, we have now demonstrated in medaka: (1) largenumbers of STR loci are available, (2) in controlbreeding pairs about 10% of the loci yield >1%offspring with mutant alleles, and (3) STR loci respondin the expected direction due to low dose-rateirradiation. We are, therefore, poised to determinethe response of STR mutations in medaka under avariety of experimental conditions.

We also completed work for 7 publications, includingSREL reprints 2782, 2838, 2839, 2840, 2841, and2870, in addition to one paper that is press (Croshaw,D.A., N.A. Schable, M.B. Peters, and T.C. Glenn. Inpress. Isolation and characterization of micro-satellite DNA loci from Ambystoma salamanders.Conservation Genetics.

(C) Development of fish models for understanding humanand ecological risksInvestigators: T.G. Hinton, T.C. Glenn, W.A. Hopkins,and collaborators from The University of Georgia

See report under section 2A above.

Question 3:How do spatial and temporal variation in contamination,variation introduced by remediation practices, and species-specific differences in ecology and physiology alter risksto humans and other biota?

(A) Waterfowl as vectors of contaminant dispersalInvestigators: I.L. Brisbin, Jr., C.H. Jagoe, and C.S.RomanekIn order to adequately assess the potential of migratorywaterfowl to serve as long-distance vectors of SRScontaminants to the food chain of the hunting public,more specific information is needed concerning theexact ecological/trophic roles of these birds in SRSfood chains and the specific origins/destinations ofmigratory movements of the birds which winter in SRSwetlands and thus are potentially vulnerable to


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exposure to site contaminant releases. During the pastyear we addressed these issues through analyses ofpreviously collected SRS waterfowl samples forcontaminant burdens (radiocesium and mercury) andassociated levels of stable isotopes of hydrogen,carbon, and nitrogen in the same individuals. Theseanalyses were conducted on samples of 26 Americancoots (Fulica americana), 16 lesser scaup (Aythyaaffinis), and 16 ring-necked ducks (Aythya collaris).

Hydrogen isotope analyses of muscle samplesindicated that while away from the SRS, coots tendedto spend time at more northerly latitudes (further awayfrom the SRS) than did lesser scaup. Mercuryconcentrations in both muscle and liver increasedfrom coots (largely herbivorous) through ring-neckedducks (more omnivorous) with highest levelsoccurring in lesser scaup (more carnivorous), thussupporting the idea of general biomagnification withincreasing contamination levels at increasingly highertrophic levels. Levels of stable isotopes of nitrogenhowever failed to confirm that lesser scaup werenecessarily feeding at higher trophic levels than theother two species, suggesting the need for more in-depth dietary analyses for this species while winteringat the SRS site. Neither mercury nor radiocesiumcontamination levels showed any discerniblerelationship to latitudinal origin of the birds assuggested by hydrogen stable isotope profiles.However, differing frequency distributions of hydrogenstable isotopes in muscle vs. liver samples within thesame species indicated the need to consider relativerates of isotope turnover within different tissues before,during, and after the period of migration.

As was the case during the previous year, a nearlycomplete absence of the formerly abundant Americancoot from the abandoned SRS cooling reservoirsduring the winter of 2004-2005 again prevented thecollection of an annual sample of this species as partof a long-term study of radoicesium contaminationlevels in wintering SRS waterfowl. Coots have beenroutinely used over the years as the “worst possiblecase” indicator for radiocesium because of theelevated levels of this contaminant that are routinely

shown by this species when it is present on the SRS.

(B) Risk associated with remediationInvestigator: L.A. Newman

The student working on this project has completedher work and has written up the paper on the results.We have seen that exposure to TCE by C. elegans atconcentrations approximating that seen on aremediation site did not result in increased lethalityor a decrease in mobility. Exposure of earthwormsdid not result in any external affects, and the wormsdid not show a physiologically relevant increase inthe levels of metabolites.

The work with C. elegans will be continued, lookingat other parameters, such as reproductive success andfeeding patterns with TCE. These parameters, as wellas movement and lethality, will also be studied for othersolvents. This work is being done in conjunction withfaculty at the University of South Carolina-Aiken.

(C) Determining the most sensitive species to organiccontaminantsInvestigators: W. Hopkins and scientists from USGS-Patuxent Wildlife Center and Michigan StateUniversity

For the risk assessment process to be maximallyeffective, it is important to understand which receptororganisms are most sensitive to the effects ofenvironmental contamination. It has been repeatedlysuggested that reptiles are the most sensitivevertebrates to organic contaminants due to their poorlydeveloped detoxification system, but little empiricalevidence supports this contention. In collaborationwith scientists from USGS-Patuxent Wildlife Center andMichigan State University, we completed two studiesin FY04 that characterized aspects of the cytochromeP450 system in snakes (family Colubridae). Thisresearch was completed in FY05 and two manuscriptsdetailing our findings have been completed (one underinternal review, the other in review at Toxicol AppliedPharmacol). We anticipate that both will be publishedin FY06.


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(D) Fish as vectors of contaminant dispersalInvestigators: D. Fletcher and collaborators (M.Paller, Savannah River National Laboratory; J. Isely,Clemson University; W. Littrell and S. Dyer, WSRC-SGCP)

Consumption of contaminated fish by anglers ispotentially an important offsite pathway for humanexposure to 137Cs originating from the SRS. To betterunderstand this problem, a radiotelemetry study wasconducted from June 1999 through April 2003 tocharacterize the movements of potentiallycontaminated largemouth bass in Steel Creek, arestricted-access SRS stream, and the publiclyaccessible Savannah River. Results from the radiotracking study indicated that mobility varied amongindividual largemouth bass and was affected by season,water level, and habitat. Largemouth bass usuallyestablished comparatively small home ranges, butoften left them and sometimes traveled long distances,especially during February through May, their expectedpre-spawning and spawning periods. High water levelsduring this time appeared to produce the greatestnumber of long distance movements by largemouthbass. After long distance movements, bass frequentlyset up new home ranges; however in the SavannahRiver, homing to the Steel Creek area was alsoobserved.

In spite of the occasional long distance movements,largemouth bass tended to be relatively sedentarymuch of the time. This was particularly true in upperSteel Creek, where movement was more restricted andhome ranges smaller than in lower Steel Creek andthe Savannah River. As a result, upper Steel Creekcontributed few largemouth bass to the SavannahRiver. Most of the interchange of largemouth bassbetween Steel Creek and the Savannah River wasrestricted to the lower reaches of Steel Creek,especially that on the Savannah River floodplain.Seasonal and diurnal interchange occurred betweenthese habitats. The tendency to occupy relatively smallhome ranges much of the time also restrictedlargemouth bass dispersal in the Savannah River.Approximately 90% of the largemouth bass relocations

in the Savannah River were within 10 km of the mouthof Steel Creek.

A study of the escapement of fishes from SRS reservoirsidentified critical influences of outlet structure typeand lake levels on movement of fishes though outletstructures. Species composition of escapement catchalso varied temporally. Fish that passed through thedams likely presented a larger ecological risk thanhuman risk because of their size and speciescomposition. However, escapement during criticalhigh water discharges remains to be ascertained.

A mark-recapture population survey conducted inconjunction with the radiotelemetry study showed thatafter two summers of hypolimnetic releases from LLake and several years of reduced stream discharge,small numbers of largemouth bass were resident inSteel Creek. Although bass populations reboundedwhen warm water discharge from L Lake was restored,the small number of bass present in Steel Creek at theconclusion of our study was likely contributingrelatively few individuals to the Savannah River. Acomparison of the total amount of 137Cs transportedannually from Steel Creek by largemouth bass and theamount transported by water and suspendedsediments clearly showed the predominance of thelatter in terms of total quantity of 137Cs moved fromthe SRS into public areas. This conclusion wassupported by a published dispersal model (J. Environ.Radioactivity, 2005), which showed that 137Cs levelsin Savannah River largemouth bass predicted on thebasis of largemouth bass dispersal from SRS streamswere below actual levels measured in Savannah Riverlargemouth bass. Although this discrepancy couldresult from erroneous assumptions in the dispersalmodel, an alternative explanation is that some 137Cs isaccumulated by largemouth bass within the river as aresult of the discharge of 137Cs in water and sedimentfrom SRS creeks. We conclude that during our study,the emigration of largemouth bass from SavannahRiver Site streams was unlikely to be responsible forlong distance dispersal of substantial radiologicalcontamination in the Savannah River.


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(E) Endangered species using contaminated sitesInvestigators: A.L. Bryan Jr., I.L. Brisbin, Jr., and C.H.Jagoe

To document Bald Eagle utilization and behavior onthe SRS, we conducted studies which resulted in amanuscript regarding eagle food habits, feeding rates,and nesting behavior (Bryan, A.L. Jr., et al.. In Press.Behavior and food habits of breeding Bald Eaglesin inland South Carolina. Southeastern Naturalist).This manuscript provides baseline informationnecessary for predictive models of dietary contaminantexposure. Models concerning contaminant exposurevia the diet by nesting eagles on the SRS have beenconstructed and subsequent manuscripts are inpreparatory stages, with submission to appropriatescientific journals scheduled for the latter half ofcalendar 2005

Studies regarding contaminant exposure (mercury)to another endangered species, the Wood Stork(Mycteria americana), relative to variation in wetand dry seasons are addressed under Characterization3(B).

Question 4:What are the potential interactions (additive, synergistic,antagonistic) from exposures to mixed (radioactive andnon-radioactive) contaminants, as well as otherenvironmental stressors?

(A) Complex interactions between biotic and abiotic factorsand assessments of riskInvestigators: W. Hopkins, T. Hinton, and scientistsfrom USGS-Columbia Environmental ResearchCenter

See report under section 2A above. In addition, SREL’sLow Dose-rate Irradiation Facility was the core for aproposal that was submitted to the National Institutesof Environmental Health Sciences (NIEHS). Theproposal is very relevant to contaminant issues on allDOE sites: “Contaminant Mixtures: MultigenerationalEffects from Chronic Exposures.” The proposal, which

involves 12 research groups from three countries,requested $13M from the NIEHS over a 5-year period.Successful applicants will be notified in the fall of 2005.

Question 5:What are the risks from low dose-rate, chronic exposuresto radiation?

(A) Determining the risk from chronic low-dose radiationexposureInvestigator: T. Hinton

See report under section 2A above.

Question 6:What are the most cost-effective, yet still valid, biomarkersof meaningful impacts to biota?

(A) Development of biomarkers to asses the effects ofcontaminants on biotaInvestigators: C. Romanek, C.H. Jagoe, and W.Hopkins

Chronic exposure to contaminants causes stress inorganisms, which often manifests itself as increasedmetabolic demand because energy is used to: (1)metabolize, sequester, or excrete contaminants, (2)repair contaminant-induced damage, and (3)maintain homeostasis. Because more energy is usedfor these functions, less is available for other activitiessuch as growth and reproduction. Biochemicalmarkers, or biomarkers, are commonly used toindicate the effects of contaminants on impactedorganisms as they potentially provide an early warningof ecological damage to exposed biota. Conventionalbiomarkers like metallothionein or glutathioneindicate exposure to various pollutants, but bettermeasures of increased energetic costs of pollutantexposure are needed. Our preliminary studies haveshown that changes in the ratio of the stable isotopesof nitrogen in tissues of fish and birds exposed to toxicmetals (e.g. Hg) provide this measure. These changesindicate that rates of protein turnover increase with


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exposure to pollutants. We have shown that nitrogenisotope shifts in proteinaceous tissues precede changesin metallothionein and glutathione biomarkers whenanimals are exposed to contaminants (e.g. Hg).However, the exact causes and relationships to proteinturnover remain to be clarified.

In FY2005, we published a manuscript inEnvironmental Science & Technology (Shaw-Allenet al., In press) that documents these findings.Increases in nitrogen isotope ratios were observed inthe whole liver and the acid soluble fraction of theliver of animals fed diets relatively high in Hgcompared to a reference group. Meanwhile, significantdifferences were not detected in hepatic levels of themetal-binding biomarkers metallothionein andglutathione. These shifts are due to increased rates ofprotein breakdown relative to synthesis and reflect afundamental shift in protein metabolism. Because theshift in the acid-soluble fraction was stronger thanthe bulk liver, the increase is probably driven by anincrease in bodily derived amino acids in the freeamino acid pool.

This work forms the basis for continued research todevelop the techniques to analyze the nitrogen isotoperatios of specific biomarkers and the amino acidresidues that comprise them. It is our hope thatnitrogen isotopes can be used to determine themetabolic pathways and fluxes that are altered byexposure to contaminants (e.g. Hg). Presently, ourlaboratory is developing procedures to hydrolyzeproteins and retain amino acid groups that aresensitive to oxidation such as cysteine and methionine.These amino acids contain sulfur and are active sitesfor mercury binding. Progress has been made in thedevelopment of techniques related to compoundspecific isotope ratio analysis. GC-C-IRMSinstrumentation has been used to separate derivitizedcarbon-bearing compounds and analyze themindividually for their carbon isotope composition. Thiswork will be extended to include the derivitization ofamino acids for nitrogen isotope analysis to furtherelucidate how contaminant exposure affectsorganisms, populations and communities in impacted

environments.

Question 7:What are the mechanisms and consequences of trophicand maternal transfer of contaminants?

(A) Maternal transfer of contaminants in amphibiansInvestigators: W. Hopkins and B. Jackson, withscientists from University of Maryland-ChesapeakeBiological Laboratory

Although there is now general consensus that manyamphibian populations around the globe are decliningat alarming rates, the cause of most declines remainsunknown. Environmental contamination is one ofseveral factors implicated in declines, and may beparticularly important for sensitive developmental lifestages. Developmental pathways in embryonicamphibians can be altered when embryos are exposedto contaminants via two primary mechanisms: uptakefrom their surroundings (e.g., water), and transferfrom mother to offspring. To date, no studies haveexamined maternal transfer of contaminants inamphibians, despite its devastating effects ondevelopment in every other major vertebrate lineage.In FY05, we determined that adult female narrowmouth toads (Gastrophryne carolinensis) breedingnear a near a coal burning power plant transfer highconcentrations of Se and Sr to their eggs. Contaminatedeggs produced 20% fewer viable offspring due todecreased hatching success and an increasedprevalence of developmental malformations. Our studydemonstrated that maternal transfer is an importantroute of contaminant exposure in amphibians andwarrants future study.

In FY05 we presented our findings at the annualmeeting of the Ecological Society of America and theWorld Congress of the Society of EnvironmentalToxicology and Chemistry. The manuscript describingthis work is under internal review and will besubmitted to Environmental Science & Technologyin early FY06.


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(B) Indirect effects of contaminantsInvestigators: W. Hopkins, D. Scott, S. Harper, B.Jackson, and B. Metts

Recent evidence suggests that the effects ofcontaminants on wildlife often exceed those estimatedin laboratory toxicity tests because otherenvironmental factors (e.g., predators, resourceabundance) can influence contaminant toxicity. Weconducted a series of three outdoor mesocosmexperiments with either pesticides or complexmixtures of metals to examine the effects that thesecontaminants have on complex assemblages of aquaticorganisms. In the first of these studies, we manipulatedpesticide (carbaryl) concentration (control, 3.5 mg/L, 7.0 mg/L) and larval density (low and high) of twospecies of Ambystoma salamanders to examine howthe natural stress of competition might influencecontaminant toxicity. Carbaryl negatively affectedsalamander snout-vent length (SVL), growth rate, lipidreserves, time to metamorphosis, percent survival, andpercent metamorphosis. The intensity of this effect onSVL, lipid reserves, and growth rate was influencedby density. Additionally, the two species respondeddifferently to the treatments. The effects of carbaryland increased density on percent metamorphosis werenearly additive, but were generally less than additiveon other variables. The negative effects of chemicalcontamination on salamanders were likely due topesticide-induced reductions of food resources, aszooplankton abundance decreased by as much as 97%following carbaryl application. Our first studydemonstrated the importance of the interactive effectsthat chemical contamination and natural environ-mental factors have on salamander assemblages. Thesecond two studies used a similar approach todetermine whether complex mixtures of metals hadsimilar detrimental effects on zooplanktonassemblages and if predators reliant upon zooplanktonwould be adversely impacted. Preliminary resultssuggest that metal contamination does decreasezooplankton abundance, but not nearly to the extentthat pesticides do. Effects on salamander assemblagesalso appear less pronounced, but may still be sufficientto reduce the number of viable recruits to terrestrial

habitats. Interestingly, concentrations of metalsaccumulated in salamander tissues were extremelyhigh, suggesting that they may act as important vectorsof contamination from aquatic to terrestrial habitats.

The first of these studies was published (Metts, B.S.,et al. 2005. Interaction of an insecticide with larvaldensity in pond-breeding salamanders(Ambystoma). Freshwater Biology 50:685-696; SRELreprint 2815). Data are currently being statisticallyanalyzed from the latter two studies and manuscriptswill be submitted to peer-reviewed journals in FY06.

(C) Use of stable isotopes to elucidate exposure pathwaysInvestigators: C. Romanek, C.H. Jagoe, I.L. Brisbin,Jr., W. Hopkins, and B. Jackson


Question 8:What are the risks associated with the indirect selectionfor antibiotic resistance by microbial exposure to heavymetals?

(A) Importance of heavy metal exposure in promotingantibiotic resistance in bacteriaInvestigators: JV. McArthur, C.H. Jagoe, and T.C.Glenn

We have established that native aquatic bacteria thatare exposed to heavy metal contamination on theSavannah River Site have increased levels of antibiotic-resistance towards commonly used antibiotics. We haveeffectively shown that indirect selection caused byexposure to metals actually increases the proportionof bacterioplankton carrying antibiotic resistant traits.Likewise, bacterioplankton exposed to antibiotic showincreased levels of metal resistance. We seek todetermine how wide-spread this phenomenon is byincreasing our survey of new habitats. Majoraccomplishments of FY05 include:

Performed a microcosm experiment using coastalwater collected from the ACE Basin to determine the


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indirect selection of antibiotic resistant bacteria whenexposed to heavy metals.1. Amended triplicate bottles with varying

concentrations of mercuric chloride, cadmiumchloride and potassium dichromate.

2. Performed flow cytometric analysis to quantifytotal number of bacteria at the beginning and endof the experiment.

3. Cultured nearly 300 bacterial isolates that werescreened for antibiotic and metal resistance.

4. DNA from all isolates was amplified by PCR andsequenced.

5. Samples also collected from the microcosms foranalysis of metal content, dissolved organiccarbon, antibiotic content, and nutrient analysis.

Gene transfer experiments:1. Tested GFP (green fluorescent plasmid) marked

recipients for use in gene transfer experiments.2. Performed filter mating experiments with GFP

marked recipients and bacteria exposed to metalsfrom the sediment and water of the D Area ashsettling basins.

3. Performed a GFP-plasmid transfer experiment atthe Skidaway Institute of Oceanography to developa flow-cytometric assay for high throughputdetermination of model plasmid transfer ratesunder various conditions such as metal exposure.

(B) Mechanisms that link heavy metal and antibioticresistanceInvestigators: JV. McArthur, C.H. Jagoe, and T.C.Glenn

Bacterial traits that have environmental importanceare often carried on mobile elements called plasmids.It is not clear how, when, and how often plasmid DNAis exchanged among bacteria in the environment. Weseek to determine the risks associated with plasmidtransfer of plasmids carrying antibiotic resistancegenes in native populations of bacteria. Majoraccomplishments of FY05 include:� Re-tested resistance of 300 Aeromonas isolates

from bullheads (Ameiurus spp.) and sedimentfrom Four Mile Creek and Meyers Branch to

chloramphenicol, gentamycin, kanamycin,streptomycin, and tetracycline.

� Extracted plasmids from 150 Aeromonas isolatesfrom bullheads, bass, and sediment from FourMile Creek, Meyers Branch, and Par Pond andobtained 6 plasmids. Grew up and extractedplasmids from 43 previously frozen isolates fromthe cadmium-tetracycline and nickel-ampicillinmicrocosm experiments. These isolates wereresistant to 3 or more selective agents (antibioticsor metals).

� Collected sediment and water from the SavannahRiver, extracted DNA, and amplified using 6 ORIspecific primer sets specific to a variety of plasmidincompatibility groups. Got amplification withprimers specific to the trfA2 region of the IncPplasmid group in both water and sediment.

� Set up microcosm experiment with SavannahRiver water and sediment spiked with cadmiumand nickel.

� Plasmids have been isolated from environmentalbacteria. The number and size of plasmids hasbeen measured. A sub-sample of plasmids is beingtransformed into laboratory strains of E. coli forfurther characterization.

� Initial gene transfer experiments have beenconducted between indigenous bacteria (genedonors) from the ash settling basins of a coal-fired power plant and a Pseudomonas sp. B13-GFP2 strain (gene recipient). These experimentsindicate that ash-settling basins containhorizontally transferable genes encodingresistances to tetracycline, gentamycin,streptomycin, and mercury. Such gene transferexperiments will be a major focus of FY05research and further characterization of themobile elements identified from theseexperiments will be the focus of additionalresearch in FY05 and FY06.


41

Question 9:What statistical techniques provide the most power andhighest confidence when estimating total dose or exposure?

(A) Bootstrap methodsInvestigators: M.D. Wilson, T.G. Hinton, and C.H.Jagoe

Several data sets were tested using a traditionalgoodness-of-fit test, (Lilliefors test for normality)before and after log transformation, as well as using abootstrap goodness-of-fit test. A peer-reviewed journalarticle was published in the Journal of RadiologicalProtection that illustrated the bootstrap hypothesistesting and power analysis on the Sellafield (UK) dataset.

The bootstrap goodness-of-fit test was developed andapplied to several data sets. The Bootstrap goodness-of-fit test was positive when the Lilliefors test waspositive, indicating that the bootstrap methodproduced robust estimates of the distributions.However, the benefit of this test is that a test of thedistribution of the mean can be applied. This isbeneficial because it is most often the distribution ofthe mean that is of interest in hypothesis testing. If adata set fails distributional tests, but the mean can beshown to pass, then classical distributional theory canbe applied to conduct inference and hypothesis testing.

For the data sets failing both normal and log-normal,the bootstrap test was applied to both the raw dataand the log-transformed data. Some additional datasets were shown to have log-normal means by theresults of the bootstrap test. The data sets that areidentifiable as normal or lognormal will be used toconfirm results of the non-parametric methods, whichshould prove to be consistent with methods based ondistributional assumptions for data sets which meetthese assumptions. This work is integrated with workon the distributional properties of contaminant datasets described in the Characterization section, question5.

(B) Bayesian methodsInvestigators: M.D. Wilson, T.G. Hinton, and C.H.Jagoe

The software package WinBUGS has been installed andelementary code has been written. Work currentlyfocuses on learning the use of WinBUGS and thespecific implementation of both Bayesian mixturemodel estimation and traditional Bayesian methodswhen prior distributions can be specified.

(C) Mixture modelsInvestigators: M.D. Wilson, T.G. Hinton, and C.H.Jagoe

We have begun writing and testing code in themathematical programming language, Matlab toestimate the parameters of a mixture of normal andlog-normal distributions on simulated data where thetrue parameters are known. This method is quitecomputationally intensive and developing code is notstraightforward. This work continues.

Because of recommendations from DOE, newproposals were developed. This work wasincorporated into a proposal to characterizeradiocesium on the SRS. This work proposes usingstable isotope analysis to identify the position of anorganism in a food chain and to use mixture modelmethods to characterize the degree of mixing betweennormal and log-normal.


Remediation and Restoration

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The SREL Remediation and Restoration Group conducts multidisciplinary research designed to assist DOE in thedevelopment, evaluation, and stakeholder acceptance of remediation and restoration efforts that are protective of humanas well as ecosystem health. The following discussion summarizes research accomplishments for FY05.

Report on FY05 Remediation and Restoration Milestones

Question 1:How can solute transport and retention within the variablysaturated vadose zone, which serves as the long-termsource for many contaminated systems, be incorporatedwithin conceptual and predictive models of contaminantmigration?

(A) Field studies of vadose zone solute transport cyclingmodelsInvestigators: J.C. Seaman and collaborators fromClark Atlanta and Cornell Universities

We continued the development and refinement ofvadose transport model within the HYDRUS1D codefor describing tritium movement through the soilprofile, including parameterization for tritium lossesdue to evapotranspiration. We determined waterretention properties for a number of distinct soilhorizons reflecting the typical soil pedon observedwithin the irrigation site using the UFA system. Twoseparate field scale tracer experiments wereconducted (i.e., pulse tritium applications) and wecontinued sampling additional irrigation plots as partof collaboration with Cornell University (Drs. Riha andRebel) and the U.S. Forest Service-Savannah River(Drs. Blake, Hitchcock, and Barton). Laboratoryexperiments were initiated to determine the utility ofSurface Complexation Modeling (SCM) to variablysaturated systems. SCM addresses some of the inherentuncertainties in the Kd approach to predicting solute

partitioning through the use of a limited number ofindependent parameters that can account for changesin partitioning under variable geochemical conditions.In SCM, contaminant sorption behavior is describedin terms of generic surface functional groups, withformation constants, reaction stoichiometries, and insome cases sorption site densities determined by fittingbatch sorption data, factors that may change withfluctuating moisture conditions.

Question 2:What are the primary hydrogeochemical processes thatdetermine contaminant migration under the kineticallycontrolled, heterogeneous conditions encountered in thefield?

(A) Development of an automated vadose monitoringsystemInvestigators: J.C. Seaman and collaborators fromClark Atlanta University and DYNAMAX, Inc.

During FY05 we installed radiotelemetry equipment,incorporated their function within the AVM datalogging/instrument control program, and developedthe initial web-based data reporting system (http://loggernet) with the help of Jeff Harris and DenoKarapatakis. We also installed an additional set of soilmoisture and tension sensors on the second irrigationplot, for which custody was returned to S&GCP so that


43

it may be included in the normal irrigation scheduleused for the majority of plots. The original AVM soillysimeter pumping system proved unreliable underfurther field testing, failing to consistently pull therequired vacuum to draw sample into the lysimeter.To address this shortcoming, the system wasredesigned to include an additional vacuum pumpseparate from the ISCO pumping system that will nowbe dedicated to moving the sample from the lysimeterto the fraction collector and purging the sampling linesas necessary. A prototype of the new system has proveneffective under limited laboratory testing. The revisedpatent application, including the new radiotelemetrycapabilities, will be submitted following successfulfield testing of the new AVM design.

Question 3:What hydrogeochemical factors control permeable reactivebarrier function?

(A) In situ redox manipulation studiesInvestigators: G.L. Mills, J.C. Seaman, and A. Neal

Over the past decade, permeable reactive barriers havebeen developed as an alternative to traditional pumpand treat systems for groundwater remediation. Thistechnology involves construction of permanent, semi-permanent, or replaceable units across the flow pathof a dissolved phase contaminant plume. As thecontaminated groundwater moves through thepermeable reactive zone, contaminants areimmobilized or degraded and uncontaminatedgroundwater emerges down gradient. The reactivematerials employed within the barrier should: (1) benon-toxic, (2) remain effective for immobilizing ordegrading contaminants for several years, (3) besufficiently insoluble to remain in place, (4) retainpermeability following reaction, and (5) be cost-effective relative to other treatment technologies. In-situ redox manipulation (ISRM) using sodiumdithionite to create a subsurface reactive barrier forthe treatment of reducible groundwater contaminants,including PCE, and Cr, is being implemented at DOE’sHanford Site. To date, the ISRM approach has not been

widely tested on highly-weathered geological materialslike the Upper Coastal Plain sediments present on theSRS. The fact that dithionite is commonly used in theextraction of Fe oxides suggests that such a treatmentmay be deleterious to the physical integrity of thesediments, an aspect that has not been adequatelyaddressed in previous studies. Therefore, the objectiveof this study is to use dynamic column techniques toevaluate the effectiveness of dithionite reduced SRSaquifer materials in degrading or immobilizing CCl

4,

PCE, TCE, and Cr(VI) by chemical reduction focusingon the behavior of Fe and the physical and hydrologicalproperties of the aquifer sediments.

The installation of a chemically reducing reactivebarrier using dithionite solution includes an injectionphase during which dithionite permeates the treatmentzone, a reaction phase that reduces the Fe within theaquifer sediments, and an extraction phase, whenunreacted dithionite is removed from the system.Results from the column experiments indicated thatsignificant amounts of Fe are mobilized only afterinjection of 12 pore volumes of dithionite solution(34.5 mmol S

2O

42-; pH 11). The mobilized Fe is

predominately from oxide, carbonate, and sulfideminerals. Structurally bound and exchangeable Fe2+

changed little after 2 pore volumes. A study on theeffect of reaction time on Fe mobility showedsignificant Fe2+ solubilization and transport after 10hours that the Fe was derived from the oxide,carbonate, and sulfide phases.

The reducing capacity of the dithionite treated aquifersediments was assessed by determining the amountof soluble Cr(VI) sequestered from groundwaterwithin the reduced sediments. The results indicate thatthe injection of 12 pore volumes of dithionite solutionand a subsequent reaction time of 6 hours were theoptimum conditions for creating an effective treatmentbarrier while minimizing the dissolution of Fe intothe effluent remediated groundwater. No significantdecrease in hydraulic conductivity or increase ingroundwater turbidity (i.e., colloid mobility) wereobserved using these conditions, suggesting that theseconditions would be suitable for treatment systems


44

using ISRM in South Eastern Coastal Plain sediments.

Question 4:What are the primary mechanisms by which amendmentscontrol immobilization of contaminants, and what are theappropriate geochemical and biological endpoints toevaluate the stability of immobilized contaminants?

(A) Immobilization of contaminants through the additionof amendmentsInvestigators: T.G. Hinton, P. Bertsch, H. Dion, B.Koo, C. Romanek, R. Sharitz and collaborators fromSavannah River National Laboratory (D. Kaplan,A. Knox)

Three remediation-oriented manuscripts werepublished in FY05 from our research on the SavannahRiver Site. In the first manuscript (Hinton, T.G., etal. 2005. Phytoextraction of uranium and thoriumby native trees in a contaminated wetland. J.Radioanalytical and Nuclear Chem. 264:417-422;SREL reprint 2829) we explored the potential of nativetrees to remediate a uranium- and thorium-contaminated wetland. This work examined the firststep of phytoimmobilization by determining the abilityof native trees to extract U and Th from a contaminatedwetland located on the SRS. The focus on evaluatingnative plants was prompted by the desire not tointroduce foreign species into the ecologically sensitivearea and because intrusive remediation approachesare not feasible in legislatively protected wetlands.Based on measurements of the annual biomass ofleaves and their contaminant concentrations, weestimated the reduction in soil contamination overtime. Significant differences among tree species werefound, with tupelo (Nyssa sylvatica) and sweetgum(Liquidambar styraciflua) having a significantlygreater capacity to remove U and Th from the soilthan the other tree species found on the site.Phytoextraction rate constants were developed andrevealed that although U and Th phytoextraction wasexceptionally high at the site—an order of magnitudegreater than published literature would predict—thecommunity of native trees would lower the soil

inventory of U and Th by only 1% over the next 100years.

A second manuscript (Kaplan, D. I., et al. 2005.Cesium-137 partitioning to wetland sediments anduptake by plants. J. Radioanalytical and NuclearChem. 264:393-399; SREL reprint 2830) examinedthe partitioning of 137Cs in wetland sediments andplants in a contaminated canal used to dischargeeffluents from the R-reactor in the 1960s on the SRS.The physical location of 137Cs as a function of depthwithin the sediment, as well as among sedimentcomponents (clay, sand, organic matter) revealed thatthe maximum 137Cs concentrations generally occurred2.5 to 7.5 cm below the sediment surface. About 60%of the total 137Cs inventory in each sampled sedimentcore existed at the organic matter-mineral interface.Sequential extractions revealed that most of the 137Csin this interface layer was bound by amorphous Fe-oxyhydroxides or organic matter. Almost half of the137Cs was weakly held to these soils, compared to only10% similarly held in sediments from the Hanford Site.The Hanford sediments contain a higher percentageof illite clays, and illite strongly binds Cs. The lack ofstrong binding on the SRS sediments was also observedin the propensity for plants to take up high percentagesof 137Cs from the sediments. These data reveal yet againthat site specific sampling is needed to understandthe dynamics and resulting risks associated withcesium contaminated environments.

The third manuscript (Dion, H.M., et al. 2005.Cesium-137 in floodplain sediments of the LowerThree Runs Creek on the DOE Savannah River Site.J. Radioanalytical and Nuclear Chem. 264:481-488;SREL reprint 2842) explored 137Cs in sediments of afloodplain contaminated by releases from the SRS.Examination of 137Cs within different sediment texturalclasses revealed that the highest concentrations of 137Cs(Bq kg-1) were in the clay fractions of the sediments.Clay, however, comprised a tiny percentage (< 2%)of the sediment. Sand was the dominant component(79 to 99%). Although the concentration of 137Cs inthe sand fraction was four times less that in the clay,the much larger abundance of sand resulted in it


45

containing the largest percentage of the creek’s total137Cs inventory (total Bq). Thus, the dynamics of Cswithin the watershed are likely influenced more bythe sand fraction than the clay. This observation iscounter-intuitive to the widely held belief that theenvironmental behavior of Cs is governed by clayminerals. The sand-sized fraction contained specificsorption sites, which may be due to nanostructuralmica present as coatings on sand-sized particles. Bulkmineralogy was inadequate to discern the presenceof these mica phases. This research suggests that 137Cstransport in these highly weathered, sand dominatedsediments may be governed by mechanisms notpreviously explored.

(B) Testing immobilization capacity of soil amendmentsfor use in phytoextractionInvestigators: D.C. Adriano and collaborators fromUniversitaat fur Bodenkultur, Vienna, Austria


(C) The role of biosolids in the remediation of coal reject/fly ashInvestigators: D.C. Adriano, B. Koo, T. Punshon, andcollaborators from the University of Kentucky


(D) Effects of hydroxylapatite on the toxicity of nickel anduranium to microorganismsInvestigators: P.M. Bertsch, B.P. Jackson, T. Punshon(CRESP), and collaborators at the MedicalUniversity of South Carolina and the University ofChicago

We have demonstrated a pH-dependent Ni-toxicity toa constitutive TCE degrader, Burkholderia cepaciaPR1

301. PR1 is able to grow at higher concentrations

of Ni at pH 5 (17 mM) as compared to pH 6 (3.4mM) and pH 7 (0.85 mM). This trend was notobserved in the Ni-resistant strains Wautersiametallidurans CH34 and 31A, which have well-studied Ni efflux transporters. While possessing similar

efflux transporters, different trends were notedbetween CH34 and 31A. CH34 was most resistant toNi at pH 6 (12.8 mM) and least resistant at pH 7 (8.5mM) while 31A was most resistant at pH 6 (34.1 mM)but least resistant at pH 5 (17 mM). To determine ifthe transporters were influenced by pH-dependent Ni-toxicity, two transconjugants were created by Dr. D.van der Lelie (Brookhaven National Laboratory) byinserting the ncc-nre Ni-resistance genes from 31Ainto PR1 and BuST, another constitutive TCE degradercreated from B. cepacia G4 (the parent strain of PR1).Insertion of these efflux transporters increased Niresistance in PR1:ncc-nre as compared to PR1. Growthwas observed at 51.1, 17 and 1.7 mM Ni at pH 5, 6and 7, respectively, displaying at least a two-fold levelof increase in Ni resistance as compared to PR1.BuST:ncc-nre was able to grow in the presence of atleast 51.1, 17 and 3.4 mM Ni at pH 5, 6 and 7,respectively. Both transconjugants displayed the samepH-dependent trend in Ni toxicity as observed withPR1. Further work is underway to determine themechanism of Ni-tolerance displayed by PR1. Niaccumulation by PR1 does not explain changes in Nitoxicity with change of pH as PR1 accumulated 1.49(+ 0.25), 1.12 (+ 0.09) and 3.88 (+ 0.93) mg Ni-1 gdry weight at pH 5, 6 and 7, respectively. Examinationof PR1 culture supernatants by size exclusionchromatography coupled to inductively coupledplasma mass spectrometry to detect potential Ni-binding substances detected a small (<500 Da) Ni-binding substance. This same peak was detected inabiotic controls, suggesting that it is a mediumcomponent and not produced by PR1. Two-dimensional gel electrophoresis has been used toexamine changes in protein expression in PR1 afterexposure to Ni at pH 5 and 7. A chaperonin protein,GroEL, which is involved in protein folding/refolding,was identified by peptide mass fingerprint analysis.GroEL showed the highest level of expression at pH 5after a 3 h exposure to 3.4 mM Ni. An additional 12proteins showing altered levels of expression underthe conditions examined have recently been identifiedby protein sequencing using a tandem massspectrometer. Ni transport assays are currentlyunderway to determine if Ni is transported out of the


46

cell and if pH influences Ni transport. In addition,genomic DNA of PR1 and four Gram-positive, Ni-tolerant isolates from Steed Pond, a U and Nicontaminated wetland at the SRS, will be examinedusing a metals-related microarray in the laboratoryof Dr. J. Zhou at Oak Ridge National Laboratory. Themicroarray consists of almost 2000 genes dealing withmetal homeostasis (i.e., transport, resistance,sequestration, etc.) with approximately 60 of theserelating to Ni.

Publications:Van Nostrand, J.D., A.G. Sowder, P.M. Bertsch, and P.J.

Morris. In press. The effect of pH on the toxicityof nickel and other divalent metals toBurkholderia cepacia PR1

301. Environ. Toxicol.

Chem.Presentations:Van Nostrand, J.D., A. Sowder, J. Arthur, P.M. Bertsch,

and P.J. Morris. 2005. Ni tolerance inBurkholderia cepacia PR1

301, American Society

for Microbiology General Meeting, Atlanta, GA,(poster presentation).

Van Nostrand, J.D., J. Arthur, P.M. Bertsch, and P.J.Morris. 2004. Protein expression changes in B.cepacia PR1

301 with exposure to nickel at pH 5

and 7. EPA STAR Graduate Fellowship Conference,Washington, DC, (poster presentation).

Van Nostrand, J.D., J. Arthur, P.M. Bertsch, and P.J.Morris. 2004. Changes in protein expression inB. cepacia PR1

301 after exposure to nickel at pH

5 and 7. Student Research Day, Medical Universityof South Carolina, Charleston, SC, (posterpresentation).

Question 5:Can plant genes involved in the remediation of groundwatercontaminants be identified?

(A) Searching for genes involved in the degradation ofhalogenated solventsInvestigators: L.A. Newman and researchers at theUniversity of Washington

Work is continuing on this project. We are currentlyscreening a cDNA library from tobacco, which isknown to metabolize TCE at a higher rate thatArabidopsis. This screening is being done by lookingfor the release of bromide ions from the metabolismof ethylene dibromide. This compound is transformedby the same enzymes involved in TCE degradation, andthe bromide can be tracked easily in suspensioncultures where chloride cannot be tracked.

We have also identified five P450 genes fromArabidopsis which have the same reaction domain asseen in P450 2E1, which is the major TCE degradingenzyme in mammals. These genes have been clonedout of Arabidopsis and linked to strong constitutivepromoters, and inserted back into both Arabidopsisand tobacco. We have multiple transformants intobacco, and have transformed Arabidopsis and arewaiting for those plants to germinate. Once we havesufficient plant material, we will screen for changesin metabolic patterns as well as sensitivity to high levelsof TCE. We also have Arabidopsis lines that have hadthe genes knocked out, and we are currently screeningthem for changes in TCE degradation patterns. Thiswork has been presented by the student at theAssociation for Environmental Health Sciences annualconference (2004), and the EPA InternationalPhytotechnologies meeting (2005). The poster at theAEHS conference won the Best Student Presentationaward for the conference.

Question 6:How do microbes affect metal speciation transformations,and what is the sustainability of these transformations insitu?

(A) Comparing microbial communities in the rhizospherevs. bulk soilInvestigators: C.L. Zhang, D. Adriano, and B. Koo

It is hypothesized that root exudates stimulatemicrobial growth and affect community structure inthe rhizosphere. These microorganisms serve as focalpoints for metal accumulation and immobilization


47

using cell walls as templates for adsorption andtransformation. The overarching goal is to determinethe microbial community structure affected by rootexudates, and co-existing heavy metals andradionuclides and to determine the rolemicroorganisms play in the speciation, uptake, andimmobilization of co-contaminant metals andradionuclides in the rhizosphere. We characterizedthe difference in microbial community structuresbetween the bulk soil and the root hair in the samesample location. Samples were collected from theLower Tims Branch on the SRS. DNAs were extractedfrom these samples using a commercial kit andprimers specific for Bacteria (27F/1492R) were usedfor PCR amplification of these DNA samples. The PCRproducts were screened using Denaturing GradientGel Electrophoresis (DGGE). Individual bands in theDGGE gel were excised and purified and the DNAfragments were sequenced. All the sequences werethen clustered and analyzed phylogenetically usingsoftware packages. Our preliminary results indicatethe difference in microbial diversity is small betweenthe bulk soil and the root hair for a particular samplelocation. This study was terminated and our effortshave been focusing on other areas of research inmicrobial ecology at the Savannah River Site.

Question 7:What traits of ‘native’ plants and populations determinetheir suitability for use in remediation and site restoration?

(A) Use of fluorometry in appraising plant health duringphytoremediationInvestigator: K.W. McLeod

Phytoextraction, the use of plants to removecontaminants from soil, is widely recognized as morecost-effective than excavation and disposal. Lead is acontaminant of concern that is very costly and difficultto remove from the environment. When lead is takenup in leaves, it causes damage to the photosyntheticapparatus. This damage causes an increase influorescence, which can be detected using afluorometer. But other plant stresses can also cause

changes in fluorometric measurements and thesesecondary stresses must be also recognized. Theobjectives of this recent experiment were: (1) todetermine if lead stress can be detected in kenaf(Hibiscus cannabinus L.) before visible symptomsoccur, and (2) compare two different fluorometers.If fluorometry can detect stress before visiblesymptoms are apparent, then possibly the soilchemical environment can be controlled to maximizecontaminant uptake, minimize plant damage, andultimately maximize contaminant extraction.

Kenaf plants were grown in potting soil artificiallycontaminated with lead. A constant concentration ofacetic acid was used to acidify the soil (necessary toextract the lead), while a variable concentration ofchelate (EDTA, ethylenediamine tetraacetic acid) wasused to bind the metal. A LICOR 6400 portablephotosynthesis system fitted with a leaf chamberfluorometer and the CFM-636973 fluorometer wereused to take fluorometric measurements at specificintervals after chelate/acid addition. Rfd, an indicatorof stress, was well correlated between both machines(r=0.83, n=162). It was possible to detect stressbefore the appearance of visible stress (spotting) onthe leaves with either fluorometer. However, becauselead stress caused severe leaf senescence to occurwithin 48 h after treatment, fluorometric analysis didnot give sufficient advance time to permit modificationof the soil contaminant environment and prevent leafdeath.

(B) Evaluating native woody plants for efficiency ofphytoremediation of chlorinated solventsInvestigators: L.A. Newman and researchers fromthe U.S. Forest Service and the University ofWashington

We have completed the study on the hardwoods. Whilethe poplars have always been used as a ‘standard’ forremediation potential, we found that sweetgum andsycamore performed as well if not better as far as wateruptake rates. We also saw higher levels of metabolitesin these plants than in the poplar. Regulators we spokewith showed a much greater interest in evergreen


48

species as an alternative to poplar, so we looked at avariety of evergreen plants rather than the herbaceousplants. As might be expected, there were much loweruptake rates in the evergreen species. This work hasbeen presented by the student at the Association forEnvironmental Health Sciences annual conference(2004), and the EPA International Phytotechnologiesmeeting (2005). We plan to run the herbaceous plantsthis fall, when greenhouse temperatures havemoderated.

(C) Comparing poplar cultivars for use in phyto-remediation in the SoutheastInvestigators: L.A. Newman and researchers fromthe U.S. Forest Service

Field work has been completed for this project. Welooked at parameters including survival, height andtruck diameter, as well as insect and disease resistance.We found that there was a great deal of variationbetween the plants, but that three were consistentlybetter suited for growth in the Southeast. This workhas been written up as two articles and submitted toBiomass and Bioenergy, and have been accepted forpublication. We will be collecting hardwood cuttingsthis fall from these clones and growing them in thegreenhouse to determine their water uptakeefficiencies.

Question 8:Can natural processes, such as succession, be directed oraccelerated to restore sustainable vegetation on impactedor remediation sites, including closure caps, floodplains,and isolated wetlands?

(A) Use of native vegetation on waste site closure capsInvestigators: B.S. Collins and K.W. McLeod

In October 2003, we initiated research to examineestablishment and maintenance of alternativevegetation on closure caps. Currently, caps on the SRSare vegetated by seeding or sodding turf grasses, whichmust be maintained by frequent mowing. In springand summer 2004, we surveyed existing caps to

identify extant vegetation. In addition, we began anexperiment to compare vegetation establishment usingalternative species (a common native grass[broomsedge, Andropogon virginicus] vs. a turf grass[centipede], sown with or without a naturalizedlegume [Lespedeza cuneata] that is common inoldfields on the SRS) planted in mesocosmsconstructed to mimic closure caps. Additionaltreatments, including watering, fertilizer, lime, mulch,and season of planting (spring vs. summer) wereapplied to test their effects on vegetation establishment.We also began a field plot study to determine if turfgrass or native broomsedge vegetation is moresusceptible to invasion by undesirable deep-rootedspecies. Plots were randomly assigned to mowing(mowing, no mowing) and loblolly pine addition(seeding, no seeding) treatments. The vegetationsurvey revealed that ‘natural’ oldfield herbaceousvegetation and woody species found in oldfields,sandhills, pine plantations, and upland pine forestscan establish within the planted grass community onclosure caps. The presence of the legume, mulch, andconsistent watering each resulted in significantlyhigher vegetation cover in the experimental capcommunities established in spring 2004; for summer2004 plantings, consistent watering did not increasevegetative cover. With consistent watering, sectionssown with turf grass seed had lower vegetation coverthan other watered combinations, though still muchhigher cover than any non-watered combination.Through winter and spring, 2005, no loblolly seedshad germinated in plots where they were sown. Weare continuing to monitor these plots. Overall, theinitial results indicate ‘natural’ vegetation is a suitablealternative to turf grass communities on closure caps,although consistent watering and presence of a legumemay increase cover with spring-planted native grassor turf grass vegetation.

(B) Succession in disturbed floodplain wetlands andsandhillsInvestigators: R R. Sharitz, K.W. McLeod, S.J. Harper,and B.S. Collins

From 1992 to 1994, species trials were initiated in


49

the Fourmile Branch delta to investigate the bestmethods of reestablishing tree species in a severelydisturbed, thermally-affected stream delta. Treatmentsexamined included planting stock type, habitat, treeshelters, root pruning, and competition controls.Survival of most species, as determined in 1994 or1996 and 2003, changed little over the past decade,and was not strongly affected by the treatments withina trial, except for root pruning. Trees in manytreatments have grown tremendously, but individualswith no competition controls generally grew moreslowly. For example, baldcypress (Taxodiumdistichum Richard) has had a high survival rate,regardless of whether they were planted as barerootor balled-and-burlapped saplings, and have grownto 8-12 meters in height. Overcup oak (Quercuslyrata Walter), water hickory (Carya aquatica(Michaux f.) Nuttall), Nuttall oak (Q. nuttalliiPalmer), and willow oak (Q. phellos L.) planted inlater trials, also had adequate survival rates and havegrown to 5+ meters. Low mortality rates after theinitial 3-4 years suggest that these species areappropriate for restoration. In contrast, survival ofwater tupelo (Nyssa aquatica L.) and green ash(Fraxinus pennsylvanica Marshall) have continuallydeclined over time, making these species less suitablefor successful restoration. These results werepresented at the Thirteenth Biennial SouthernSilviculture Research Conference and will bepublished in the proceedings of this meeting.

Sandhills, nutrient-poor habitats with sandy, xericsoils, support a unique flora and fauna, including asuite of threatened, endangered, and sensitive (TES)plant and animal species. Many of these habitats havebeen disturbed by former and current land-usepractices and by SRS management activities. We usedhigh-resolution satellite images and aerialphotography in a geographic information system(GIS) database to extract sandhills from surroundingpine woodlands and managed forests of the SRS.These sandhills sites were surveyed for vegetation andsoil characteristics (moisture, texture, pH, andnutrients) and were compared with sandhills habitatsfrom other federal installations in the region. During

FY04 we completed TES plant surveys and lab analysesof soils. We now are using this information to developpreliminary TES habitat models. In addition, we haveplanted focal TES plants into experimental gardens toexamine the effects of land uses (logging, human footand vehicle traffic) on plant survival and growth. Wealso have implemented an experiment to study theeffects of forest management (fire, herbicide,shredding) on a sandhills community.

(C) Survey of plants colonizing coal ash basinsInvestigators: L.A. Newman and faculty from theUniversity of Kentucky

We have completed the survey of the coal reject basin,looking at types, locations, and frequency of vegetation,metal levels in the growth matrix, and metal levels inthe plants. What we found was that early successionplants such as blackberry and bayberry did well onthe site, and were found in multiple areas, even thosewith low pH. More sensitive plants such as poplar wereonly found in areas where pH levels were morestabilized. None of the plants studied showed metaltissue levels that would characterize them asaccumulator plants, and thus potential issues with foodchain transfer do not appear to be an issue. This workis in the process of being written up for publication.

(D) Restoration of isolated depression wetlandsInvestigators: R.R. Sharitz and B.E. Taylor

Many Carolina bays and other isolated wetland pondson the SRS were ditched, cleared, and otherwisealtered by landowners prior to the establishment ofthe SRS. In 1998, the Carolina Bay Restoration Projectwas initiated by SREL, the U.S. Forest Service, the U.S.Fish and Wildlife Service, and several universities.Sixteen severely degraded Carolina bays areundergoing experimental restoration, and DOE-SR willreceive credits to its wetland mitigation bank for thisproject. After pre-treatment studies in 1998-2000, thehydrology of these bays was restored in 2000-2001by plugging drainage ditches. Vegetation treatmentsincluded clear-cut removal of invasive woody speciesand planting of wetland herbaceous or forest species


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into the basins. In FY04, the third year of post-restoration, vegetation analysis revealed an increasein obligate and facultative wetland species to 50%(compared with 28% prior to restoration treatments),coupled with a decline in overall plant species richnessas invasive upland species were extirpated.Comparison of pre- and post-restoration invertebrateassemblages revealed increases of 5-10 or moremicrocrustacean species in most ponds wherehydroperiod was affected substantially. Other predictedeffects (increase of species richness with conversionfrom forested to herbaceous vegetation, loss ofephemeral habitat specialists with increasedhydroperiod) have not yet been observed. Post-treatment monitoring of the vegetation andinvertebrates was completed, providing the dataneeded for assessment of restoration success.

Vegetation research was supported by a grant fromUSDA Forest Service. Papers characterizing plantcommunities of reference depression wetlands werepublished in Wetlands and Journal of the TorreyBotanical Society. A manuscript on vegetationresponse to restoration treatments was submitted inFY05.

Research on aquatic invertebrates was also supportedby a grant from USDA Forest Service. Bimonthlyinvertebrate monitoring (3 years pre-treatment; 4years post-treatment) was completed in December2005. Sample processing is complete for micro-crustaceans, nearly complete for macroinvertebrates.Stable isotope studies to compare trophic position ofinvertebrates between forested and herbaceouswetlands will be completed in FY06. We published apaper in Journal of the North AmericanBenthological Society that uses the pre-treatmentmacroinvertebrate data in a comparison of wetlandcommunities on a continental-scale gradient (Canadato Florida). We published an invited research reportin Ecological Restoration showing the rapid responseof microcrustacean species richness in the treatmentwetlands. We submitted a manuscript to Wetlands ontrajectories of change in microcrustacean assemblagesin Bay 93, another restored wetland, over an 8-year

post-treatment period. We also published two generalaccounts of aquatic invertebrates in the SRS: a NationalEnvironmental Research Park report onmicrocrustaceans of the SRS and a chapter on aquaticinvertebrates of the SRS.


Research Support Programs

� Environmental Health and Safety Program� Distance Learning Program� Quality Assurance Program� Research Data Archive Activities� SREL Undergraduate and Graduate Education Programs� Environmental Outreach Program� DOE Research Set-Aside Areas

Several SREL programs provide critical support to the research, outreach, andeducation missions of the Laboratory. These support programs include:

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Environmental Health and SafetyProgramDonald R. Mosser and Vivian G. Dicks

The Savannah River Ecology Laboratory continues tooperate successfully under the work-smart safety andenvironmental standards that resulted from SREL’sparticipation in DOE’s Necessary and Sufficient process.These standards continue to address the hazards associatedwith SREL operations by permitting a focused effort on thehealth and safety issues most pertinent to SREL operations.

SREL maintains a commitment to two full-time employeepositions dedicated to the support of the SRELEnvironmental Health and Safety (EH&S) Program.Approximately 23 laboratory research technicians alsoprovide support to the SREL EH&S Program by serving aslaboratory Chemical Coordinators. Chemical Coordinatorsare responsible for maintaining chemical inventoryinformation and providing support in the identification,accumulation, and storage of hazardous wastes.

In an effort to increase the efficiency and effectiveness ofthe SREL EH&S Program, an emphasis continues to beplaced on safety and environmental training of SRELpersonnel. New personnel safety and environmentalorientation was presented to approximately 40 individualsduring the past year. Additionally, training was providedfor Chemical Coordinators and hazardous waste workers.WSRC provided Radiological Worker and radiationgenerating device training to SREL personnel as necessary.

SREL’s internal computer network was used to providetargeted safety information to specific work groups withinthe laboratory. Lessons learned and health and safety topicswere distributed via e-mail throughout the year. Safetytraining literature was also made available in break roomsand hallway literature racks.

Facility inspections remain a cornerstone of the SREL SafetyProgram. SREL personnel conducted regularly scheduledfacility inspections. SREL also conducted assessments inthe areas of chemical and radiological air emissions,community right-to-know, and the Georgia Right-to-Knowlaw in compliance with state and federal requirements.

Safety Services personnel reviewed and approved morethan 265 chemical purchases. SREL continues to supportthe SRS Environmental Management System as a signatoryto the SRS Environmental Management System PolicyStatement.

Waste minimization and chemical disposal issues continueto be emphasized to increase efficiency and costeffectiveness. Waste minimization techniques such assource reduction and bench-top treatment continue to beincorporated into experimental protocols, reducing theburden associated with waste disposal procedures whilesupporting SREL’s pollution prevention efforts. Incooperation with WSRC Solid Waste Division, SRELsuccessfully shipped approximately 234 pounds of excesshazardous laboratory chemicals and laboratory hazardouswastes to a hazardous waste disposal firm (May 2005).

In support of SRS safety programs, SREL participated inthe development of corrective action plans related to theSRS Lessons Learned program and the development of thenew SRS visitor control programs. As part of the new SRSvisitor control program, SREL participated in a sitewidereview of each organization’s work activities and associatedwork hazards as they relate to visitors and subcontractors.SREL continues to move forward with implementation ofthe new SRS visitor control requirements.

Distance Learning ProgramLaura Janecek and Marie Hamilton

The Savannah River Ecology Laboratory maintains a state-of-the-art Distance Learning (DL) facility that delivers two-way audio and visual transmissions via a T1 line. This facilityis part of the Georgia Statewide Academic and MedicalSystems (GSAMS), a cooperative and collaborative distanceeducation network in the state of Georgia with about 300interactive audio and videoconference classrooms. SREL’sDL Facility provides the capability to communicate withother distance learning users throughout the country. SRELuses DL for classroom instruction for a Master’s degreeprogram, other graduate courses, outreach presentations,graduate student committee meetings, faculty meetings,and staff briefings. The primary distance learning program

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for SREL is the multidisciplinary Master of Science degreein Environmental Toxicology, offered in cooperation withthe UGA School of Pharmacy. This is the first degree offeredby UGA through any distance learning site. Eight studentshave completed all required coursework and are workingon the research component of the degree, and fourstudents have graduated from the program. Because ofincreased costs associated with administration of thisdegree program, it is likely that it will no longer be offeredafter FY05.

The SREL Outreach Program has been using DL technologyto extend their programming because they can reachmultiple classrooms as well as minimize travel and animalhandling time by using this facility. Outreach personnelpresented 5 lectures on various ecological topics tostudents in South Carolina and Georgia K-12 schools. ManyGeorgia schools have not been able to maintaincompatibility with GSAMS and the Georgia audiences aredeclining.

Quality Assurance ProgramLaura Janecek

SREL has continued to maintain a formal, U.S. Departmentof Energy (DOE)-approved Quality Assurance (QA)program. The program is devoted to assuring thecontinuing quality of SREL research. These SREL “GoodResearch Practices” highlight research concepts andcontext, research logistics, and the conduct of researchand are available to all SREL personnel on the Lab’s intranetweb site. All new Laboratory research personnel arerequired to familiarize themselves with this material priorto beginning work at SREL.

Research Data Archive ActivitiesLaura Janecek and Debbie Reese

Responsible management of research data holdings playsan important role in preserving the SREL’s corporatememory. Since 1989, SREL has been actively building acentralized repository of research data files and theassociated “metadata” necessary to make these data fully

accessible. The goals of SREL’s Research Data Archiveactivity are to avoid the inadvertent loss of data and to useadvanced electronic computer/communication technology,including the use of computer networks and the Internet,to provide access to important data as efficiently aspossible. Inclusion of new and historical researchinformation into the SREL data archives continued duringFY05 and the Central Archive Data Repository now hasinformation covering over 511 separate studies.

The web-based SREL data archive system that allows usersto upload metadata information and actual data filesdirectly from their office desktop computers continued towork well during FY05. Anyone at SREL or on the SRS cansearch for data using this new web-based system, howeverdecisions about releasing original data to third parties areretained by the principal investigators.

SREL Undergraduate and GraduateEducation ProgramTravis Glenn

The objective of the SREL Education Program is to promoteprofessional development and enhance environmentalawareness among undergraduate and graduate studentsthrough research participation and training programs withemphasis on conducting ecological research important tothe Savannah River Site mission. Undergraduate andgraduate student participants in FY05 are listed in Table 1(pages 54-55).

The SREL Education Program has averaged 18undergraduate students per year since 1968. Thesestudents, from over 275 different colleges and universities,have been co-authors on 150+ peer reviewed researchpublications; more than 200 of these students have goneon to pursue careers in science. The Undergraduate-Research Experience for Undergraduates, funded by theNational Science Foundation, sponsored 11 students thispast year. In addition, we sponsored three student fundedby NOAA, two students funded by SREL, and two funded bySouth Carolina State University.

Since 1967, an average of six students a year have

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Student Academic Institution Faculty AdvisorAdam David Borden Louisiana State University, LA Steven HarperDanielle Denise Britt University of South Carolina, Aiken Gary MillsCaleb Ryan Caton University of South Dakota, Vermillion I Lehr BrisbinLatasha Yolanda Cofer Voorhees College, SC Travis GlennAnthony Brian DeMarco University of Georgia, Athens John SeamanCeleste Tara Holz-Schietinger The Evergreen State University, WA Travis GlennTravis Johnson Reinhart College, GA Charles Jagoe/J Vaun McArthurRamirez Ezell Lewis Fort Valley State University, GA Charles Jagoe/J Vaun McArthurRachel Diane Mahan University of Missouri, Columbia J. Whitfield GibbonsDeidra Marie McCray South Carolina State University Gary MillsNina Marie Menichino University of Wales, Bangor, United Kingdom Andrew NealAndrea LaToya Richardson South Carolina State University J Vaun McArthurKaren Jean Schlatter McGill University, Montreal, Canada J. Whitfield GibbonsAlexa Michel Schmitz Oberlin College, OH Lee NewmanJennifer Marlene Summerlin Augusta State University, GA Charles JagoeTara Denice Sumter South Carolina State University Gary MillsJennifer Marie Thomsen Clemson University, SC Christopher RomanekMarshall Therdrick Washington Claflin University, SC Charles Jagoe/J Vaun McArthur

Table 1A. SREL Undergraduate Student Program Participants

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completed graduate studies at SREL and over 315dissertations and theses have been written. During FY05,eight students completed their degree requirements (fourM.S. and four Ph.D.). Since 1985, our graduate studentshave won over 200 awards from regional, national, andinternational competitions at numerous professional

SREL Graduate Students Completing Degree Requirements in FY05:Heather Brant M.S. University of Georgia 2004 Charles JagoeSarah Drake M.S. University of Georgia 2004 Rebecca SharitzSusanne Hauswaldt Ph.D. University of South Carolina 2004 Travis GlennOlga Tsyusko Ph.D. University of Georgia 2004 Michael Smith/Travis GlennSusan Turner Ph.D. University of Georgia 2004 Rebecca SharitzAngel Kelsey Wall M.S. University of Georgia 2004 Charles Jagoe/John SeamanNeera Young M.S. University of Georgia 2004 Michael SmithYong Jin Lee Ph.D. University of Georgia 2005 Christopher Romanek

societies and foundations. During the past year, ourgraduate students continued to compete successfully forvarious national and regional awards. Some of these arelisted in the section on Special Accomplishments (page5).


Student Degree Institution Faculty AdvisorKimberly Andrews Ph.D. University of Georgia, Athens J. Whitfield GibbonsElizabeth Burgess Ph.D. University of Georgia, Athens Andrew NealMonica Carroll Ph.D. University of Georgia, Athens Christopher Romanek*Mara Cea Ph.D. La Frontera University, Chile Gary Mills*Alcides Cintra Ph.D. Universidade Estadual Paulista, Brazil Domy AdrianoDana Cook Ph.D. University of Georgia, Athens Andrew NealSarah DuRant M.S. University of Georgia, Athens William HopkinsWilliam Duval Ph.D. University of Georgia, Athens Rebecca SharitzCarol Flaute M.S. University of Georgia, Athens Steve HarperGabrielle Graeter M.S. University of Georgia, Athens J. Whitfield GibbonsAaliyah Green M.S. University of Georgia, Athens Charles JagoeEllen Hepfer M.S. University of Georgia, Athens Machelle WilsonRyan Holem M.S. University of Georgia, Athens William HopkinsCori-Alice Holladay M.S. Appalachian State University, NC Beverly CollinsGlenn Kirkland M.S. University of Georgia, Athens I Lehr BrisbinYong Jin Lee Ph.D. University of Georgia, Athens Christopher RomanekFrantisek Majs Ph.D. University of Georgia, Athens John Seaman*Mirela Matioc Ph.D. University of Georgia, Athens Travis Glenn*Clint Page Ph.D. University of Georgia, Athens Andrew NealGretchen Peltier Ph.D. University of Georgia, Athens William HopkinsJohn Peterson M.S. Auburn University, Alabama William HopkinsSteven Schaff Ph.D. University of Georgia, Athens Kenneth McLeodJulian Singer Ph.D. University of Georgia, Athens John SeamanAmy Squire M.S. University of Georgia, Athens Rebecca SharitzKarolina Stark Ph.D. University of Stockholm, Sweden Thomas Hinton*Steven Stoddard Ph.D. University of Illinois Rebecca SharitzBrian Todd Ph.D. University of Georgia, Athens J. Whitfield GibbonsOlga Tsyusko Ph.D. University of Georgia, Athens Michael Smith/Travis GlennJoy Van Nostrand Ph.D. Medical University of South Carolina Paul BertschQin Wang Ph.D. University of Georgia, Athens Machelle WilsonArlena Wartell Ph.D. University of Georgia, Athens Travis GlennJamie Williams M.S. University of Georgia, Athens Barbara TaylorJohn Willson Ph.D. University of Georgia, Athens J. Whitfield GibbonsChristopher Winne Ph.D. University of Georgia, Athens J. Whitfield GibbonsMeredith Wright Ph.D. University of Georgia, Athens J Vaun McArthurQi Ye Ph.D. University of Georgia, Athens Chuanlun ZhangWeidong Zhao Ph.D. University of Georgia, Athens Chuanlun Zhang

* Short-term students – did not complete degree while part of the SREL Education Program

Table 1B. SREL Graduate Student Program Participants

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Environmental OutreachKenneth McLeod

As a unit of The University of Georgia, the Savannah RiverEcology Laboratory has the tripartite missions of research,outreach, and education. These three missions arecomplimentary and have strongly contributed to SREL’sposition as a respected, unbiased source of informationin the Central Savannah River Area. The intellectualindependence of the academician assures the localcommunity of objective research on the impacts of siteoperations on SRS and regional ecosystems. Added to thisindependence is the dedication and enthusiasm of the SRELEnvironmental Outreach Program, which can be easilyassessed by their Vision – To translate ecologicalknowledge into public passion for science – and Mission– To increase the understanding of science bycommunicating ecological knowledge to the publicthrough educational programs, products, publications,and the media.

SREL uses information from its own research efforts toeducate the public locally, regionally, and nationally. Issuesas diverse as amphibian and reptile population declines,potential responses of organisms to contamination,distribution and abundance of sensitive species, monitorednatural attenuation, and dispersal of organisms fromradioactively or chemically contaminated sites all areimportant beyond SREL.

The Outreach Program is designed to enhance SREL’soverall mission of acquiring and communicatingenvironmental knowledge and addresses the U.S.Department of Energy’s (DOE) current focus onenvironmental issues. Public education, especially for K-12 audiences, is accomplished through a variety ofprograms.� During the past year, SREL scheduled 247 talks, 14

tours, 19 exhibits, and 62 workshops, reaching a totalof 64,761 people. Topics for these presentationsincluded biodiversity, the process of science, animaladaptation, plants and wetlands, chemistry andenvironmental science, local ecosystems andconservation, classification, environmental steward-ship, and careers in ecology and research.

� Over 325 4th and 5th grade students and 16 teachersat three Aiken County and one Lexington Countyelementary schools participated in 5 individual classscience workshops and a daylong field trip. Scientificinquiry and process skills were stressed; studentsworked in groups, conducted experiments, andrecorded and analyzed data.

� School groups enjoy field trips to the Laboratory’sConference Center to participate in the Ecologist-for-a-Day program.

� Teachers are trained in methods of teaching ecologyduring workshops and are provided with materialsproduced by the Outreach staff.

� Outreach speakers have made 5 presentations tomultiple classrooms through the use of the DistanceLearning Facility. Many Georgia schools have not beenable to maintain compatibility with GSAMS and theGeorgia audiences are declining.

� An internal laboratory newsletter, The Grape Vine, isdistributed electronically, 12 times per year.

Outreach programs include “Ecotalk,” an opportunity forstudents to have nature brought into their classroom for aface-to-face lesson on a variety of live animals found inlocal habitats. These presentations, offered in schools,emphasize hands-on scientific learning using activities inthe environmental sciences. “Ecologist for a Day” visitsallow students to spend the day in the field gaining “hands-on” knowledge of the plants and animals of the uniqueUpper Three Runs Creek area. Participants get anopportunity to work with SREL researchers catching,marking, and measuring various species of reptiles,amphibians, small mammals, and invertebrates. Inaddition, the Outreach Program offers tours of SRELfacilities and surrounding field sites, as well as exhibitsand workshops for the general public.

During the past year SREL completed its new “research-to-classroom” hands-on science program for elementaryschool students. With funding from the American HondaFoundation (AHF) and partial funding from TheChristensen Fund , SREL presented five workshops to every5th grade class at East Aiken and Greendale Elementaryschools, and to every 4th grade class at J.D. LeverElementary and Midland Valley Charter Schools in Aiken

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County; workshops were also presented to two 4th gradeclasses at Batesburg-Leesville Elementary School inLexington County. In each two-hour classroom session,SREL staff showcased one or two SREL researchersconducting studies in subject areas related to the topic ofthe day, and students learned about on-going research byscientists working in their community. Students thenperformed hands-on activities that reinforced the content,as well as scientific inquiry and process skills. All activitieswere linked in some way to local habitats as well as toSouth Carolina and national science standards. At theconclusion of the program, all students participated in adaylong field trip and acted as “junior scientists” by usingthe skills taught in the classroom in an outdoor setting.Funding for this program was not renewed for FY06 byThe American Honda Foundation, so SREL is seekingfunding to continue this popular program from othersources.

Associated with the AHF funding, SREL continued a website(www.kidsdoscience.org) that provides all the necessarymaterials for the 10 hands-on activities used in theclassroom workshops. This website serves as an excellentresource for any teacher who may want to use the activities/materials. Also as part of the AHF funding, additionalprograms were also given to general audiences such ascivic, scout, and church groups, as well as at communityand regional events. Many of these programs continued toemphasize “the methods of science,” but additional topicsincluded watershed processes and pollutants, groundwatercontamination, phytoremediation, radioecology, andcreation and restoration of seasonal wetlands.

SREL is a founding member of the Central Savannah RiverArea Environmental Science Education Cooperative (CSRAESEC), which sponsors an annual EcoMeet as well as otherscience-related programs throughout the year. This year’sEcoMeet had 28 teams from regional middle schoolsparticipating in an ecological science-bowl. Next year, theRichmond County (Georgia) Board of Education willrequire all county middle schools to participate in theEcoMeet. Participation in ESEC has provided theopportunity to showcase SREL at Augusta’s Fort DiscoveryScience Museum and also has provided an opportunityfor speakers from the Lab to address groups at Fort

Discovery.

SREL Outreach members have also provided expertise tothe Aiken County Watershed Alliance (ACWA), a localcommunity group, and the Central Savannah River AreaRegional Science and Engineering Fair, Inc., anorganization that serves an 18-county area of SouthCarolina and Georgia. In addition, Outreach memberseducated local and state leaders on the ecological value ofseasonal wetlands, and accompanied personnel of theSouthern Environmental Law Center on visits to regionalnewspapers to highlight the importance of seasonalwetlands.

Thousands of copies of educational products and materialsare distributed nationwide to schools, organizations, andthe general public. Educational materials include two six-foot-long full-color posters describing the importance ofwetlands to reptiles and amphibians, along with teachers’guides. The full-color brochure Snakes of Georgia andSouth Carolina (currently in its fifth printing) has provedto be an extremely successful educational product thatreflects positively on DOE and the SRS. The book has beenplaced at no charge in every public library in Georgia andSouth Carolina and is also widely distributed at no cost tohospital emergency rooms, veterinary clinics, ambulanceservices, classrooms, scout leaders, and to various otherorganizations such as the Boys and Girls Clubs in Aikenand Augusta. Articles referencing the book have appearedin numerous newspapers and magazines includingpublications in Florida and Texas.

The Outreach Program also continued to distributeeducational materials including fliers on CarnivorousPlants and Their Habitats; An Amphibian’s Eye View ofWetlands; and Is it a Water Moccasin?; a children’s comicbook entitled Stepping into Ecology: the EcologicalAdventures of Mud E. Boot; a Chemistry – it’s all aboutthe nature of things sticker, the Metric System Rapbookmark; and an emergency services calendar thatdepicts plants of the SRS (produced in cooperation withWestinghouse Savannah River Company). All of theseproducts have been extremely popular and thousands ofcopies have been distributed during the past year. Alsodistributed was The National Environmental Research

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Park at Savannah River Site: Serving an EssentialMission for 25 Years. The National EnvironmentalResearch Park (NERP) brochure highlights 25 years ofresearch associated with the NERP program at theSavannah River Site. Full-color fact sheets and research“snapshots” on a wide variety of research topics arepublished and distributed as well.

The public relations component of the Outreach Programincludes the distribution of news releases on a variety oftopics to selected media affiliates, officials of DOE, andThe University of Georgia. In FY05 SREL researchersprovided information to such diverse media outlets asScience, New York Times, The Washington Post, Fortune,MSNBC, National Geographic News, The Times-Union,Nature (Jacksonville, FL) The Christian Science Monitor,The Chronicle of Higher Education, the Associated Press,Reptiles, The Geology Society News, Skirt (a woman’sweekly), Wildlife Magazine, National Wildlife, OutdoorLife, Georgia Magazine, The Vivarium, Hi (an Arabiclanguage magazine),and many local news outlets in theSoutheast such as The Atlanta Journal-Constitution, TheAugusta Chronicle, The Charlotte Observer, The AthensBanner-Herald, Beaufort Gazette, The Charleston Postand Courier, The State, The Aiken Standard, SavannahMorning News, The Island Packet, and the AshevilleCitizen News. Online coverage has included such sites asCharlotte.com, Audubon, Environmental News Network,Lowcountry Now, EurekAlert, Topix.net. AccessNorthGA.com, Ascribe, dateline.columbia, GoUpstate.comand others. Topics in the news ranged from animalbehavior to environmental impacts.

The Public Relations office screens most inquiries fromthe press, directing reporters to the most appropriateresearchers for their stories. In addition, SREL initiatespress contacts, maintains a website for reporters andsubmits releases and stories to appropriate websites andmagazines, including alumni magazines.

DOE Research Set-Aside AreasCharles E. Davis

The Savannah River Site (SRS) is a National EnvironmentalResearch Park (NERP) and its large land area andcontrolled public access provide a diverse and protectedoutdoor laboratory where researchers study theenvironmental impacts of the SRS’s industrial and forestmanagement operations. Because these studies are usuallylong-term, they require relatively undisturbed areas as“control” sites where reference, baseline data can beobtained. Known as Research Set-Aside Areas, thesereference sites have been withdrawn from the SRS’scommercial forest and set aside by the Department ofEnergy (DOE) primarily for non-manipulative ecologicalresearch and educational outreach activities. These areasalso serve as “reserve” areas that represent excellentexamples of both the typical and unique plant communitiesindigenous to the SRS while providing critical habitat forthe Site’s threatened, endangered, or sensitive (TES) floraand fauna. Currently, there are 30 Set-Aside Areas on theSRS that collectively account for approximately 14,100acres (5,706 ha), or 7% of the Site. Individually, they rangein size from 10 acres (4.05 ha) to 7,400 acres (2.995ha), and are located in 43 of the Site’s 89 timber resourcecompartments. There are approximately 270 miles (435km) of posted boundary line associated with these Set-Aside Areas.

Periodic inspections of Set-Aside boundary postings areconducted by SREL where potential land-use conflicts orimpacts are anticipated. SREL and the USFS-SR cooperatein verifying and updating Set-Aside boundary lines that areconcordant with prescribed forest stand treatments. Todate, boundaries for 12 of the 30 Set-Aside Areas havebeen updated using GPS (Global Positioning System)technology; significant portions of the E. P. Odum Wetland(Area No. 30) and Ruth Patrick/Meyers Branch (Area No.11) Set-Asides have been completed as well. Because thisGIS boundary layer continues to be a dynamic, workinglayer, SREL provides updates of the Set-Aside boundarylayer to the USFS-SR upon request. SREL updated thisboundary layer, including the metadata, and released itinternally in FY05.

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Management of the Set-Aside Areas

SREL’s Set-Aside Research Coordinator is responsible fordeveloping, writing, and implementing stewardshipmanagement plans for each of the 30 Set-Asides. Set-AsideAreas that have an anticipated management need in thenear future (e.g., plantation first thinning, adjacentproperty management concern) are scheduled formanagement plan development. Management plans for Set-Asides are generally developed by a team of individualsfrom various SRS organizations. When necessary,researchers with individual expertise or with a long-termresearch interest in a Set-Aside may be requested toparticipate as a core team member.

During the past year, SREL worked on five managementplans, completing plans for Rainbow Bay AmphibianReserve Set-Aside Area (Area No. 16) and the Risher PondSet-Aside. Plans for the Craig Pond/Sarracenia Bay Set-Aside (Area No. 17), UGA Old Lab Site (Area No. 2), andGinger’s Bay Set-Aside (Area No. 19) were also workedon. These plans were expanded this year to addressresearcher safety, nuisance flora and fauna, and ecolitterissues.

Some accomplishments for the Set-Aside program for thisFY included marking 17 acres (6.9 ha) of pine for removalas a first thinning treatment in the Rainbow Bay Set-Aside.A major milestone was accomplished this year when a 30-acre (12.2 ha) pine thinning treatment was conducted inthe Flamingo Bay Set-Aside (Area No. 21), marking theactual implementation of a management plan. In theGinger’s Bay Set-Aside, a pine stand was approved for firstthin marking pending Set-Aside Task Group approval.Although researchers requested that Steel Creek Bay (AreaNo. 08) and Ellenton Bay (Area No. 01) be burned thispast year, the treatments were not done due to renewedrains. In the Craig Pond/Sarracenia Bay Set-Aside, adefensive fire line was installed within the Set-Aside as ameasure of control when Chem Nuclear conducted aprescribed burn in the spring. The road running throughEllenton Bay was cleared of vegetation to facilitate tours;researchers initiated trapping to control beaver and ottersin this Set-Aside because of the impact these animals werehaving on herpetofaunal research. Finally, additional

acreage (yet to be determined) was added to the OakHickory Forest (Area No. 12) and the Upper Three RunsCreek Set-Asides following a USFS-SR ID teamrecommendation. It was suspected that the BeechHardwood Set-Aside (Area No. 6) is being impacted by Pbcontaminants from an upslope 1950s pistol range andmonitoring was initiated by WSRC.

Research in Set-Aside Areas

Set-Asides continued to be increasingly used by researchersthis FY both as research study sites and as reference sitesfor collections of uncontaminated plants, animals, soils,or water. Nine theses and dissertations were published thisyear with study sites that included Set-Asides. Manypublications were devoted to research in depressionalwetland Set-Asides. A new plant study was initiated this FYin which two Set-Aside Areas are being used as controls toevaluate impacts from military training and forestmanagement on TES species. A number of publicationsused Set-Aside areas as controls when evaluating ecologicalrisks, contaminant transport, and site remediation.

Groups other than SREL also used Set-Aside Areas. SRNLused organisms collected in Set-Asides to test methods ofevaluating remediation and restoration actions as well asthe development of terrestrial bio-assessment protocolsat DOE sites. Archaeologists with the USC-Savannah RiverArchaeologist Research Program expanded theirinvestigations at the Flamingo Bay Set-Aside this year. Incooperative efforts, SREL and USFS-SR researcherscontinued to study coarse woody debris decomposition,softmast production in bottomland hardwood forests, andthe role of fleshy fruit production, consumption, anddispersal on promoting biological diversity.

FY05 SREL documents, publications, theses, anddissertations that used DOE Set-Aside Areas

Thirty-two documents were published this year reportingactivity and research from the Set-Aside Areas. Recent SRELand other SRS agency documents, scientific publications,theses, and dissertations that used or referenced DOE Set-Aside Areas during FY05 include:

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Andrews, K. 2004. Interspecific comparisons of behavioral responsesof southeastern snakes to roads. M.S. Thesis. University ofGeorgia, Athens, Georgia, USA.

Batzer, D.P., S.E. Dietz-Brantley, B.E. Taylor and A.E. DeBiase. 2005.Evaluating regional differences in macroinvertebratecommunities from forested depressional wetlands across easternand central North America. Journal of North AmericaBenthological Society 24:403-414.

Bertsch, P.M. and C.L. Strojan. 2004. Science in support of assessingecological risk and implementing novel remediation strategiesat the Savannah River Site, SC. Waste Management 2004Conference, February 29-March 4, 2004, Tucson, AZ.

Burbage, L.E. 2004. Environmental gradients and plant speciesdistributions with Carolina bay wetlands. M. S. Thesis. Universityof Georgia, Athens, Georgia, USA.

Comer, C.E. 2005. An analysis of spatial and genetic populationstructure in white-tailed deer and implications for management.Ph.D. Dissertation. University of Georgia, Athens, Georgia, USA.

Collins, B.S., JV. McArthur and R.R. Sharitz. 2004. Plant effects onmicrobial assemblages and remediation of acidic coal pile runoffin mesocosm treatment wetlands. Ecological Engineering23:107-115.

Davis, C.E. 2005. Stewardship management plans for the RainbowBay Amphibian Reserve Area Set-Aside on the Savannah RiverSite Site. Savannah River Ecology Laboratory, University ofGeorgia, Aiken, SC.

Davis, C.E. 2005. Stewardship management plans for the Risher PondSet-Aside on the Savannah River Site Site. Savannah River EcologyLaboratory, University of Georgia, Aiken, SC.

De Steven, D., and M.M. Toner. 2004 Vegetation of Upper CoastalPlain depression wetlands: Environmental templates and wetlanddynamics within a landscape framework. Wetlands 24:23-42.

Harmon, S.M., J.K. King, J.B. Gladden, G.T. Chandler and L.A. Newman.2005. Mercury body burdens in Gambusia holbrooki andErimyzon sucetta in a wetland mesocosm amended with sulfate.Chemosphere 59:227-233.

Harper, S.J. and R.R. Sharitz.2005. Delineating Sandhills communities:The use of advanced techniques to extract features from satelliteimagery. In Bettinger, P.C.C Hyldahl, S.D. Danskin, J. Zhu, Y.Zhang, W.G. Hubbaard, T. Lowe, M. Wimberly, and B. Jackson.(eds.) Proceedings of the 4th Southern Forestry and NaturalResources GIS Conference, December 16-17, 2004. Athens, GA.Warnell School of Forest Resources, University of Georgia, AthensGA.

Hopkins, W.A., C.T. Winne and S.E. DuRant. 2004. Differentialswimming performance of two natricine snakes exposed to acholinesterase-inhibiting pesticide. Environmental Pollution133:531-540.

Jenkins, C.A. 2004. Investigating growth patterns in Kinosternomsubrubrum species of turtle. M.S. Thesis. University of NorthCarolina at Chapel Hill. Chapel Hill, North Carolina, USA.

Jin, V.L. 2004. Soluable organic nitrogen in plant and soil processesin the upper coastal plain. Ph.D. Dissertation. University ofGeorgia, Athens, Georgia, USA.

Kelsey-Wall, A. 2004. Evaluation of a Tritium Irrigation Site: A FieldStudy, Assessment of the Biological Half-Life and evaluation ofOxidative Stress Effects in Mice with Low-Level, Oral Exposureto Tritium. M.S. Thesis. University of Georgia, Athens, Georgia,USA.

Kelsey-Wall, A., J.C. Seaman, C.H. Jagoe, C.E. Dallas and K.F. Gaines.2005. Rodents as receptor species at a tritium disposal site.Journal of Environmental Radioactivity 82:95-104.

Kilgo, J.C. and J.I Blake. (In press). Ecology and management of aforested landscape: Fifty years of natural resource stewardshipon the Savannah River Site. A publication of the U.S ForestService-Savannah River.

Kwit, C., D.J. Levey and C.H. Greenberg. 2004.Contagious seeddispersal beneath heterospecific fruiting trees and itsconsequences. Oikos. 107:303-308.

Marcy, B.C., D.E. Fletcher, F.D. Martin, M.H. Paller, and M.J.M. Reichert.Fishes of the Middle Savannah River Basin: With Emphasis onthe Savannah River Site. University of Georgia Press.

Metts, B.S., W.A. Hopkins and J.P. Nestor. 2005. Interaction of aninsecticide with larval density in pond-breeding salamanders(Ambystoma). Freshwater Biology 50:685-696.

Mulhouse, J.M. 2004. Vegetation change in herbaceous Carolina baysof the Upper Coastal Plain: Dynamics during drought. M.S. Thesis.University of Georgia, Athens, Georgia, USA.

Paller, M.H., C.H. Jagoe, H. Bennett, H.A. Brant and J.A. Bowers. 2004.Influence of methylmercury from tributary streams on mercurylevels in Savannah River Asiatic clams. Science of the TotalEnvironment 325:209-219.

Paller, M.H., D.E. Fletcher, T. Jones, S.A. Dyer, J.J. Isely and J.W. Littrell.2005. Potential of largemouth bass as vectors of 137Cs dispersal.Journal of Environmental Radioactivity 80:27-43.

Parresol, B.R. 2004. Point and fixed plot sampling inventory estimatesat the Savannah River Site, South Carolina. Report. USDA ForestService, Savannah River, Aiken, SC.

Rinehart, B.D. 2004. The use of small mammals as indicators of heavymetal bioavailability in a contaminated riparian zone. M.S. Thesis.University of Georgia, Athens, Georgia, USA.

Roe, J.H., W.A. Hopkins and B.P. Jackson. 2005. Species- and stage-specific differences in trace element tissue concentrations inamphibians: implications for the disposal of coal-combustionwastes. Environmental Pollution 136:353-363.

Sever, D.M. and W.A. Hopkins. 2004. Oviductal sperm storage in theground skink scincella laterale holbrook (Reptilia: scincidae).Journal of Experimental Zoology 301A:599-611.

Taylor, B. E. and A. E. DeBiase. 2005. Are microcrustaceans usefulfor assessing success of wetland pond restoration? (SouthCarolina). Ecological Restoration 23:56-57.

Turner, S.A. 2005. Facilitation and competition between a nitrogen-fixing perennial legume, Lespedeza cuneata, and an annual,Heterotheca subaxillaris, in a South Carolina old field. Ph.D.dissertation. University of Georgia, Athens, Georgia, USA.

Unrine, J.M., C.H. Jagoe, A.C. Brinton, H.A. Brant and N.T. Garvin.2005. Dietary mercury exposure and bioaccumulation inamphibian larvae inhabiting Carolina bay wetlands.

60


61

Environmental Pollution 135:245-253.Willson, J.D., C.T. Winne and L.A. Fedewa. 2005. Unveiling escape

and capture rates of aquatic snakes and salamanders (Sirenspp. and Amphiuma means) in commercial funnel traps.Journal of Freshwater Ecology 20:397-404.

Winne, C.T., M.D. Dorcas and S.M. Poppy. 2005. Population structure,body size, and seasonal activity of black swamp snakes(Seminatrix pygaea). Southeastern Naturalist 4:1-14.


Externally Funded Grants

PI Domy AdrianoProject Title SGER: Regulation of Metal Bioavailability in Floodplain Continuum by Carbon and Sulfur CyclingFunding Agency National Science FoundationBudget $99,906Period August 1, 2003–July 31, 2005

PI Paul M. BertschProject Title Linking Chemical Speciation, Desorption Kinetics, and Bioavailability of U and Ni in Aged-Contaminated Sediments: A

Scientific Basis for Natural Attenuation and Risk AssessmentFunding Agency US Department of Energy/Office of Science EMSPBudget $239,000Period February 1, 2005–July 31, 2006

PI Paul M. BertschProject Title Examination of Coastal Aquaculture Effluent and Receiving Water Quality Throughout the Tidal CycleFunding Agency US Department of Commerce/NOAABudget $94,094Period July 1, 2003–May 31, 2005

PI Paul M. BertschProject Title Tidal Creek Materials Loading for the South Carolina-Georgia LU-CES ProgramFunding Agency South Carolina Sea Grant ConsortiumBudget $67,255Period July 1, 2003–November 30, 2004

PI Paul M. BertschProject Title Tidal Creek Materials Loading for the South Carolina-Georgia LU-CES ProgramFunding Agency South Carolina Sea Grant ConsortiumBudget $60,514Period July 1, 2004–June 30, 2005

PI I. Lehr Brisbin, Jr.Project Title Ecological Studies of Birds in the Vicinity of the Augusta Regional Airport at Bush Field and the Messerly Wastewater

Treatment PlantFunding Agency The Augusta-Richmond County Consolidated GovernmentBudget $154,915Period October 1, 2003–September 30, 2005

PI I. Lehr Brisbin, Jr.Project Title CLEARMADDFunding Agency Centers for Disease Control and PreventionBudget $25,000Period August 1, 2002–July 31, 2005

PI I. Lehr Brisbin, Jr.Project Title Mercury studies on wading birds on the Georgia coastFunding Agency Savannah Presbytery MK Penetcostal FundBudget $8,200Period July 1, 2004–May 31, 2005

62


63

PI A. Lawrence Bryan, Jr.Project Title Determination of Wood Stork breeding success in Georgia in 2004Funding Agency USDI Fish and Wildlife ServiceBudget $8,325Period April 1, 2004–September 30, 2004

PI A. Lawrence Bryan, Jr.Project Title Determination of Use of Aquatic Roadside Habitats for Foraging by Endangered Wood Storks (Mycteria Americana)Funding Agency Georgia Department of TransportationBudget $24,964Period April 18, 2005–April 17, 2006

PI Beverly CollinsProject Title Thresholds of Disturbance: Land Management Effects on Vegetation and Nitrogen DynamicsFunding Agency Strategic Environmental Research and Development Program (SERDP)Budget $1,331,765 total ($33,500 in FY05)Period January 1, 2000–February 28, 2005

PI Beverly CollinsProject Title SEMP Integration ProgramFunding Agency Strategic Environmental Research and Development Program (SERDP)Budget $20,000 in FY05Period July 1, 2003–June 30, 2005

PI Beverly CollinsProject Title On-site Field Study Coordination, Data Acquisition, Data Analysis, Monitoring Support, and Technology Integration

Assistance in Support of Continuing Ecological StudiesFunding Agency US Department of DefenseBudget $90,000Period June 1, 2005–October 31, 2005

PI J. Whitfield GibbonsProject Title PARC ProgramFunding Agency USDA Forest ServiceBudget $39,000 in FY05Period July 2003–June 2005

PI J. Whitfield GibbonsProject Title Effects of Forestry Practices on the Gopher TortoiseFunding Agency Strategic Environmental Research and Development Program (SERDP)Budget $50,000Period January 2004–December 2004

PI J. Whitfield GibbonsProject Title PARC ProgramFunding Agency USDI Fish and Wildlife ServiceBudget $8,730 in FY04Period January 2004–December 2004

PI J. Whitfield GibbonsProject Title Development of Habitat Guidelines for HerpetofaunaFunding Agency USDA Forest ServiceBudget $53,400 in FY04Period corresponds to Cooperative Agreement (expires September 30, 2005)


64

PI J. Whitfield GibbonsProject Title Development, Production, and Distribution of Environmental Education Materials for Indigo Snake ProtectionFunding Agency USDI Fish and Wildlife ServiceBudget $39,916Period May 1, 2001–September 30, 2005

PI J. Whitfield GibbonsProject Title Cooperative Agreement: The Inventory Report for the Southeast Coastal NetworkFunding Agency USDI National Park ServiceBudget total award of $262,770Period April 1, 2001–April 24, 2006

PI J. Whitfield Gibbons and William HopkinsProject Title Sublethal Effects of Pesticide ExposureFunding Agency US Golf AssociationBudget $87,600 total awardPeriod February 1, 2001–January 31, 2005

PI J. Whitfield GibbonsProject Title A Primary Understanding of Our Environment: Teaching Kids the Science of EcologyFunding Agency The Christensen FundBudget $105,000Period August 1, 2002–August 1, 2005

PI J. Whitfield GibbonsProject Title Inventory of Herpetofauna for the Appalachian Highlands and Cumberland Piedmont Networks of the National Park

ServiceFunding Agency USDI National Park ServiceBudget $37,226Period September 9, 2002–December 31, 2005

PI J. Whitfield GibbonsProject Title A Primary Understanding of Our Environment: Teaching Kids the Science of EcologyFunding Agency American Honda FoundationBudget $69,080Period August 1, 2003–November 30, 2004

PI J. Whitfield GibbonsProject Title Collaborative Research: Land-use Practices and Persistence of Amphibian PopulationsFunding Agency National Science FoundationBudget $149,903Period May 15, 2003–April 30, 2006

PI J. Whitfield GibbonsProject Title Operation of the NARCAM WebsiteFunding Agency US Department of InteriorBudget $85,000Period August 1, 2003–August 31, 2005

PI J. Whitfield GibbonsProject Title Turtle Management on National Wildlife RefugesFunding Agency US Department of InteriorBudget $60,000Period March 1, 2004–December 15, 2005


65

PI J. Whitfield GibbonsProject Title Synthesis Report and Scientific Literature Review on Impacts of Roads on Reptiles and AmphibiansFunding Agency US Department of TransportationBudget $45,000Period September 10, 2004–September 26, 2005

PI J. Whitfield GibbonsProject Title Incidence of Chytrid Fungal Parasitism in Southeastern National ParksFunding Agency US Department of InteriorBudget $10,000Period September 13, 2004–September 40, 2006

PI J. Whitfield GibbonsProject Title Support of the PARC WebsiteFunding Agency National Fish and Wildlife FoundationBudget $3,243Period April 1, 2005–June 30, 2006

PI J. Whitfield GibbonsProject Title History, Ecology, and Demography of the Gopher Tortoise at Tillman Sandridge Preserve (TSP) and Public Service

AuthorityFunding Agency SC Department of Natural ResourcesBudget $9,600Period May 1, 2005–October 31, 2005

PI Travis GlennProject Title DNA Research to Support Management of American Alligators in LouisianaFunding Agency Louisiana Department of Wildlife and FisheriesBudget $30,000Period September 1, 2003–June 30, 2006

PI Travis GlennProject Title Molecular Phylogeny of North American Spiranthes OrchidsFunding Agency American Orchid SocietyBudget $6,450Period January 1, 2004–December 31, 2005

PI Travis GlennProject Title Support for Turtle ConservationFunding Agency National Fish and Wildlife FoundationBudget $14,206Period July 1, 2003–June 30, 2005

PI Travis GlennProject Title Development of Peromyscus genomicsFunding Agency University of South CarolinaBudget $115,028Period August 1, 2004–July 31, 2005

PI Thomas Hinton and Travis GlennProject Title Transgenerational Effects of Chronic Low-Dose Irradiation in a Medaka Fish Model SystemFunding Agency Department of Energy Low Dose ProgramBudget $1,033,705 total ($128,373 for SREL in FY05; award pending)Period March 1, 2005–February 28, 2008


66

PI William HopkinsProject Title Modeling the Individual and Interactive Risks to an Amphibian Population Resulting from Breeding Site Contamination

and Terrestrial Habitat LossFunding Agency US Environmental Protection Agency/University of MarylandBudget $68,730Period December 19, 2001–December 18, 2004

PI William HopkinsProject Title Effects of Maternal Transfer of Contaminants on Amphibian Development and FitnessFunding Agency The Eppley Foundation for Research, Inc.Budget $11,500Period February 11, 2004–February 10, 2005

PI Charles H. Jagoe and J Vaun McArthurProject Title REU: The Impact of Energy Technologies on Natural Environmental SystemsFunding Agency National Science FoundationBudget $193,800 total awardPeriod May 1, 2002–April 30, 2005

PI Charles H. JagoeProject Title Environment Cooperative Science Center: Regional Studies in Sustainable Management of Coastal and Marine Habitats

for Decision MakingFunding Agency South Carolina State UniversityBudget $172,444 total awardPeriod January 1, 2001–September 30, 2005

PI Charles H. JagoeProject Title REU-Effects of Energy Technologies on Environmental SystemsFunding Agency National Science FoundationBudget $175,000Period May 1, 2005–April 30, 2007

PI Andrew NealProject Title Uranium Speciation on Sediment and Grouted SurfacesFunding Agency Bechtel BWXT Idaho, LLCBudget $15,180Period August 6, 2003–August 15, 2005

PI Christopher S. RomanekProject Title Controlled Growth of Biologic and Abiotic Carbonates and Fe-oxidesFunding Agency National Aeronautics and Space AdministrationBudget $50,000 total awardPeriod May 14, 1999–July 31, 2004

PI Christopher S. RomanekProject Title Alumni Initiatives Award between SREL and University of GranadaFunding Agency Council for International Exchange of ScholarsBudget $11,720Period June 1, 2003–May 31, 2005

PI Christopher S. RomanekProject Title Aging of Coral Specimens from NOAA-SCDNR 2003 Ocean Exploration Mission to the Charleston BumpFunding Agency South Carolina Department of Natural ResourcesBudget $11,596


67

Period September 1, 2003–August 31, 2004

PI Christopher S. RomanekProject Title Aging of Coral Specimens from NOAA-SCDNR Project: “From the Estuary to the Abyss: Exploring along the Latitude 31-

30 Transect”Funding Agency South Carolina Department of Natural ResourcesBudget $8,400Period January 23, 2004–August 31, 2005

PI John C. SeamanProject Title Tritium Distribution, Mixing, and Transport at the Tritiated Water Management FacilityFunding Agency USDA Forest ServiceBudget $88,160Period April 15, 2003–December 31, 2005

PI Rebecca SharitzProject Title Impacts of Military Training and Land Management in Threatened and Endangered Species in the Southeastern Fall

Line/Sandhills CommunityFunding Agency Strategic Environmental Research and Development Program (SERDP)Budget $939,523 total ($188,700 in FY05)Period 2002–2005

PI Rebecca R. SharitzProject Title Vegetation Establishment Success in Restored Carolina Bay Depressions on the Savannah River Site, South CarolinaFunding Agency USDA Forest ServiceBudget $43,750Period August 6, 2001–May 31, 2004

PI Rebecca R. SharitzProject Title Effects of Altered Flows in the Congaree River on the Floodplain of the Congaree Swamp National MonumentFunding Agency Cooperative Ecosystem Study Unit-PiedmontBudget $50,005Period September 10, 2003–September 30, 2005

PI Rebecca R. SharitzProject Title Vegetation Establishment Success in Restored Carolina Bay Depressions on the SRSFunding Agency USDA Forest ServiceBudget $35,500Period September 1, 2003–May 31, 2006

PI Ramunas StephanauskasProject Title The Role of Metal Contamination in the Proliferation of Antibiotic Resistance in Coastal Water-Borne PathogensFunding Agency US Department of Commerce/NOAABudget $534,311Period September 1, 2004–August 31, 2007

PI Carl StrojanProject Title Technical Review/Comments–SRS Environmental Report for 2004Funding Agency Education Research and Development Association (ERDA)Budget $4,454Period January 18, 2005–July 17, 2005

PI Barbara E. TaylorProject Title Aquatic Invertebrates in Carolina Bays and Other Wetland Ponds Before and After Restoration Treatments


68

Funding Agency US Department of AgricultureBudget $10,000Period September 1, 2003–September 30, 2005

PI Chuanlun ZhangProject Title Carbon Isotope Fractionations Associated with Bacterial Methane Oxidation: Implications for Carbonate Buildups at

Hydrocarbon SeepsFunding Agency American Chemical SocietyBudget $54,358Period August 1, 2002–August 31, 2005

PI Chuanlun ZhangProject Title Microbial Interactions and Processes: Diversity, Function, and Biogeochemical Consequences of

Chemolighoautotrophic Archaea in Nevada Hot SpringsFunding Agency National Science FoundationBudget $147,053Period May 1, 2004–July 31, 2005


Publications

2771 Sever, D.M. and W.A. Hopkins. 2004. Oviductal sperm storagein the ground skink Scincella laterale holbrook (Reptilia:Scincidae). Journal of Experimental Zoology 301A:599-611.

2772 Bolan, N., D. Adriano and S. Mahimairaja. 2004. Distributionand bioavailability of trace elements in livestock and poultrymanure by-products. Critical Reviews in EnvironmentalScience and Technology 34:291-338.

2773 Jackson, B.P., P.V. Winger and P.J. Lasier. 2004. Atmosphericlead deposition to Okefenokee Swamp, Georgia, USA.Environmental Pollution 130:445-451.

2774 Battaglia, L.L., B.S. Collins and P.B. Weisenhorn. 2004.Quercus michauxii regeneration in and around agingcanopy gaps. Canadian Journal of Forest Research 34:1359-1364.

2775 Hopkins, W.A., B.P. Staub, J.A. Baionno, B.P. Jackson, J.H.Roe and N.B. Ford. 2004. Trophic and maternal transfer ofselenium in brown house snakes (Lamprophis fuliginosus).Ecotoxicology and Environmental Safety 58:285-293.

2776 Unrine, J.M. and C.H. Jagoe. 2004. Dietary mercury exposureand bioaccumulation in larvae of the southern leopard frog,Rana sphenocephala. RMZ-Materials and Geoenvironment51:1418-1421.

2777 Unrine, J.M., C.H. Jagoe, W.A. Hopkins and H.A. Brant. 2004.Adverse effects of environmentally relevant dietary mercuryexposure in larvae of the southern leopard frog, Ranasphenocephala. RMZ-Materials and Geoenvironment51:1422-1425.

2778 Li, Y., H. Vali, S.K. Sears, J. Yang, B. Deng and C.L. Zhang.

69

2004. Iron reduction and alteration of nontronite NAu-2 bya sulfate-reducing bacterium. Geochimica et CosmochimicaActa 68:3251-3260.

2779 Zhang, C.L., B.W. Fouke, G.T. Bonheyo, A.D. Peacock, D.C.White, Y. Huang and C.S. Romanek. 2004. Lipid biomarkersand carbon-isotopes of modern travertine deposits(Yellowstone National Park, USA): implications forbiogeochemical dynamics in hot-spring systems. Geochimicaet Cosmochimica Acta 68:3157-3169.

2780 Roe, J.H., W.A. Hopkins, J.A. Baionno, B.P. Staub, C.L. Roweand B.P. Jackson. 2004. Maternal transfer of selenium inAlligator mississippiensis nesting downstream from a coal-burning power plant. Environmental Toxicology andChemistry 23:1969-1972.

2781 Glaudas, X. 2004. Do cottonmouths (Agkistrodonpiscivorus) habituate to human confrontations? SoutheasternNaturalist 3:129-138.

2782 Schable, N.A., B.C. Faircloth, W.E. Palmer, J.P. Carroll, L.W.Burger, L.A. Brennan, C. Hagen and T.C. Glenn. 2004.Tetranucleotide and dinucleotide microsatellite loci from thenorthern bobwhite (Colinus virginianus). MolecularEcology 4:415-419.

2783 Jørgensen, N.O.G., R. Stepanauskas, A.U. Pedersen, M.Hansen and O. Nybroe. 2003. Occurrence and degradationof peptidoglycan in aquatic environments. FEMS MicrobiologyEcology 46:269-280.

2784 Stepanauskas, R., M. Moran, B.A. Bergamaschi and J.T.Hollibaugh. 2003. Covariance of bacterioplanktoncomposition and environmental variables in a temperate delta

Books Published in FY05

NERP Documents Published in FY05

Gibbons, W. and M. Dorcas. 2005. Snakes of the Southeast. University of Georgia Press, Athens, GA. 253 pages.

Marcy, B.C., Jr., D.E. Fletcher, F.D. Martin, M.H. Paller, and M.J.M. Reichert. 2005. Fishes of the Middle Savannah River Basin With Emphasis onthe Savannah River Site. University of Georgia Press, Athens, GA. 462 pages.

DeBiase, A.E. and B.E. Taylor. 2005. Microcrustaceans (Branchiopoda and Copepoda) of Wetland Ponds and Impoundments on the SavannahRiver Site, Aiken, South Carolina. SRO-NERP-28. SRS National Environmental Research park Program, Aiken, SC.

Journal Articles Published in FY05


70

system. Aquatic Microbial Ecology 31:85-98.

2785 Kekli, A., A. Aldahan, M. Meili, G. Possnert, N. Buraglio andR. Stepanauskas. 2003. 129I in Swedish rivers: distributionand sources. The Science of Total Environment 309:161-172.

2786 Duncan, L., J. Dilustro and B. Collins. 2004. Avian responseto forest management and military training activities at FortBenning, Georgia. Georgia Journal of Science 62:95-103.

2787 Shao, Q., M.D. Wilson, C.S. Romanek and K.A. Hobson. 2004.Time series analysis of elemental and isotopic data frombiomineralized whale tissue. Environmental and EcologicalStatistics 11:323-337.

2788 Bertsch, P.M. and C.L. Strojan. 2004. Science in support ofassessing ecological risk and implementing novel remediationstrategies at the Savannah River Site, SC. Waste Management2004 Conference, February 29-March 4, 2004, Tucson, AZ.

2789 DeVault, T.L., B.D. Reinhart, I.L. Brisbin, Jr. and O.E. Rhodes,Jr. 2004. Home ranges of sympatric black and turkey vulturesin South Carolina. The Condor 106:706-711.

2790 Adriano, D.C., W.W. Wenzel, J. Vangronsveld and N.S. Bolan.2004. Role of assisted natural remediation in environmentalcleanup. Geoderma 122:121-142.

2791 Hinton, T.G., J.S. Bedford, J.D. Congdon and F.W. Whicker.2004. Effects of radiation on the environment: A need toquestion old paradigms and enhance collaboration amongradiation biologists and radiation ecologists. RadiationResearch 162:332-338.

2792 Zhao, D., B.E. Borders and M.D. Wilson. 2003. Individual-tree diameter growth and mortality models for bottomlandmixed-species hardwood stands in the lower Mississippialluvial valley. Forest Ecology and Management 199:307-322.

2793 Pearson, A., Z. Huang, A.E. Ingalls, C.S. Romanek, J. Wiegel,K.H. Freeman, R.H. Smittenberg and C.L. Zhang. 2004.Nonmarine crenarchaeol in Nevada hot springs. Applied andEnvironmental Microbiology 70:5229-5237.

2794 Rothermel, B.B. 2004. Migratory success of juveniles: apotential constraint on connectivity for pond-breedingamphibians. Ecological Applications 14:1535-1546.

2795 Wilson, M., W.P. McCormick and T.G. Hinton. 2004. Themaximally exposed individual—comparison of maximumlikelihood estimation of high quantiles to an extreme valueestimate. Risk Analysis 24:1143-1151.

2796 Vali, H., B. Weiss, Y. Li, S.K. Sears, S.S. Kim, J.L. Kirschvink

and C.L. Zhang. 2004. Formation of tabular single-domainmagnetite induced by Geobacter metallireducens GS-15.Proceedings of the National Academy of Sciences 101:16121-16126.

2797 Hinton, T.G., D.P. Coughlin, Yi Yi, and L.C. Marsh. 2004. Lowdose rate irradiation facility: initial study on chronicexposures to medaka. Journal of Environmental Radioactivity74:43-55.

2798 Punshon, T., A.L. Neal and B.P. Jackson. 2004. Cadmium.pp. 171-208. In: Trace and Ultratrace Elements in Plants andSoil. I. Shtangeeva (ed.). WIT Press.

2799 Paller, M.H., C.H. Jagoe, H. Bennett, H.A. Brant and J.A.Bowers. 2004. Influence of methylmercury from tributarystreams on mercury levels in Savannah River Asiatic clams.Science of the Total Environment 325:209-219.

2800 Collins, B.S., JV. McArthur and R.R. Sharitz. 2004. Plant effectson microbial assemblages and remediation of acidic coal pilerunoff in mesocosm treatment wetlands. EcologicalEngineering 23:107-115.

2801 Hopkins, W.A., C.T. Winne and S.E. DuRant. 2004. Differentialswimming performance of two natricine snakes exposed toa cholinesterase-inhibiting pesticide. Environmental Pollution133:531-540.

2802 Wickliffe, J.K., A.M. Bickham, B.E. Rodgers, R.K. Chesser,C.J. Phillips, S.P. Gaschak, J.A. Goryanaya, I. Chizhevsky andR.J. Baker. 2003. Exposure to chronic, low-dose rate ã-Radiation at Chornobyl does not induce point mutations inBig Blue Mice. Environmental and Molecular Mutagenesis42:11-18.

2803 Roe, J.H., W.H. Hopkins, J.W. Snodgrass and J.D. Congdon.2004. The influence of circadian rhythms on pre- and post-prandial metabolism in the snake Lamprophis fuliginosus.Comparative Biochemistry and Physiology Part A 139:159-168.

2804 Unrine, J.M. and C.H. Jagoe. 2004. Dietary mercury exposureand bioaccumulation in southern leopard frog (Ranasphenocephala) larvae. Environmental Toxicology andChemistry 23:2956-2963.

2805 Unrine, J.M., C.H. Jagoe, W.A. Hopkins and H.A. Brant. 2004.Adverse effects of ecologically relevant dietary mercuryexposure in southern leopard frog (Rana sphenocephala)larvae. Environmental Toxicology and Chemistry 23:2964-2970.

2806 Dilustro, J., B. Collins, L. Duncan and C. Crawford. 2005.


71

Moisture and soil texture effects on soil C02 efflux components

in southeastern mixed pine forests. Forest Ecology andManagement 204:85-95.

2807 Newman, L.A. and C.M. Reynolds. 2004. Phytodegradationof organic compounds. Current Opinion in Biotechnology15:225-230.

2808 Magnuson, T.S., A.L. Neal and G.G. Geesey. 2004. Combiningin situ reverse transcriptase polymerase chain reaction,optical microscopy, and x-ray photoelectron spectroscopyto investigate mineral surface-associated microbial activities.Microbial Ecology 48:578-588.

2809 Hopkins, W.A., J.W. Snodgrass, J.A. Baionno, J.H. Roe, B.P.Staub and B.P. Jackson. 2005. Functional relationships amongselenium concentrations in the diet, target tissues, andnondestructive tissue samples of two species of snakes.Environmental Toxicology and Chemistry 24:344-351.

2810 Collins, B.S., R.R. Sharitz and D.P. Coughlin. 2005. Elementalcomposition of native wetland plants in constructedmesocosm treatment wetlands. Bioresource Technology96:937-948.

2811 Hopkins, W.A., B.P. Staub, J.A. Baionno, B.P. Jackson andL.G. Talent. 2005. Transfer of selenium from prey to predatorsin a simulated terrestrial food chain. Environmental Pollution134:447-456.

2812 Jackson, B.P., J.F. Ranville and A.L. Neal. 2005. Applicationof flow field flow fractionation-ICPMS for the study of uraniumbinding in bacterial cell suspensions. Analytical Chemistry77:1393-1397.

2813 Sawyer, R.H., L. Rogers, L. Washington, T.C. Glenn and L.W.Knapp. 2005. Evolutionary origin of the feather epidermis.Developmental Dynamics 232:256-267.

2814 Mou, X., M.A. Moran, R. Stepanauskas, J.M. Gonzalez andR.E. Hodson. 2005. Flow-cytometric cell sorting andsubsequent molecular analyses for culture-independentidentification of bacterioplankton involved indimethylsufoniopropionate transformations. Applied andEnvironmental Microbiology 71:1405-1416.

2815 Metts, B.S., W.A. Hopkins and J.P. Nestor. 2005. Interactionof an insecticide with larval density in pond-breedingsalamanders (Ambystoma). Freshwater Biology 50:685-696.

2816 Glaudas, X., T.M. Farrell and P.G. May. 2005. Defensivebehavior of free-ranging pygmy rattlesnakes. Copeia 1:196-200.

2817 Winne, C.T. and M.B. Keck. 2005. Intraspecific differencesin thermal tolerance of the diamondback watersnake(Nerodia rhombifer): effects of ontogeny, latitude, and sex.Comparative Biochemistry and Physiology, Part A 140:141-149.

2818 Pinder, J.E., III, T.G. Hinton and F.W. Whicker. 2005. Theinfluence of a whole-lake addition of stable cesium on theremobilization of aged 137Cs in a contaminated reservoir.Journal of Environmental Radioactivity 80:225-243.

2819 Unrine, J.M., C.H. Jagoe, A.C. Brinton, H.A. Brant and N.T.Garvin. 2005. Dietary mercury exposure and bioaccumulationin amphibian larvae inhabiting Carolina bay wetlands.Environmental Pollution 135:245-253.

2820 Hauswaldt, J.S. and T.C. Glenn. 2005. Population genetics ofthe diamondback terrapin (Malaclemys terrapin).Molecular Ecology 14:723-732.

2821 Smith, R.B., T.D. Tuberville, A.L. Chambers, K.M. Herpich andJ.E. Berish. 2005. Gopher tortoise burrow surveys: Externalcharacteristics, burrow cameras, and truth. AppliedHerpetology 2:161-170.

2822 Taylor, B.E. and A.E. DeBiase. 2005. Are microcrustaceansuseful for assessing success of wetland pond restoration?(South Carolina). Ecological Restoration 23:56-57.

2823 Croshaw, D.A. 2005. Cryptic behavior is independent of dorsalcolor polymorphism in juvenile northern leopard frogs(Rana pipiens). Journal of Herpetology 39:125-129.

2824 Zhao, D., M. Wilson and B.E. Borders. 2005. Modelingresponse curves and testing treatment effects in repeatedmeasures experiments: a multilevel nonlinear mixed-effectsmodel approach. Canadian Journal for Research 35:122-132.

2825 Zhang, C.L., Z. Huang, J. Cantu, R.D. Pancost, R.L. Brigmon,T.W. Lyons and R. Sassen. 2005. Lipid biomarkers and carbonisotope signatures of a microbial (Beggiatoa) mat associatedwith gas hydrates in the Gulf of Mexico. Applied andEnvironmental Microbiology 71:2106-2112.

2826 Cooper, D.C., A L. Neal, R.K. Kukkadapu, D. Brewe, A. Cobyand F.W. Picardal. 2005. Effects of sediment iron mineralcomposition on microbially mediated changes in divalentmetal speciation: Importance of ferrihydrite. Geochimica etCosmochimica Acta 69:1739-1754.

2827 Jackson, B.P., J.F. Ranville, P.M. Bertsch and A.G. Sowder.2005. Characterization of colloidal and humic-bound Ni andU in the “dissolved” fraction of contaminated sedimentextracts. Environmental Science & Technology 39:2478-2485.


72

2828 Winne, C.T., M.D. Dorcas and S.M. Poppy. 2005. Populationstructure, body size, and seasonal activity of black swampsnakes (Seminatrix pygaea). Southeastern Naturalist 4:1-14.

2829 Hinton, T.G., A.S. Knox, D.I. Kaplan and R. Sharitz. 2005.Phytoextraction of uranium and thorium by native trees in acontaminated wetland. Journal of Radioanalytical and NuclearChemistry 264:417-422.

2830 Kaplan, D.I., T.G. Hinton and A.S. Knox. 2005. Cesium-137partitioning to wetland sediments and uptake by plants.Journal of Radioanalytical and Nuclear Chemistry 264:393-399.

2831 Adriano, D.C., N.S. Bolan, J. Vangronsveld and W.W. Wenzel.2005. Heavy Metals. pp. 175-182. In: Encyclopedia of Soilsin the Environment, edited by D. Hillel. Elsevier AcademicPress. Amsterdam, The Netherlands.

2832 Bolan, N.S., D. Curtin and D.C. Adriano. 2005. Acidity. pp.11-17. In: Encyclopedia of Soils in the Environment, editedby D. Hillel. Elsevier Academic Press. Amsterdam, TheNetherlands.

2833 Koo, B-J, D.C. Adriano, N.S. Bolan and C.D. Barton. 2005.Root exudates and microorganisms. pp. 421-428. In:Encyclopedia of Soils in the Environment, edited by D. Hillel.Elsevier Academic Press. Amsterdam, The Netherlands.

2834 Stepanauskas, R., T.C. Glenn, C.H. Jagoe, R.C. Tuckfield, A.H.Lindell and J McArthur. 2005. Elevated microbial toleranceto metals and antibiotics in metal-contaminated industrialenvironments. Environmental Science & Technology 39:3671-3678.

2835 Zhao, D., B.E. Borders and M.D. Wilson. 2005. A density-dependent matrix model for bottomland hardwood standsin the lower Mississippi Alluvial Valley. Ecological Modelling184:381-395.

2836 Jiménez-López, C., C.S. Romanek, F.J. Huertas, H. Ohmotoand E. Caballero. 2004. Oxygen isotope fractionation insynthetic magnesian calcite. Geochimica et CosmochimicaActa 68:3367-3377.

2837 Zhang, C. 2005. Genome-empowered stable-isotopebiogeochemistry: integrating genomics, proteomics andmetabolomics. Logical Biology 5:79-80.

2838 Weston, J.L., N.A. Schable and T.C. Glenn. 2004.Characterization of six microsatellite primers for the greyfox (Urocyon cinereoargenteus). Molecular Ecology Notes4:503-505.

2839 Koopman, M.E., N.A. Schable and T.C. Glenn. 2004.Development and optimization of microsatellite DNA primersfor boreal owls (Aegolius funereus). Molecular EcologyNotes 4:376-378.

2840 Otero-Arnaiz, A., A. Schnabel, T.C. Glenn, N.A. Schable, C.Hagen and L. Ndong. 2005. Isolation and characterization ofmicrosatellite markers in the East African tree, Acaciabrevispica (Fabaceae: Mimosoideae). Molecular EcologyNotes 5:366-368.

2841 Faircloth, B.C., A. Reid, T. Valentine, S.H. Eo, T.M. Terhune,T.C. Glenn, W.E. Palmer, C.J. Nairn and J.P. Carroll. 2005.Tetranucleotide, trinucleotide, and dinucleotide loci from thebobcat (Lynx rufus). Molecular Ecology Notes 5:387-389.

2842 Dion, H.M., C.S. Romanek, T.G. Hinton and P.M. Bertsch.2005. Cesium-137 in floodplain sediments of the Lower ThreeRuns Creek on the DOE Savannah River Site. Journal ofRadioanalytical and Nuclear Chemistry 264:481-488.

2843 Winne, C.T. and M.B. Keck. 2004. Daily activity patterns ofWhiptail Lizards (Squamata: Teiidae: Aspidoscelis): aproximate response to environmental conditions or anendogenous rhythm? Functional Ecology 18:314-321.

2844 Li, Y., Y. Zheng and B. Fu. 2005. Mössbauer spectroscopy ofomphacite and garnet pairs from eclogites: Application togeothermobarometry. American Mineralogist 90:90-100.

2845 Kevbrin, V. V. 2004. Alkalithermophiles: A double challengefrom extreme environments. pp. 1-16. In: Origins: Genesis,Evolution and Diversity of Life, edited by J. Seckbach. KluwerAcademic Publishers, Dordrecht, NL.

2846 Bolan, N.S., L. Wong and D.C. Adriano. 2004. Nutrientremoval from farm effluents. Bioresource Technology 94:251-260.

2847 Ye, Q., Y. Roh, S.L. Carroll, B. Blair, J. Zhou, C.L. Zhang andM.W. Fields. 2004. Alkaline anaerobic respiration: isolationand characterization of a novel alkaliphilic and metal-reducing bacterium. Applied and EnvironmentalMicrobiology 70:5595-5602.

2848 Harmon, S.M., J.K. King, J.B. Gladden, G.T. Chandler and L.A.Newman. 2005. Mercury body burdens in Gambusiaholbrooki and Erimyzon sucetta in a wetland mesocosmamended with sulfate. Chemosphere 59:227-233.

2849 Zhang, C.L. and B. Lanoil. 2004. Geomicrobiology andbiogeochemistry of gas hydrates and cold seeps. ChemicalGeology 205:187-194.


73

2850 Sassen, R., H.H. Roberts, R. Carney, A.V. Milkov, D.A.DeFreitas, B. Lanoil and C. Zhang. 2004. Free hydrocarbongas, gas hydrate, and authigenic minerals in chemosyntheticcommunities of the northern Gulf of Mexico continental slope:relation to microbial processes. Chemical Geology 205:195-217.

2851 Formolo, M.J., T.W. Lyons, C. Zhang, C. Kelley, R. Sassen, J.Horita and D.R. Cole. 2004. Quantifying carbon sources inthe formation of authigenic carbonates at gas hydrate sites inthe Gulf of Mexico. Chemical Geology 205:253-264.

2852 Sassen, R., S.T. Sweet, D.A. DeFreitas, N.L. Eaker, H.H. Robertsand C. Zhang. 2004. Brine vents on the Gulf of Mexico slope:hydrocarbons, carbonate-barite-uranium mineralization, redbeds, and life in an extreme environment. 2004 Gulf CoastSection Society of Economic Paleontologists and Mineralogists444-463.

2853 Batzer, D.P., S.E. Dietz-Brantley, B.E. Taylor and A.E. DeBiase.2005. Evaluating regional differences in macroinvertebratecommunities from forested depressional wetlands acrosseastern and central North America. Journal of North AmericaBenthological Society 24:403-414.

2854 Roe, J.H., W.A. Hopkins and B.P. Jackson. 2005. Species-and stage-specific differences in trace element tissueconcentrations in amphibians: implications for the disposalof coal-combustion wastes. Environmental Pollution 136:353-363.

2855 Kelsey-Wall, A., J.C. Seaman, C.H. Jagoe, C.E. Dallas and K.F.Gaines. 2005. Rodents as receptor species at a tritiumdisposal site. Journal of Environmental Radioactivity 82:95-104.

2856 Punshon, T., B.P. Jackson, A. Lanzirotti, W.A. Hopkins, P.M.Bertsch and J. Burger. 2005. Application of synchrotron x-ray microbeam spectroscopy to the determination of metaldistribution and speciation of biological tissues. SpectroscopyLetters 38:343-363.

2857 DeVault, T.L., B.D. Reinhart, I.L. Brisbin, Jr. and O.E. Rhodes,Jr. 2005. Flight behavior of black and turkey vultures:implications for reducing bird-aircraft collisions. Journal ofWildlife Management 69:592-599.

2858 Paller, M.H., D.E. Fletcher, T. Jones, S.A. Dyer, J.J. Isely andJ.W. Littrell. 2005. Potential of largemouth bass as vectors of137Cs dispersal. Journal of Environmental Radioactivity 80:27-43.

2859 Snodgrass, J.W., W.A. Hopkins, B.P. Jackson, J.A. Baionnoand J. Broughton. 2005. Influence of larval period on

responses of overwintering green frog (Rana Clamitans)larvae exposed to contaminated sediments. EnvironmentalToxicology and Chemistry 24:1508-1514.

2860 Mahimairaja, S., N.S. Bolan, D.C. Adriano and B. Robinson.2005. Arsenic contamination and its risk management incomplex environmental settings. pp. 1-82. In: Advances inAgronomy, edited by D. L. Sparks. Elsevier/Academic Press.San Diego, CA.

2861 Tague, R.T. and S.A. Foré. 2005. Analysis of the spatial geneticstructure of Passiflora incarnata in recently disturbed sites.Canadian Journal of Botany 83:420-426.

2862 Harper, S.J. and R. Sharitz. 2005. Delineating sandhillcommunities: the use of advanced techniques to extractfeatures from satellite imagery. pp. 123-136. In: Proceedingsof the 4th Southern Forestry and Natural Resources GISConference. Warnell School of Forest Resources, Universityof Georgia, Athens, GA.

2863 Moen, D.S., C.T. Winne and R.N. Reed. 2005. Habitat-mediatedshifts and plasticity in the evaporative water loss rates of twocongeneric pit vipers (Squamata, Viperidae, Agkistrodon).Evolutionary Ecology Research 7:759-766.

2864 Hitchcock, D.R., C.D. Barton, K.T. Rebel, J. Singer, J.C. Seaman,J.D. Strawbridge, S.J. Riha and J.I. Blake. 2005. A containmentand disposition strategy for tritium-contaminatedgroundwater at the Savannah River Site, South Carolina,United States. Environmental Geosciences 12:17-28.

2865 Barton, C.D., L.S. Paddock, C.S. Romanek, S. Maharaj and J.Seaman. 2005. Metal attenuation processes in a landfillcontaining coal combustion waste: implications forremediation. Environmental Geosciences 12:45-55.

2866 Punshon, T., A. Lanzirotti, S. Harper, P.M. Bertsch and J.Burger. 2005. Distribution and speciation of metals in annualrings of black willow. Journal of Environmental Quality34:1165-1173.

2867 McCay, T.S. and M.J. Komoroski. 2004. Demographicresponses of shrews to removal of coarse woody debris in amanaged pine forest. Forest Ecology and Management189:387-395.

2868 Willson, J.D., C.T. Winne and L.A. Fedewa. 2005. Unveilingescape and capture rates of aquatic snakes and salamanders(Siren spp. and Amphiuma means) in commercial funneltraps. Journal of Freshwater Ecology 20:397-404.

2869 Sawyer, R.H., T.C. Glenn, J.O. French and L.W. Knapp. 2005.Developing antibodies to synthetic peptides based on


74

comparative DNA sequencing of multigene families.Methods in Enzymology 395:636-652.

2870 Glenn, T.C. and N.A. Schable. 2005. Isolating microsatelliteDNA loci. Methods in Enzymology 395:202-222.

2871 Wenzel, W.W., E. Lombi and D.C. Adriano. 2004.Biogeochemical processes in the rhizosphere: role inphytoremediation of metal-polluted soils. pp. 273-303. In:Heavy Metal Stress in Plants: From Biomolecules toEcosystems, edited by M. Prasad and J. Hagemeyer. SpringerVerlag Berlin Heidelberg New York.

2872 Wilson, M. and D. Jackson. 2005. Uncertainty and powerat low levels of incurred radiation dose. Journal ofRadiological Protection 25:51-66.


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75