exposure of game species to trace elements and …

EXPOSURE OF GAME SPECIES TO TRACE ELEMENTS AND

RADIOCESIUM ON THE SAVANNAH RIVER SITE IN SOUTH CAROLINA

by

RICKI ELAINE OLDENKAMP

(Under the Direction of James C. Beasley)

ABSTRACT

Despite the widespread harvest and consumption of game by recreational hunters,

there are few data available regarding contaminant burdens in many commonly harvested

wildlife species. I sampled wild pigs (Sus scrofa), gray squirrels (Sciurus carolinensis),

and waterfowl from a contaminated Department of Energy site to quantify concentrations

of trace elements and radiocesium in muscle and liver tissues for assessment of potential

human health risks from the consumption of game, and contaminant accumulation rates

in tissues. Concentrations varied among collection locations and species, although

waterfowl collected from a coal ash basin consistently had high levels of trace element

burdens (especially Selenium) and those from areas with known radiological

contamination had elevated radiocesium concentrations, often exceeding limits

established by the European Economic Community for safe human consumption.

INDEX WORDS: Coal Combustion Waste, Game Meat, Gray Squirrels, Human Consumption Risk, Waterbirds, Waterfowl, Wild Pigs, Radiocesium, Trace Elements



by


B.S., Biology, Northern Michigan University, 2013

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2016

© 2016

Ricki Elaine Oldenkamp

All Rights Reserved



by


Major Professor: James C. Beasley

Committee: Karl V. Miller William A. Hopkins Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia May 2015

iv

DEDICATION

I dedicate this to my family, and my friends that have become my family.

v

ACKNOWLEDGEMENTS

I have learned so much during the last two years, and am extremely thankful to

Jim Beasley for giving me a chance when I didn’t have a traditional wildlife background.

My first friend upon starting the program has been by my side throughout this journey

and also been my “bird dog” for duck research, Chris Leaphart I never would have gotten

through this without you. I am extremely thankful for my friends, Becky, Betsy, and

Nathan, y’all have been very supportive and taught me things from hunting to Cajun

cooking and I will forever cherish those memories. I am also thankful to Larry Bryan and

Bobby Kennamer for their support and advice. Also to Bill Hopkins, whose brain I envy

and who is super kind to share his smarts with me. To my favorite professor, Karl Miller,

you are funny and so damn smart, I loved your habitat class and that course made me feel

I had truly found the right path for my career, I hope I make you proud. I am also grateful

for the SREL folks, there have been many fun times and trying times out there in the

middle of nowhere South Carolina, glad we had each other for both. If I did not have so

much love and support from my wonderful father, Rick Oldenkamp, and sister, Amanda

Szabo, I most definitely would have given up in some of my most stressful moments, I

love you both more than I can explain. Also to my favorite person in the world, my

brother TJ Oldenkamp, you have inspired me greatly with all that you have accomplished

over these last few years, I am so proud of the man you are and it has made me want to

meet you in that place of excellence, I am extremely glad we are friends and your no-

nonsense advice propelled me forward to the finish, thank you.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS .................................................................................................v

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES ......................................................................................................... xiii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW .........................................1

LITERATURE CITED ................................................................................7

2 TRACE ELEMENTS AND RADIOCESIUM IN GAME SPECIES AND

HUMAN CONSUMPTION RISKS ................................................................18

ABSTRACT ...............................................................................................19

INTRODUCTION .....................................................................................20

METHODS ................................................................................................24

RESULTS ..................................................................................................35

DISCUSSION ............................................................................................41

MANAGEMENT IMPLICATIONS .........................................................47

LITERATURE CITED ..............................................................................67

3 WATERFOWL EXPOSURE TO COAL COMBUSTION WASTES AND

HUMAN CONSUMPTION RISKS ................................................................85

ABSTRACT ...............................................................................................86

INTRODUCTION .....................................................................................87

v

METHODS ................................................................................................91

RESULTS ..................................................................................................99

DISCUSSION ..........................................................................................101

MANAGEMENT IMPLICATIONS .......................................................104

LITERATURE CITED ............................................................................116

4 RADIOCESIUM IN WATERFOWL/WATERBIRDS FROM A RETIRED

NUCLEAR REACTOR COOLING RESERVOIR .......................................129

ABSTRACT .............................................................................................130

INTRODUCTION ...................................................................................131

METHODS ..............................................................................................134

RESULTS ................................................................................................141

DISCUSSION ..........................................................................................143

CONCLUSION ........................................................................................146

LITERATURE CITED ............................................................................154

5 CONCLUSION ..............................................................................................160

vi

LIST OF TABLES

Page

Table 2-1: Sample sizes of game species collected from the Savannah River Site (SRS)

and analyzed for trace elements and radiocesium in 2012-2015; sex ratios are also

indicated. ................................................................................................................48

Table 2-2: Comparisons of trace element concentrations (ppm, dry mass) in muscle and

liver tissues of wild pigs collected from the Savannah River Site (SRS) and those

collected from five counties in Georgia (GA) in 2012-2015. See Table 1 for

sample sizes of muscle and liver tissues analyzed. ................................................49

Table 2-3: Correlations among trace element concentrations in muscle (above diagonal)

and liver (below diagonal) tissues of wild pigs collected from the Savannah River

Site (SRS) in 2012-2015 (n=88 muscle, n=30 liver). Only those elements with

more than 50% of the values above detectable limits are included. Correlations

between muscle and liver samples for individual elements are presented on the

diagonal in bold. .....................................................................................................50


and liver (below diagonal) tissues of wild pigs collected from five counties in

Georgia (GA) in 2012-2015 (n=20 muscle, n=20 liver). Only those elements with

more than 50% of the values above detectable limits are included. Correlations

between muscle and liver samples for individual elements are presented on the

diagonal in bold. .....................................................................................................51

vii

Table 2-5: Descriptive statistics for radiocesium concentrations in muscle and liver

tissues of wild pigs collected from the Savannah River Site (SRS) and from five

counties in Georgia (GA) in 2012-2015. ...............................................................52

Table 2-6: Radiocesium concentrations (Bq/g, dry mass) in muscle and liver tissues of

wild pigs collected from the Savannah River Site (SRS) and from five counties in

Georgia (GA) from 2012-2015. .............................................................................53

Table 2-7: Comparisons of trace element concentrations (ppm, dry mass) in muscle of

squirrels collected near the D-Area ash basins and squirrels collected from all

other locations on the Savannah River Site (SRS) in 2012-2015. .........................54

Table 2-8: Descriptive statistics for radiocesium concentrations in muscle tissue of

squirrels collected from the Savannah River Site (SRS) in 2012-2015. ................55

Table 2-9: Comparison of radiocesium concentrations (Bq/g, dry mass) in muscle tissue

of squirrels collected from four different locations on the SRS in 2012-2015.a ....56

Table 2-10: Waterfowl and waterbird species collected on the Savannah River Site (SRS)

in 2012-2015, with the scientific names, alpha codes, guild groupings, and sample

sizes in various analyses. .......................................................................................57

Table 2-11: Comparisons of trace element concentrations (ppm, dry mass) in muscle and

liver tissues of diving ducks collected from D-Area ash basins and diving ducks

collected from other water bodies on the Savannah River Site (SRS) in 2012-

2015. ....................................................................................................................58

Table 2-12: Concentrations of trace elements (ppm, dry mass) in muscle and liver tissues

of dabbling ducks collected from the Savannah River Site (SRS) in 2012-2015. .59

viii

Table 2-13: Concentrations of trace elements (ppm, dry mass) in muscle and liver tissues

of other water birds collected from the Savannah River Site (SRS) in 2012-2015.60

Table 2-14: Correlationsa among trace element concentrations in muscle (above diagonal)

and liver (below diagonal) tissues of all waterfowl/waterbirds collected from the

Savannah River Site (SRS) in 2012-2015 (n=66 muscle, n=70 liver). Only those

elements with more than 50% of the values above detectable limits are included.

Correlations between muscle and liver samples for individual elements are

presented on the diagonal in bold. .........................................................................61

Table 2-15: Descriptive statistics for radiocesium concentrations measured in waterfowl

collected from the Savannah River Site (SRS) in 2012-2015. ...............................62

Table 2-16: Comparisons of whole-body and tissue radiocesium concentrations (Bq/g,

wet mass) in diving ducksa collected from four different locations of the SRS in

2012-2015. .............................................................................................................63

Table 2-17: Monthly allowances of ½ lb. meals for adults and ¼ lb. for children before

exceeding the EPA’s oral reference dose ratings for selenium (Se) and mercury

(Hg) for muscle tissue of wild pigs collected from the Savannah River Site (SRS)

(n=88) and five counties in Georgia (GA) (n=20) 2012-2015. Consumption limits

based on average concentrations are presented with consumption limits based on

the maximum concentration found in an individual in parentheses. Levels of As

were all BDL so consumption limits are not included. ..........................................64

Table 2-18: The monthly allowances of ½ lb. meals for adults and ¼ lb. for children

before exceeding the EPA’s chronic oral reference dose limits for arsenic (As),

selenium (Se), and mercury (Hg) for muscle tissue of diving ducks collected from

ix

the D-Area ash basins (n=24) and other water bodies (n=18) on the Savannah

River Site (SRS) 2012-2015. Consumption limits based on average concentrations

are presented with limits based on the maximum concentration found in an

individual for each trace element in parentheses. Levels of As for Dabbling ducks

were all BDL so consumption limits are not included. ..........................................65

Table 3-1: Tissue and blood concentrations of arsenic (As), selenium (Se), and mercury

(Hg) of each recollected ring-necked duck restricted to the D-Area ash basins

(n=33) on the Savannah River Site (SRS) in the winter of 2014-2015 with

between 3 and 92 days of exposure. ....................................................................106

Table 3-2: Trace elements from ring-necked ducks (n=33) before and after being

restriction to the D-Area ash basins on the Savannah River Site (SRS) for between

3 and 92 days in winter of 2014-2015. Data are shown in approximately 15-day

increments of exposure with the mean±SE concentrations for each element. .....107

Table 3-3: Selenium (Se) linear regression with days of exposure for recollected ring-

necked ducks restricted to the D-Area ash basins (n=33) between 3 and 92 days

on the Savannah River Site (SRS) in the winter of 2014-2015. ..........................109

Table 3-4: Arsenic (As) linear regression with days of exposure for recollected ring-



Table 3-5: Mercury (Hg) linear regression with days of exposure for recollected ring-



x


and liver (below diagonal) tissues for recollected ring-necked ducks restricted to

the D-Area ash basins (n=33) between 3 and 92 days on the Savannah River Site

(SRS) in the winter of 2014-2015. Correlations between muscle and liver samples

for individual elements are presented on the diagonal in bold. ...........................110

Table 3-7: The monthly allowances of ½ lb. meals for adults and ¼ lb. for children before

exceeding the EPA’s chronic oral reference dose limits for arsenic (As), selenium

(Se), and mercury (Hg) for muscle tissue of ring-necked ducks collected from the

D-Area ash basins on the Savannah River Site (SRS) after being restricted

between 3 and 92 days of exposure. Consumption limits based on average

concentrations of cooked ducks muscle are presented with limits based on the

maximum concentration found in an individual for each trace element in

parentheses. ..........................................................................................................111

Table 4-1: Ecological half-life estimates for species from Pond B or Par Ponda on the

Savannah River Site (SRS). For the current study American coots were restricted

to Pond B for between 33 and 173 days of exposure to radiocesium in that system.147

Table 4-2: Descriptive statistics for a random sampling of American coots and ring-

necked ducks that were trapped from L-Lake and whole-body counted for

radiocesium prior to release onto Pond B on the Savannah River Site (SRS) over

the winter of 2013-2015. ......................................................................................148

Table 4-3: Descriptive statistics for radiocesium concentrations of American coots and

ring-necked ducks that were released to Pond B on the Savannah River Site (SRS)

for between 33 and 173 days of exposure before being collected. ......................149

xi

LIST OF FIGURES

Page

Figure 2-1: Savannah River Site (SRS) locations targeted for sample collections (wild

pigs, squirrels, waterfowl/waterbirds) for trace elements and radiocesium

quantification in 2012-2015, included the D-Area ash basins, Fourmile Branch,

Tim’s Branch, Pond A/R-Canal, Pond B, and L-Lake. .........................................66

Figure 3-1: D-Area ash basins on the Savannah River Site (SRS), SC. Basin 1, the largest

basin is partially filled in and has extensively revegetated. The smaller enclosed

wetland formed by revegetation in this basin was utilized as the release and

exposure area for the ring-necked ducks in this study in winter of 2014-2015. ..112

Figure 3-2: Muscle concentrations of arsenic (As), selenium (Se), mercury (Hg), and days

of exposure of recollected ring-necked duck restricted to the D-Area ash basins

(n=33) on the Savannah River Site (SRS) in the winter of 2014-2015 between 3

and 92 days of exposure. ......................................................................................113

Figure 3-3: Liver concentrations of arsenic (As), selenium (Se), mercury (Hg), and days




Figure 3-4: Blood concentrations of arsenic (As), selenium (Se), mercury (Hg), and days


xii



Figure 4-1: The Par Pond Reservoir system on the Savannah River Site (SRS) that

includes P- and R-reactors with depictions of canals that carried the radionuclide

contaminated cooling water to Ponds B and C and Par Pond during several reactor

releases. ................................................................................................................150

Figure 4-2: Historical and current data for whole-body radiocesium concentrations in

American coots collected from Pond B on the Savannah River Site between the

winters of 1975-1976 and 2014-2015; a.) includes a dashed line for the whole-

body equivalent (0.324 Bq/g) to the European Economic Community limit for

radiocesium in fresh meat (0.600 Bq/g) and b) is a linear regression of natural log-

transformed data from collections between winters 1986-1987 and 2014-2015,

estimates utilized in ecological half-life calculations. .........................................151

Figure 4-3: Whole-body radiocesium concentrations in a.) American coots and b.) ring-

necked ducks collected from Pond B on the Savannah River Site (SRS) after

exposure between 32 and 173 days. Day 0 whole-body concentrations are counts

done on live-captured birds from L-Lake before release onto Pond B. Solid lines

show non-linear fits to the data and dashed lines represent our calculated whole-

body equivalent (0.324 Bq/g) to the European Economic Community limit for

radiocesium in fresh meat (0.600 Bq/g). ..............................................................152

Figure 4-4: Radiocesium concentrations in muscle and liver tissue graphed against

whole-body concentrations of a.) American coots and b.) ring-necked ducks

xiii

collected from Pond B on the Savannah River Site (SRS) after exposure between

32 and 173 days. Lines are linear fits with intercepts constrained to zero. .........153

1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Wildlife face numerous anthropogenic pressures globally, especially direct and

indirect threats from human development and the resulting reduction and degradation of

remaining native habitats. In particular, anthropogenic activities have released numerous

pollutants into the environment, resulting in potential adverse health effects in both

wildlife and human consumers of wildlife. Wildlife can be exposed to contaminants via

physical contact with, or ingestion of, pollutants, raising a concern because many wildlife

species are harvested for food and these pollutants could be passed on to human

consumers. The continued exposure through ingestion of contaminated food can result in

the accumulation of contaminants in the tissues of exposed individuals, resulting in

greater body burdens in higher tropic level consumers through biomagnification (USEPA

2012). Because humans are apex predators in many systems, it is important to elucidate

patterns of bioaccumulation of contaminants within food webs.

Hunting and consumption of wildlife is important culturally but also as source of

food globally, including in the U.S. In the last U.S. Fish and Wildlife census in 2011

there were 13.7 million hunters, an increase of 9 percent from 2006 (USFWS and USCB

2011). Despite the potential for many pollutants to bioaccumulate or biomagnify within

food webs, game animals are not subject to the same scrutiny and testing as farm raised

livestock and poultry, leaving a gap in knowledge of contaminant exposure resulting

2

from consumption of game, like recent concerns showing increased levels of lead in

hunters using lead ammunition to hunt game animals (Pain et al. 2010).

Human generated pollution can affect wildlife worldwide but game species with

large home ranges or migratory behaviors are of particular interest for monitoring

(Taggart et al. 2011). Of specific concern are waterfowl and waterbirds, which can travel

hundreds to thousands of miles during migration (Kennamer 2003, Cristol et al. 2012,

Conder and Arblaster 2016). Despite the fact that the U.S. has lost approximately 52% of

its original wetlands over the last few centuries, populations of many waterfowl species

have been increasing in recent decades and currently are 43% higher than the long-term

average since population estimates first originated in 1955 (Dahl 2000, USFWS 2014).

As conversion of wetlands continues, increasing numbers of birds will need to use bodies

of water that have a history of anthropogenic disturbances (Foley and Batcheller 1988).

In particular, use of aquatic habitats contaminated with toxic substances such as trace

elements, PCBs, organochlorines, and radiocesium (137Cs) is a serious concern. Wildlife

using contaminated sites may suffer deleterious effects and potential exists for human

exposure to contaminants through consumption of contaminated meat even far from sites

of contamination (Brisbin et al. 1973; Fendley et al. 1977; Brisbin and Vargo 1982; Foley

and Batcheller 1988; Kennamer et al., 1998; Sajwan et al., 2009; Cristol et al. 2012,

Kalisinska et al., 2013). However, despite increases in waterfowl numbers and the

potential for human exposure, there is limited data on the extent and types of

contamination waterfowl could be passing on to hunters, as well as how long waterfowl

have to forage in a contaminated area before they become a risk to hunters.

3

There are many ways in which wildlife can be exposed to contaminants in the

environment. Of increasing interest is exposure to trace elements found in coal

combustion wastes (CCW). Coal fly ash effluent discharged into Belews Lake and Hyco

Reservoir in North Carolina and in Martin Creek Reservoir in Texas has resulted in trace

element accumulation in aquatic biota (Lemly 1996, 2002). Further, studies associated

with a large fly ash effluent spill from the Kingston Plant in Tennessee showed that even

terrestrial animals can be exposed to elevated levels of elements found in fly ash

deposited in settling basins (Ruhl et al. 2009). Coal-fly ash contains many trace elements,

such as Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Se, and Zn, which can leach out of the ash

into the water and combine with sediments (Cherry and Guthrie 1977; Evans and Giesy

1978; Alberts et al. 1985; Sandhu et al. 1993). Trace elements such as As, Se, and Hg are

of special concern in ash basins because of the potentially deleterious effects to human

consumers of exposed wildlife (Luther 2010).

One of the most toxic metals affecting organisms is Hg (Diez et al. 2008), which

is often found within coal fly ash. Hg accumulates in tissues and organs such as muscle,

kidney, liver, and the brain (Graeme and Pollack 1998). Methylmercury, (MeHg) the

most toxic form of Hg, can cause severe neurological damage to humans and wildlife

(Grandjean et al. 1999, Clarkson et al. 2003). Acute effects of Hg at high concentrations

have been well-documented, but there are also adverse effects identified at lower tissue

concentrations, representing chronic exposure (Wolfe et al. 1998). In humans Hg affects

neurobehavioral functioning (Goyer 1991); high doses cause problems with coordination

of movement, muscle weakness, as well as sensibility, language, vision and hearing

(Duchesne et al. 2004). Exposure to Hg is especially important for sensitive populations

4

like expectant mothers and children. Tracking the transfer to humans through

consumption of fish and game is vital to prevent overexposure to this pollutant (USEPA

2014).

Selenium is often found in CCW settling basins where it is primarily found in

sediments (Lemy 2002, Luther 2010) but is readily transferred through food webs.

Therefore, wildlife can be exposed to Se through consumption of vegetation grown from

contaminated sediments, or incidental ingestion of sediments while foraging, as well as

through consumption of other species that are bioaccumulating contaminants (Heinz

1996). In humans and wildlife exposure to Se may result in disruption of endocrine

function, neurotoxicity, hepatotoxicity, and effects on reproductive capabilities (USEPA

1999). Similarly, As is often found in fly ash (Ruhl et al. 2009, Luther 2010) and also

poses a threat to wildlife and human health, resulting in increasing blood pressure leading

to heart attacks. As exposure can also result in increased risk of skin, lung, bladder, and

kidney cancer in humans (USEPA 1998).

Interactive effects among trace elements can influence the rates of uptake,

elimination, and sometimes the area of deposition within the body (Lorentzen et al. 1998,

Kehrig et al. 2009, García-Barrera et al. 2012). In mallards (Anas platyrhynchos) it

appears Hg and As act antagonistically with Se (Hoffman et al. 1992, Heinz and Hoffman

1998). In mallard embryos MeHg and selenomethionine were cumulative or synergistic

in toxicity in regards to survival and teratogenesis (Hoffman and Heinz 1998). Moreover,

the adverse effects from Se exposure such as mortality, impaired growth,

histopathological lesions, and resultant oxidative stress have been found to be

5

ameliorated by interactions with Hg and As in laboratory experiments (Heinz 1979,

Heinz 1996, Heinz and Hoffman 1998).

Radioactive materials from nuclear activities such as atmospheric fallout from

nuclear weapons testing and nuclear accidents like those in Chernobyl and Fukushima are

another source of contamination that can affect wildlife (UNSCEAR 1988; Gudiksen et

al. 1989; Mishra 1990; Bennet 1995; Dreicer et al. 1996; Masson et al. 2011; Evangeliou

et al. 2013; Masson et al. 2013, Tagami and Uchida 2013).Within the U.S. there are

dozens of Department of Energy (DOE) sites where nuclear weapons activities have

contaminated the environment with radionuclides (e.g. Willard 1960, Fitzner and Rickard

1975, Halford et al. 1981). For example, radionuclides and chromium (Cr) discharged

into the Columbia River in Washington from the Hanford Nuclear Reservation

contaminated breeding grounds of Chinook salmon (Farag 2006).

Release of radionuclides can have significant effects on the environment and

humans for many years given the long half-lives of many isotopes (Skuterud et al. 2005,

Hinton et al. 2007, Christodouleas et al. 2011). In particular, 137Cs has a radioactive half-

life of 30.2 years and is slowly mobilized from sediments (Evans et al. 1983).

Radiocesium is of biological importance because its chemical behavior is similar to

potassium and thus can accumulate in skeletal muscle, particularly in potassium deficient

environments (Brisbin 1991).

Clearly, there is a growing importance to elucidate the potential health effects of

contaminant exposure to wildlife and the levels of contaminant accumulation within

consumable tissues of game species to assess human risks. Despite potential risks to

wildlife and human consumers, there are limited data available on contaminant levels

6

within many common game species in North America (Wolkers et al. 1994; Kennamer et

al. 1998; Braune and Malone 2006; Dvořák et al. 2010; Taggart et al. 2011, Tagami and

Uchida 2013). Thus, these data could have applications in regulating the allowable

release and remediation efforts of pollutants, as well as the consumption of game in

specific areas. Wildlife of higher trophic levels or that spend a greater portion of the year

in contaminated areas would be expected to have higher levels of trace elements or

radiocesium and thus be a greater potential risk to human consumers, but knowledge of

how residence times influence bioaccumulation in animals also is lacking (Hall et al.

2009 and Vest et al. 2009).

The goals of this research were to elucidate levels of common trace elements in

CCW and radiocesium in several common game species, the accumulation rates of these

contaminants, and how observed contaminant burdens relate to human consumption risks.

To accomplish this, several popular game species in the U.S. [wild pigs (Sus scrofa), gray

squirrels (Sciurus carolinensis), and waterfowl/waterbirds] were collected between 2012

and 2015 from the Savannah River Site (SRS), a DOE property in South Carolina, which

contains uncontaminated habitats and areas of known contamination, allowing for

collection of animals across a diversity of contaminated and uncontaminated areas.

Various tissue samples were taken from the game animals and tested for trace elements

and/or radiocesium.

Specifically, in Chapter 2 I quantified trace element and radiocesium levels in

free-ranging game animals inhabiting both contaminated and reference areas and

compared observed contaminant concentrations of free-ranging game animals to

recommended limits for human consumption and levels known to cause deleterious

7

effects to the game animals. In Chapter 3 my objectives were to 1) quantify trace element

uptake in blood, muscle, and liver tissues over known periods of time by waterfowl

exposed in situ to a coal ash settling basin and investigate potential accumulation rates, 2)

develop a model to predict muscle/liver burdens based on concentrations in blood as a

potential non-destructive sampling method and test the performance of the model against

a subset of our data, and 3) calculate human consumption limits based on concentrations

of recognized elements of human health concern (As, Se, and Hg) over known time

periods of exposure. For Chapter 4 I quantified uptake of radiocesium at multiple time

points by migrating American coots (Fulica americana) and ring-necked ducks (Aythya

collaris) subsequent to translocation to a radiocesium contaminated reservoir and

compared observed accumulation patterns between the species. I also calculated the

ecological half-life for radiocesium in American coots at this site by comparing whole-

body burdens to historical data collected from the same location. Collectively, this

research seeks to present levels of contaminants for several common mammalian and

avian game species and how certain waterfowl/waterbird species accumulate

contaminants over time when present in polluted ecosystems. These results will have

implications for wildlife inhabiting sites with similar contaminants around the world.

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material from pond water and sediment. Organic Geochemistry 8:55-64.

8

Bennett, B. G. 1995. Exposures from worldwide releases of radionuclides.

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Braune, B. M., and B. J. Malone. 2006. Organochlorines and trace elements in upland

game birds harvested in Canada. Science of the Total Environment 363:60-69.

Brisbin Jr., I. L., R. A. Geiger, and M. H. Smith. 1973. Accumulation and redistribution

of radiocesium by migratory waterfowl inhabiting a reactor cooling reservoir.

Environmental behavior of radionuclides released in the nuclear industry,

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Austria, pp. 373-384.

Brisbin Jr., I. L., and M. J. Vargo. 1982. Four-year declines in radiocesium

concentrations of American coots inhabiting a nuclear reactor cooling reservoir.

Health Physics 43:266-269.

Brisbin Jr., I. L. 1991. Avian radioecology. Current Ornithology, Volume 8. (Ed. D.M.

Power) Plenum Publishing Corporation, New York, NY, pp. 69-140.

Cherry, D. S., and R. K. Guthrie. 1977. Toxic metals in surface waters from coal ash.

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Christodouleas, J. P., R. D. Forrest, C. G. Ainsley, Z. Tochner, S. M. Hahn, and E.

Glatstein. 2011. Short-term and long-term health risks of nuclear-power-plant

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Clarkson, T. W., L. Magos, and G. J. Meyers. 2003. Human exposure to mercury: The

three modern dilemmas. Journal of Trace Elements in Experimental Medicine

16:321-343.

9

Conder, J. M., and J. A. Arblaster. 2016. Development and use of wild game

consumption rates in human health risk assessments. Human and Ecological Risk

Assessment 22:251-264.

Cristol, D. A., L. Savoy, D. C. Evers, C. Perkins, R. Taylor, and C. W. Varian-Ramos.

2012. Mercury in waterfowl from a contaminated river in Virginia. Journal of

Wildlife Management 76:1617-1624.

Dahl, T. E. 2000. Report to Congress on the status and trends of wetlands in the

conterminous United States 1986 to 1997. United States Department of Interior,

Fish and Wildlife Service, Washington, DC, pp. 82.

Diez, S., C. Barata, and D. Raldua. 2008. Exposure to mercury: A critical assessment of

adverse ecological and human health effects. Trace elements as contaminants and

nutrients: Consequences in ecosystems. (Ed. M.N.V. Prasad) John Wiley & Sons,

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Dreicer, M., A. Aarkrog, R. Alexakhin, L. Anspaugh, N. P. Arkhipov, and K. J.

Johansson. 1996. Consequences of the Chernobyl accident for the natural and

human environments. One Decade after Chernobyl: Summing up the

Consequences of the Accident. International Atomic Energy Agency (IAEA),

Vienna, Austria, pp. 319-361.

Duchesne, J. F., B. Levesque, D. Gauvin, B. Braune, S. Gingras, and E. Dewailly. 2004.

Estimating the mercury exposure dose in a population of migratory bird hunters in

the St. Lawrence River region, Quebec, Canada. Environmental Research 95:207-

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10

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CHAPTER 2

TRACE ELEMENTS AND RADIOCESIUM IN GAME SPECIES AND HUMAN

CONSUMPTION RISKS 1

____________________

1 Oldenkamp, R. E., A. L. Bryan Jr., R. A. Kennamer, S. C. Webster, and J. C. Beasley. To be submitted to the Journal of Wildlife Management.

19

ABSTRACT

Mercury (Hg), Selenium (Se), and Arsenic (As) found in coal combustion wastes

(CCW) and radionuclides released from anthropogenic activities are an environmental

and human health concern. Despite the widespread harvest and consumption of wildlife

by recreational hunters, game species are not subject to the same safety testing as

livestock and thus there are few data available regarding contaminant concentrations in

many commonly harvested wildlife. We sampled wild pigs (Sus scrofa), gray squirrels

(Sciurus carolinensis), and waterfowl from uncontaminated habitats as well as areas of

known contamination and quantified levels of trace elements and radiocesium in muscle

and liver tissues for assessment of potential human health risks from the consumption of

game. Our results revealed substantive variability in contamination burdens within and

among species, tissue types, elements, and sampling locations, likely reflecting

differences in resource selection, diet, and behavior. Species collected at a CCW ash

basin consistently had elevated levels of trace elements, particularly Se, suggesting CCW

may be an important pathway for wildlife and subsequently human exposure to this

element. Similarly, we observed elevated concentrations of radiocesium in individuals

from locations with histories of operational releases of radionuclides. The majority of

tissue samples analyzed were below levels known to adversely affect wildlife health and

radiocesium levels were below established EEC limits for human consumption.

Waterfowl consistently had elevated levels of several measured elements of interest (Se

and Hg), especially individuals collected from areas with known contamination. Given

the high levels of trace element burdens we observed in waterfowl collected from the ash

basin site, and the common occurrence of similar surface impoundments in the U.S.

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additional studies are needed to more clearly elucidate potential risks to both wildlife and

waterfowl hunters.

INTRODUCTION

Anthropogenic pollution can come from a variety of sources including

atmospheric deposition of metals or releases of wastes from factories, run-off of

pesticides and herbicides from farms, and burning of fossil fuels. The U.S. Environmental

Protection Agency (EPA) cites coal and oil-fired electric generating units as the dominant

emitters of mercury, acid gases, and many toxic metals in the U.S. (USEPA 2012a). As

of 2015, in the U.S. alone the EPA identified more than 500 coal power facilities using

735 surface impoundments to store coal combustion waste (CCW; USEPA 2015).

Furthermore, coal use is on the rise in developing countries, making CCW important at a

global scale (Humphries, 1999). The millions of tons of CCW deposited into surface

impoundments across the U.S. (Luther 2010a) contain trace elements that the EPA

considers environmental and human health risks and represent potential pathways for

contaminant exposure for wildlife and human consumers of wildlife (Rowe et al. 2002,

Luther 2010a).

Surface impoundments (hereafter referred to as ash basins) used to store CCW

represent potential habitat for foraging and reproduction for many wildlife species,

especially birds and amphibians, which can result in trace element exposure and

bioaccumulation (Yudovich and Ketris 2005a,b; Yudovich and Ketris 2006; Reash 2012;

Otter et al. 2012). Wildlife can be affected through the consumption of contaminated

water, sediment/soil, and/or food (USEPA 2012b) and examples of trace element

bioaccumulation in wildlife exposed to CCW are well documented (Dorman et al. 2010,

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Lemly and Skorupa 2012, Ruhl et al. 2012, Mayfield et al. 2013, Rice et al. 2014). For

example, coal fly ash effluent discharged into Belews Lake and Hyco Reservoir in North

Carolina, in Martin Creek Reservoir in Texas, and the largest ash release in U.S. history

into the Clinch and Emory Rivers in Tennessee have resulted in trace element

accumulation in aquatic and some terrestrial biota (Lemly 1996, 2002; Ruhl et al. 2009;

Beck et al. 2013; Van Dyke et al. 2013; Beck at al. 2015).

Although numerous trace elements exist within fly ash that accumulate in tissues

of exposed wildlife, Mercury (Hg), Selenium (Se), and Arsenic (As) are of particular

concern for environmental and human health (Luther 2010b). Exposure to toxicants

found in coal ash such as As, cadmium (Cd), lead (Pb), and Hg can impact an animals’

central nervous system, cardiovascular, hematopoietic, gastrointestinal, urinary, and

reproductive systems (Chmielnicka et al. 1989, Domingo 1994, Hughes et al. 2011). In

particular, effects of Hg at high concentrations have been well-documented, but even

chronic low-dose exposure can have severe adverse effects (Wolfe et al. 1997). In

humans, Hg affects neurobehavioral functioning (Goyer 1991); high doses cause

problems with coordination of movement, muscle weakness, sensibility, language, vision

and hearing (Duchesne et al. 2004), and is of particular concern for expectant mothers

and children (USEPA 2014). Exposure to essential elements such as Cr, copper (Cu), Se,

and zinc (Zn) in high doses can affect hematopoietic, gastrointestinal, and reproductive

systems of animals (Ohlendorf and Flemming 1988, Domingo 1994, Eisler 1998) and in

humans exposure to excessive Se can disrupt endocrine function as well as cause

neurotoxicity and hepatotoxicity (USEPA 1999b). Similarly, accumulation of As can

22

increase blood pressure, leading to heart attacks, and can result in increased risk of skin,

lung, bladder, and kidney cancer (USEPA 1998).

In addition to contaminant exposure through CCW, wildlife also can be exposed

to radioactive materials from nuclear activities such as atmospheric fallout from nuclear

weapons testing and nuclear accidents (e.g., Chernobyl and Fukushima) that released

large amounts of radionuclides (e.g., 131I, 137Cs, 134Cs) into the local environment, as well

as dispersed and deposited radioactive material across the Northern Hemisphere

(UNSCEAR 1988, Gudiksen et al. 1989, Mishra 1990, Bennet 1995, Dreicer et al. 1996,

Masson et al. 2011, Evangeliou et al. 2013, Masson et al. 2013). Within the U.S. there

also are dozens of sites that have been impacted by the U.S. Department of Energy’s

(DOE) nuclear weapons activities. For example, radionuclides and chromium (Cr)

discharged into the Columbia River in Washington, U.S.A. contaminated breeding

grounds of Chinook salmon (Farag 2006). Although release of radionuclides into the

environment has happened at a lower frequency than CCW releases, these events can

have a profound effect on the environment and humans for many years given the long

physical half-lives of many radionuclides (Skuterud et al. 2005, Hinton et al. 2007,

Christodouleas et al. 2011).

As of 2011 there were 13.7 million hunters within the U.S. (USFWS and USCB

2011), the vast majority of whom pursue game for consumption. Consumption of wild

game meat has a reputation for being healthier than many farmed raised meats, but wild

game can present a potential route of contaminant exposure to human consumers as game

animals are not subject to the same testing as livestock (Duchesne et al. 2004, Cristol et

al. 2012, Conder and Arblaster 2016). Wildlife can be exposed to contaminants directly at

23

point sources (e.g., through foraging or other behaviors resulting in incidental sediment

ingestion; Neely 1980, Boening 2000). Moreover, game animals with large home range

sizes (e.g., wild pigs – Sus scrofa, white-tailed deer – Odocoileus virginianus) or

migratory behavior (e.g., ducks) can transport contaminants extensive distances from

point sources, exposing unknowing hunters, their families, and recipients of donated

game meat to contaminants accumulated outside the vicinity of their hunting area

(Kennamer 2003, Taggart et al. 2011, Cristol et al. 2012, Conder and Arblaster 2016).

Despite the potential exposure of human hunters to contaminants through

consumption of game, there are limited data available on contaminant levels within many

common game species in North America (Wolkers et al. 1994, Kennamer et al. 1998,

Braune and Malone 2006, Dvořák et al. 2010, Taggart et al. 2011). Moreover, although

laboratory studies are typically performed to identify lethal doses of contaminants and

measure morbidity, the complex exposure pathways and consequent levels of

contaminants found in natural systems require in situ studies to elucidate accumulation

patterns of trace elements and radiocesium. Such data are essential to inform public

health officials and wildlife managers about actual risks to the public and/or the animals

utilizing those systems.

For this study, we collected several common, popular game species in the U.S.

[wild pigs, gray squirrels (Sciurus carolinensis), and waterfowl] from the Savannah River

Site (SRS), a DOE property in South Carolina with both uncontaminated habitats and

areas of known contamination, to quantify trace element and radiocesium burdens in

consumable tissues. Our objectives were to 1) quantify trace element and radiocesium

levels in game animals inhabiting both contaminated and reference areas and to 2)

24

compare observed contaminant burdens to recommended limits for human consumption

and levels known to cause deleterious effects to the game animals themselves. These

results have implications for wildlife inhabiting sites with similar contaminants across the

globe.

METHODS

Study Area

This research primarily occurred on the Savannah River Site (SRS), a ~800 km2

limited-access former nuclear production and research facility located in the coastal plain

of South Carolina that is owned and operated by the U.S. Department of Energy (White

and Gaines 2000). The SRS was created to provide nuclear weapons materials at the

beginning of the Cold War in 1951 (Savannah River Nuclear Solutions, LLC 2011) and

now has five decommissioned nuclear reactors, radioactive materials processing

facilities, and retired coal power plants (White and Gaines 2000). The remainder of the

site is dominated by natural habitats and consists of managed pine stands (54%), wetlands

(23%), upland hardwood and mixed forest (11%), grasslands (9%), and upland scrub

forest (3%; White and Gaines 2000; DeVault et al. 2004). Wetlands and other aquatic

habitat on the SRS include bottomland and swamp forests, creeks, streams, two large

cooling reservoirs, upland depressions, and Carolina bays (Lide 1994).

Upon development of the SRS, the conversion of farmland to forest created a

restricted access wildlife refuge currently occupied by 54 species of mammals, 255

species of birds, 100+ species of reptiles and amphibians, and almost 100 species of

freshwater fish (Wike et al. 2006). However, the construction and operation of the

nuclear facilities affected approximately 3,000 hectares of land, created ~2,000 hectares

25

of cooling reservoirs, and all but one stream on site received releases of thermal effluents

(White and Gaines 2000). Contamination from nuclear production, processing, waste

disposal, and power generation is known to exist on the SRS, including radiocesium from

leaking fuel elements and reprocessing operations (White and Gaines 2000). Trace

element contamination also exists on site, primarily in the form of coal fly-ash (Cherry et

al. 1979), and waste mercury from production activities that leaked from seepage basins

(Horton 1974).

The D-Area coal-fired power plants on the SRS were operational from 1953-2012

and during this time deposited sluiced fly ash into a series of basins that drain into Beaver

Dam Creek, a tributary of the Savannah River, and the surrounding wetlands (Gaines et

al. 2002, Bryan et al. 2012, USDOE 2012). Water and biota in the basins and creek

watershed contain elevated levels of aluminum (Al), As, Cd, Cr, iron (Fe), Hg,

manganese (Mn), nickel (Ni), Se, and Zn (Cherry et al. 1979, Rowe et al. 1996) and

numerous studies have documented bioaccumulation and adverse effects from

contaminant exposure in this system for a variety of organisms such as bacteria, aquatic

invertebrates, amphibians, fish, turtles, alligators, and birds (Hopkins et al. 1999, Hopkins

et al. 2000, Rowe et al. 2002, Stepanauskas et al. 2005, Wright et al. 2006; Bryan et al.

2012).

In addition, more than 2.09e+13 Bq of radiocesium was discharged into aquatic

ecosystems on the SRS, representing an important pathway of exposure for numerous

wildlife species (Carlton et al. 1992). In particular, between 1961 and 1964

approximately 5.7 X 1012 Bq of radiocesium was released into Pond B, a reactor cooling

reservoir on the SRS, with documented use by 12 waterfowl species (Ashley and Zeigler

26

1980, Mayer et al. 1986). Past work has quantified levels of radiocesium in Pond B

sediments (Brisbin et al. 1974) and related levels to those found in a variety of animals,

including migratory game birds that can disperse contaminants extensive distances

outside SRS boundaries (Brisbin et al. 1973, Fendley et al. 1977, Brisbin and Vargo

1982, Kennamer et al. 1998).

Collection of Samples

We collected muscle and liver samples from wild pigs and waterfowl/waterbirds

and muscle samples from gray squirrels between late 2012 through early 2015 throughout

various contaminated and uncontaminated areas on the SRS. Sampling locations varied

by species depending upon their abundance and distribution around specific areas of

known SRS operational activities and history of contamination and included the D-Area

ash basins, Fourmile Branch, Pond B, Pond A/R-canal, and various uncontaminated or

generalized areas including L-Lake (Figure 2-1). Additional muscle and liver samples

from wild pigs were collected in Georgia (from Dooly, Macon, Pulaski, Terrel, and

Randolph Counties; courtesy of Jager Pro, Inc.) to serve as reference samples for this

species. We collected squirrels (D-Area ash basins, Fourmile Branch, Pond B/R-canal,

and Tim’s Branch Beaver Pond) and waterfowl/waterbirds (D-Area ash basins, Fourmile

Branch, Pond B/R-canal, Tim’s Branch Beaver Pond, and L-Lake) via shot gun whereas

wild pigs were trapped in box or corral traps throughout the SRS (largely by SRS pig

control contractors) and central GA and euthanized via gunshot to the head. All animal

handling practices and euthanasia were carried out with accompanying federal and state

collecting permits and in accordance with University of Georgia Animal Care and Use

guidelines under protocol A2012 12-010-Y3-A5.

27

Seasonal timing of collections varied by species with most SRS pigs collected in

late summer through fall (GA wild pigs in spring), waterfowl/waterbirds from late fall

through winter, and squirrels in fall. Upon collection, all squirrels and

waterfowl/waterbirds were weighed, sexed, and frozen at -20°C for later dissection. We

collected a muscle sample from the upper hind leg of all collected squirrels.

Waterfowl/waterbirds were whole-body counted for radiocesium and a subset were

dissected to collect breast muscle and liver tissues for additional radiocesium and trace

element analyses to provide data for comparison studies as well as calculations for

consumption limits of muscle tissue. Wild pigs were sexed and weighed in the field

where we also collected tissue samples from the upper hind leg muscle and liver for

contaminant analyses. Wet weights of all muscle and liver samples were recorded before

samples were freeze-dried and weighed again, then homogenized into a powder using a

coffee grinder. Grinder canisters were cleaned with a 5% nitric acid solution and dried

between uses. All wild pig and squirrel samples were tested, along with the subset of

waterfowl for which we collected muscle and liver tissue, for trace elements and

radiocesium concentrations for comparison against published effects levels when

available. We also used these data to calculate human consumption limits for muscle

tissue.

Elemental Analysis

Trace element [Cr, Ni, Cu, Zn, As, Se, Cd, Pb, and uranium (U)] analysis was

conducted on all muscle and liver samples; samples were also analyzed for total mercury

(THg) content. For trace element analysis approximately 250 mg of dry sample was

microwave digested (MARSX Xpress, CEM Corporation, Matthews, NC) with 10.0 ml

28

trace metal-grade nitric acid (70% HNO3). Following digestion, samples were brought to

a final volume of 15.0 ml with Milli-Q (18MΩ) water before analysis by inductively

coupled plasma mass spectroscopy (Nexlon 300X ICP-MS; Perkin Elmer, Norwalk, CT)

according to QA/QC protocols outlined in EPA Method 6020A (USEPA 2007). The

minimum detection limits (ppm) for each element was: Cr (0.54), Ni (0.84), Cu (0.67),

Zn (7.47), As (0.39), Se (0.49), Cd (0.40), Pb (0.36), U (0.44). For quality control

purposes certified reference material (TORT-3 lobster hepatopancreas; National Research

Council, Ottawa, ON, Canada), a blank, and a digestion replicate were run for every 20

samples. Mean percent recoveries ranged from 85%-105% for elements in certified

reference materials and all element concentrations are presented as parts per million

(ppm) on a dry mass basis.

To analyze total mercury (THg), 30-50 mg subsamples of the freeze-

dried/homogenized tissues were analyzed by thermal decomposition, catalytic

conversion, amalgamation, and atomic absorption spectrophotometry (DMA 80;

Milestone, Shelton, CT, USA) according to U.S. EPA) method 7473. The instrument

detection limit (IDL) for this method is 0.01 nanograms (ng) of total mercury. Within

each set of 10 samples we included a replicate, blank, and two standard reference

materials (SRM; TORT-2 lobster hepatopancreas or TORT-3 lobster hepatopancreas, and

PACS-2 marine sediment, National Research Council of Canada, Ottawa, ON) to ensure

quality assurance and solid SRMs were used to calibrate the instrument. Method

detection limits (MDLs; threefold the standard deviation of procedural blanks) averaged

0.0004 ppm dry mass. Mean percent recoveries of THg for the SRMs TORT-2, TORT-3,

29

or PACS-2 were 102.1 ± 1.6, 106.0 ± 3.8, and 102.6 ± 5.0, respectively. Concentrations

are presented as parts per million (ppm) on a dry mass basis.

Radiocesium Analysis

Waterfowl/waterbirds were whole-body counted with a 10·2-cm x 15·2-cm NaI

(Tl) gamma detector (Bicron Model:6H3Q/5; S/N:BJ-124R) coupled to an IBM 300-GL

Personal Computer (Windows 98 OS) containing an onboard Canberra MCA card and

controlled by Canberra Genie 2000 gamma spectroscopy software (Version 1.3; entire

system located in SREL Lab 120). A counting window (Region of Interest-ROI) of 596–

728 kiloelectron-Volts (keV) centered on 662 keV was used to record total detector

absorption events from the radiocesium emission of 662 keV photons. The system was

calibrated daily, as counting took place, with a traceable radiocesium calibration disc

(New England Nuclear Gamma Reference Disc Source Set; Catalogue No. NES-101S;

radiocesium disc; 1.04 microCuries on 10/2/1985) by adjusting the system amplifier gain

control to center the disc-generated peak on channel 331 (661.7keV). Generally, 30-min

count times (1800 sec) were used for counting collected/sacrificed birds (whole-body;

frozen) and backgrounds (empty chamber), while 15-min count times (900 sec) were

used for counting aqueous standards (the ILB Series of standards; containing known

radiocesium quantities [Becquerels (Bq)]; decay corrected to count dates). Background-

corrected count rates (counts per second; cps) from the ILB Series of standards were used

to produce mass-specific count yields which were in turn used to produce a predictive

equation of expected yields from bird mass (in grams; yield=0.4449*mass-0.343;

R2=0.97). Finally, background-adjusted bird count rates and the birds' mass-specific

30

yields were used to determine radiocesium content of birds (whole-body Bq, Bq/g,

pCi/g).

All liver and/or muscle samples were freeze-dried and powdered then packed into

scintillation vials. We analyzed these samples for radiocesium activity using a Packard

Cobra II Auto-Gamma Counter (Model Cobra II 5003) with a single 3-inch through-hole

NaI detector. A counting window (Region of Interest-ROI) of 580–754 kiloelectron-Volts

(keV) centered approximately on 662 keV was used to record total absorption events

from the radiocesium emission of 662 keV photons. The system was auto-calibrated

daily, as counting took place, with a traceable radiocesium source (SREL-0113; 0.1

microCuries on 10/2012). Generally, 60-min count times (3600 sec) were used for

counting dry, powdered samples (packed into tubes) and backgrounds (empty tubes) that

were arranged in every fifth counting position. Four standards were prepared from

commercially-available chicken breast muscle tissue that was dried, homogenized into

powder, spiked with known quantities of radiocesium (745 Bq in each spiked standard;

decay-corrected for the date of preparation), and then loaded/packed into 4 scintillation

tubes in 1-gram increments ranging from 1-4g. Background-corrected count rates (net

counts per second; ncps) recorded for these spiked standards were used to produce

estimates of mass-specific count yields (a ratio of measured net count rate [ncps] to

expected disintegration rates [dps] for the known radiocesium activity) at each of 4

available sample height settings relative to the NaI detector of the Cobra II system.

Sample position #4 produced count yields that varied little across all sample masses (SD

< 0.007) and so samples/backgrounds were all counted using sample height #4 setting,

with an averaged count yield value of 0.2213 used as a constant in radiocesium content

31

determinations for unknowns. Specifically, background-adjusted dry tissue count rates

divided by the yield constant were estimated as the radiocesium content of dry tissue

samples (total Bq, Bq/g [dry mass], and Bq/g [wet mass]). Minimum Detectable

Concentrations (MDCs) were calculated for all radiocesium analyses using the equation

of Lloyd Currie (Currie 1968).

Statistical Analysis

SRS wild pig samples (for trace elements and radiocesium) were compared to

samples collected in central Georgia (see description above); also sex differences

between wild pigs were explored. Trace elements in SRS squirrels were compared

between the D-Area ash basin and all other SRS areas combined due to previous

determination of high concentrations of certain elements (e.g., Se) in that system for

some biota. Radiocesium in squirrel muscle samples was compared between the

radiocesium-contaminated locations combined (Fourmile Branch beaver pond and Pond

B/R-canal) against two separate areas without radiocesium contamination (D-Area ash

basins and Tim’s Branch). Preliminary analyses of trace element concentrations in

waterfowl/waterbirds suggested the D-Area ash basin samples (collections only of diving

ducks and waterbirds) were distinct from the remaining SRS locations. Therefore, we ran

separate analyses on diving and dabbling ducks and for diving ducks compared elemental

concentrations (Cr, Cu, Zn, Se, Hg, Cd) in tissues of individuals collected from the D-

Area ash basins to tissue concentrations in individuals collected from other SRS sites

combined. Concentrations of trace elements found in dabbling ducks and waterbirds are

presented for reference but no statistical comparisons were made between tissues

concentrations because of small sample sizes and that there were not dabbling ducks

32

represented from collections at the D-Area ash basins. Radiocesium in

waterfowl/waterbirds was quantified for all birds but only compared between locations

where we had sufficient numbers of diving ducks: Pond B/R-Canal, Fourmile Beaver

Pond, L-Lake, and D-Area ash basins.

Trace elements for which >50% samples were below detection limits (BDL) were

excluded from analyses; all remaining concentrations BDL were replaced with 50% of

the respective minimum detection limit (MDL; Hall et al. 2009; Fletcher et al. 2014). We

tested all element results distributions for normality (Shapiro-Wilk test p<0.05) in R (R

Core Team 2012) and subsequently log-transformed all data prior to inclusion in analyses

to improve distributions. We used Multiple Analysis of Variance (MANOVA) models

with the Pillai–Bartlett statistic in R (Package stats version 3.3.1) to test whether element

concentrations differed between locations. Muscle and liver samples were tested

separately because trace element concentrations have been shown to differ between these

tissue types in previous toxicology studies (Scheuhammer 1987, Boening 2000, Mason et

al. 2000, Farkas et al. 2003, Ikem et al. 2003, Coğun et al. 2006, Havelková et al. 2008).

Potential correlations between tissues were assessed for each element and species group

(e.g., Se in muscle and liver for SRS wild pigs) and between elements for each tissue

(e.g., Se vs Hg in SRS wild pig muscle samples) with Spearman Rank correlations on

non-transformed data. Spearman’s correlation test ranks the data, controlling for the non-

normal distribution before making comparisons.

Radiocesium concentrations in several muscle and liver samples were below

background levels, resulting in negative values. Negative values and the very small

positive concentrations in samples below the MDCs are often not reported or are set to

33

some arbitrary indicator value, which biases the mean and variance of such data (Gilbert

and Kinnison 1981, Newman et al. 1989). Therefore, we included all negative values, as

well as those below the MDCs, when determining average radiocesium concentrations.

These data were non-normally distributed, therefore, we minimally scaled the data by

adding a number sufficient to remove all negative values and log transformed values

prior to inclusion in models; data were back-transformed and scaling removed before

inclusion in tables. We created separate Analysis of Variance (ANOVA) models for each

taxa of animals and if there were significant differences found in radiocesium

concentrations between study locations we conducted Tukey post-hoc tests to elucidate

differences among sites. We present geometric means and associated standard errors due

to unbalanced sample sizes between groups.

Human Consumption Limits

Based on observed concentrations of key trace elements known to have

deleterious effects on human health (As, Se, Hg; see results), we derived human

consumption limits for wild pigs, squirrels, as well as dabbling and diving ducks

independently. The EPA provides chronic oral reference dose limits for each of these

trace elements; these are levels at which an individual could be exposed daily over the

course of their life and not expect negative health consequences (citation). Human

consumption limits were calculated with both average and maximum concentrations of

the aforementioned trace elements found in muscle samples.

Nearly all Hg in muscle of higher trophic level organisms such as birds and fish is

in the methylmercury (MeHg) form; 80-100% in muscle of piscivorous birds and up to

98% in fish muscle (Wiener et al. 2003; Evers et al. 2005). We tested for total THg,

34

which would show all mercury, but since the majority of the human exposure is to MeHg

instead of elemental mercury and this is the most bioavailable form for humans to absorb,

calculations of consumption limits were done with EPA limits for MeHg (USEPA 1999a,

2001; Hall et al. 2009). MeHg also is the most toxic form of mercury and can cause

severe neurological damage to humans and wildlife (Grandjean et al. 1999, Clarkson et

al. 2003).

The EPA established chronic oral reference doses of 0.0001 milligrams per

kilograms per day (mg/kg/day) for methylmercury, 0.005 mg/kg/day for selenium

(USEPA 1999b) and 0.0003 mg/kg/day for arsenic (USEPA 1998) were used in

calculating consumption limits. These levels are daily dose exposures that alone are

unlikely to produce appreciable deleterious effects over a lifetime of exposure; as

exposures increase above the reference doses so does the risk of adverse health effects.

We used average standard weights for adults and children (70 kg or 154 lbs and 16 kg or

35 lbs, respectively), as well as meal sizes (227g or ½ lb for adults and 113g or ¼ lb for

children) for our calculations based on established EPA methods for fish advisory

calculations.

Since most meat is neither eaten raw (wet mass) or completely devoid of water

(dry mass) we also calculated trace element concentrations based on what the average

person would be exposed to from consuming cooked meat. For wild pig calculations we

utilized the average percent moisture loss during cooking found for squirrel and deer

muscle (17.9%; Holben 2002) and for ducks we utilized the average percent moisture loss

in cooked duck breast muscle (28.2%; Omojola 2007). We used these values to amend

concentrations of trace elements that would be found in cooked muscle for consumption

35

limit calculations. This gives a higher concentration for the trace element than normally

reported wet weight concentrations, but more accurate to real world scenarios for human

consumption of game meat. Mean radiocesium concentrations (Bq/g) in raw muscle

samples for wild pigs, squirrels, and waterfowl/waterbirds that were above MDCs were

compared to the European Economic Community limit of 0.600 Bq/g for fresh meat

(EEC 1986).

RESULTS

For this study 108 wild pigs, 24 squirrels, and 130 waterfowl/waterbirds were

collected; the number of muscle and liver samples tested for trace elements and

radiocesium by taxa and the sex ratios of those groups is presented in Table 2-1.

Wild Pigs

We quantified trace element concentrations in muscle for 88 SRS wild pigs and

further analyzed liver element concentrations for a subset of 30 of these individuals. In

addition, we quantified trace element concentrations in muscle and liver tissues for 20

non-SRS wild pigs (Table 2-2). Concentrations of six trace elements (Cr, Cu, Zn, Se, Hg,

Cd) were found to be above the MDLs in either muscle or liver and were used in

statistical analyses. MANOVA’s testing for differences in trace element concentrations in

muscle and liver between wild pigs captured on the SRS and those in Georgia revealed

some differences between the two locations. Post-hoc analyses indicated that

concentrations of three elements (Hg, Se, and Zn) were higher in SRS samples in one or

both tissues whereas chromium concentrations were higher in GA pigs (GA muscle Cr

= 3.10 ppm vs. SRS muscle Cr = 0.70 ppm, dry mass; Table 2-2).

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Several trace element concentrations were correlated for both SRS and Georgia

samples (Tables 2-3 and 2-4). In particular, for SRS wild pigs, which had higher Se

concentrations than Georgia wild pigs, Se in muscle was positively correlated, though not

strongly, to all the other elements (Table 2-3). For SRS wild pigs, Se and Cd in liver were

moderately and positively correlated (Table 2-3). For Georgia wild pigs, where Cr was

higher than in SRS wild pigs, Cr was positively related to Cu and Zn in muscle (Table 2-

4). In addition, Se and Hg were both positively correlated between muscle and liver

samples for both SRS and Georgia wild pigs (Tables 2-3 and 2-4).

Comparisons of radiocesium tissue concentrations (Table 2-5) revealed SRS

muscle samples were nearly five times higher (geometric = 0.295 Bq/g, dry mass) than

those sampled in Georgia (geometric = 0.057 Bq/g, dry mass). Higher concentrations

were observed in liver samples as well (Tables 2-5 and 2-6). No differences in

radiocesium tissue concentrations were observed between male and female pigs (Table 2-

6).

Squirrels

Twenty four gray squirrels were collected across the SRS. Concentrations of four

trace elements (Cr, Cu, Zn, Hg) were found to be above the MDLs in squirrel muscle and

were used in statistical analyses. Results of our MANOVA analysis comparing element

concentrations between squirrels collected near the D-Area ash basins versus all other

SRS locations failed to detect any significant differences between sites (Table 2-7). No

correlations were found among trace elements in squirrel muscle tissue.

Radiocesium concentrations in squirrel muscle tissue from locations on the SRS

(Table 2-8) suggested among-site differences, with samples from locations having known

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releases of radiocesium tending to have higher concentrations (Table 2-9). Statistical

comparisons with ANOVA indicated that muscle radiocesium concentrations in squirrels

collected from the Fourmile Branch and Pond B/R-Canal area were highest, (geometric

= 1.27 Bq/g, dry mass), and significantly higher than squirrels from either Tim’s

Branch beaver pond (geometric = 0.38 Bq/g, dry mass) or the D-Area ash basins

(geometric = 0.29 Bq/g, dry mass; Table 2-9).

Waterfowl/Waterbirds

Twelve species of waterfowl and three species of waterbirds collected on the SRS

were included in various analyses; muscle and liver samples for 66 and 70

waterfowl/waterbirds, respectively, were tested for trace elements (Table 2-10). Our

MANOVA indicated Se (muscle and liver) and Zn (muscle only) concentrations were

elevated in D-Area ash basin diving ducks compared to diving ducks collected from other

SRS locations, although ducks collected from other SRS locations had higher Cu

concentrations in liver and higher Cr in muscle (Table 2-11). Arsenic is an important

element found in coal ash and of interest for regulation of wastes, remediation efforts,

and human health risk assessment. More than 50% of the diving ducks sampled in this

study had As concentrations in muscle and liver that were BDL. Therefore, we did not

compare As tissue concentrations among sites. However, of the diving ducks collected

from the D-Area ash basins, 29% and 16% of muscle and liver samples, respectively had

levels that were BDL, compared to 91% and 86% from other SRS locations. Here we

report As concentrations in both muscle and liver for diving ducks collected at the ash

basins (muscle = 0.78±0.10, 0.20-2.07 ppm, dry mass; liver = 2.04±0.35, 0.20-6.95

ppm, dry mass) and other SRS locations (muscle = 0.29±0.07, 0.20-2.74 ppm, dry

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mass; liver = 0.42±0.13, 0.20-4.80 ppm, dry mass) for comparison purposes with other

studies.

Dabbling ducks were collected from three sites on the SRS. Mallards (Anas

platyrhynchos) were collected on the Fourmile Branch beaver pond (n=10) and Wood

Ducks (Aix sponsa) were collected from a beaver pond on Tim’s Branch (n=5), Fourmile

Branch beaver pond (n=1), and R-Canal (n=1). Concentrations of those elements (Cr, Cu,

Zn, Se, Hg, Cd) with the majority of samples > MDL are presented in Table 2-12.

Waterbirds analyzed for elemental concentrations included American Coots

(Fulica Americana) (n=4) and Pied-billed Grebes (Podilymbus podiceps) (n=3) from the

D-Area ash basins, and Double-crested Cormorants (Phalacrocorax auritus) (n=4) from

Pond B. Concentrations of elements (Cr, Cu, Zn, As, Se, Hg, Cd) found in these tissue

samples are presented in Table 2-13. The highest Hg level found during this study was in

the liver tissue of a cormorant collected at Pond B (169.4 ppm, dry mass; Table 2-13).

Due to small sample sizes no statistical testing was performed on dabbling ducks or

waterbirds.

Spearman’s rank correlation revealed several trace element concentrations were

correlated in waterfowl/waterbird tissue samples (Table 2-14). Se concentrations in

muscle were positively correlated to Cu, Zn, and Hg, whereas liver Se concentrations

were positively related to Cr, Cd, and Hg (Table 2-14). Cu concentrations also were

positively correlated to Zn in muscle. Only concentrations of Zn were correlated between

muscle and liver, positively, albeit weakly (Table 2-14).

All 130 waterfowl and water birds collected from the SRS were whole-body

counted for radiocesium. Muscle and liver tissues also were counted for a subset of 98 of

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individuals (Table 2-15). Comparisons of whole-body, muscle, and liver tissues of diving

ducks among four SRS locations indicated radiocesium concentrations in individuals

from Pond B were significantly higher (whole-body: geometric = 0.113 Bq/g, fresh

mass; muscle: geometric = 0.163 Bq/g, wet mass; liver: geometric = 0.149 Bq/g,

wet mass; Table 2-16) than concentrations from the three other SRS locations. Sample

concentrations from Fourmile Branch beaver pond, L-Lake, and the D-Area ash basins

were not statistically different from each other.

Human Consumption Limits

After accounting for average moisture loss during cooking based on studies by

Holben (2002) and Omojola (2007), we estimated a retained moisture content of 56.4%

for wild pigs, 58.8% for squirrels, and 43.2% for waterfowl which we utilized in

subsequent calculations to obtain concentrations of As, Se, and Hg in cooked muscle. The

values for each group for were obtained from calculating the actual moisture lost in the

drying process and then adjusting the element concentration for estimated moisture lost

during cooking instead of complete drying. Muscle concentrations of Hg resulted in

consumption limits that allowed fewer meals than As and Se so Hg results are used as an

overall guide to the number of meals allowed.

SRS wild pigs had lower allowable meals than Georgia wild pigs based on muscle

concentrations of trace elements, although allowances were still well above average

numbers consumed by hunters in the region (Smith, unpublished manuscript) based on

average levels observed in our samples. Consumption limits based on average

concentrations of Hg found in SRS wild pigs (muscle = 0.062 ppm, cooked mass)

calculated for human adults were 15.2 (1/2 lb) meals per month, while limits calculated

with the maximum observed Hg concentration (muscle = 0.350 ppm, cooked mass)

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reduced the limit to 2.7 meals per month (Table 2-17). The meals allowable for children

calculated utilizing the average concentration of Hg were 7.0 (1/4 lb) meals per month,

but only 1.2 meals per month at the maximum concentration (Table 2-17). Element

concentrations in squirrels collected from the D-Area ash basins were not statistically

different from those collected at other sites. Concentrations of trace elements in squirrel

muscle were low such that at the average level of Hg (muscle = 0.017 ppm, cooked

mass) a child could consume ~25 meals per month and an adult ~50 meals before

approaching the EPA’s oral reference dose limit.

We calculated that diving ducks from the D-Area ash basin had the lowest

allowable number of monthly meals of all taxa. Consumption limits based on the average

Hg concentrations in diving duck muscle from the ash basins ( = 0.447 ppm, cooked

mass) were 2.1 (1/2 lb) meals per month for adults and only 1/3 of a meal per month at

the maximum Hg concentration (muscle = 3.153 ppm, cooked mass; Table 2-18). For

children these consumption limits are less, only 1.0 (1/4 lb) meal per month at the

average level and 1/10 of a meal at the maximum Hg concentration (Table 2-18).

Calculations for consumption of sampled dabbling ducks from other locations are also

included for comparison (Table 2-18).

Even though radiocesium concentrations in wild pigs on the SRS ( = 0.459

Bq/g, dry mass) were nearly five times higher than GA pigs, only 21% of the SRS muscle

samples were above their respective MDCs. No wild pig muscle sample exceeded the

European Economic Community (EEC 1986) limit of 0.600 Bq/g for fresh meat, although

the maximum we observed (0.579 Bq/g, wet mass) approached the limit. The maximum

radiocesium concentration in a squirrel muscle sample (from Pond B/R-Canal; 0.595

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Bq/g, wet mass) approached the limit as well, but levels in uncontaminated areas (D-Area

ash basins and Tim’s Branch) and known radiocesium contaminated areas (Pond B/R-

canal and Fourmile Branch) only averaged 0.1002 and 0.2829 Bq/g, wet mass,

respectively. In the present study, 19 of 98 (19.4%) waterfowl/waterbirds had muscle

radiocesium concentrations in excess of the EEC limit. All 19 of these birds were

collected from Pond B, an area with known radiocesium contamination, and included 1

ring-necked duck (Aythya collaris), 2 double-crested cormorants, and 16 American coots;

63.3% of waterfowl/water bird muscle samples collected from Pond B exceeded the EEC

limit. The maximum muscle concentration of radiocesium observed in any animal was

2.14 Bq/g, wet mass, in an American coot from Pond B.

DISCUSSION

Here we present trace element and radiocesium concentrations in consumable

tissues of North American game species that occur in both contaminated and

uncontaminated areas on the SRS. However, the SRS is but one of many such

contaminated sites as anthropogenic disturbances are pervasive throughout ecosystems

across the globe (Sanderson et al. 2002, Ellis and Ramankutty 2008). Our results revealed

substantive within and among species variability in tissue contamination, likely reflecting

underlying differences in resource selection, diet, behavior, and distribution. Species

collected at a CCW ash basin, for example, consistently had elevated levels of trace

elements known to occur in coal-fly-ash, particularly Se, supporting prior studies that

demonstrated CCW is an important source of exposure to this element. Similarly, we

often observed elevated concentrations of radiocesium in individuals collected from SRS

sites with known histories of operational releases of radionuclides (e.g., Pond B and

Fourmile Branch).

42

We examined concentrations of trace elements in game animals relative to

concentrations known to impact wildlife health and/or reproduction. Knowledge of

effects levels/concentrations for many elements and/or many game species (particularly

for most non-carnivorous mammals) is lacking, but does exist for those elements of

concern relative to human health (e.g., Hg and Se). These “effects levels” are generally

associated with liver concentrations as liver often accumulates greater concentrations of

contaminants than muscle tissue, thus providing the highest detection probability.

Elemental concentrations for game mammals (i.e. wild pigs and squirrels) were generally

below levels known to affect wildlife health and/or reproduction, and we typically saw

higher trace element burdens in wild pigs. Since wild pigs commonly root within soil for

subsurface food items, exposure to contaminated soil is likely higher than for squirrels

who are liable to have less sustained contact with contaminated soils (Taggert et al.

2009).

Of particular interest are our results for waterfowl as they are highly mobile and

migratory, and thus able to disperse contaminants hundreds or thousands of kilometers

from point sources, increasing the risk of exposure for hunters beyond local communities

surrounding contaminated sites. Trace element concentrations found in diving duck

tissues suggested differences in potential exposure/risk among elements and locations. Se

concentrations in livers of diving ducks from the CCW basins averaged > 35 ppm dry

mass, with a maximum observed concentration of 71.5 ppm; these levels are above

concentrations considered potentially toxic (10-20 ppm dry mass) or toxic (> 20 ppm dry

mass; Ohlendorf and Heinz 2011) to waterfowl, although currently little is known about

the potential health effects to individuals of populations of waterfowl/waterbirds naturally

43

exposed to suites of trace elements. Average Se concentration in diving ducks collected

from other SRS sites with no known releases of Se was only 9.33 ppm. However, four

samples from non-CCW sites were within the potentially toxic (n=3) and toxic (n=1)

ranges. It is unknown whether these individuals acquired high Se burdens from other SRS

locations or off-site areas within their breeding range or along their migratory route.

Interestingly, the highest concentration of Se (83.65 ppm) was observed in the liver of a

Ring-necked duck collected from Pond B. Ring-necked ducks were infrequently sighted

at the ash basins in our study, suggesting this individual likely accumulated Se elsewhere

within its breeding or migratory range. Liver Se levels in dabbling ducks (mallards and

wood ducks) from areas with no known Se contamination did not exceed 1.5 ppm,

although Se in livers of the waterbird species averaged near or above the level considered

toxic. Given the high trace element concentrations we observed in waterfowl collected

from the ash basin site, and that there are currently 735 similar surface impoundments for

CCW in the U.S. (and many more worldwide), additional studies of a broader scope are

needed to more clearly elucidate potential risks to both wildlife and waterfowl hunters.

Concentrations of Hg in avian liver tissue were generally below levels of concern,

although liver Hg concentrations for a small number of diving ducks from the ash basins

(n=2) and non-ash basin sites (n=1) exceeded levels of concern (2 ppm wet mass, ~8-10

ppm dry mass) thought to result in adverse health effects (Shore et al. 2011). All dabbling

ducks, coots, and grebes had liver Hg concentrations below the level of concern.

However, highly piscivorous cormorants from Pond B had the highest average and

maximum Hg liver concentrations (~60 ppm and ~169 ppm Hg dry mass, respectively) of

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any animal analyzed in this study, levels that greatly exceeded the threshold at which

adverse health effects could be expected.

Across all taxa surveyed, our results suggest that for humans Hg was the element

that produced the most restrictive allowance of meals per month before exceeding EPA

chronic oral exposure limits. At average Hg concentrations for wild pigs, a relatively high

number of meals for adults and children (15.2 and 7.0 respectively) would be permitted,

although when the maximum concentration was considered allowances dropped to 2.7

meals/month for adults and 1.2 for children, consumption rates relevant to average levels

reported for hunters in the southeastern U.S. (Smith, unpublished manuscript). GA wild

pigs had meal allowances that greatly exceeded those from the SRS (51.6 and 23.7 meals

per month for adults and children, respectively at average Hg levels in muscle). At

maximum concentrations meal allowances of 30.0 and 13.7 meals per month were

calculated for adults and children, respectively. Foraging habits of squirrels appeared to

limit contaminant uptake at sites with higher known concentrations of trace elements in

sediments (i.e. D-Area and Fourmile Branch) and thus consumption of squirrels appear to

be less of a risk for human exposure as evidenced by the large number of meals allowed

(50+ for adults and 25+ for children) when compared to wild pigs and waterfowl. In

contrast to the liberal meal allowances for wild pigs and squirrels even from areas with

known contaminant releases, consumption limits of diving ducks for adults and children

based on average Hg concentrations from the ash basin were 2.1 and 1.0 meal(s) per

month respectively. This was further reduced to 0.3 meals for adults and 0.1 meals for

children when considering maximum observed concentrations in muscle tissue. These

data suggest waterfowl use of areas contaminated with CCW pose a potential risk to not

45

only the waterfowl themselves, but also hunters and their families, especially pregnant

mothers and children (Duchesne et al. 2004). Use of CCW impoundments by waterfowl

as well as uptake rates of contaminants for waterfowl that use contaminated

impoundments within either their breeding or migratory ranges are largely unknown, but

represent an important area of future study.

Unfortunately, as with certain trace elements, “effects” levels or concentrations of

radiocesium relative to wildlife health is lacking (Hinton 1998). Thus, as a conservative

approach, we discuss the observed concentrations and trends relative to human

consumption levels of concern established by the European Economic Community for

radiocesium in fresh meat. Unlike other elements, radiocesium generally accumulates to

higher concentrations in muscle tissue than in liver tissue (e.g., Brisbin and Smith 1975,

Potter et al. 1989), as was observed for wild pigs and waterfowl in our study.

Radiocesium concentrations in wild pigs on the SRS were about five times higher than in

pigs collected from central Georgia. Still, only 21% of the SRS pig muscle samples were

above their respective MDCs. No wild pig muscle sample exceeded the EEC limit for

fresh meat and thus currently consumption of wild pigs from the SRS does not appear to

be a danger to human health. However, pig behavior may increase exposure to and

subsequent accumulation of numerous contaminants including radiocesium; thus in areas

of suspected contamination wild pigs that may be taken as game meat should be tested.

Levels of radiocesium in squirrel muscle tissue from areas with known

radiocesium releases (i.e., Fourmile Branch and Pond B/R-Canal) were higher than in

squirrels from locations with no such known contaminant releases (i.e., D-Area ash

basins and Tim’s Branch beaver pond). The maximum radiocesium concentration in a

46

squirrel muscle sample (from Pond B/R-Canal; 0.595 Bq/g, wet mass) approached the

EEC (1986) limit of 0.600 Bq/g in fresh meat. Squirrels have smaller home ranges than

larger or more mobile species, like wild pigs and waterfowl, and therefore are more

localized threats when considering contamination sources.

Radiocesium concentrations were higher in diving ducks from Pond B (whole-

body geometric = 0.113 Bq/g, fresh mass) than other areas of SRS. However, as

previously reported by Brisbin et al. (1973), American coots had the highest radiocesium

levels among the waterfowl species surveyed. In our study, 19 of 98 (19.4%)

waterfowl/waterbirds sampled had muscle radiocesium concentrations in excess of the

0.600 Bq/g, fresh mass, EEC (1986) limit, all from Pond B; thus 63.3% of the Pond B

birds exceeded the consumption limit. It should be noted that cormorants are not eaten

and coots are less popularly consumed than many other species of waterfowl/waterbirds.

Whole-body radiocesium concentrations in American coots on Pond B ( = 0.94 Bq/g,

fresh mass) were about 7 times higher than Pond B diving ducks. These differences

between coot and diving duck radiocesium levels have been attributed to species

differences in diet (e.g., Brisbin et al. 1973). However, species differences in residence

time on contaminated sites, which is not well understood at this point, has the potential to

explain substantial variation as well. Additional studies are needed to elucidate the effects

of residence time on radiocesium accumulation for a greater diversity of

waterfowl/waterbirds and other wildlife known to utilize radionuclide-contaminated sites

worldwide.

€

x

€

x

47

MANAGEMENT IMPLICATIONS

Our results revealed differences in contaminant concentrations among species,

tissue types, elements, and sampling locations. Although our sampling occurred on the

SRS, a location with known contamination, the majority of tissue samples analyzed were

below levels known to adversely affect wildlife health and in the case of radiocesium fell

below established EEC limits for human consumption. However, some samples collected

from locations with known elevated levels did exceed wildlife health thresholds or EEC

limits. These data fill critical knowledge gaps regarding trace element and radiocesium

accumulation in multiple game species utilizing contaminated areas.

While we continue to deal with environmental contamination from recent

catastrophes like the nuclear accident at Fukushima and the failed ash basin

impoundment that sent coal-fly-ash into the Emory-Clinc-Tennessee River System, we

need to consider how these and future environmental practices affect wildlife that can

expose humans hunters and their families to contaminants. The research presented herein

represents an important advance in our understanding of potential exposure levels of

wildlife inhabiting contaminated areas, however, long-term monitoring of contaminant

exposure in game species and the subsequent effects on resident or migrant wildlife and

human hunters across larger spatial scales is essential. Species with large home ranges or

that are migratory should be a priority for monitoring as they present risks to the public

far from contaminant sources (Taggart et al. 2011). Also most often we analyze human

consumption risks on an individual species basis; but we do know that most hunters

consume multiple species so future studies should treat consumption limit calculations

more holistically, incorporating fish, as well as multiple game species.

48

Table 2-1. Sample sizes of game species collected from the Savannah River Site (SRS)

and analyzed for trace elements and radiocesium in 2012-2015; sex ratios are also

indicated.

Species Sampled Analyses Muscle M/F Liver M/F SRS Wild pigsa Trace Elements 88 (50/37) 30 (14/15)

Radiocesium 82 (49/33) 26 (12/14) GA Wild pigs Trace Elements 20 (12/8) 20 (12/8)

Radiocesium 19 (11/8) 20 (12/8) SRS Squirrels Trace Elements 24 (9/15) - -

Radiocesium 24 (9/15) - - SRS Waterfowlb Trace Elements 66 (41/25) 70 (45/25) Radiocesium 98 (60/38) 98 (60/38)

aSex was not determined for one SRS wild pig.

b130 total waterfowl were whole-body counted for radiocesium.

49

Table 2-2. Comparisons of trace element concentrations (ppm, dry mass) in muscle and liver tissues of wild pigs collected

from the Savannah River Site (SRS) and those collected from five counties in Georgia (GA) in 2012-2015. See Table 2-1 for

sample sizes of muscle and liver tissues analyzed.

!! ______SRS Wild Pigs___ ______GA Wild Pigs___ Elementa Tissue Mean SE Range Mean SE Range F Pb Cr Muscle 0.70 0.04 0.27-1.62

3.10 0.65 0.76-10.82

57.25 <0.0001

Liver 0.70 0.11 0.27-3.02

1.58 0.21 0.77-4.02

16.63 <0.001

Cu Muscle 9.87 1.42 2.85-84.60

7.03 1.28 3.24-29.98

0.87 0.35

Liver 24.30 3.96 0.34-89.99

19.94 2.71 10.08-63.64

0.67 0.42

Zn Muscle 86.38 3.38 42.56-211.91

69.08 3.95 40.38-106.15

5.53 <0.05

Liver 113.83 7.31 3.74-203.74

112.51 4.58 90.74-170.66

0.02 0.89

Se Muscle 1.79 0.07 0.25-3.56

0.96 0.12 0.25-1.99

27.27 <0.0001

Liver 2.95 0.16 1.37-4.81

2.21 0.21 1.11-4.38

7.75 <0.01

Hg Muscle 0.14 0.01 0.01-0.80

0.04 0.00 0.02-0.07

21.06 <0.0001

Liver 0.35 0.04 0.06-0.87

0.07 0.01 0.02-0.15

36.44 <0.0001

Cdc Liver 1.14 0.24 0.20-5.22 0.44 0.09 0.20-1.83 ― ― aOver 50% of the following elements were below method detection limits and are not presented in this table: As, Ni, Pb, U,

and Cd (muscle).

bNS=not significant (P > 0.05).

cNot compared statistically due to a low percentage (~45%) of above detection concentrations for the GA wild pigs.

50

Table 2-3. Correlationsa among trace element concentrations in muscle (above diagonal) and liver (below diagonal) tissues of wild

pigs collected from the Savannah River Site (SRS) in 2012-2015 (n=88 muscle, n=30 liver). Only those elements with more than 50%

of the values above detectable limits are included. Correlations between muscle and liver samples for individual elements are

presented on the diagonal in bold.

Cr Cu Zn Se Hg Cd Cr NS 0.29 (0.006) NS 0.21 (0.05) NS ― Cu NS NS NS 0.26 (0.02) NS ― Zn NS NS NS 0.37 (0.0004) NS ― Se -0.50 (0.005) NS NS 0.62 (0.0004) 0.25 (0.02) ― Hg NS NS NS NS 0.45 (0.01) ― Cd -0.43 (0.02) NS NS 0.76 (<0.0001) NS ―

aSpearman correlation coefficients and P values in parentheses; NS=not significant (P > 0.05).

51

Table 2-4. Correlationsa among trace element concentrations in muscle (above diagonal) and liver (below diagonal) tissues of wild

pigs collected from five counties in Georgia (GA) in 2012-2015 (n=20 muscle, n=20 liver). Only those elements with more than 50%

of the values above detectable limits are included. Correlations between muscle and liver samples for individual elements are

presented on the diagonal in bold.

Cr Cu Zn Se Hg Cd

Cr NS 0.59 (0.007) 0.50 (0.03) NS NS ―

Cu 0.48 (0.03) NS NS NS NS ―

Zn NS NS NS NS NS ―

Se NS NS NS 0.84 (<0.0001) NS ―

Hg NS NS NS 0.66 (0.002) 0.50 (0.02) ―

Cd NS NS NS NS NS ― aSpearman correlation coefficients and P values in parentheses; NS=not significant (P > 0.05).

52

Table 2-5. Descriptive statistics for radiocesium concentrations in muscle and liver tissues of wild pigs collected from the

Savannah River Site (SRS) and from five counties in Georgia (GA) in 2012-2015.

Group Compartment Variable N % below

MDCa Mean SE Min Max Median CV (%) SRS Pigs Muscle Bq/g; dry mass 82 49 0.289 0.034 -0.053 2.211 0.209 105.2

Bq/g; wet mass

0.075 0.009 -0.014 0.579 0.054 107.9

MDC; dry mass

0.207 0.010 0.128 0.795 0.185 44.1

wet:dry ratio 76

3.905 0.045 2.218 4.676 3.905 10.5

GA Pigs Muscle Bq/g; dry mass 19 100 0.051 0.013 -0.062 0.151 0.044 113.5

Bq/g; wet mass

0.020 0.006 -0.028 -0.070 0.016 123.6

MDC; dry mass

0.202 0.019 0.128 0.431 0.176 40.2

wet:dry ratio 17

2.585 0.058 2.124 2.955 2.585 9.9

SRS Pigs Liver Bq/g; dry mass 26 85 0.085 0.017 -0.031 0.234 0.081 101.8

Bq/g; wet mass

0.026 0.005 -0.010 0.073 0.023 103.0

MDC; dry mass

0.198 0.014 0.121 0.405 0.168 36.4

wet:dry ratio 18

3.278 0.056 2.805 3.869 3.278 8.7

GA Pigs Liver Bq/g; dry mass 20 100 -0.026 0.012 -0.147 0.097 -0.029 -201.8

Bq/g; wet mass

-0.012 0.005 -0.067 0.046 -0.012 -208.6

MDC; dry mass

0.227 0.013 0.122 0.320 0.235 26.6

wet:dry ratio 19 2.363 0.056 2.026 3.080 2.338 10.6 aMDC=minimum detectable concentration; calculated from Currie (1968).

53

Table 2-6. Radiocesium concentrations (Bq/g, dry mass) in muscle and liver tissues of

wild pigs collected from the Savannah River Site (SRS) and from five counties in

Georgia (GA) from 2012-2015a.

Tissue Location Geometric Mean

Lower 95% CI

Upper 95% CI

Muscle SRS Pigs 0.295 0.222 0.384 GA Pigs 0.057 0.006 0.136 Liver SRS Pigs 0.097 0.042 0.167 GA Pigs -0.048 -0.076 -0.010 Muscle F (N=41) 0.149 0.083 0.241 M (N=60) 0.147 0.088 0.225 Liver F (N=22) 0.040 -0.008 0.101 M (N=24) -0.014 -0.048 0.028

aRadiocesium concentrations differed by location for muscle (ANOVA: F(1,98) = 16.023,

P = 0.0001) and liver (ANOVA: F(1,43) = 22.329, P = <0.0001) tissues; radiocesium

levels did not differ by sex.

54

Table 2-7. Comparisons of trace element concentrations (ppm, dry mass) in muscle of squirrels collected near the D-Area ash

basins and squirrels collected from all other locations on the Savannah River Site (SRS) in 2012-2015.

Ash Basin Squirrels (N=9) Other Squirrels (N=15) Elementa Tissue Mean SE Range Mean SE Range F Pb Cr Muscle 1.20 0.15 0.55-1.86

1.18 0.16 0.27-2.42

0.01 0.94

Cu Muscle 5.21 0.64 3.55-9.84

4.93 0.44 3.20-10.15

0.14 0.71

Zn Muscle 35.94 1.47 31.86-45.57

35.34 1.28 27.78-43.24

0.09 0.77

Hg Muscle 0.04 0.01 0.02-0.10 0.05 0.01 0.01-0.17 0.39 0.54 aOver 50% of the following elements were below method detection limits and were not presented in this table: As, Cd, Ni, Pb,

Se, and U.


55

Table 2-8. Descriptive statistics for radiocesium concentrations in muscle tissue of squirrels collected from the Savannah

River Site (SRS) in 2012-2015.

Tissue Squirrel Location Variable N

% below MDCa Mean SE Min Max Median CV (%)

Muscle D-Area Ash Basins Bq/g; dry mass 9 56 0.275 0.035 0.163 0.439 0.258 37.9

Bq/g; wet mass 9

0.117 0.015 0.073 0.199 0.098 39.7

MDC; dry mass 9

0.268 0.023 0.186 0.343 0.301 25.8

wet:dry ratio 5

2.370 0.104 1.959 3.112 2.370 13.2

Four-mile/Pond B/R-canal Bq/g; dry mass 9 44 1.159 0.290 0.202 2.181 1.178 75.1

Bq/g; wet mass 9

0.283 0.068 0.059 0.595 0.284 72.6

MDC; dry mass 9

0.188 0.004 0.170 0.203 0.195 6.3

wet:dry ratio 9

2.521 0.181 2.873 4.582 3.874 14.0

Tim's Branch Beaver Pond Bq/g; dry mass 6 17 0.281 0.045 0.096 0.404 0.307 39.4

Bq/g; wet mass 6

0.075 0.014 0.023 0.122 0.078 45.7

MDC; dry mass 6

0.202 0.019 0.159 0.290 0.191 23.2

wet:dry ratio 6 3.878 0.208 2.930 4.310 4.074 13.1 aMDC=minimum detectable concentration; calculated from Currie (1968).

56

Table 2-9. Comparison of radiocesium concentrations (Bq/g, dry mass) in muscle tissue

of squirrels collected from four different locations on the SRS in 2012-2015.a

Location Geometric Mean

Upper 95% CI

Lower 95% CI

D-Area Ash BasinsA 0.292 0.524 0.141

Fourmile/Pond B/R-canalB 1.269 2.027 0.776

Tim's Branch Beaver PondA 0.383 0.746 0.168 aRadiocesium concentrations differed among locations for muscle (ANOVA: F(2,21) =

9.088, P < 0.001). Post-hoc Tukey HSD tests were used for determining pairwise

locational differences. Locations with the same capital letter were not significantly

different (P > 0.05).

57

Table 2-10. Waterfowl and waterbird species collected on the Savannah River Site (SRS) in 2012-2015, with the scientific

names, alpha codes, guild groupings, and sample sizes in various analyses.

Common Name Scientific Name Alpha Code Guild Group Muscle

Elements Liver

Elements Muscle /

Liver 137Cs Whole-body

137Cs

Wood Duck Aix sponsa WODU Dabbling Duck 7 7 10 10 Green-winged Teal Anas crecca GWTE Dabbling Duck 0 0 0 3 Mallard Anas platyrhynchos MALL Dabbling Duck 10 10 10 14 Gadwall Anas strepera GADW Dabbling Duck 0 0 0 2 American Wigeon Anas americana AMWI Dabbling Duck 0 0 0 2 Northern Pintail Anas acuta NOPI Dabbling Duck 0 0 0 1

Canvasback Aythya valisineria CANV Diving Duck 1 1 1 1 Ring-necked Duck Aythya collaris RNDU Diving Duck 8 8 14 23 Lesser Scaup Aythya affinis LESC Diving Duck 13 13 13 14 Bufflehead Bucephala albeola BUFF Diving Duck 8 8 8 9 Hooded Merganser Lophodytes cucullatus HOME Diving Duck 5 5 5 5 Ruddy Duck Oxyura jamaicensis RUDU Diving Duck 7 7 7 9 American Coot Fulica americana AMCO Rail 4 4 23 29

Pied-billed Grebe Podilymbus podiceps PBGR Diver 3 3 3 4 Double-crested Cormorant Phalacrocorax auritus DDCO Diver 0 4 4 4

TOTALS 66 70 98 130 aResidence status on the SRS: R=resident, on the SRS in all seasons; M=migrant, only on the SRS in the fall/winter.

58

Table 2-11. Comparisons of trace element concentrations (ppm, dry mass) in muscle and liver tissues of diving ducks collected

from D-Area ash basins and diving ducks collected from other water bodies on the Savannah River Site (SRS) in 2012-2015.

Ash Basin Divers (n=24) Other Divers (n=18) Elementa Tissue Mean SE Range Mean SE Range F Pb Cr Muscle 0.58 0.06 0.27-1.09

0.94 0.23 0.27-4.05 4.69 <0.01

Liver 1.13 0.11 0.27-2.75

1.18 0.35 0.27-6.53 0.03 0.87

Cu Muscle 34.04 2.87 18.85-78.06

29.62 1.75 7.01-40.05 2.99 0.09

Liver 61.50 10.04 12.57-244.92

226.07 48.51 26.30-699.14 14.37 <0.001

Zn Muscle 39.23 2.10 25.18-64.00

31.12 1.77 8.01-43.72 7.94 <0.01

Liver 118.59 7.03 68.62-211.01

141.25 15.11 66.10-308.93 2.18 0.1500

Se Muscle 12.90 1.29 2.52-23.45

4.40 2.25 0.53-42.04 12.06 <0.001

Liver 35.58 3.65 5.05-71.48

9.33 4.42 2.03-83.65 21.25 <0.0001

Hg Muscle 0.79 0.24 0.07-5.55

0.37 0.11 0.02-1.39 2.05 0.16

Liver 2.67 0.64 0.23-11.50

1.52 0.61 0.09-10.89 1.60 0.21

Cd Liver 2.73 0.69 0.49-13.06 2.37 0.95 0.20-17.18 0.09 0.76 aOver 50% of the following elements were below method detection limits and were not presented in this table: As, Ni, Pb, U,

and Cd (muscle).


59

Table 2-12. Concentrations of trace elements (ppm, dry mass) in muscle and liver tissues of dabbling ducks collected from the

Savannah River Site (SRS) in 2012-2015.

Elementa Tissue Mallard (n=10)b Wood Duck (n=7)c Mean SE Range Mean SE Range

Cr Muscle 0.88 0.19 0.27-2.28 0.75 0.18 0.27-1.40

Liver 0.69 0.16 0.27-1.66 - - Cu Muscle 19.51 1.51 14.43-31.13 16.57 1.04 13.33-20.31

Liver 110.01 14.44 51.32-176.06 28.55 3.42 21.04-42.26

Zn Muscle 33.46 1.24 27.81-41.73 29.39 2.43 21.91-41.33

Liver 120.07 10.12 84.01-181.69 150.95 14.85 87.26-203.23

Se Muscle 1.57 0.24 0.57-3.13 1.04 0.2 0.24-1.97

Liver 3.84 0.16 2.96-4.63 2.72 0.42 1.54-4.81

Hg Muscle 0.09 0.02 0.04-0.23 0.04 0.01 0.02-0.10

Liver 0.34 0.09 0.13-1.05 0.13 0.04 0.05-0.33

Cd Liver 1.14 0.28 0.20-3.00 0.9 0.23 0.20-2.10 aOver 50% of the following elements were below method detection limits and were not presented in this table: Cr (liver of Wood

Ducks), Cd (muscle of both species), As, Ni, Pb, U.

bAll 10 Mallards were collected at Fourmile Branch beaver pond.

cWood Ducks were collected at Tim's Branch beaver pond (n=5), Fourmile Branch beaver pond (n=1), and R-Canal (n=1).

60

Table 2-13. Concentrations of trace elements (ppm, dry mass) in muscle and liver tissues of other water birds collected from

the Savannah River Site (SRS) in 2012-2015.

Elementa Tissue American Coot (n=4)b Pied-billed Grebe (n=3)c Double-crested Cormorant (n=4)d

Mean SE Range Mean SE Range Mean SE Range Cr Muscle 0.92 0.2 0.55-1.48 0.84 0.09 0.69-1.02 - - -

Liver 1.29 0.18 0.84-1.71 1.35 0.1 1.15-1.49 1.28 0.11 1.05-1.53

Cu Muscle 63.56 3.83 52.79-70.90 37.74 5.91 28.35-48.65 - - -

Liver 41.75 22.09 10.39-105.38 26.91 8.8 14.61-43.96 18.72 1.53 16.21-22.46

Zn Muscle 60.3 3.37 54.81-70.13 56.11 2.58 50.98-59.04 - - -

Liver 114.56 36.09 58.84-216.90 105.95 8.28 94.98-122.17 74.86 2.78 69.96-82.49

As Muscle 0.85 0.27 0.20-1.50 - - - - - -

Liver 2.04 1.32 0.20-5.89 0.37 0.09 0.20-0.48 - - -

Se Muscle 15.33 2.32 10.44-21.59 28.12 4.51 19.18-33.52 - - -

Liver 17.41 3.7 9.72-25.49 41.79 8.62 24.71-52.35 19.52 12.06 3.71-55.39

Hg Muscle 0.06 0.01 0.03-0,08 0.72 0.08 0.61-0.87 - - -

Liver 0.37 0.07 0.24-0.51 1.33 0.2 0.97-1.66 60.64 37.19 5.75-169.39

Cd Liver 0.36 0.09 0.20-0.56 2.49 1.63 0.79-5.75 0.65 0.09 0.47-0.81

a >50% of elements were BDL thus not presented: Cd (muscle all species), As (grebe muscle and cormorant liver), Ni, Pb, U.

bAll 4 coots were collected from D-Area ash basins.

cAll 3 grebes were collected from D-Area ash basins.

dAll 4 cormorants were collected from Pond B.

61

Table 2-14. Correlationsa among trace element concentrations in muscle (above diagonal) and liver (below diagonal) tissues of all

waterfowl/waterbirds collected from the Savannah River Site (SRS) in 2012-2015 (n=66 muscle, n=70 liver). Only those elements

with more than 50% of the values above detectable limits are included. Correlations between muscle and liver samples for individual

elements are presented on the diagonal in bold.

Cr Cu Zn Se Hg Cd Cr NS NS NS NS NS ―

Cu NS NS 0.63 (<0.0001) 0.53 (<0.0001) NS ―

Zn -0.27 (0.02) 0.52 (<0.0001) 0.29 (0.02) 0.52 (<0.0001) NS ―

Se 0.47 (<0.0001) NS NS NS 0.57 (<0.0001) ―

Hg 0.31 (0.009) -0.29 (0.01) -0.37 (0.002) 0.62 (<0.0001) NS ―

Cd NS NS 0.26 (0.03) 0.33 (0.006) NS ― aGiven are Spearman correlation coefficients and P values in parentheses; NS=not significant (P > 0.05).

62

Table 2-15. Descriptive statistics for radiocesium concentrations measured in waterfowl collected from the Savannah River

Site (SRS) in 2012-2015.a

Compartment Variable N % below MDCb Mean SE Min Max Median CV (%)

Whole-body Bq/g; fresh mass 130 56 0.159 0.029 -0.005 1.683 0.01 207.7

! MDCb; fresh mass 130 ! 0.0057 0.0001 0.0023 0.0103 0.0056 26.7

! ! ! ! ! ! ! ! ! !Muscle Bq/g; dry mass 98 62 1.228 0.224 -0.083 8.027 0.1 180.3

! Bq/g; wet mass 98 ! 0.328 0.059 -0.024 2.145 0.028 177.8

! MDC; dry mass 98 ! 0.163 0.002 0.123 0.223 0.161 12.9

! wet:dry ratio 87 ! 3.603 0.021 3.269 4.397 3.581 5.3

! ! ! ! ! ! ! ! ! !Liver Bq/g; dry mass 98 66 0.797 0.151 -0.087 7.566 0.058 187.8

! Bq/g; wet mass 98 ! 0.225 0.042 -0.026 2.07 0.016 186.7

! MDC; dry mass 98 ! 0.152 0.003 0.11 0.281 0.15 17.8 wet:dry ratio 87 3.539 0.029 2.829 4.411 3.514 7.6

aIncludes all waterfowl collected from 6 locations on the SRS: D-Area ash basins, Tim’s Branch beaver pond, L-Lake,

Fourmile Branch beaver pond, R-Canal/Pond A, and Pond B.

bMDC=minimum detectable concentration; calculated from Currie (1968).

63

Table 2-16. Comparisons of whole-body and tissue radiocesium concentrations (Bq/g, wet mass) in diving ducks (Bufflehead,

Canvasback, Lesser Scaup, Ring-necked Duck and Ruddy Duck) collected from four different locations of the SRS in 2012-

2015.a

Compartment Location Geometric Mean

Lower 95% CI

Upper 95% CI Max

Whole-body (N=55) Pond BA 0.1127 0.0693 0.1815 0.2651

Fourmile Beaver PondB 0.0063 0.0016 0.0143 0.0391

L-LakeB 0.0005 -0.0012 0.0031 0.012

D-Area Ash BasinsB -0.0002 -0.0016 0.0018 0.0389

Muscle (N=42) Pond BA 0.1634 0.0663 0.3689 0.6362

Fourmile Beaver PondB 0.0134 -0.0061 0.0584 0.0935

L-LakeB -0.0029 -0.0147 0.0311 0.0204

D-Area Ash BasinsB -0.0091 -0.0141 -0.0005 0.0764

Liver (N=42) Pond BA 0.1485 0.0818 0.2565 0.3036

L-LakeB 0.0047 -0.0105 0.0347 0.0341

Fourmile Beaver PondB -0.0009 -0.0113 0.017 0.0281 D-Area Ash BasinsB -0.0027 -0.0096 0.007 0.0551

aANOVA tests were conducted separately by compartment for a location effect on log-transformed (and scaled) radiocesium

levels (Whole-body: F(3,51) = 45.62, P < 0.0001; Muscle: F(3,38) = 11.57, P < 0.0001; Liver: F(3,38) = 16.19, P < 0.0001), and

followed by post-hoc Tukey HSD tests for determining pairwise locational differences. Locations within compartments with

the same capital letter were not significantly different (P > 0.05). Geometric means are back-transformations of least-squares

means of log-transformed (with scaling removed) radiocesium concentrations.

64

Table 2-17. Monthly allowances of ½ lb. meals for adults and ¼ lb. for children before exceeding the EPA’s oral reference dose

ratings for selenium (Se) and mercury (Hg) for muscle tissue of wild pigs collected from the Savannah River Site (SRS) (n=88) and

five counties in Georgia (GA) (n=20) 2012-2015. Consumption limits based on average concentrations are presented with

consumption limits based on the maximum concentration found in an individual in parentheses. Levels of As were all BDL so

consumption limits are not included.

SRS Wild Pigs GA Wild Pigs Element Age Dry Cooked Raw Dry Cooked Raw Se Adult 26.3 (13.2) 60.2 (30.2) 102.3 (51.2)

48.6 (23.6) 111.5 (54.1) 189.3 (91.9)

Child 12.1 (6.1) 27.7 (13.9) 47.0 (23.5)

22.3 (10.8) 51.2 (24.9) 86.9 (42.2)

Hg Adult 6.6 (1.2) 15.2 (2.7) 25.8 (4.5)

22.5 (13.1) 51.6 (30.0) 87.6 (50.9) Child 3.0 (0.5) 7.0 (1.2) 11.8 (2.1) 10.3 (6.0) 23.7 (13.7) 40.2 (23.4)

65

Table 2-18. The monthly allowances of ½ lb. meals for adults and ¼ lb. for children before exceeding the EPA’s chronic oral

reference dose limits for arsenic (As), selenium (Se), and mercury (Hg) for muscle tissue of diving ducks collected from the D-

Area ash basins (n=24) and other water bodies (n=18) on the Savannah River Site (SRS) 2012-2015. Consumption limits based

on average concentrations are presented with limits based on the maximum concentration found in an individual for each trace

element in parentheses. Levels of As for Dabbling ducks were all BDL so consumption limits are not included.

Ash Basin Diving Ducks Other SRS Diving Ducks Dabbling Ducks Element Age Dry Cooked Raw Dry Cooked Raw Dry Cooked Raw As Adult 3.4 (1.4) 5.9 (2.4) 11.8 (4.8) 9.6 (1.0) 16.9 (1.8) 33.5 (3.6) ― ― ―

Child 1.5 (0.6) 2.7 (1.1) 5.4 (2.2) 4.4 (0.5) 7.8 (0.8) 15.4 (1.6) ― ― ―

Se Adult 3.6 (2.0) 6.4 (3.5) 12.2 (7.0) 16.1 (1.1) 28.3 (2.0) 56.2 (3.9) 34.7 (15.0) 61.1 (26.4) 121.3 (52.4)

Child 1.7 (0.9) 2.9 (1.6) 5.8 (3.2) 7.4 (0.5) 13.0 (1.0) 25.8 (1.8) 15.9 (6.9) 28.1 (12.1) 55.7 (24.1)

Hg Adult 1.2 (0.2) 2.1 (0.3) 4.2 (0.6) 4.2 (0.7) 7.3 (1.2) 15.0 (2.4) 13.0 (4.0) 23.0 (7.1) 45.6 (14.1) Child 0.6 (0.1) 1.0 (0.1) 1.9 (0.3) 2.0 (0.3) 3.4 (0.6) 6.7 (1.1) 6.0 (1.9) 10.5 (3.3) 20.9 (6.5)

66

Figure 2-1. Savannah River Site (SRS) locations targeted for sample collections (wild

pigs, squirrels, waterfowl/waterbirds) for trace elements and radiocesium quantification

in 2012-2015, included the D-Area ash basins, Fourmile Branch, Tim’s Branch, Pond

A/R-Canal, Pond B, and L-Lake.

67

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85

CHAPTER 3

WATERFOWL EXPOSURE TO COAL COMBUSTION WASTES AND HUMAN

CONSUMPTION RISKS 1

____________________

1 Oldenkamp, R. E., A. L. Bryan, Jr., R. A. Kennamer, and J. C. Beasley. To be submitted to the Journal of Wildlife Management.

86

ABSTRACT

Waterfowl are important and popular game species and thus represent a potential

pathway for human exposure to anthropogenic pollutants through the consumption of

contaminated meat. In particular, settling basins containing coal combustion waste

(CCW) enriched in trace elements such as arsenic (As), selenium (Se), and mercury (Hg)

are often utilized by free-ranging migratory waterfowl, representing potential sources for

contaminant uptake. We developed an experiment to restrict waterfowl to a CCW

contaminated basin then quantified levels of contaminants in waterfowl and modeled

trace element burdens in blood, muscle, and liver for known time periods of exposure

(between 3 and 92 days of exposure). We developed equations to predict muscle/liver

burdens based on concentrations in blood as a potential non-destructive sampling method

and used muscle tissue to calculate human consumption limits based on concentrations of

recognized elements of human health concern (As, Se, and Hg). We observed a

significant increase in Se concentrations in muscle, liver, and blood tissues over the

duration of our experiment. Consumption limits of waterfowl breast muscle based on

EPA chronic oral reference dose guidelines for As, Se, and Hg. Consumption limits were

lowest for As, with an allowance as low as 2.9 meals per month, although given the lack

of As accumulation observed through time these results may reflect elevated levels of As

for ring-necked ducks prior to the initiation of this study. Consumption limits based on

observed Se accumulation decreased from 26.7 to 6.2 meals per month during the course

of this study. Children’s allowances based on average concentrations were approximately

half that of adults for each element. These data provide unique insights into accumulation

rates of contaminants for waterfowl utilizing habitats contaminated with CCW and

87

suggest more comprehensive, long-term monitoring of contaminant burdens in waterfowl

is needed across a broad geographic scale to assess waterfowl exposure (and human

hunter exposure risk) to pollutants.

INTRODUCTION

Waterfowl are an important game species globally. In the U.S. alone ~2.5 million

hunters spend nearly $1.8 billion on waterfowl hunting annually (USFWS and USCB

2011). The vast majority of hunters pursue game for consumption, and waterfowl hunters

consume numerous birds each year due to large daily federal bag limits (daily max

allowed during hunting season; Duchesne et al. 2004; Smith et al. unpublished

manuscript). Despite the widespread consumption of waterfowl and other game, wild

game is not subjected to the same regulatory testing as livestock and thus consumption of

free-ranging wildlife could potentially expose unknowing hunters, their families, and

recipients of donated game to environmental pollution (Cristol et al. 2012, Conder and

Arblaster 2016). Waterfowl, in particular, could represent an important pathway for

contaminant exposure in humans because they are vulnerable to uptake of contaminants

due to extensive use of aquatic habitats, where many pollutants occur, and foraging

behaviors that disturb sediments (Bryan et al. 2012). Moreover, waterfowl are highly

mobile and migratory and thus can transport pollutants hundreds or thousands of

kilometers from point sources (Kennamer 2003, Cristol et al. 2012, Conder and Arblaster

2016).

Although numerous pollutants exist within aquatic ecosystems, surface

impoundments containing coal combustion wastes (CCW) represent a potential pathway

for wildlife exposure to several trace elements flagged by the EPA as environmental and

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human health risks (Luther 2010a,b; Rowe et al. 2002). Millions of tons of CCW are

deposited into surface impoundment ponds across the US and as of 2015 the EPA

identified more than 500 coal power facilities with 735 surface impoundments to store

CCW in the US alone (USEPA 2015). Furthermore, coal use is on the rise in developing

countries, making CCW important at a global scale (Humphries 1999). Many of these

storage areas are unlined and present a risk of environmental pollution from the

multifarious combustion byproducts that are enriched in potentially toxic trace elements

such as arsenic (As), selenium (Se), and mercury (Hg; Greeley et al. 2016). Indeed, more

than 20 surface impoundment accidents have caused substantive fish, wildlife, and

economic losses (Lemly 1996, 2002; Ruhl et al. 2009; Lemly and Skorupa 2012; Rigg et

al. 2015). This does include exposure at these impoundments when there is not a

structural failure or release of effluents into nearby waterways. Waterfowl and waterbirds

may be particularly susceptible to CCW exposure as open surface impoundments often

have wooded or herbaceous areas of cover along edges supporting a diversity of prey

(e.g., aquatic vegetation, insects, amphibians, and fish; Rowe et al. 2002). Yet,

comparatively little is known about exposure of waterfowl to CCW compared to fish,

amphibians, and reptiles (Hopkins et al. 1999, 2000; Yang et al. 2010; Van Dyke 2013).

Bioaccumulation, the build-up of contaminants within the tissues of the body, can

occur through the consumption of the source pollutant or through movement in trophic

levels within the food web (USEPA 2012b). Trace elements in or around CCW storage

areas have been found to bioaccumulate or biomagnify in wildlife, especially aquatic or

semi-aquatic organisms (Yudovich and Ketris 2005a,b; Yudovich and Ketris 2006; Reash

2012; Otter et al. 2012), posing potential health risks to aquatic organisms, terrestrial

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wildlife, and humans (Dorman et al. 2010, Lemly and Skorupa 2012, Ruhl et al. 2012,

Mayfield et al. 2013, Rice et al. 2014). Examples of CCW pollution affecting wildlife are

well documented. For instance, coal fly ash effluent discharged into Belews Lake and

Hyco Reservoir in North Carolina and in Martin Creek Reservoir in Texas have resulted

in trace element accumulation in aquatic biota, with Se contamination resulting in

significant increases in developmental abnormalities in fish larvae, salamanders, and toad

tadpoles and local extinctions of sensitive species (Lemly 1996, 2002; Hopkins et al.

2006; Roe et al. 2006; Metts et al. 2013). The largest industrial waste spill in U.S. history,

from Kingston (TVA) plant in Tennessee, resulted in elevated levels of As, Se, and Hg in

fish and resident raccoons, signifying transfer of contamination to terrestrial organisms

(Ruhl et al. 2009, Beck et al. 2013, Van Dyke et al. 2013, Beck et al. 2015, Rigg et al.

2015).

Most laboratory toxicology studies that have sought to identify lethal doses of

contaminants and measure morbidity, used liver tissue as the standard to explain health

effects of exposure. Liver and kidney, accumulate substantially greater concentrations of

most trace elements than muscle tissue (Farkas et al. 2003, Ikem et al. 2003, Coğun et al.

2006). While exceptions exist, (Mason et al. 2000, Havelková et al. 2008), this has meant

that detoxifying organs have been used most often as biological indicators of contaminant

presence in water. However, this common approach in laboratory toxicology studies

typically ignores element concentrations in muscle tissue and thus is insufficient to

facilitate calculations of human exposure risk for consumption of wildlife inhabiting

contaminated ecosystems. This approach also ignores complications with natural

exposure (e.g. fluctuations in exposure through time and space and through food items

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with differing levels of contamination) and multiple contaminant exposure scenarios (i.e.

coal ash has many different trace elements of differing toxicity that may interact) that are

the norm in polluted ecosystems.

Data on contaminant concentrations in wild waterfowl is surprisingly scarce

despite their importance as a consumed game species and their potential to accumulate

trace elements or other contaminants through feeding in sediments of aquatic habitats

where pollutants often concentrate (Chapter 2, see Cristol et al. 2012). Moreover,

interpretations of contaminant levels in many studies can be problematic because wild

birds can potentially transition between contaminated and uncontaminated habitats during

both migratory and resident periods. Testing of wild birds from contaminated areas can

provide a snapshot of contaminant levels within tissues; however, the length of time birds

were present within the contaminated habitat is generally unknown and thus experimental

research is needed to elucidate accumulation rates of contaminants to inform observed

concentrations in free-ranging waterfowl (Chapter 2). In this study we characterized

accumulation rates of several trace elements of importance to human and wildlife health

in waterfowl restricted to a coal fly ash surface impoundment over a 3-month period, and

use these data to assess potential risks to waterfowl and human hunters consuming

waterfowl that have utilized CCW impoundment facilities for varying periods of time.

Specifically, our objectives were to 1) quantify trace element uptake in blood, muscle,

and liver tissues over known periods of time by waterfowl exposed in situ to a coal ash

settling basin and elucidate potential interactions among elements, 2) develop a model to

predict muscle/liver burdens based on concentrations in blood as a potential non-

destructive sampling method and test the performance of the model against a subset of

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our data, and 3) calculate human consumption limits based on concentrations of

recognized elements of human health concern (As, Se, and Hg) over known time periods

of exposure.

METHODS

Study Area

This study occurred on the Savannah River Site (SRS), a ~800 km2 limited access

former nuclear production and research facility owned and operated by the U.S.

Department of Energy. The SRS is located in the coastal plain of South Carolina and is

classified as a National Environmental Research Park (White and Gaines 2000). Created

in 1951 to provide nuclear weapons materials at the beginning of the Cold War

(Savannah River Nuclear Solutions, LLC 2011), the SRS now contains five

decommissioned nuclear reactors, radioactive materials processing facilities, and nine

retired coal power plants (White and Gaines 2000). These facilities encompass <5% of

the SRS; the remaining area is comprised of a mosaic of natural habitats consisting of

managed pine stands (54%), wetlands (23%), upland hardwood and mixed forest (11%),

grasslands (9%), and upland scrub forest (3%; Lide 1994, White and Gaines 2000,

DeVault et al. 2004).

The ash basins associated with the SRS’ D-area coal fired power plant represent

the most thoroughly studied ash disposal system in the world. From 1953-2012 the D-

Area coal-fired power plants on the SRS were operational and during this time sluiced fly

ash was deposited into settling impoundments that flowed into Beaver Dam Creek, a

tributary of the Savannah River, and nearby wetlands (Halverson et al. 1997, Gaines et al.

2002, USDOE 2012). These settling impoundments are unlined earthen basins located

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approximately 0.5 km from the Savannah River. Vegetation has grown in and around

edges of the basins and several species of waterfowl are frequently observed foraging in

these artificial habitats (Chapter 2). In particular, following cessation of discharge, basin

1 (15 ha), the focal area of this research, became partially filled and vegetation expanded

inward from already established wooded edges creating a small semi-vegetated wetland

enclosed by the rest of the basin (Figure 3-1). Numerous studies have reported

bioaccumulation and adverse effects from exposure to elevated levels of aluminum (Al),

As, cadmium (Cd), chromium (Cr), iron (Fe), Hg, manganese (Mn), nickel (Ni), Se, and

zinc (Zn) for a wide array of organisms inhabiting the basins and creek watershed

including bacteria, aquatic invertebrates, amphibians, fish, turtles, alligators, and birds

(Cherry et al. 1979; Hopkins et al. 1999, 2000; Rowe et al. 1996, 2002; Stepanauskas et

al. 2005; Hopkins et al. 2006; Roe et al. 2006; Wright et al. 2006; Bryan et al. 2012;

Metts et a;. 2013). There are abundant populations of waterfowl that overwinter or are

resident on SRS water bodies and several species are commonly observed using the D-

area CCW impoundments (Chapter 2, Mayer et al. 1986, Kennamer 2005).

Trapping and Sample Collection

Male ring-necked ducks were selected as a target species for this research because

of their extensive use of the SRS as a stop-over and over-wintering location during

migration (Mayer et al. 1986) and because of their varied diet, which includes aquatic

vegetation, insects, snails, and mussels (Hoppe et al. 1986). In December 2014-February

2015 ducks were trapped with swim-in box traps baited with corn placed at L-lake, an

uncontaminated waterbody on the SRS. Upon capture, ducks were banded with uniquely

numbered USFWS bands and fitted with a colored nasal saddle to distinguish unique

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cohorts of birds during collections. Approximately 1 ml of blood was collected from the

basilica or brachial vein in the wing, or less often the metatarsal vein in the leg, and

placed into tubes without anticoagulant and allowed to clot before being frozen at -80°C

for later trace element analysis. This blood served as a baseline level of contamination to

determine if any birds had high levels of any trace elements in their blood prior to our

experiment, indicating possibility of accumulation in the muscle and liver from previous

locations along their migratory routes. All birds were translocated to ash basin 1 (Figure

3-1) where we clipped the ends of all flight feathers on one wing prior to release to

prevent them from leaving the basin but allowing them to freely move around the basin

and forage.

After release at the ash basin, ducks were lethally collected (with a shotgun)

periodically, aiming to spread out the collections every few days between 3 and 92 days

of exposure. After collection, we collected a 1 ml blood sample via cardiac puncture and

placed samples into tubes without anticoagulant that we subsequently stored in a -80°C

freezer for later trace element analysis. We also collected a weight for each bird and froze

them at -20°C for later dissection. All animal handling practices and euthanasia were

carried out with accompanying federal and state collecting permits and in accordance

with University of Georgia Animal Care and Use guidelines under protocol A2013 06-

004-Y3-A1. After thawing, we dissected all ducks to collect breast muscle and liver

tissues for trace element analyses. We collected wet weights for these tissue samples,

which we subsequently freeze-dried and re-weighed prior to homogenizing them into a

powder using a coffee grinder. We cleaned grinder canisters with a 5% nitric acid

solution and dried canisters between uses.

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Elemental Analysis

We conducted analyses for trace elements [V, Cr, Ni, Cu, Zn, As, Se, Cd, and Pb]

and total mercury (THg) content on muscle, liver, and blood samples. For trace element

analysis approximately 250 mg of dry muscle or liver sample was microwave digested

(MARSX Xpress, CEM Corporation, Matthews, NC) with 10.0 ml trace metal-grade

nitric acid (70% HNO3). Following digestion, samples were brought to a final volume of

15.0 ml with Milli-Q (18MΩ) water before spectroscopy (Nexlon 300X ICP-MS; Perkin

Elmer, Norwalk, CT) according to QA/QC protocols outlined in EPA Method 6020A

(USEPA 2007). The minimum detection limits (ppm) for each element for muscle and

liver was: V (0.03), Cr (0.03), Ni (0.04), Cu (0.11), Zn (0.11), As (0.05), Se (0.33), Cd

(0.04), and Pb (0.04).

For trace element analysis of blood, we placed approximately 0.50 ml of blood

into a trace metal free tube and weighed samples before placing them in an -80°C freezer

for a few hours to make sure the sample was solid before transferring to the freeze-drier.

After freeze-drying for 12 hours the sample was weighed again before being placed into a

sand bath (~75-85°C) with 2 ml of trace metal-grade nitric acid (70% HNO3) and 1 ml of

hydrogen peroxide (30% H202) added slowly over 90 minutes while still on the heat.

Samples were brought to a final volume of 5.0 ml with Milli-Q (18MΩ) water before

spectroscopy with the same protocol as tissue samples. For blood samples the minimum

detection limits (ppm) for each element was: V (0.06), Cr (0.09), Ni (0.04), Cu (0.25), Zn

(2.15), As (0.05), Se (0.38), Cd (0.04), and Pb (0.47). For quality control purposes,

certified reference material (TORT-3 lobster hepatopancreas; National Research Council,

Ottawa, ON, Canada), a blank, and a digestion replicate were run for every 20 samples.

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Mean percent recoveries ranged from 86-222% for tissues and 77-146% for blood for

elements in certified reference materials and all element concentrations are presented as

parts per million (ppm) on a dry mass basis.

Following U.S. Environmental Protection Agency (EPA) method 7473 to analyze

total mercury (THg), 30-50 mg subsamples of the freeze-dried/homogenized tissues, and

50 uL of digested, diluted blood, were analyzed by thermal decomposition, catalytic

conversion, amalgamation, and atomic absorption spectrophotometry (DMA 80;

Milestone, Shelton, CT, USA). The instrument detection limit (IDL) for this method was

0.01 nanograms (ng) of total mercury. Quartz sample boats were utilized for the acid-

digested blood samples. For each set of 10 samples we included a replicate, blank, and

two standard reference materials (SRMs; TORT-3 lobster hepatopancreas, and PACS-2

marine sediment, National Research Council of Canada, Ottawa, ON) to ensure quality

assurance and solid SRMs were used to calibrate the instrument. For muscle and liver

tissues method detection limits (MDLs; threefold the standard deviation of procedural

blanks) averaged 0.0004 ppm dry mass; mean percent recoveries of THg for the SRMs

TORT-3 and PACS-2 were 93.6 ± 5.3 and 101.6 ± 3.9 respectively. Blood MDLs

averaged 0.00004 ppm dry mass with mean percent recovery of THg in the SRM, TORT-

3, of 91.2 ± 15.1. Concentrations for tissues and blood are presented as parts per million

(ppm) on a dry mass basis.

Statistical Analysis

Any ducks that exhibited pre-exposure As, Se, or Hg levels two standard

deviations above the mean were excluded from all analyses as they were assumed to

potentially have elevated burdens of these elements prior to translocation to the ash basin.

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Trace element concentrations have been shown to differ between tissue types in previous

toxicology studies; therefore, we performed tests on muscle and liver samples separately

(Scheuhammer 1987, Boening 2000, Mason et al. 2000, Farkas et al. 2003, Ikem et al.

2003, Coğun et al. 2006, Havelková et al. 2008). For each tissue, when >50% samples

were below detection limits (BDL) for individual elements those elements were excluded

from subsequent analyses. When <50% of samples of a particular tissue were BDL, we

replaced them with 50% of the respective minimum detection limit (MDL; Hall et al.

2009, Fletcher et al. 2014). We tested distributions of all elements for normality (Shapiro-

Wilk test p<0.05) in R (R Core Team 2012) and subsequently transformed data prior to

inclusion in analyses; specific transformations are detailed for individual analyses.

Although we quantified concentrations of several trace elements, we limited our

statistical analyses to those elements commonly reported in the D-area ash basins where

our study was conducted that are a concern to human health and to the health and survival

of organisms occupying ash basins (As, Se, Hg; Luther 2010a,b). Other element levels

are presented for descriptive purposes only. As, Se, and Hg are bioavailable to all trophic

levels in D-area and have potential for antagonistic or synergistic interactions, which can

affect accumulation patterns (Gaines et al. 2002, Hopkins et al. 1999, Unrine et al. 2007).

Interactions among these three elements have been found in laboratory studies of

waterfowl and studies of different organisms in situ in contaminated areas. To determine

tissue-specific accumulation patterns of these elements over the course of our experiment,

we log-transformed concentrations of As, Se, and Hg to improve distributions and

subsequently developed separate linear regression models for each element over time. To

assess potential interactions among elements for naturally exposed waterfowl we utilized

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a Spearman correlation test among As, Se, and Hg with days of exposure included as a

covariate to control for expected change over time.

To verify the utility of blood samples as a non-destructive sampling technique to

predict element concentrations in muscle and liver in future studies, we first compared 4

common data transformations (inverse, square root, log, and exponential of the

concentrations) with the untransformed concentrations (raw data) and chose the best fit

(R2 value) linear regression model for each tissue relative to post-experiment blood levels

for As and Se (Hodgman et al. 1996). Hg was not included because >50% of the blood

values were BDL. We then ran a k-fold cross validation to confirm our predictive

equations for As and Se would work on novel data (Package DAAG version 1.2). We

choose to evaluate three folds of the data, testing 1/3 of the data at a time against the

model developed for each element. The overall mean sums of squares (MS) for the 3-

folds was compared to the original model residual standard error (RSE) to determine how

closely the MS and RSE aligned to assess whether the equations generated were reliable

for predictions on unseen data.

To assess human consumption limits for ducks utilizing ash basins over time

periods relevant to migratory waterfowl, we binned collections into 6 time periods of

exposure, each ~15 days. The Environmental Protection Agency (EPA) has established

chronic oral reference dose limits for As, Se, and Hg based on consumption of fish;

concentrations a human could theoretically be exposed to daily over the course of their

life and not expect detrimental health consequences. Although we quantified total THg,

which includes all mercury in a given sample, nearly all Hg in tissues of higher trophic

level organisms such as birds and fish is in the methylmercury (MeHg) form; 80-100% in

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muscle of piscivorous birds and up to 98% in fish muscle (Wiener et al. 2003, Evers et al.

2005). Thus, THg can be assumed to approximate MeHg concentrations for consumption

risk analyses (Cristol et al. 2012). MeHg also is the most bioavailable form for humans

and wildlife to absorb and the most toxic form of mercury, which can cause severe

neurological damage. Therefore, calculations of consumption limits were performed with

EPA limits for MeHg (USEPA 1999a, 2001; Grandjean et al. 1999; Hall et al. 2009;

Clarkson et al. 2003). We used the average and maximum concentrations of the

aforementioned trace elements found in muscle samples to calculate human consumption

limits and provide estimates of the average and worst-case exposure risk scenarios for

consuming waterfowl with exposure in the range of days represented in each exposure

group.

For our calculations of consumption limits of waterfowl muscle, we utilized

established EPA equations for fish advisory limits; including average standard weights

for adults and children (70 kg or 154 lbs and 16 kg or 35 lbs, respectively) and meal sizes

(227g or ½ lb for adults and 113g or ¼ lb for children). When choosing a metric to set

our threshold, we used the EPA established chronic oral reference doses of 0.0001

milligrams per kilograms per day (mg/kg/day) for MeHg (USEPA 1999a), 0.005

mg/kg/day for Se (USEPA 1999b) and 0.0003 mg/kg/day for As (USEPA 1998). These

levels are daily dose exposures that alone are unlikely to produce appreciable deleterious

effects over a lifetime of exposure; as exposures increase above the reference doses so

does the risk of adverse health effects.

Most toxicology studies dealing with tissues report either wet (raw sample) or dry

mass (devoid of water), but neither are representative of how most people consume

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muscle tissue. Therefore, we calculated trace element concentrations for consumption

limits based on what we would expect in a cooked sample. We utilized the average

percent moisture loss in cooked duck breast muscle (28.2%; Omojola 2007) to amend

concentrations of trace elements that would be found in cooked muscle. This gives a

higher concentration of trace elements than the raw sample and lower concentrations than

the dry sample, but a more accurate assessment of real world scenarios for human

consumption of game meat.

RESULTS

We released 90 ring-necked ducks onto ash basin 1 in D-Area in two release

periods (December 3rd-18th 2014 and February 3rd-10th 2015). Between January 24th and

March 12th 2015 we collected 38 ducks that ranged between 3 and 92 days of exposure

(Table 3-1). Cd and Pb in muscle and blood and Hg in post-experiment blood had >50%

of samples BDL and thus were excluded from presented descriptive statistics (Table 3-2).

Pre-experiment blood values indicated 5 birds exhibited As, Se or Hg levels two standard

deviations above the mean and thus were excluded from analysis.

For muscle, liver, and blood the relationship between log-transformed As, Se, and

Hg concentrations and time of exposure showed Se had a significant positive relationship

with days of exposure for muscle and blood (Table 3-3). As concentrations in muscle,

liver and blood did not differ significantly over the course of our experiment days of

exposure (Table 3-4). Hg had a significant positive relationship with days of exposure in

muscle and a significant negative relationship with days of exposure in liver (Table 3-5).

When assessing potential interactions among elements we found that none of the

elements were significantly correlated in muscle, but in liver Se and As were significantly

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correlated, as were Se and Hg in liver (Table 3-6). When comparing element levels

between muscle and liver tissues, we observed a significant correlation between tissues

for both As and Se, but not Hg (Table 3-6). Investigation into whether element

concentrations in post-experiment blood were correlated with levels observed in other

tissues revealed that blood concentrations of As were significantly correlated with muscle

and liver. Blood concentrations of Se were significantly correlated with muscle.

Our regression models quantifying the relationship between post-collection blood

element concentrations and those observed in muscle and liver tissue showed that for As

the raw data gave a better fit than any of the transformations we tried for muscle, and for

liver the inverse of the concentration provided the best fit. The regression equation for

muscle was and liver was , both with R2 values

of 0.56. For Se the inverse concentration was the best transformation for muscle, giving

the equation with a R2 of 0.96; none of the transformations

improved the fit beyond the raw concentrations for Se in liver, giving the equation

(R2 of 0.14).

Results of our k-fold cross validation models showed that for As in muscle the

overall mean sums of squares (MS; 0.17) for the 3 folds of the data was less than the

model residual standard errors (RSE; 0.40). This indicated that the equation was reliable

for prediction on unseen data because the error expected on unseen data would have been

close to or below our original error value for the RSE. For liver, the equation for As was

also reliable with the overall MS of 0.54 less than the RSE of 0.66. Overall MS for Se in

muscle (1.06) was very close to the RSE (0.99), and indicates this equation would

produce estimates with about a 7% increase in error expected with novel data. In liver Se

y = 0.5648x +0.3897 y −1 = 0.2872*e −1 +0.2414

y −1 =1.3145* x −1 +0.0272

y = 0.4916x +10.529

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had a much higher overall MS (57.80) than RSE (7.32) and therefore would not be

sufficient to provide reliable predictions on unseen data.

Moisture loss in muscle from freeze-drying ranged between 66.6-77.9% and our

calculated estimate for moisture content for cooked muscle of 43.0% was used in all

consumption limit calculations (Table 3-7). For As, the number of meals allowed per

month for adults based on average concentrations found in every group ranged from 2.9-

11.8 meals, but varied inconsistently through time, with the lowest levels observed in

Group 2, and the highest in Group 3. Children allowances based on average As

concentrations were between 1.5 and 5.4 meals per month, with 4 out of 6 groups at or

below 1.4 meals per month at the maximum concentrations found. Adult allowances

based on average Se concentrations declined over time from 26.7 meals per month in

Group 1 down to 6.2 meals per month in Group 6; children’s meals allowed also declined

through time from 12.2 meals in Group 1 to 2.8 meals in Group 6. For Hg, adults would

be allowed between 27.5-33.1 meals per month compared to 12.6-15.2 for children at the

average concentrations across all sampling groups.

DISCUSSION

This study provides the most comprehensive assessment of rates of trace element

accumulation in waterfowl utilizing CCW impoundments to date (White et al. 1986). The

results of this study are expected to assist in elucidating potential risks to waterfowl

utilizing coal fly ash settling basins and also human hunters and their families that could

eat these birds. We focused on three common trace elements found in CCW (As, Se, Hg)

that can have deleterious consequences for exposed wildlife and human consumers

(Heinz and Hoffman 1998, Rowe et al. 2002). Of the elements evaluated, only Se clearly

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accumulated through time, a pattern observed in muscle, liver, and blood tissues.

Surprisingly, Hg concentrations in muscle increased and in liver decreased during the

course of this study, possibly due to interactive effects with Se (Hopkins et al. 2007) as

both As and Hg were correlated with Se in liver tissue.

The accumulation of Se in muscle tissue through time, resulted in a corresponding

decrease in the number of allowable meals per month, decreasing in adults from 26.7

meals per month in our first exposure group to 6.2 in our last exposure group (decreasing

from 12.2 to 2.8 meals per month in children) which had exposures of 76-92 days, a

scenario akin to ducks overwintering in a contaminated area. Despite this decrease, for

adults these estimates still exceed average numbers of waterfowl meals consumed per

month by hunters in the southeastern U.S., but are within the reported range consumed

(Smith et al. Unpublished Manuscript).

Average levels of As in muscle ranged from 0.42-1.70 ppm dw and consumption

limit calculations from these concentrations resulted in adult meal allowances ranging

from 2.9-11.8 meals per month. The European Food Safety Authority (EFSA) advises

that there is no “safe” threshold for As in food, so exposure to As from CCW should be

carefully monitored in game species (EFSA 2009). Although As accumulation in muscle

tissue did not increase over the course of our experiment, As concentrations in waterfowl

should be quantified and sources of As need to be investigated given waterfowl ability to

move extensive distances from point sources. Moreover, our results indicated As had a

negative relationship with Se in muscle tissue, so presence of Se may have ameliorated

accumulation of As, which has been seen previously in laboratory experiments with

mallards (Heinz 1979, Heinz 1996, Hoffman et al. 1992, Stanley et al. 1994).

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In a previous study (Chapter 2) we opportunistically collected ducks from this

same ash basin (with no known residence times) and reported higher levels of Hg than

were observed in the present study, with average muscle levels of 0.79 ppm dw and

levels ranging from 0.07-5.55 ppm dw. Thus while our current study had high average

meal allowances of 33.1 meals per month for adults, the previous opportunistically

collected ducks from this same basin had average meal allowances of only 2.1 meals per

month for adults and 1.0 meal for children, reducing to 0.3 meals per month for adults

and 0.1 meals for children at maximum observed concentrations (Chapter 2). The

underlying reason for this discrepancy is unknown but likely due to a combination of two

factors. First, as has been seen in laboratory studies for waterfowl, the presence of Se can

ameliorate uptake of Hg concentrations (Heinz 1979, Heinz 1996, Heinz and Hoffman

1998), a pattern also documented in fish and amphibians in CCW contaminated areas

(Southworth et al. 1994, 2000; Hopkins et al. 2006; Peterson et al. 2009; Yang et al.

2010; Metts et al. 2013, Penglase et al. 2014). The significant interaction between Hg and

Se concentrations in muscle tissue further support the likelihood that substantial uptake of

Se limited uptake of Hg in our experiment. Secondly, species sampled in (Chapter 2)

included a variety of diving ducks but no ring-necked ducks and thus low uptake of Hg in

the present study may reflect species-level differences due to diet. Correspondingly, our

results indicated the importance that mensurative studies alone may not give the clearest

possible picture of wildlife exposure risks and thus experimental in situ exposure studies

are needed to elucidate accumulation patterns in free-ranging populations.

For future studies, our data suggest there is a reasonable correlation between

blood As levels and concentrations found within muscle and liver tissues, suggesting

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blood samples may be used as a non-lethal evaluation tool for quantifying As levels in

free-ranging waterfowl. The relationship between Se in muscle and post-exposure blood

was slightly weaker, with approximately a 7% increase in error expected if the equation

were used on unseen data. The addition of more samples from future studies may be

needed to thoroughly elucidate this relationship. Unfortunately the equation with the best

fit for the relationship of Se liver to post-collection blood did not provide good predictive

value for unseen data.

Taggart et al. (2011) have remarked that game meat may represent a poorly

regulated risk to human health, as game meat can contain levels of toxic elements that

pose a potential risk especially for “at risk” groups like pregnant women, children, or

subsistence hunters (who may consume higher than average amounts of game meat;

Duchesne et al. 2004). Our efforts to connect length of exposure with organ/tissue

burdens for a variety of common trace elements associated with coal ash may be able to

be combined with behavioral data on use of ash basins by free-ranging populations to get

representative estimates of exposure. The data we present on rates of accumulation for

several common CCW contaminants will aid potential monitoring programs to assess

contamination burdens in waterfowl across broader geographical scales to more clearly

elucidate potential risks to wildlife health and human consumers of waterfowl.

MANAGEMENT IMPLICATIONS

Surface impoundments are a potential threat to wildlife because they are often

attractive habitat for many species, which can result in trace element exposure and

increased risk of adverse health effects. We found pollution from coal combustion wastes

has introduced appreciable concentrations of some trace elements into game meat.

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Although levels were generally below those that likely could result in adverse health

effects to the birds or trigger human consumption advisories, the suggested average daily

Se intake for adult humans (0.06 mg; Rayman 2004) is equal to 1.8 mg per month,

approximately the cooked muscle tissue concentrations found in birds in the first

exposure time period of this experiment. This coupled with the fact that the average daily

intake of Se for women (0.09 mg) and men (0.13 mg; Fairweather-Tait et al. 2011)

already exceeds the suggested intake should warrant at least concern about waterfowl

exposure to Se and monitoring what levels could be passed on to human consumers.

There are few studies of this nature that focus on muscle tissue, rather than liver and

kidney, and because of the mobility of waterfowl the scale of this contamination pathway

is difficult to assess. Our data suggest assessments of trace element concentrations in

blood could be reliable predictors of contaminant burdens in muscle and liver for some

elements. Although the relationship between element accumulation in muscle/liver and

feathers was not evaluated in this study, such a comparison could provide an additional

useful non-lethal metric (or hunter assisted method) for assessing waterfowl exposure to

contaminants. Such methodology could be integrated into a comprehensive study

assessing contaminant exposure patterns across the 3 main flyways, from Canada across

the U.S. and down to Mexico could facilitate long-term monitoring of contaminant

exposure patterns. Evers et al. (2005) conducted a meta-analysis focusing on Hg (or

MeHg) in waterfowl in the Northeastern U.S., but there remains a need for a more

comprehensive and extensive survey of multiple elements of animal and human health

concern to facilitate assessment of potential risks of contaminant exposure.

106

Table 3-1. Tissue and blood concentrations of arsenic (As), selenium (Se), and mercury

(Hg) of each recollected ring-necked duck restricted to the D-Area ash basins (n=33) on

the Savannah River Site (SRS) in the winter of 2014-2015 with between 3 and 92 days of

exposure.

Muscle Liver Blood Id Days Exp. As Se Hg As Se Hg As Se Hg

195 3 0.15 0.72 0.04 1.63 20.79 0.15 0.24 0.90 0 197 3 0.33 0.84 0.02 1.76 20.56 0.25 0.53 1.33 0 189 8 0.80 7.63 0.06 3.69 21.04 0.15 1.42 14.07 0 180 9 1.57 3.19 0.06 4.62 9.42 0.07 2.35 6.26 0 178 19 2.20 4.91 0.03 4.91 15.76 0.12 2.78 14.04 0 188 22 1.84 5.28 0.05 3.41 16.10 0.10 2.18 18.92 0 190 22 1.49 6.03 0.04 2.51 17.21 0.25 2.32 15.81 0.02 179 23 1.84 5.96 0.06 5.13 15.08 0.13 2.29 15.75 0 192 29 1.14 6.77 0.06 1.37 21.24 0.22 0.94 17.72 0.02 110 32 0.25 9.13 0.06 0.47 11.78 0.15 0.17 15.08 0 194 34 1.11 8.69 0.03 1.89 20.34 0.15 1.63 22.08 0.02 171 40 0.22 5.15 0.06 0.35 6.52 0.11 0.16 7.75 0.06 150 43 0.33 8.62 0.04 0.62 9.42 0.09 0.29 12.28 0 148 44 0.20 5.69 0.06 0.27 6.10 0.11 0.21 9.03 0 34 48 0.17 7.04 0.08 0.40 7.29 0.08 0.23 8.54 0.07 15 52 0.35 9.36 0.05 0.84 10.89 0.12 0.41 10.03 0 3 55 0.27 9.46 0.03 0.27 5.64 0.07 0.09 9.64 0 32 56 1.03 10.33 0.06 2.46 25.00 0.11 1.47 14.71 0.04 39 56 0.46 9.56 0.07 1.28 16.86 0.12 0.57 11.96 0 169 64 0.73 13.08 0.05 1.89 30.52 0.07 0.91 20.98 0.09 136 67 1.68 6.88 0.06 1.48 14.88 0.09 2.24 17.17 0.02 144 67 0.83 10.05 0.06 1.70 21.75 0.07 0.90 20.70 0.02 158 67 0.89 8.72 0.03 2.41 14.66 0.11 0.77 14.98 0 160 67 0.34 7.18 0.07 0.70 10.23 0.10 0.35 10.58 0 164 70 1.26 10.54 0.06 2.40 17.95 0.08 1.37 20.70 0.02 95 73 0.99 12.18 0.05 1.98 20.26 0.19 1.01 19.18 0 26 75 0.58 14.33 0.06 1.32 22.52 0.11 0.52 16.64 0 168 78 2.13 11.57 0.07 3.83 24.01 0.11 0.28 7.09 0 41 79 1.26 13.46 0.05 2.42 18.16 0.07 1.18 20.41 0 43 79 1.31 11.92 0.05 3.00 26.06 0.19 0.21 9.61 0 97 85 1.06 12.32 0.06 2.48 17.99 0.07 1.19 19.60 0.02 85 90 1.03 15.64 0.08 2.57 38.99 0.11 0.15 20.01 0 36 92 1.20 15.03 0.06 1.00 7.29 0.07 0.84 25.41 0

107

Table 3-2. Trace elements in ring-necked ducks (n=33) before and after restriction to the D-Area ash basins on the Savannah

River Site (SRS) for between 3 and 92 days in winter of 2014-2015. Concentration mean±SE at ~15-day exposure increments.

Element Tissue 3-15 days (n=4) 16-30 days (n=5) 31-45 days (n=5) 46-60 days (n=5) 61-75 days (n=8) 76-92 days (n=6) V Muscle 0.05±0.00 0.07±0.01 0.08±0.01 0.09±0.01 0.08±0.01 0.10±0.02

Liver 0.35±0.04 0.34±0.07 0.28±0.04 0.42±0.10 0.41±0.04 0.42±0.06

Blood 0.06±0.01 0.05±0.00 0.13±0.04 0.08±0.01 0.08±0.02 0.10±0.02

! Cr Muscle 0.70±0.05 0.74±0.06 0.73±0.04 0.75±0.05 0.76±0.02 0.85±0.06

Liver 2.22±1.11 1.00±0.08 1.03±0.10 1.14±0.14 1.34±0.07 1.22±0.16

Blood 1.12±0.15 1.06±0.05 1.28±0.12 1.05±0.11 1.09±0.06 1.48±0.17

! Ni Muscle 0.05±0.00 0.06±0.02 0.07±0.01 0.10±0.02 0.07±0.01 0.11±0.02

Liver 4.59±4.29 0.39±0.09 0.14±0.06 0.13±0.05 0.19±0.04 0.28±0.03

Blood 0.18±0.04 0.20±0.01 0.22±0.04 0.19±0.03 0.17±0.01 0.30±0.06

! Cu Muscle 34.12±1.93 34.38±1.73 32.09±3.27 29.94±1.86 33.99±1.91 45.41±6.03

Liver 346.20±43.13 286.93±54.47 227.06±42.70 262.44±56.75 288.42±46.88 184.60±70.66

Blood 2.26±0.08 2.41±0.13 2.16±0.26 2.10±0.36 2.67±0.34 2.60±0.36

! Zn Muscle 36.15±2.94 37.86±1.95 34.32±2.69 32.03±1.10 35.85±1.65 43.25±2.76

Liver 171.05±28.20 178.53±8.19 135.38±8.93 147.13±21.62 158.92±5.49 152.29±22.26

Blood 18.46±0.44 20.25±0.87 20.59±0.82 17.43±1.18 19.70±0.72 19.07±0.84

! As Muscle 0.71±0.32 1.70±0.18 0.42±0.17 0.45±0.15 0.91±0.15 1.33±0.17

Liver 2.92±0.74 3.47±0.71 0.72±0.30 1.05±0.40 1.73±0.20 2.55±0.38

Blood 1.13±0.48 2.10±0.31 0.49±0.29 0.55±0.24 1.01±0.21 0.64±0.20

! Se Muscle 3.10±1.61 5.79±0.32 7.45±0.84 9.15±0.56 10.37±0.96 13.32±0.69

Liver 17.95±2.85 17.08±1.10 10.83±2.59 13.14±3.54 19.09±2.19 22.08±4.31

Blood 5.64±3.06 16.45±0.85 13.25±2.55 10.98±1.09 17.62±1.27 17.02±2.89

! Hg Muscle 0.05±0.01 0.05±0.01 0.05±0.01 0.06±0.01 0.05±0.00 0.06±0.00

Liver 0.16±0.04 0.17±0.03 0.12±0.01 0.10±0.01 0.10±0.01 0.10±0.02

!

108

Cd Liver 1.16±0.48 1.26±0.10 0.92±0.31 0.83±0.16 1.14±0.22 1.24±0.17

! Pb Liver 0.51±0.35 1.07±0.87 0.21±0.10 0.12±0.05 0.08±0.03 0.26±0.09 a Specific days of exposure within groups were (3-15 days, 19-29 days, 31-44 days, 48-56 days, 64-75 days, 78-92 days)

b For Hg in blood 22 (66%) were BDL

c For Cd in muscle 28 (85%), and all blood samples, were BDL

d For Pb in muscle 30 (91%) and in blood 27 (82%) were BDL

109

Table 3-3. Selenium (Se) linear regression with days of exposure for recollected ring-necked ducks restricted to the D-Area

ash basins (n=33) between 3 and 92 days on the Savannah River Site (SRS) in the winter of 2014-2015.

Muscle Liver Blood Source Estimate SE t P Estimate SE t P Estimate SE t P

As Intercept -0.60 0.30 -1.99 0.05 0.58 0.32 1.83 0.08 -0.03 0.37 -0.08 0.93 DaysExp 0.01 0.01 1.05 0.30 0.00 0.00 -0.50 0.62 -0.01 0.01 -1.21 0.24

Table 3-4. Arsenic (As) linear regression with days of exposure for recollected ring-necked ducks restricted to the D-Area ash

basins (n=33) between 3 and 92 days on the Savannah River Site (SRS) in the winter of 2014-2015.

Muscle Liver Blood Source Estimate SE t P Estimate SE t P Estimate SE t P

Se Intercept 1.00 0.17 6.02 <0.0001 2.61 0.18 14.20 <0.0001 1.79 0.24 7.60 <0.0001 DaysExp 0.02 0.00 6.91 <0.0001 0.00 0.00 0.75 0.46 0.01 0.00 3.36 0.002

Table 3-5. Mercury (Hg) linear regression with days of exposure for recollected ring-necked ducks restricted to the D-Area

ash basins (n=33) between 3 and 92 days on the Savannah River Site (SRS) in the winter of 2014-2015. Blood Hg

concentrations had >50% BDL and thus were not tested statistically.

Muscle Liver Source Estimate SE t P Estimate SE t P

Hg Intercept -3.17 0.12 -29.63 <0.0001 -1.86 0.13 -14.13 <0.0001 DaysExp 0.004 0.00 2.27 0.03 -0.01 0.00 -2.75 0.01

110

Table 3-6. Correlationsa among trace element concentrations in muscle (above diagonal)

and liver (below diagonal) tissues for recollected ring-necked ducks restricted to the D-

Area ash basins (n=33) between 3 and 92 days on the Savannah River Site (SRS) in the

winter of 2014-2015. Correlations between muscle and liver samples for individual

elements are presented on the diagonal in bold.

As Se Hg As 0.81 (<0.0001) NS NS Se 0.53 (<0.001) 0.46 (0.005) NS Hg NS 0.35 (0.04) NS

aSpearman correlation coefficients and P values in parentheses; NS=not significant (P >

0.05)

111

Table 3-7. The monthly allowances of ½ lb. meals for adults and ¼ lb. for children before exceeding the EPA’s chronic oral

reference dose limits for arsenic (As), selenium (Se), and mercury (Hg) for muscle tissue of ring-necked ducks collected from

the D-Area ash basins on the Savannah River Site (SRS) after being restricted between 3 and 92 days of exposure a.

Consumption limits based on average concentrations of cooked ducks muscle are presented with limits based on the maximum

concentration found in an individual for each trace element in parentheses.

Element Group 3-15 days

(n=4) 16-30 days

(n=5) 31-45 days

(n=5) 46-60 days

(n=5) 61-75 days

(n=8) 76-92 days

(n=6) As Adult 7.0 (3.2) 2.9 (2.3) 11.8 (4.5) 11.0 (4.8) 5.4 (3.0) 3.7 (2.3)

Child 3.2 (1.4) 1.3 (1.0) 5.4(2.1) 5.1 (2.2) 2.5 (1.4) 1.7 (1.1)

! ! !Se Adult 26.7 (10.8) 14.3 (12.2) 11.1 (9.1) 9.0 (8.0) 8.0 (5.8) 6.2 (5.3)

Child 12.2 (5.0) 6.6 (5.6) 5.1 (4.2) 4.1 (3.7) 3.7 (2.6) 2.8 (2.4)

! ! !Hg Adult 33.1 (33.1) 33.1 (33.1) 33.1 (33.1) 27.5 (20.7) 33.1 (23.6) 27.5 (20.7) !! Child 15.2 (15.2) 15.2 (15.2) 15.2 (15.2) 12.6 (9.5) 15.2 (10.8) 12.6 (9.5)

a Specific days of exposure within groups were (3-15 days, 19-29 days, 31-44 days, 48-56 days, 64-75 days, 78-92 days)

112

Figure 3-1. D-Area ash basins on the Savannah River Site (SRS), SC. Basin 1, the largest

basin is partially filled in and has extensively revegetated. The smaller enclosed wetland

formed by revegetation in this basin was utilized as the release and exposure area for the

ring-necked ducks in this study in winter of 2014-2015.

113

Figure 3-2. Muscle concentrations of arsenic (As), selenium (Se), mercury (Hg), and

days of exposure of recollected ring-necked duck restricted to the D-Area ash basins

(n=33) on the Savannah River Site (SRS) in the winter of 2014-2015 between 3 and 92

days of exposure.

R² = 0.003

R² = 0.76

R² = 0.14 0.0 2.0 4.0 6.0 8.0

10.0 12.0 14.0 16.0 18.0

0 15 30 45 60 75 90

Con

cent

ratio

n (p

pm d

w)

Days of Exposure

Muscle Tissue

As

Se

Hg

114

Figure 3-3. Liver concentrations of arsenic (As), selenium (Se), mercury (Hg), and days

of exposure of recollected ring-necked duck restricted to the D-Area ash basins (n=33) on

the Savannah River Site (SRS) in the winter of 2014-2015 between 3 and 92 days of

exposure.

R² = 0.05

R² = 0.05

R² = 0.19 0.0 5.0

10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0

0 15 30 45 60 75 90

Con

cent

ratio

n (p

pm d

w)

Days of Exposure

Liver Tissue

As

Se

Hg

115

Figure 3-4. Blood concentrations of arsenic (As), selenium (Se), mercury (Hg), and days

of exposure of recollected ring-necked duck restricted to the D-Area ash basins (n=33) on

the Savannah River Site (SRS) in the winter of 2014-2015 between 3 and 92 days of

exposure.

R² = 0.11

R² = 0.24

R² = 0.0002 0.0

5.0

10.0

15.0

20.0

25.0

30.0

0 15 30 45 60 75 90

Con

cent

ratio

n (p

pm d

w)

Days of Exposure

Blood Levels Over Time

As

Se

Hg

116

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CHAPTER 4

RADIOCESIUM IN WATERFOWL/WATERBIRDS FROM A RETIRED NUCLEAR

REACTOR COOLING RESERVOIR 1

____________________

1 Oldenkamp, R. E., R. A. Kennamer, A. L. Bryan, Jr., and J. C. Beasley. To be submitted to the Journal of Radioactivity.

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ABSTRACT

Low-levels of radiocesium contamination is distributed throughout much of the

globe due to nuclear accidents, nuclear weapons testing, and releases from nuclear

facilities. Radiocesium can be accumulated in the bodies of wildlife species, particularly

in skeletal muscle. Thus gamebirds, including waterfowl and water birds, can be a

conduit of human exposure to radiocesium, even from closed access contaminated sites

when birds travel hundreds to thousands of km from the contaminated sources. We

released American coots (Fulica americana) and ring-necked (Aythya collaris) ducks

onto a radiocesium contaminated reservoir on the Savannah River Site in Aiken, SC,

USA, then recollected birds after 33-173 days of exposure. We quantified uptake of

radiocesium over time and compared uptake between species in whole-body relationships

as well as concentrations in muscle and liver tissue. For American coots we also

calculated the ecological half-life for radiocesium in our study system by comparing

whole-body burdens to historical data collected from the same location. Coots maintained

equilibrium whole-body radiocesium concentrations from the first collection event at 32

days through 90 days of exposure, while concentrations in ring-necked ducks continued

to increase between 33 and 90 days of exposure. Beyond 90 days tissue concentrations

declined in both species. Ring-necked ducks had higher concentrations of radiocesium

levels in muscle tissue (t = 2.64, P = 0.01), but liver levels were not significantly

different between species. Ninety-seven percent (34 of 35) of coots and 92% (33 of 36) of

ring-necked ducks had muscle concentrations above the European Economic Community

(EEC 1986) limit of 0.600 Bq/g in fresh meat established for human consumption. We

estimated the ecological half-life of radiocesium in American coots at our study site to be

131

~17 years (with a possible range between approximately 13-24 years). Our data suggest

waterfowl/waterbirds represent a possible vector for human exposure to radionuclides

from contaminated areas, especially habitats where birds may stop-over on migration or

areas in which they over-winter.

INTRODUCTION

Releases of radiocesium into the environment can have wide-ranging

consequences for ecosystems and potentially human health. Although low levels of

radiocesium contamination are distributed throughout much of the globe due to nuclear

accidents and nuclear weapons testing, releases from nuclear facilities also have

occurred, often with inputs directly into aquatic ecosystems. Radiocesium is of biological

importance because its chemical behavior is similar to potassium and can be accumulated

in the bodies of wildlife species, particularly in skeletal muscle (Potter et al. 1989).

Moreover, 137Cs, an important isotope of radiocesium, has a relatively long physical half-

life of 30.2 years and thus can remain in aquatic and terrestrial ecosystems for prolonged

periods (Evans et al. 1983).

Gamebirds, including waterfowl and water birds can be a conduit of human

exposure to radiocesium, even from closed-access contaminated sites. Birds that forage in

restricted contaminated areas may be harvested by hunters hundreds to thousands of kms

from the contaminated sources (Kennamer 2003, Cristol et al. 2012). The Savannah River

Site (SRS), a U.S. Department of Energy (DOE) former nuclear production site in South

Carolina has abundant wetlands and large abandoned cooling reservoirs. These resources

are protected from public disturbance and provide important inland breeding, stop-over,

and overwintering habitat for thousands of resident and migratory waterfowl/waterbirds

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representing as many as 28 species (Kennamer 2005). Many species use the SRS as a

migratory stopover or overwintering location, including ring-necked ducks (Aythya

collaris) that have later been harvested throughout much of eastern North America, as far

north as Manitoba to Nova Scotia, and as far south as Cuba (Kennamer 2003).

Multiple low-level releases of radiocesium have occurred since the SRS was

established in the 1950s (Carlton et al. 1992, 1994), with radiocesium released into

cooling waters from four of the five nuclear reactors (Paller et al. 1999). During the past

three decades principles of radionuclide distribution and decline in SRS aquatic habitats

have been studied in sediments (Mohler et al.1997) and numerous biota such as wood

ducks (Aix sponsa; Fendley et al. 1977), American coots (Brisbin and Vargo 1982,

Brisbin 1991a, Brisbin and Kennamer 2000), snakes (Bagshaw and Brisbin 1984), and

fish (Paller et al. 1999, Peles et al. 2000).. In particular, Pond B on the SRS has been an

important study system over the last several decades as it received the greatest inputs of

radiocesium of the SRS reservoirs (Brisbin 1991a). Previous work in Pond B determined

American coots (Fulica americana) consistently accumulated levels of radiocesium

exceeding other wintering species investigated, including carnivorous, omnivorous, and

piscivorous species (Brisbin et al. 1973). Thus, coots have been used as a model species

for ecological risk assessments in this system (Brisbin et al 1973, Brisbin 1993). Diet is a

likely biological explanation given for this differentiation in accumulation, as coots

primarily consume algae and aquatic vascular plants that are known to accumulate high

levels of radiocesium (Potter 1987, Brisbin et al. 2002). However, these data were

derived from opportunistically collected individuals, with unknown residence times.

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Understanding the rate of radiocesium decline in affected aquatic systems is

imperative for elucidating potential risks to wildlife and humans who may consume these

vectors, as well as for assessing remediation efforts and determining when human

exclusion from contaminated areas is no longer necessary. Historical data for coots in the

SRS Pond B system provides a unique long-term data set for the calculation of the

decline of radiocesium in a natural system through the development of an ecological half-

life in biota. An ecological half-life delineates the decrease in contaminant concentration

over time within an ecosystem, and is thus reflected as a decrease in levels observed

within organisms as it becomes biologically unavailable (Brisbin 1993, Paller et al.

1999). The ecological half-life for radionuclides, like radiocesium, can be influenced by a

myriad of chemical, physical, and biological processes (Whicker and Schultz 1982,

Brisbin 1991a, Paller et al. 1999). Ecological half-life differs from the physical half-life,

which is the rate of radioactive decay to daughter products, and biological half-life,

which is the rate at which the isotope is turned over and eliminated from the body

(Brisbin 1993). A relatively long ecological half-life has previously been calculated for

Pond B biota (Table 4-1), possibly due to the low rate of water turnover in that system,

low potassium concentrations (thus radiocesium has less competition for binding in biota

and will remain available for accumulation instead of bound in the sediments), large

amounts of rooted aquatic macrophytes that can translocate cations from the sediment

into the water column, and the radiocesium remaining in the system is the 137Cs isotope

which has the 30.2 year half-life (Whicker et al. 1990).

As a variety of wildlife species can be exposed to radiocesium from Pond B,

investigating the ecological half-life is relevant not only for future access issues at SRS,

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but also other locations dealing with the legacy of past nuclear pollution. Wetlands at

other nuclear weapons production sites (e.g. Willard 1960, Fitzner and Rickard 1975,

Halford et al. 1981) and in regions contaminated by nuclear accidents such as the

Chernobyl site (Brisbin 1991b) and Fukushima, Japan (Tagami and Uchida 2013) all

support populations of migratory waterfowl which can transfer radiocesium away from

these sites (Brisbin 1991a).

For this study, we collected American coots and ring-necked ducks from an

uncontaminated lake on SRS and released them on Pond B, then recollected birds

between approximately one and six months of radiocesium exposure. We included ring-

necked ducks because they can travel thousands of kilometers from the site (Kennamer

2003), and provide a comparative species with a more varied diet (Brisbin et al. 1973,

Bergan and Smith 1986, Hoppe et al. 1986). Our objectives were to 1) quantify uptake of

radiocesium at multiple time points of exposure by coots and ring-necked ducks

subsequent to translocation to Pond B and compare whole-body uptake between species

as well as concentrations in muscle and liver tissue, and 2) calculate the ecological half-

life for radiocesium in American coots on Pond B by comparing whole-body burdens to

historical data collected from the same location. These results have implications for

choosing focal species for experimental restriction to contaminated areas as well as for

long-term mensurative monitoring programs.

METHODS

Study Area

Research occurred on the Savannah River Site (SRS), an ~800 km2 limited-access former

nuclear production and research facility owned and operated by the U.S. Department of

135

Energy in South Carolina (White and Gaines 2000). The SRS was created in 1951 to

provide nuclear weapons materials (Savannah River Nuclear Solutions, LLC 2011), but

all five nuclear reactors have since been decommissioned (White and Gaines 2000).

Wetlands and other aquatic habitats on the SRS are abundant, including creeks, streams,

upland depressions, Carolina bays, bottomland and swamp forests, as well as two large

cooling reservoirs; these areas are important habitat for migrating waterfowl/waterbirds

(Lide 1994).

Pond B, a former recipient of heated effluent from R reactor on the SRS, is an 87-

ha reservoir with a mean depth of 4.3 m. Precipitation is the only input and the water has

low potassium levels and is slightly acidic (Figure 4-1; Alberts and Dickson 1985,

Alberts et al. 1988). Between 1961 and 1964, approximately 5.7 X 1012 Bq of

radiocesium was released into the Pond B reservoir (Ashley and Zeigler 1980). Use of

Pond B by 12 waterfowl species has been documented (Mayer et al. 1986). Radiocesium

in Pond B sediments (Brisbin et al. 1974) has been quantified and related to levels found

in migratory game birds with potential to disperse contaminants extensive distances from

the contaminated reservoir (Brisbin et al. 1973, Fendley et al. 1977, Brisbin and Vargo

1982, Kennamer et al. 1998). L-Lake, another cooling reservoir on the SRS, was used as

our uncontaminated source site for this research.

Trapping and Sample Collection

Many birds that reside or overwinter on the SRS are exposed to unknown

amounts of radiocesium contamination. Testing captured wild birds from contaminated

water sources does not evaluate residence time and birds can utilize multiple water

resources (contaminated and uncontaminated) during resident and migratory periods,

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confounding estimates of contaminant uptake and exposure. To quantify accumulation of

radiocesium in coots and ring-neck ducks inhabiting Pond B, birds were trapped (coots:

December 7th 2013–January 13th 2014, coots and ring-necked ducks: December 8th–18th

2014) with welded-wire traps baited with corn (Haramis et al. 1982) or dip-netted from a

boat on L-Lake, an uncontaminated reservoir on the SRS. Captured birds were banded

with U.S. Fish and Wildlife Service aluminum leg bands, coots were fitted with colored

neck collars and ducks with colored nasal saddles for ease of identification during

collections, and a subset were whole-body counted for radiocesium to establish a

background level before release on Pond B. Before release we wing-scissored flight

feathers on one wing to render birds flightless, restricting individuals to the contaminated

waterbody.

Previous work in the Pond B system with coots found that they reach an

asymptotic radiocesium level (maximal radiocesium body burden) in approximately 20-

25 days (Brisbin et al. 1989, Brisbin 1991a). Therefore, birds were left on Pond B a

minimum of 30 days before lethal collection (using shotguns) to ensure maximum body

burdens would be reached in coots for comparison with previous data. Collections were

conducted at approximately 30, 60, and 90 days (with a few birds left for later recovery

up to 173 days) to verify achievement of asymptotic body burdens in coots and ring-

necked ducks at approximately 30 days of exposure. Upon collection birds were weighed

and frozen at -20°C for later whole-body counting and dissection. All birds were whole-

body counted for radiocesium and then dissected to collect breast muscle and liver tissues

for radiocesium quantification in specific tissues. Wet weights of all muscle and liver

samples were recorded before samples were freeze-dried, then weighed again before

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being homogenized into a powder using a coffee grinder. Grinder canisters were cleaned

(5% nitric acid solution) and dried between uses. All animal handling practices and

euthanasia were carried out with accompanying federal and state collecting permits and

in accordance with University of Georgia Animal Care and Use guidelines under protocol

A2013 06-004-Y3-A1.

Radiocesium Analysis

Birds were whole-body counted with a 10·2-cm x 15·2-cm NaI (Tl) gamma

detector (Bicron Model:6H3Q/5; S/N:BJ-124R) coupled to an IBM 300-GL Personal

Computer (Windows 98 OS) containing an onboard Canberra MCA card and controlled

by Canberra Genie 2000 gamma spectroscopy software (Version 1.3; entire system

located in SREL Lab 120). A counting window (Region of Interest-ROI) of 596–728

kiloelectron-Volts (keV) centered on 662 keV was used to record total detector

absorption events from the radiocesium emission of 662 keV photons. The system was

calibrated daily, as counting took place, with a traceable radiocesium calibration disc

(New England Nuclear Gamma Reference Disc Source Set; Catalogue No. NES-101S;

radiocesium disc; 1.04 microCuries on 10/2/1985) by adjusting the system amplifier gain

control to center the disc-generated peak on channel 331 (661.7keV). Generally, 30-min

count times (1800 sec) were used for counting collected/sacrificed birds (whole-body;

frozen) and backgrounds (empty chamber), while 15-min count times (900 sec) were

used for counting aqueous standards (the ILB Series of standards; containing known

radiocesium quantities [Becquerels (Bq)]; decay corrected to count dates). Background-

corrected count rates (counts per second; cps) from the ILB Series of standards were used

to produce mass-specific count yields which were in turn used to produce a predictive

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equation of expected yields from bird mass (in grams; yield=0.4449*mass-0.343;

R2=0.97). Finally, background-adjusted bird count rates and the birds' mass-specific

yields were used to determine radiocesium content of birds (whole-body Bq, Bq/g,

pCi/g).

All muscle and liver samples were freeze-dried, powdered, then packed into

scintillation vials for quantification of radiocesium activity using a Packard Cobra II

Auto-Gamma Counter (Model Cobra II 5003) with a single 3-inch through-hole NaI

detector. A counting window (Region of Interest-ROI) of 580–754 kiloelectron-Volts

(keV) centered approximately on 662 keV was used to record total absorption events

from the radiocesium emission of 662 keV photons. The system was auto-calibrated

daily, as counting took place, with a traceable radiocesium source (SREL-0113; 0.1

microCuries on 10/2012). Generally, 60-min count times (3600 sec) were used for

counting dry, powdered samples (packed into tubes) and backgrounds (empty tubes) that

were arranged in every fifth counting position. Four standards were prepared from

commercially-available chicken breast muscle tissue that was dried, homogenized into

powder, spiked with known quantities of radiocesium (745 Bq in each spiked standard;

decay-corrected for the date of preparation), and then loaded/packed into 4 scintillation

tubes in 1-gram increments ranging from 1-4g. Background-corrected count rates (net

counts per second; ncps) recorded for these spiked standards were used to produce

estimates of mass-specific count yields (a ratio of measured net count rate [ncps] to

expected disintegration rates [dps] for the known radiocesium activity) at each of 4

available sample height settings relative to the NaI detector of the Cobra II system.

Sample position #4 produced count yields that varied little across all sample masses (SD

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< 0.007) and so samples/backgrounds were all counted using sample height #4 setting,

with an averaged count yield value of 0.2213 used as a constant in radiocesium content

determinations for unknowns. Specifically, background-adjusted dry tissue count rates

divided by the yield constant were estimated as the radiocesium content of dry tissue

samples (total Bq, Bq/g [dry mass], and Bq/g [wet mass]). Minimum Detectable

Concentrations (MDCs) were calculated for all radiocesium analyses using the equation

of Lloyd Currie (Currie 1968).

Data Analysis

Many of the whole-body counts of radiocesium for the birds that were captured

on L-Lake were below background (or blank count) rates, which gave us negative values

for radiocesium concentrations. A common practice is to not report these values, or the

small positive values that happen to be below MDCs, or set them to an arbitrary indicator

value, thus biasing the mean and variance of the concentration data (Gilbert and Kinnison

1981, Newman et al. 1989). We chose to report all values, including negatives and those

below the MDCs, when determining average radiocesium concentrations and standard

errors (SE).

All statistical tests were performed with JMP Pro Version 10.0.1; SAS Institute

Inc., Cary, NC. We used t-tests to compare whole-body, muscle, and liver radiocesium

concentrations between coots and ring-necked ducks, after testing for equality in the

variances. To evaluate the relationship between whole-body radiocesium concentrations

and concentrations in muscle and liver, we used linear regression (with forced intercepts

of zero). We compared the slope estimates of the relationships between whole-body and

tissue burdens for each species with homogeneity of slopes models (Analysis of Variance

140

models using least squares fits). Using the common muscle slope for both species we

calculated the whole-body radiocesium equivalent, having this standard for the muscle to

whole-body relationship allows a comparison to the EEC limit for radiocesium in fresh

meat (0.600 Bq/g), with only having to whole body count birds, which is much less labor

intensive than completing muscle counts.

From historical coot collection data on Pond B during winters of 1975-76, 1983-

84, 1986-87, 1987-88, and 1991-92 along with our coot data collected from Pond B in

winters 2013-2014 and 2014-2015 (Figure 4-2a), we modeled the long-term natural

attenuation of radiocesium body burdens (Brisbin and Kennamer 2000). The last input of

radiocesium was in 1965, and we would expect a decline of radiocesium through time

since then; however investigation of historical data revealed that radiocesium burdens in

coots increased between the winters of 1975-76 and 1986-87, possibly from residual

radiocesium flushing out of the canal that leads from the reactor to the reservoir during

flooding events. Therefore, we chose to model the ecological half-life in the system from

the (natural log-transformed) whole-body radiocesium data for 96 coots starting at the

winter of 1986-87 through our present day collection data, in winters 2013-2014 and

2014-2015 (Figure 4-2b). Using linear regression we estimated model parameters and

95% confidence intervals (CI) to describe the decline of radiocesium whole-body burdens

(Bq/g wet mass; y) for Pond B coots sampled during different years (x; continuous

variable). Our estimated parameters included the slope (β) and the intercept (ea , e is the

base of the natural log). Since 1965 was the initial winter year migratory coots would

have been exposed to the maximum radiocesium concentration on Pond B, we set the

intercept to this year (Ashley and Zeigler 1980). The slope acquired from our regression

141

model was used in the half-life equation for radiocesium (T1/2 =ln(2) / β) to produce an

ecological half-life (Te) estimate and a 95% CI around the estimate.

RESULTS

Upon initial capture from L-Lake in 2013-2015 a random selection of 32 birds (25

coots and 7 ring-necked ducks) were whole-body counted to establish a background

concentration of radiocesium before moving them to Pond B. These birds had average

radiocesium concentrations of 0.0006 Bq/g (coots) and -0.0014 Bq/g (ring-necked

ducks), with ranges of -0.0022–0.0051 Bq/g and -0.0046–0.0001 Bq/g, respectively

(Table 4-2). All whole-body concentrations were below individual corresponding MDCs

of 0.012 Bq/g for coots and 0.011Bq/g fresh mass for ring-necked ducks (Table 4-2).

In the winter of 2013–2014 we released 34 coots to Pond B and re-collected 16

between 44 to 88 days of exposure. During winter of 2014-2015 we released 50 coots and

55 ring-necked ducks on Pond B and re-collected birds between 32 to 173 days of

exposure. In total, we were able to recollect 35 coots and 36 ring-necked ducks, whose

whole-body radiocesium concentrations averaged 0.94±0.064 SE Bq/g and 1.18±0.097

SE Bq/g, respectively (Table 4-3). All collected birds had whole-body radiocesium

concentrations above their respective MDCs (0.014 Bq/g for coots and 0.012 Bq/g fresh

mass for ring-necked ducks; Table 4-3). A t-test (unequal variances), showed that whole-

body radiocesium concentrations differed significantly between species (t = 2.04, P =

0.05), with ring-necked ducks having higher concentrations.

Congruent with previous studies of coot attenuation of radiocesium on Pond B, in

our study coots maintained equilibrium whole-body concentrations from the first

collection event at 32 days through 90 days of exposure (Figure 4-3a). A small sample

142

size of coots (N=5) collected beyond 90 days of exposure suggested eventual decline in

whole-body radiocesium concentrations (Figure 4-3a). Contrary to the equilibrium

reached at approximately 30 days in coots, whole-body radiocesium concentrations in

ring-necked ducks continued to increase between 33 and 90 days of exposure, before we

saw eventual decline beyond 90 days (Figure 4-3b).

Muscle and liver radiocesium concentrations for the 35 coots averaged

1.64±0.118 SE Bq/g and 1.02±0.062 SE Bq/g wet mass, respectively (Table 4-3). In the

36 ring-necked ducks muscle and liver concentrations averaged 2.24±0.195 SE Bq/g and

1.25±0.097 SE Bq/g wet mass, respectively (Table 4-3). Ring-necked ducks had higher

concentrations of radiocesium levels in muscle tissue (t = 2.64, P = 0.01), but liver levels

were not different between species (t = 1.94, P = 0.06). All coots and ring-necked ducks

had muscle and liver radiocesium levels above their respective MDCs (Table 4-3).

Ninety-seven percent (34 of 35) of coots and 92% (33 of 36) ring-necked ducks had

muscle concentrations above the European Economic Community (EEC 1986) limit of

0.600 Bq/g in fresh meat established for human consumption (Figure 4-3).

In coots and ring-necked ducks radiocesium concentrations in wet muscle and

liver tissues were positively correlated to whole-body radiocesium burdens (Figure 4-4).

Results indicated that radiocesium concentrated to a greater extent into muscle (β =

1.74±0.036 SE, β = 1.91±0.030 SE) than into liver (β = 1.06±0.028 SE, β = 1.03±0.034

SE) for both coots and ring-necked ducks, respectively. In comparing slopes for the

relationships of tissue concentrations to whole-body radiocesium between species we

found no significant relationship for muscle (overall model: F3,67 = 441.1, P < 0.0001, R2

= 0.95; slopes: F1,67 = 0.002, P = 0.10) or liver (overall model: F3,67 = 101.5, P <0.0001,

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R2 = 0.82; slopes: F1,67 = 0.002, P = 0.97). Because the slopes were not different we

developed a common slope for both species combined (muscle: β = 1.85±0.025 SE, liver:

β = 1.04±0.023 SE). We estimated the whole-body radiocesium equivalent for both

species using the common muscle slope (β = 1.85), suggesting a whole-body burden of

0.324 Bq/g, would be necessary to exceed the EEC limit (0.600 Bq/g) for radiocesium in

fresh muscle. Using linear regression (R2 = 0.31) we obtained an α estimate of 1.84±0.25

SE which allowed us to estimate the mean radiocesium concentration in Pond B coots

after the releases into the cooling water in 1965 at 6.30 Bq/g (95% CI = 3.86-10.28). The

β estimate (-0.0412±0.0064 SE) when incorporated into the half-life equation (T1/2 =ln(2)

/ β) produced an ecological half-life (Te) estimation of 16.82 (95% CI = 12.91-24.19)

years.

DISCUSSION

We verified historical research reporting that whole-body radiocesium burdens in

coots reached equilibrium with the environment in ≤30 days of exposure (Brisbin et al.

1989), although our data suggest burdens may have begun to decline after about 90 days

of exposure. In contrast, whole-body radiocesium concentrations in ring-necked ducks

continued to increase up to approximately 90 days before also experiencing a gradual

decline. Past research with free-ranging coots observed a decline in radiocesium burdens

in late spring, speculating this trend may have been due to the “dilution” of those birds

that had spent the winter on the contaminated water body (but were leaving north on

migration) with migrating birds returning north from uncontaminated waters during

migration (Brisbin et al. 1991a). However, given that we observed a similar decline

towards the end of our experiment (spring) in both species of restricted birds, we

144

hypothesize this decline may be due to excretion through a seasonal change in physiology

or bioavailability through altered dietary habits. Additional research into temporal

patterns of radiocesium uptake and elimination is therefore needed to more clearly

elucidate potential seasonal and dietary effects on radiocesium accumulation to improve

risk assessment calculations of free-ranging birds.

Our findings revealed ring-necked ducks had higher concentrations of

radiocesium in muscle tissue than coots, possibly due to differences in diet or other

behaviors that may influence exposure, but liver concentrations did not differ between

species. Muscle, where radiocesium accumulates to a higher degree then organs (Potter et

al. 1989), is used when determining human consumption risks for contaminated meat.

When comparing muscle concentrations from both species to the EEC recommended

limits for consumption of radiocesium in fresh meat, we found that 97% of coots and

92% of ring-necked ducks in our study were above these levels. In a previous study with

free-ranging birds opportunistically sampled from Pond B, only 63.3% were above this

threshold and coots from Pond B had whole-body radiocesium levels about 7 times

higher than diving ducks [ruddy ducks (Oxyura jamaicensis) and bufflehead (Bucephala

albeola)] sampled at the same time (Chapter 2). These data suggest that by restricting

birds we increased residency time (and thus radiocesium accumulation) beyond what

would normally be expected in free-ranging populations. These data also suggest that

coots may spend more time foraging in this habitat than diving ducks, although ring-

necked ducks were not included in the previous assessment of free-ranging birds on Pond

B (Chapter 2). Given the variation observed in uptake and accumulation between species

and discrepancies with previous mensurative studies, our data suggest a combination of

145

mensurative and experimental research with more than one species are needed in

ecological monitoring of radiocesium.

A previous study used muscle concentrations of a small sample of Pond B coots

to estimate the whole-body equivalent concentration of radiocesium (0.47 Bq/g), this

value is the amount of radiocesium which if counted in the whole-body of subsequently

collected coots then equates to the EEC suggested limit for fresh meat of 0.600 Bq/g

(Potter 1987). However, the whole-body radiocesium equivalent calculated from the

combined muscle slope for both species in our study was 0.324 Bg/g. This shows that the

updated whole-body radiocesium concentration (lower in radiocesium than the past

calculation) equates to the EEC limit.

With the physical half-life of 137Cs being approximately 30 years, an estimate for

how long the isotope would be elevated above background levels in the system is

generally 5 times as long as the physical half-life (in this case ~150 years after release

before 137Cs would no longer be detectable because of normal radioactive decay

processes; Brisbin 1991a). However, because of later release of radiocesium bound in

sediments or vegetation or its disappearance from the local environment through

migration, weathering, and removal by organisms, the ecological half-life can differ from

the physical half-life. In the SRS Pond B system, our estimated ecological half-life

suggest it will be approximately 17 years (possible range between 13-24 years) before the

whole-body equivalent for radiocesium reaches a level that is under the EEC limit for

fresh meat. Thus, if the ecosystem remains stable (i.e. the bioavailability of 137Cs does not

change), and residence times remain similar for birds, it will not be until approximately

146

2030 before whole-body radiocesium fall below possible restrictive limits for human

consumption.

CONCLUSION

The risks associated with radionuclides are of ecological concern because of the

long physical half-lives of certain isotopes like radiocesium (137Cs), remaining as

environmental contaminants available for uptake by biota (Hinton 1998). Given that

radiocesium concentrates in skeletal muscle and thus may be consumed by hunters,

understanding rates of uptake and elimination of radionuclides requires basic and applied

research, through mensurative and experimental exposure studies in natural conditions, to

fully comprehend potential effects on wildlife and risks to human consumers. Research in

areas with a long history of contamination can serve as a model for areas just beginning

long-term ecological risk assessments, such as Fukushima, Japan and long-term data used

to derive ecological half-life calculations can be useful in predicting the length of time

and degree to which contaminated systems pose health or environmental risks. Additional

studies are needed to elucidate the effects of residence time on radiocesium accumulation

for a greater diversity of waterfowl/waterbirds and other wildlife known to utilize

radionuclide-contaminated sites worldwide. Collectively, the data presented herein

suggest that waterfowl/waterbirds represent a possible vector for human exposure to

radionuclides from contaminated areas, especially areas in which birds may stop-over on

migration or areas in which they over-winter for months at a time.

147

Table 4-1. Ecological half-life estimates for species from Pond B or Par Ponda on the

Savannah River Site (SRS). (For the current study American coots were restricted to

Pond B for between 33 and 173 days of exposure to radiocesium in that system.)

Species Location Ecological Half-Life

(yrs)

95% CI (yrs) Citation

American coot Pond B 16.8 12.9-24.2 Current study Largemouth bass Pond B 16.7 14.3-20.3 Paller et al. 1999, 2002

Largemouth bass Pond B 13.6 N/A Peles et al. 2000

Sunfishes Pond B 13.4 8.1-47.2 Paller et al. 1999, 2002

American coot Par Pond-North Arm 4.92 4.4-5.6 Brisbin and Kennamer 2000

American coot Par Pond-Hot Arm 3.87 3.5-4.4 Brisbin and Kennamer 2000

American coot Par Pond-West Arm 4.36 3.7-5.3 Brisbin and Kennamer 2000

Largemouth bass Par Pond 4.99 3.7-7.9 Paller et al. 1999, 2002

Sunfishes Par Pond 4.78 3.5-7.4 Paller et al. 1999, 2002 a All ecological half-life estimates for Par Pond were from periods before the 1991 drawdown of

the reservoir.

148

Table 4-2. Descriptive statistics for a random sampling of American coots and ring-necked ducks that were trapped from L-

Lake and whole-body counted for radiocesium prior to release onto Pond B on the Savannah River Site (SRS) over the winter

of 2013-2015.

Species Variable n % below MDC a Mean SE Min Max Median CV (%) American coot Bq/g; fresh mass 25 100 0.0006 0.0004 -0.0022 0.0051 0.0002 294.6

MDC; fresh mass 25 100 0.012 0.0003 0.011 0.016 0.012 11.2

Ring-necked duck Bq/g; fresh mass 7 100 -0.0014 0.0007 -0.0046 0.0001 -0.0007 -123.3 MDC; fresh mass 7 100 0.011 0.0002 0.01 0.012 0.011 4.2

a MDC=minimum detectable concentration; calculated from Currie (1968)

149

Table 4-3. Descriptive statistics for radiocesium concentrations of American coots and ring-necked ducks that were released to

Pond B on the Savannah River Site (SRS) for between 33 and 173 days of exposure before being collected.

Species Sample Variable n % below MDC a Mean SE Min Max Median CV (%)

American coot Whole-body Bq/g; fresh mass 35 0 0.941 0.064 0.19 1.85 0.885 39.9

MDC; fresh mass 35 0 0.014 0.0002 0.012 0.017 0.014 10.5

Muscle Bq/g; dry mass 35 0 6.258 0.448 1.248 11.805 5.95 42.4

Bq/g; wet mass 35 0 1.636 0.118 0.279 3.006 1.559 42.7

MDC; dry mass 35 0 0.352 0.0057 0.303 0.436 0.353 9.5

wet:dry ratio 35 0 3.814 0.043 3.423 4.53 3.756 6.7

Liver Bq/g; dry mass 35 0 3.554 0.213 0.668 5.711 3.484 35.4

Bq/g; wet mass 35 0 1.024 0.062 0.192 1.69 1.014 36

MDC; dry mass 35 0 0.318 0.0052 0.273 0.388 0.31 9.7

wet:dry ratio 35 0 3.478 0.03 3.16 3.874 3.448 5.2

Ring-necked duck Whole-body Bq/g; fresh mass 36 0 1.178 0.097 0.122 2.18 1.226 49.4

MDC; fresh mass 36 0 0.012 0.0001 0.011 0.014 0.012 4.2

Muscle Bq/g; dry mass 36 0 7.495 0.661 0.944 15.447 7.339 52.9

Bq/g; wet mass 36 0 2.238 0.195 0.28 4.55 2.282 52.4

MDC; dry mass 36 0 0.321 0.0043 0.273 0.369 0.314 8.1

wet:dry ratio 36 0 3.347 0.018 3.157 3.691 3.323 3.3

Liver Bq/g; dry mass 36 0 4.311 0.331 0.707 7.583 4.159 46

Bq/g; wet mass 36 0 1.247 0.097 0.184 2.235 1.211 46.5

MDC; dry mass 36 0 0.317 0.0048 0.257 0.368 0.316 9

wet:dry ratio 36 0 3.455 0.031 3.137 3.987 3.439 5.5 a MDC=minimum detectable concentration; calculated from Currie (1968)

150

Figure 4-1. The Par Pond Reservoir system on the Savannah River Site (SRS) that

includes P- and R-reactors with depictions of canals that carried the radionuclide

contaminated cooling water to Ponds B and C and Par Pond during several reactor

releases.

151

a.)

b.)

Figure 4-2. Historical and current data for whole-body radiocesium concentrations in

American coots from Pond B on the Savannah River Site between the winters of 1975-

1976 and 2014-2015; a.) dashed line for the whole-body equivalent (0.324 Bq/g) to the

European Economic Community limit for radiocesium in fresh meat (0.600 Bq/g) and b)

a linear regression of natural log-transformed data from collections between winters

1986-1987 and 2014-2015, estimates utilized in ecological half-life calculations.

0.0

2.0

4.0

6.0

8.0

10.0

12.0 W

hole

-bod

y 137C

s (B

q/g

fres

h m

ass)

Winter of Collections

EEC fresh meat 137

Cs limit (whole-body equivalent)

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

Log

Who

le-b

ody

137 C

s (B

q/g

fres

h m

ass)

Winter*of*Collections

α estimate: 1.84 ± 0.25 SE β estimate: -0.0412 ± 0.0064 SE R

2 = 0.31

152

a.)

b.)

Figure 4-3. Whole-body radiocesium concentrations in a.) American coots and b.) ring-

necked ducks collected from Pond B on the Savannah River Site (SRS) after exposure

between 32 and 173 days. Day 0 whole-body concentrations are counts done on live-

captured birds from L-Lake before release onto Pond B. Solid lines show non-linear fits

to the data and dashed lines represent our calculated whole-body equivalent (0.324 Bq/g)

to the European Economic Community limit for radiocesium in fresh meat (0.600 Bq/g).

0.0

0.5

1.0

1.5

2.0

2.5

0 30 60 90 120 150 180

Who

le-b

ody

137 C

s (B

q/g

fres

h m

ass)

Days post-release

EEC fresh meat 137


0.0

0.5

1.0

1.5

2.0

2.5

0 30 60 90 120 150 180

Who

le-b

ody

137 C

s (B

q/g

fres

h m

ass)

Days post-release

EEC fresh meat 137


153

a.)

b.)

Figure 4-4. Whole-body radiocesium concentrations in a.) American coots and b.) ring-

necked ducks collected from Pond B on the Savannah River Site (SRS) after exposure

between 32 and 173 days. Day 0 whole-body concentrations are counts done on live-

captured birds from L-Lake before release onto Pond B. Solid lines show non-linear fits

to the data and dashed lines represent our calculated whole-body equivalent (0.324 Bq/g)

to the European Economic Community limit for radiocesium in fresh meat (0.600 Bq/g).

0.0

0.5

1.0

1.5

2.0

2.5

0 30 60 90 120 150 180

Who

le-b

ody

137 C

s (B

q/g

fres

h m

ass)

Days post-release

EEC fresh meat 137


0.0

0.5

1.0

1.5

2.0

2.5

0 30 60 90 120 150 180

Who

le-b

ody

137 C

s (B

q/g

fres

h m

ass)

Days post-release

EEC fresh meat 137


154

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160

CHAPTER 5

CONCLUSION

To expand our knowledge of the levels of trace elements and radiocesium that

game animals accumulate from contaminated areas on the Savannah River Site (SRS),

and similarly contaminated sites worldwide, the overarching objectives of my research

were to: 1) quantify levels of contaminants in common game species from areas with a

range of levels of contaminants, 2) investigate trace element/radiocesium accumulation

rates in waterfowl/water birds inhabiting contaminated habitats, and 3) relate mensurative

and experimental contaminant burdens to levels potentially harmful to wildlife and to

derive human consumption limits for several taxa inhabiting contaminated ecosystems.

To accomplish this research I studied contaminant burdens in both lethal (muscle, liver)

and non-lethal tissues (blood, whole-body burdens) for several species with the additional

goal of developing predictive relationships to facilitate non-destructive sampling in future

studies.

In Chapter 2, I conducted a mensurative study to quantify levels of trace elements

and radiocesium in wild pigs (Sus scrofa), gray squirrels (Sciurus carolinensis), and a

variety of waterfowl/waterbird species on the SRS. Contaminant burdens for SRS wild

pigs were further compared to wild pigs collected from a non-contaminated area of

central Georgia. I found SRS pigs had significantly higher burdens of Hg, Se, and Zn,

while Georgia pigs had significantly higher Cr. SRS pigs also had significantly higher

levels of radiocesium in liver and muscle, with muscle burdens being 5 times higher in

161

SRS pigs. Evaluation of contaminant burdens in squirrels revealed there were no

differences in trace element levels among individuals collected across my four sampling

sites on the SRS, but radiocesium concentrations were significantly higher in areas with

known radiocesium contamination (Pond B and Fourmile branch beaver pond).

For diving ducks, I compared trace element tissue concentrations between D-Area

(CCW settling basin site), and all other SRS locations and found that D-Area waterfowl

had significantly higher Se (muscle and liver) and Zn (muscle) burdens, while diving

ducks from the other locations had higher Cu in liver and Cr in muscle. Radiocesium

concentrations from birds collected from Pond B, a reservoir contaminated with

radionuclides, were significantly higher than for birds collected at non-radiologically

contaminated sites (Fourmile, L-Lake, and D-Area; no differences existed between these

locations). Human consumption limits based on As, Se, and Hg burdens in muscle and

Hg EPA dose limits showed that for wild pigs and squirrels the number of allowed meals

exceeded numbers that hunters in the region generally consume. However, diving ducks

sampled from the CCW settling basins had much lower meal allowances; at average trace

element concentrations in muscle adults would be allowed 2.1 meals per month and

children 1 meal per month; while at maximum concentrations 1/3 of a meal and 1/10 of a

meal would be allowed for adults and children, respectively. No wild pigs or squirrels

exceeded the European Economic Community limit for radiocesium in fresh meat, but 19

of the 98 tested waterfowl/waterbirds did exceed the limit, all of whom were collected

from Pond B.

In Chapter 3, I conducted an experiment where I trapped ring-necked ducks

(Aythya collaris) from an uncontaminated lake and after banding, affixing colored nasal

162

saddles, and clipping their flight feathers on one wing released them into the D-Area ash

basins. I subsequently re-collected these birds between 3 to 92 days of exposure to

elucidate accumulation rates of several contaminants of concern found in CCW. I

observed a positive linear trend through time in the accumulation of Se in muscle, liver,

and blood, with the strongest relationship in muscle (R2 0.76). Hg had a significant

positive relationship with days of exposure in muscle (R2 0.14) and a significant negative

relationship with days of exposure in liver (R2 0.19). Levels of Se accumulation caused

human consumption limits to drop from 26.7 to 6.2 meals per month from the first

exposure period (3-15 days exposure) to the last (76-92 days exposure) for adults and

drop from 12.2 to 2.8 meals per month for children at average muscle concentrations. Se

in liver was correlated with As and Hg, while no correlations among the elements were

found in muscle. Se and As levels in muscle were correlated to levels in observed in liver.

Also, I was able to relate blood sample concentrations of As to concentrations in muscle

and liver, as well as Se in blood to muscle, and create equations that would allow

prediction of muscle and liver concentrations from blood contaminant concentrations.

These data suggested a non-lethal blood sample, which may be able to be added to

existing USFWS banding protocols to provide a country-wide dataset for monitoring

trace element accumulation and potential risk of exposure to hunters across the 3 main

flyways, may be able to be used to test for As in muscle and liver and Se in liver with

reasonable predictive power. It is possible a predictive equation could be created for Hg,

but in my study >50% of the blood samples were BDL, which prevented inclusion in

analyses.

163

In Chapter 4, I conducted a similar contaminant uptake experiment where I

trapped American coots (Fulica americana) and ring-necked ducks from an

uncontaminated lake, clipped flight feathers on one wing, and released them on a

reservoir contaminated with radiocesium (Pond B) to model temporal patterns in uptake

of radiocesium between 32 to 173 days of exposure. Data from this experiment

corroborate a previous finding by Brisbin et al. (1989) that coots reached equilibrium

radiocesium burdens in ≤30 days of exposure. However, contrary to previous studies that

sampled free-ranging birds with unknown residence times, ring-necked ducks continued

to accumulate radiocesium up to approximately 90 days and had significantly higher

whole-body and muscle concentrations than coots. Furthermore, I observed a gradual

decline in radiocesium whole-body burdens after approximately 90 days of exposure,

suggesting potential seasonal differences in uptake due to changes in physiology or diet.

For both species, >90% sampled individuals had levels of radiocesium in muscle tissue

that exceeded the European Economic Community (EEC) limit for human consumption

of fresh meat, suggesting birds that spend only a few weeks on Pond B may accumulate

sufficient body burdens to exceed this threshold. Through the incorporation if historical

data for coots on Pond B, I was able to estimate an ecological half-life for radiocesium in

coots on Pond B of approximately 16.8 years (range of 12.9-24.2 years), suggesting it

will not be until approximately the year 2030 before the whole-body radiocesium in

coots, on average, would be below possible restrictive limits for human consumption.

An overarching theme of these three research experiments was that some game

species, and especially waterfowl, utilizing contaminated habitats on the SRS have the

capability to accumulate levels of trace elements and radiocesium that may be deleterious

164

to their own health and may pose consumption risks to human hunters. However, my data

clearly show how variability in residence time can affect the levels of accumulated

contaminants and thus it is imperative to incorporate this parameter into ecological risk

assessments whenever possible. Furthermore, animals that have access to contaminated

locations and have large home ranges (e.g. wild pigs) or are migratory (e.g.

waterfowl/waterbirds) should have special consideration when it comes to choosing focal

species for monitoring efforts, since these may act as vectors for transmission of

pollutants to humans far from containment sources.

The data presented herein build upon our understanding of contaminant

accumulation in wildlife and provide novel data on rates of contaminant uptake for

waterfowl inhabiting contaminated ecosystems. Given the high levels of several

contaminants observed in waterfowl/waterbirds in this study, including individuals that

likely accumulated these contaminants in non-SRS habitats, my data suggest a more

widespread effort is needed to better understand levels of contaminants found in game

bird muscle tissue and the potential risks (if any) to human hunters as well as the birds

themselves. Ultimately this information can help ecological lobbyists to petition for better

regulation of contaminated areas and managers to present consumption limits to the

public for common game species, like is regularly done for fish. My research results will

hopefully support wildlife conservation and management for these species that we are

stewards of and have an obligation to protect and propagate for future generations.

exposure of game species to trace elements and …

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