accumulation of environmental contaminants in wood duck valdez veliz sánchez severiche

7/31/2019 Accumulation of Environmental Contaminants in Wood Duck Valdez Veliz Snchez Severiche

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Accumulation of Environmental Contaminants in Wood Duck(Aix sponsa) Eggs, with Emphasis on Polychlorinated

Dibenzo-p-Dioxins and Polychlorinated Dibenzofurans

T. P. Augspurger K. R. Echols P. H. Peterman T. W. May

C. E. Orazio D. E. Tillitt R. T. Di Giulio

Received: 18 February 2008 / Accepted: 5 July 2008 / Published online: 26 August 2008

US Government 2008

Abstract We measured polychlorinated dibenzo-p-diox-

ins (PCDDs), polychlorinated dibenzofurans (PCDFs),polychlorinated biphenyls (PCBs), organochlorine pesti-

cides, and mercury in wood duck (Aix sponsa) eggs

collected near a North Carolina (USA) bleached kraft paper

mill. Samples were taken a decade after the mill stopped

using molecular chlorine. Using avian toxic equivalency

factors, 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity

equivalent (TEQ) concentrations were 130 pg/g fresh wet

weight in eggs (n = 48) collected near the mill in 2002

2005 and were significantly higher than those from a ref-

erence site (\1 pg/g) 25 km away. Geometric mean wood

duck egg TEQs (6 pg/g) were one-fifth those measured at

this site prior to the cessation of molecular chlorine

bleaching. Concentrations of mercury in wood duck eggs

from nests of the Roanoke River sites ranged from 0.01 to

0.14 lg/g (geometric mean, 0.04 lg/g) and were signifi-

cantly higher than those from the reference site, where

concentrations did not exceed 0.04 lg/g (geometric mean,

0.02 lg/g). All concentrations were lower than those

associated with adverse effects in birds. The congener

profiles, lack of contamination in reference site eggs, and

decline in contaminant concentrations after process chan-

ges at the mill provide strong evidence that mill dischargesinfluenced contamination of local wood duck eggs. Col-

lectively, the results indicate that the wood duck is an

effective sentinel of the spatial and temporal extent of

PCDD, PCDF, and mercury contamination.

Introduction

The avian egg is frequently used to investigate pollutant

effects on wild birds and monitor pollutant trends in the

environment (Peakall and Boyd 1987; Furness 1993). Eggs

are an important route of chemical elimination for female

birds, particularly for highly lipophilic compounds, and

measurement of contaminants in eggs allows comparison

of maternally deposited doses to those associated with

toxicological effects in field and lab investigations. Colo-

nial waterbirds are frequent subjects of such assessments

because of their high trophic status and the ease with which

large numbers of eggs and corresponding data on produc-

tivity and health of sibling embryos can be collected. The

value of using colonial waterbird reproductive outcomes,

deformity rates, and contaminant accumulation in inte-

grated pollutant assessments has been demonstrated in the

Great Lakes (Fox et al. 1991), the North American Atlantic

and Gulf coasts (Blus 1982; Custer et al. 1983), and Europe

(Bosveld et al. 1995). When the geographic scope of

assessment precludes the use of colonial waterbirds, the

ability to induce other species to nest in artificial structures

can provide some of the same monitoring benefits by

establishing home ranges at the area of interest (to increase

the time spent foraging in that area) and increasing the

number of samples and ease of obtaining them.

T. P. Augspurger (&)

U.S. Fish and Wildlife Service, 551-F Pylon Drive, Raleigh,NC 27606, USA

e-mail: [email protected]

T. P. Augspurger R. T. Di Giulio

Integrated Toxicology Program, Nicholas School of the

Environment and Earth Sciences, Duke University, Box 90328,

Durham, NC 27708, USA

K. R. Echols P. H. Peterman T. W. May

C. E. Orazio D. E. Tillitt

U.S. Geological Survey, Columbia Environmental Research

Center, 4200 New Haven Road, Columbia, MO 65201, USA

123

Arch Environ Contam Toxicol (2008) 55:670682

DOI 10.1007/s00244-008-9199-1


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Passerines such as tree swallows (Tachycineta bicolor),

house wrens (Troglodytes aedon), and European starlings

(Sturnus vulgaris) will occupy nest boxes and are used in

pollutant monitoring (Custer et al. 2002, 2003, 2005;

Arenal et al. 2004; Echols et al. 2004; Neigh et al. 2006;

Maul et al. 2006; Papp et al. 2007). Artificial nest struc-

tures for raptors such as great horned owls (Bubo

virginianus), osprey (Pandion haliaetus), and Americankestrels (Falco sparverius) have also been used for con-

taminant assessments (Lincer and McDuffie 1974; Henny

et al. 1991; Steidl et al. 1991; Strause et al. 2007). Wood

ducks (Aix sponsa) will nest in boxes intended to mimic

natural cavities, and they have been used in wetland pol-

lutant studies (Blus et al. 1993; Beyer et al. 1997;

Kennamer et al. 2005). Wood ducks are useful models for

pollutant assessments because of their wide geographic

distribution in North America, use of artificial nest boxes,

large clutch size, and well-documented breeding biology

and foraging ecology (Hepp and Bellrose 1995). The first

use of wood ducks in polychlorinated dibenzo-p-dioxins(PCDDs) and polychlorinated dibenzofurans (PCDFs) field

studies indicated that the species was sensitive to these

pollutants (White and Hoffman 1995). That led others to

use wood ducks in field assessments of dioxin-like com-

pounds (Beeman and Augspurger 1996; Custer et al. 2002;

Williams 2004). We report contaminant accumulation and

reproductive outcomes in wood duck eggs from an eastern

North Carolina hazardous waste site.

The Roanoke River between Plymouth, North Carolina,

and Albemarle Sound is contaminated by PCDDs and

PCDFs from a pulp and paper mill. Although the mill

virtually eliminated PCDD and PCDF discharge in the

mid-1990s, these contaminants persist in sediments and

biota. Prior measurement of pollutants in wood duck eggs

downstream of this source revealed 2,3,7,8-tetrachlo-

rodibenzo-p-dioxin (TCDD) toxic equivalents (TEQs) up

to 82 pg/g fresh wet weight based on U.S. Environmental

Protection Agency (USEPA 1989) toxic equivalency fac-

tors (TEFs) (Beeman and Augspurger 1996). Because those

samples were taken prior to mill process changes, this

contemporary assessment provides the first application of

wood duck eggs in monitoring PCDD and PCDF trends

over time. Also, the current assessment includes a broader

area than that evaluated in the previous study, including

Welch Creek, a tributary to the Roanoke River which

received mill effluent from 1957 to 1987, and the Eastmost

River, a distributary channel where slower flow favors

pollutant deposition.

In addition to PCDDs and PCDFs, mercury, organo-

chlorine pesticides, and PCBs were included in the current

assessment. Mercury, from a now closed chlor-alkali plant

at the mill, is a site-related bioaccumulative pollutant

which has been implicated in avian reproductive

impairment elsewhere (Thompson 1996). Organochlorine

pesticides and PCBs have not been detected at levels of

concern in fish and sediments from the Roanoke River, but

these chemicals have not been previously measured in the

regions birds. Thus, a subset of wood duck eggs was

analyzed for organochlorine pesticides and PCBs to con-

firm the expected low concentrations.

Methods

Sample Collection

Ten to 17 nest boxes were installed at each of three loca-

tions downstream of the mill: Welch Creek, the Roanoke

River main stem, and the Eastmost River (Fig. 1). During

the breeding seasons from 2002 to 2005, nest boxes were

checked for nest initiation beginning in February and then

every 710 days. One or two fresh eggs per nest box were

collected for contaminant analyses under state and federalpermits. Nests were revisited through the breeding season

to determine clutch size (number of eggs laid, including

those removed for analyses) and percentage hatch

(excluding those removed for analyses). Eggs that failed to

Fig. 1 Location of wood duck nesting boxes on Welch Creek

(squares), Roanoke River (circles), and Eastmost River (triangles)

installed downstream from a pulp and paper mill in Plymouth, North

Carolina, USA, to monitor PCDDs and PCDFs in eggs

Arch Environ Contam Toxicol (2008) 55:670682 671

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hatch were collected for examination. Eggs were also

collected for analyses from nest boxes at a reference site,

Pocosin Lakes National Wildlife Refuge, about 25 km

southeast of the study area.

Eggs were weighed to the nearest 0.01 g and their length

and breadth were measured with calipers to the nearest

0.1 mm. Shells were gently cleaned with distilled water. A

stainless-steel scalpel, rinsed with acetone, nitric acid, anddistilled water, was used to score each eggshell at its

equator, and egg contents were collected into I-Chem

Research glass jars with Teflon-lined lids.

Analytical Chemistry

The U.S. Geological Surveys Columbia Environmental

Research Center (Columbia, MO, USA) analyzed contents

of 63 eggs for the 2,3,7,8-substituted PCDDs and PCDFs

and mercury. Nine of these samples were also analyzed for

PCB congeners and organochlorine pesticides.

A mixture of15 13C-labeled PCDDs and PCDFs surrogateswas added to all samples, including quality assurance samples

(blanks and chicken egg matrix spikes), before extraction to

monitor recoveries through the cleanup procedures. Also,

surrogates consisting of PCB 029 (2,4,5-trichlorobiphenyl),

PCB 155 (2,20,4,40,6,60-hexachlorobiphenyl), and PCB 204

(2,20,3,4,40,5,6,60-octachlorobiphenyl) were added to the

subset of samples analyzed for PCBs prior to extraction.

Similarly, p,p0-DDD-d8 was added to the subset of samples

analyzed for organochlorine pesticides prior to extraction to

gauge method recovery. Mercury recovery in samples was

assessed by the preparation and analysis of inorganic and

organic mercury spikes and mercury certified reference

tissues.

For organic compound analyses, egg contents were

dehydrated by the addition of anhydrous sodium sulfate

and extracted with dichloromethane. For analysis of

PCDDs, PCDFs, and congener specific PCBs, extracts were

subjected to acid- and base-treated silica gels and adsorbent

chromatography on activated silica gel. Extracts were

further purified by high-performance size-exclusion chro-

matography and fractionated on high-performance porous

graphitic carbon into separate fractions for analyses of 17

PCDDs and PCDFs and 1-4 ortho-chlorinated PCB

congeners.

During analyses, 13C-labeled 1,2,3,4-TCDD and

1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (HxCDD) were

added to the PCDD and PCDF extracts as instrument

internal standards. The PCDFs and PCDDs were quantified

by gas chromatography/high-resolution mass spectrometry

(GC/HRMS) on either of two systems: in 20022003, a

Hewlett Packard (Wilmington, DE, USA) 5890A GC with

a 50 m 9 200 lm 9 0.11 lm Ultra-2 (HP) capillary col-

umn interfaced to a VG 70-AS HRMS (Micromass, UK)

was used; samples from 200405 were analyzed with an

Agilent (Wilmington, DE, USA) 6890 N GC with a

30 m 9 150 lm 9 0.1 lm BPX5 (SGE, Austin, TX,

USA) capillary column interfaced to an Autospec M

HRMS (Micromass) (Peterman 2006). A calibration curve

describing the response of each native congener to that of a

labeled internal standard congener was used directly in the

calculations, and its range of values was determined in thecalibration procedure. Column performance was verified

by analyzing standards of individual components and

observing the chromatographic resolution of the TCDDs,

HxCDDs, and hexachlorodibenzofurans (HxCDFs). Simi-

larly, relative retention times for other congeners were

evaluated with respect to labeled analogues. The efficiency

of the extraction and cleanup for PCDDs and PCDFs was

measured by comparing the quantity of the labeled surro-

gates detected in the final isolated extract (at GC/HRMS

analysis) with the quantity spiked into the sample at the

beginning of extraction. The instrument internal standards

spiked into the final extract were used to calculate theamounts of the surrogates recovered. Performance was also

assessed by monitoring recoveries of a mixture of native

labeled PCDDs and PCDFs spiked into chicken eggs. Peak

confirmation required that areas for selected ion responses

be[3 times background noise, that ion peaks for native

congeners with 13C-labeled analogues occur within -1 to

+3 s of those analogues, and that the ion ratio of the two

principal ion responses be within 15%.

PCBs

Nine of the 63 samples were analyzed for PCBs. Congeners

030 and 207 were used as instrumental internal standards.

Individual PCB congeners were measured using a Hewlett-

Packard 5890 Series II GC with cool on-column capil-

lary injection systems. Analytical columns were 60 m 9

0.25 mm 9 0.25 lm DB-5 (5% phenyl-, 95% methylsili-

cone; Agilent) and DB-17 (50% phenyl-, 50% methylsili-

cone; Agilent). The temperature program began at 60C,

ramped to 150C at 15C/min, then ramped to 260C at

1C/min, and finally ramped to 300C at 10C/min for a

15-min hold. The electron capture detector (ECD) tem-

perature was 330C.

A mix of several Aroclors was used to produce sec-

ondary PCB congener calibration standards which were

previously quantified based on pure primary PCB standards

(AccuStandard, New Haven, CT, USA). The PCB cong-

eners were matched and identified on one or both columns

with known PCB peaks from Aroclor standards. Up to nine

levels of calibration for each congener were used to

quantify approximately 140 congeners in the samples. The

method detection limits (MDLs) for individual PCB

congeners (0.0042.1 ng/g) and total PCBs (21 ng/g) were

672 Arch Environ Contam Toxicol (2008) 55:670682

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calculated as the mean +3 SD of 10 or more procedural

blanks. Method quantitation limits (MQLs) for congeners

were calculated similarly as the procedural blanks

mean +10 SD.

PCB recoveries were monitored with the three surro-

gates spiked into each sample at extraction and PCB-spiked

(mixed Aroclors 1242, 1248, 1254, and 1260) chicken

eggs. The three surrogates (PCBs 029, 155, and 204) werechosen to represent more volatile early-eluting PCBs

(Cl13), midrange-eluting congeners (C146), and later-

eluting PCBs (C1710), respectively.

Organochlorine Pesticides

Organochlorine pesticides were measured in 9 of the 63

samples (including at least 1 sample from each study area)

with a Hewlett-Packard 5890 Series II GC, cool on-column

capillary injection systems, and ECD. The analytical col-

umns and temperature program were the same as described

above for PCB analyses. Six levels of standards (29 com-ponents) were used for calibration. Detection limits were

calculated as discussed for PCB congeners. Analyte

recoveries were monitored by the recoveries of surrogate

standards spiked into each sample, and either a 29-pesti-

cide-mix chicken egg matrix spike or a PCB matrix spike.

Mercury

Homogenized samples were lyophilized with a Virtis

Genesis 35EL freeze-dryer. Once dried, samples were

further homogenized by mechanical grinding with a glass

rod. Mercury was determined with a Milestone (Shelton,

CT, USA) DMA-80 direct mercury analyzer. A 48- to 58-

mg subsample of each dried egg powder sample was

combusted in a stream of oxygen. Mercury was volatilized

and trapped by amalgamation on a gold substrate and

thermally desorbed and quantitated by atomic absorption

spectrophotometry.

Statistical Analyses

Because eggs dehydrate during incubation and refrigera-

tion (Stickel et al. 1973), regression equations specific to

wood duck eggs (Hoyt 1979) were used to estimate egg

fresh weight from length and breadth measurements. This

allowed calculation of egg-specific adjustment factors for

moisture loss, and we report pollutant concentrations on a

fresh wet weight basis.

Because the PCDDs and PCDFs are structurally similar,

they produce a similar pattern of toxic responses, and their

toxicological mode of action is presumed to be similar,

TEFs have been derived to approximate the toxicity of

individual congeners relative to TCDD. We calculated

TEQs using the TEFs for birds of Van den Berg et al.

(1998).

Contaminant concentrations and TEQs are compared

among sites. Concentrations were analyzed for goodness of

fit to a normal distribution by the Shapiro-Wilk test (JMP

5.1.2; SAS Institute, Inc., Cary, NC). None were normally

distributed, and log-transformations only resulted in a

normal distribution for mercury (lack of normality in thePCDDs and PCDFs was largely due to values reported at

less than MDLs at the reference site, resulting in a left-

skewed distribution; log transformations of contaminant

data for the three Roanoke basin study sites were generally

normally distributed). Accordingly, pollutant concentra-

tions were compared among sites with the nonparametric

KruskalWallis rank sum test followed by Tukeys HSD

test for mean separation (based on rankings of concentra-

tions among all sites). Statistical significance for all

comparisons was based on p\0.05. No statistical analyses

were conducted for compounds detected in less than 50%

of the samples; these included 1,2,3,6,7,8-HxCDF,2,3,4,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 1,2,3,4,6,7,8-hep-

tachlorodibenzofuran (HpCDF), 1,2,3,4,7,8,9-HpCDF, and

octachlorodibenzofuran. For analytes present in at least

50% of samples at concentrations higher than MDLs, a

value of one-half the detection limit was substituted for

nondetects in statistical comparisons. For analytes reported

as estimated values (between the MDL and the MQL), the

estimated concentration was used.

Concentrations of TCDD, TCDF, and TEQs from this

assessment were compared to those in eggs collected from

this same area a decade earlier by the Wilcoxon rank sum

test (mercury was not assessed previously). The TEQ

comparison required that the congener data from the earlier

assessment (Beeman and Augspurger 1996) be used to

recalculate TEQs based on the TEFs used in the contem-

porary assessment (Van den Berg et al. 1998).

Clutch size (number of eggs laid, including those taken

for contaminant analyses) and percent hatch (excluding

eggs taken for analyses) were also compared among sites

with the KruskalWallis test. The sample egg technique

(Blus 1982) was used to determine the degree of correla-

tion (Spearman Rho test) between contaminant

concentrations in a single embryo from a clutch and the

corresponding clutch size and percent hatch of the sibling

eggs from the clutch. There were 40 clutches for which

contaminant and productivity data were available. Statis-

tical analyses were performed on this entire dataset as well

as a dataset culled to exclude clutch sizes of[16 eggs.

This was intended to reduce the potential influence of

dump nests (nest to which two or more females contribute

eggs to a single clutch) (Hepp and Bellrose 1995) on the

summary of productivity data. Although any nest with

more than 1 egg could potentially be a dump nest and there


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is uncertainty in the maximum number of eggs in a normal

clutch, 16 eggs is often an assumed maximum clutch size

from one hen (Bellrose and Holm 1994).

Results

Quality Assurance

Procedural blanks in PCDD and PCDF analyses indicated

no appreciable background contamination of equipment or

reagents. Percentage recoveries (51105%) of 13C-labeled

surrogate PCDDs and PCDFs spiked into all samples were

acceptable (within the quality assurance range of 25

125%). Measured concentrations of PCDDs and PCDFs in

six chicken egg matrix spikes were within 20% of the

spiked values. Four of the 55 samples were analyzed in

triplicate and results indicated very good precision, with

the relative percentage difference (RPD) averaging 40 for

TCDD and 15 for TCDF (the analytes comprising themajority of TEQs in these samples).

Blanks indicated no significant PCB contamination.

Percentage recoveries of individual PCB congeners in

matrix spikes were generally within acceptable ranges for

method accuracy for these compounds (25125%) and

were between 78 and 101% for total PCBs. Recoveries of

PCB surrogates added to all samples and matrix spikes

ranged from 69 to 100%. The RPD for total PCBs in a

sample analyzed in triplicate was 16%. Of the pesticides

detected in these samples, none were present in blanks at

quantities above MDLs. Recoveries of pesticides in matrix

spikes ranged from 56 to 104% and the RPDs for the few

analytes detected in a sample analyzed in triplicate were

between 1.5 and 55.

For mercury, the blank equivalent concentration

(0.0003 lg/g dry weight) was less than the MDL of

0.005 lg/g dry weight. Percentage recoveries of mercury

from precombustion tissue spikes ranged from 91 to 111%.

Recoveries from four reference tissue materials (NIST

RM50, NIST 8415, NRCC DORM-2, CERC Striped Bass)

averaged 108%. Precision, determined as RPD from trip-

licate combustion, amalgamation, and analysis of five

samples was B2.5.

PCDDs, PCDFs, TEQs, and Mercury

There were generally only five PCDD and PCDF analytes

above 1.0 pg/g: TCDD, TCDF, 1,2,3,6,7,8-HxCDD,

1,2,3,4,6,7,8-HpCDD, and OCDD. TCDD was detected in

all samples from the Roanoke River, Welch Creek and the

Eastmost River at concentrations between 0.1 and 4.9 pg/g

but in only 27% of eggs from the reference site where

concentrations did not exceed 0.3 pg/g. TCDF was

detected in all eggs, ranging from 0.5 to 25 pg/g at the

lower Roanoke River study sites (i.e., Welch Creek, the

Roanoke River, and Eastmost River), but B0.9 pg/g in all

reference site eggs. OCDD was the analyte detected at the

highest concentration, ranging from 0.3 to 5.9 pg/g at the

reference site and 1.8 to 90 pg/g at the lower Roanoke

River sites.

Concentrations of most analytes were significantly ele-vated at the three sites associated with the lower Roanoke

River compared to those from the wildlife refuge reference

site (Table 1). While concentrations of TCDD and TCDF

tended to be higher in the eggs from boxes on Welch

Creek, they were not significantly different from those

from areas just downstream in the Roanoke River and

Eastmost River. The TEQs from PCDDs and PCDFs were

significantly elevated at the three sites associated with the

lower Roanoke River compared to those from the reference

site. The highest concentration (30 pg/g) was from a nest

along Welch Creek. TCDD and TCDF comprised the

majority of TEQs in all samples, but their relative contri-bution differed among sites (Table 1). The proportion of

TEQs from TCDD was significantly higher in Welch Creek

than the reference site. The proportion of TEQs from

TCDF was only 30% at the reference site (where TCDF,

1,2,3,7,8-PeCDD, and 2,3,4,7,8-PeCDF at concentrations

near their detection limits contributed about equally to the

overall low TEQs), but significantly higher at 70% in the

sites downstream of the paper mill. Samples in which

TCDD was elevated also tended to have elevated TCDF

(r = 0.953, p\ 0.001, n = 48) (Table 2). For those sam-

ples with higher OCDD, 1,2,3,4,6,7,8-HpCDD was also

elevated. Figure 2 shows trends in TCDD, TCDF, and

TEQs in eggs from 19921993 to 20022005. Each con-

gener and the TEQs have declined significantly and by

about fivefold over the decade.

Concentrations of mercury in wood duck eggs from

nests on Welch Creek, the Roanoke River, and Eastmost

River ranged from 0.01 to 0.14 lg/g. They were signifi-

cantly higher (Table 1) than those from the reference site,

where concentrations did not exceed 0.04 lg/g.

PCBs and Organochlorine Pesticides

All dioxin-like mono-ortho PCBs (PCBs 105, 114, 118,

123, 156, 157, 167, and 189) were detected at very low

concentrations, between 0.01 and 3 ng/g. Total PCBs in

the nine samples (all sites combined) were also low

(\20 to 80 ng/g). Pentachlorobenzene (\0.07 to 0.24 ng/

g), a-BHC (\0.08 to 0.21 ng/g), heptachlor (\0.17 to

0.22 ng/g), heptachlor epoxide (\0.22 to 1.6 ng/g),

dieldrin (0.37 to 1.8 ng/g), endrin (\0.10 to 0.30 ng/g),

oxychlordane (\0.09 to 1.6 ng/g), cis-chlordane (\0.17

to 0.55 ng/g), trans-chlordane (\0.28 to 0.46 ng/g), cis-


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nonachlor (\0.07 to 0.32 ng/g), trans-nonachlor (0.40 to

2.1 ng/g), o,p0-DDD (\0.61 to 0.77 ng/g), o,p0-DDT

(\0.10 to 0.12 ng/g), p,p0-DDE (1.7 to 230 ng/g), p,p0-

DDD (\0.33 to 0.43 ng/g), p,p0-DDT (\0.43 to 3.7 ng/

g), endosulfan I (\0.34 to 0.55 ng/g), and mirex (\0.05

to 1.5 ng/g) were the only pesticides detected. Other

organochlorine pesticides were less than their corre-

sponding MDLs: hexachlorobenzene (\2.5 ng/g),

pentachloroanisole (\0.25 ng/g), b-BHC (\0.20 ng/g),

lindane (\0.50 ng/g), d-BHC (\0.12 ng/g), aldrin

(\0.28 ng/g), dacthal (\0.50 ng/g), o,p0-DDE (\0.17 ng/

g), endosulfan II (\0.16 ng/g), endosulfan sulfate

(\0.95 ng/g), and methoxychlor (\2.0 ng/g). Concentra-

tions of pesticides were generally similar between the

Roanoke study sites and the reference site with the

exception of p,p0-DDE at the Roanoke sites (geometric

mean, 24 ng/g), which was about eight times higher than

at the reference site.

Table 1 Dioxin, furan, avian TCDD toxic equivalent, and mercury concentrations in wood duck eggs from eastern North Carolina, 20022005

Chemical p-valuea Welch

Creek

(n = 16)

Roanoke

River

(n = 18)

Eastmost

River

(n = 14)

PLNWR

(ref. site;

n = 15)

2,3,7,8-TCDD \0.0001 0.8 Ab 0.5 A 0.8 A \0.2 B

0.51.3 0.30.9 0.51.1 11 (ND)c

1,2,3,7,8-PeCDD 0.0056 0.3 A 0.3 A 0.3 A\

0.2 B0.20.5 0.20.4 0.20.5 10 (ND)

1,2,3,4,7,8-HxCDD 0.0020 0.3 A 0.3 A 0.3 A \0.2 B

0.20.4 0.20.4 0.20.5 9 (ND)

1,2,3,6,7,8-HxCDD \0.0001 0.5 A 0.6 A 0.7 A \0.2 B

0.30.8 0.50.8 0.41.0 9 (ND)

1,2,3,7,8,9-HxCDD 0.0440 0.3 A 0.3 AB 0.3 A \0.2 B

0.20.5 0.20.4 0.20.5 9 (ND)

1,2,3,4,6,7,8-HpCDD \0.0001 2.4 A 2.8 A 2.8 A 0.3 B

1.53.8 1.94.0 1.74.7 0.20.5

OCDD \0.0001 10 A 13 A 15 A 1.4 B

5.517 8.521 9.623 0.9 2.2

2,3,7,8-TCDF \0.0001 4.1 A 3.3 A 4.3 A 0.4 B

2.56.7 2.05.5 2.76.8 0.30.4

1,2,3,7,8-PeCDF 0.2812 0.2 0.2 0.2 \0.2

0.10.3 0.10.2 0.10.3 11 (ND)

2,3,4,7,8-PeCDF \0.0004 0.3 A 0.2 AB 0.4 A 0.2 B

0.20.4 0.20.3 0.30.5 0.10.3

1,2,3,4,7,8-HxCDF 0.6704 0.2 0.2 \0.2 \0.2

0.10.2 0.10.2 8 (ND) 11 (ND)

TEQs \0.0001 6 A 5 A 6 A 1 B

49 37 49 11

TCDD/TEQ (%) 0.0223 13 A 11 AB 12 AB 10 B

TCDF/TEQ (%) \0.0001 66 A 68 A 68 A 30 B

Mercury 0.0033 0.04 A 0.04 A 0.04 A 0.02 B

0.020.06 0.020.05 0.030.05 0.010.03

Note: Welch Creek, Roanoke River, and Eastmost River are downstream of a paper mill. Pocosin Lakes National Wildlife Refuge (PLNWR) is a

reference site. Dioxin and furan concentrations (geometric mean and 95% confidence intervals) are picograms per gram fresh wet weight, and

mercury concentration is micrograms per gram fresh wet weight. Other 2,3,7,8-substituted dioxins and furans were not detected in more than half

the samples. TCDD, tetrachlorodibenzo-p-dioxin; PeCDD, pentachlorodibenzo-p-dioxin; HxCDD, hexachlorodibenzo-p-dioxin; HpCDD, hep-

tachlorodibenzo-p-dioxin; OCDD, octachlorodibenzo-p-dioxin; TCDF, tetrachlorodibenzofuran; PeCDF, pentachlorodibenzofuran; HxCDF,

hexachlorodibenzofuran; TEQ, TCDD toxic equivalent concentrations, using avian toxic equivalency factors of Van den Berg et al. (1998)a KruskalWallis test (df = 3, a = 0.05)b Means followed by the same letter capital letter are not significantly differentc

Number of samples in which analyte was not detected


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Table2

CorrelationmatrixofdioxinandfurancongenersandWorldHealth

OrganizationavianTCDDtoxicequivalen

tconcentrationsinwoodduckeggsfromthelowerRoanokeRiver,

NorthCarolina,20022005

Chemical

1,2,3,7,8-

PeCDD

1,2,3,4,7,8-

HxCDD

1,2,3,6,7,8-

HxCDD

1,2,3,7,8,9-

HxCD

D

1,2,3,4,6,7,8-

HpCDD

OCDD

2,3,7,8-

TCDF

1,2,3,7,8-

PeCDF

2,3,4,7,8-

PeCDF

1,2,3,4,7,8-

HxCDF

TEQs

2,3,7,8-TCDD

0.174

-0.013

0.066

0.09

9

0.095

0.026

0.953

0.013

0.036

-0.038

0.970

p

0.238

0.930

0.653

0.50

4

0.521

0.860

\0.001

0.928

0.810

0.797

\0.001

1,2,3,7,8PeCDD

0.657

0.601

0.69

6

0.240

0.110

0.069

0.688

0.581

0.655

0.158

p

\0.001

\0.001

\0.00

1

0.100

0.454

0.642

\0.001

\0.001

\0.001

0.283

1,2,3,4,7,8-HxCDD

0.843

0.79

2

0.707

0.546

-0.104

0.337

0.372

0.385

-0.038

p

\0.001

\0.00

1

\0.001

\0.001

0.483

0.019

0.009

0.007

0.798

1,2,3,6,7,8-HxCDD

0.65

8

0.741

0.502

0.008

0.086

0.211

0.173

0.056

p

\0.00

1

\0.001

\0.001

0.958

0.559

0.150

0.240

0.706

1,2,3,7,8,9-HxCDD

0.668

0.529

-0.044

0.517

0.395

0.525

0.035

p

\0.001

\0.001

0.765

\0.001

0.005

\0.001

0.814

1,2,3,4,6,7,8-HpCDD

0.872

0.014

-0.184

0.002

-0.122

0.041

p

\0.001

0.925

0.210

0.989

0.408

0.780

OCDD

-0.064

-0.196

0.127

-0.188

-0.037

p

0.663

0.183

0.389

0.200

0.801

2,3,7,8-TCDF

-0.058

-0.004

-0.092

0.994

p

0.698

0.981

0.536

\0.001

1,2,3,7,8-PeCDF

0.636

0.897

0.019

p

\0.001

\0.001

0.899

2,3,4,7,8-PeCDF

0.600

0.072

p

\0.001

0.628

1,2,3,4,7,8-HxCDF

-0.021

p

0.886

Note:TCDD,tetrachlorodibenzo-p-dioxin;PeCDD,pentachlorodibenzo-p-diox

in;HxCDD,hexachlorodibenzo-p-dioxin;HpCDD,heptachlorodibenzo-p-dioxin;OCDD,octachlorodibenzo-p-

dioxin;TCDF,tetrachlorodibenzof

uran;PeCDF,pentachlorodibenzofuran;H

xCDF,hexachlorodibenzofuran;TEQ,TCDDtoxicequivalentconcentrations,using

aviantoxicequivalency

factorsofVandenBergetal.(1998).Valuesarecorrelationcoefficientsandp-values;allcomparisonsareforn=48.Other2,3,7,8-substitutedcongenerswerenotd

etectedinmorethanhalf

thesamples


123


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Productivity

Wood duck productivity data indicate high hatching suc-cess at all sites (Table 3). Clutch sizes were significantly

larger in Roanoke River nests when data for all nests are

evaluated; this difference is less apparent when data from

nests with clutch sizes[16 at all sites are excluded from

the analyses. One malformation was noted upon opening

unhatched eggs from these sites. A single embryo from nest

box 1 (in 2002) on the Roanoke River had exencephaly

(skull defective, causing exposure or extrusion of the

brain).

Spearman rank correlations indicated no significant

association between hatching success and either TCDD,

TCDF, or TEQs. When data for all nests at the three lowerRoanoke River sites (n = 40) were evaluated, weak but

significant negative correlations were determined for the

association between clutch size and each of these param-

eters (Spearman q and p-value: clutch size and TCDD =

-0.396 and 0.012; clutch size and TCDF = -0.435 and

0.005; and clutch size and TEQ = -0.414 and 0.008).

When data from nests with more than 16 eggs (an assumed

maximum clutch size from one hen) are excluded, none of

the correlations among clutch size and contaminants are

significant (although the Spearman q of -0.373 between

clutch size and TCDF in the culled dataset would be sig-

nificant at a = 0.06). The association is driven by two

values which had among the highest TEQs and lowest

clutch sizes (a sample from a clutch of 10 eggs from aWelch Creek nest in 2002 with a corresponding TEQ of

30 pg/g and a sample from a clutch of 8 eggs from a

Roanoke River nest in 2002 with a corresponding TEQ of

28 pg/g).

Discussion

Wood Ducks as Sentinels of PCDD/PCDF Status

and Trends

Three aspects of these results support the wood ducksutility in coplanar halogenated aromatic hydrocarbon

monitoring: (1) the significantly greater PCDD, PCDF, and

TEQ accumulation in eggs from the study areas compared

to those from the reference site, (2) the significantly lower

PCDD and PCDF concentrations in eggs collected during

20022005 compared to 19921993, and (3) the congener

profiles from all sites and all years.

Wood duck eggs from nests along Welch Creek, the

lower Roanoke River, and the Eastmost River were

0

10

20

30

40

50

60

70

80

90

100

TCDD

p=0.0016

TCDF

p=0.0005

TEQ

p=0.0005

pg/gfreshwetweight

1992-93 2002-05

Fig. 2 Trends in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-

tetrachlorodibenzofuran (TCDF), and TCDD-equivalent (TEQ) concen-

trations in wood duck eggs from nest boxes along the lower Roanoke

River, near Plymouth, North Carolina (geometric mean and upper 95%

confidence interval; n = 10 in 19921993 and n = 48 in 20022005).

Interyear comparisons by Wilcoxon rank sum test

Table 3 Productivity of wood ducks nesting in boxes erected along

the lower Roanoke River basin, 20022003

Dataseta p-valueb Welch

Creek

Roanoke

River

Eastmost

River

All nests (n = 8) (n = 18) (n = 14)

Clutch size (n) 0.031 10 ABc

16 A 10 B

522 1418 616Hatching success (%) 0.068 95 79 85

88100 5886 71100

Nests with B16 eggs (n = 7) (n = 9) (n = 10)

Clutch size (n) 0.273 9 13 8

423 1215 416

Hatching success (%) 0.250 100 75 91

89100 2593 52100

Note: Clutch sizes are geometric mean and 95% confidence intervals,

and percentage hatch data are median and interquartile range. Only

active nests (those with one or more eggs) were included in these

calculationsa

All nests: all 40 clutches for which contaminant and productivity

data were available. Nests with B16 eggs: subset of all nests, culled to

exclude results from nests with a clutch size[16 eggs (an assumed

maximum clutch size from one hen)b

KruskalWallis test (df = 2, a = 0.05)c Means followed by the same capital letter are not significantly

different


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contaminated with PCDDs and PCDFs. On a TEQ basis,

concentrations in these areas significantly exceed those of a

reference site just 25 km distant. Geometric mean TEQs in

the combined lower Roanoke River sites (Welch Creek, the

lower Roanoke River, and the Eastmost River) were six

times those of the reference site. Wood ducks are migratory

(and some are resident in the coastal Carolinas), but our

results indicate that time spent on breeding sites is suffi-cient to reflect local contamination. Female wood ducks

gain fat reserves for reproduction at the nesting site instead

of prior to migrating (Drobney 1982). Although wood

ducks are omnivorous and mainly eat vegetation, they rely

on sediment-emergent insects prior to and during egg

laying (Hepp and Bellrose 1995), increasing their suscep-

tibility to accumulating sediment-associated contaminants.

Sediment ingestion is another important route of exposure

for wood ducks at contaminated sites (Beyer et al. 1997).

Yolk formation in wood ducks takes place over a 7-day

period, followed by an approximately 24-h period of

albumin and eggshell deposition prior to egg laying(Drobney 1980). Accordingly, residues in eggs will largely

reflect those accumulated at nesting areas, as evident in this

study and others (Kennamer et al. 2005).

Wood duck eggs collected from this area in 19921993

allow a comparison to current concentrations and the first

demonstration of this species in monitoring changes in

PCDD and PCDF contamination through time. When

expressed as TEQs with the avian TEFs of Van den Berg

et al. (1998), wood duck eggs collected from lower Roa-

noke River nests in 19921993 (Beeman and Augspurger

1996) ranged from 2 to 500 pg/g. The TEQs in wood duck

eggs analyzed in 20022005 from the same area ranged

from 1 to 30 pg/g, and geometric mean concentrations of

TCDD, TCDF, and TEQs declined about four-, six-, and

fivefold respectively, over the period. About 2 years after

the 19921993 egg collections, the paper mill upstream of

the wood duck nest boxes virtually eliminated PCDD and

PCDF discharge by switching to chlorine dioxide bleaching

from molecular chlorine. Since then, concentrations of

TCDD and TCDF have declined in fish from the lower

Roanoke River. Geometric mean concentrations of TCDD

and TCDF in three whole creek chubsuckers (Erimyzon

oblongus) collected from the Roanoke in 1987 were 152

and 199 pg/g, respectively (USEPA 1992). Monitoring of

TCDD and TCDF in channel catfish (Ictalurus punctatus)

fillets from the Roanoke River downstream of the mill

shows a decline in TEQs (using a TEF of 0.1 for TCDF)

from 19891991 (when TEQs in fillets averaged about

28 pg/g) to 20022004 (when TEQs in fillets averaged

about 4 pg/g) (RMT 2005).

As documented for wood ducks here, a decline of

PCDDs and PCDFs in eggs following cessation of paper

mill bleaching with molecular chlorine has been reported

for birds in the western United States and Canada (Elliott

et al. 2001; Harris et al. 2003). Declines in concentrations

of dioxin-like compounds were evident in osprey and great

blue heron (Ardea herodias) eggs following mill process

changes that reduced PCDD and PCDF discharges (San-

derson et al. 1994; Elliott et al. 1998, 2001). Large

numbers of wood duck boxes can be accessed with less

effort than is required to obtain eggs of these other species.The proportion of wood duck egg TEQs attributed to

TCDF also declined significantly over the decade, from a

geometric mean of 82% (95% confidence interval, 73

92%) in 19921993 to 68% (95% confidence interval, 63

74%) in 20022005. Congener profiles are still dominated

by TCDF and TCDD, two compounds associated with

bleached kraft paper mills in the contamination of avian

eggs elsewhere (Elliott et al. 2001; Harris et al. 2003).

Congener profiles and interrelationships (Table 2) were

informative in assessing PCDD and PCDF sources;

although the dominant source of local PCDDs and PCDFs

in the Roanoke River is well-known, principal componentanalyses of congener profiles (Choi et al. 2001) and dioxin

fingerprinting could be employed to the residues measured

in wood duck eggs to aid in source identification. Wood

duck eggs in this assessment had detectable levels of 11 of

the 17 2,3,7,8-substituted PCDDs and PCDFs in more than

50% of the samples, providing robust data for fingerprint-

ing applications.

The congener profiles, lack of contamination in refer-

ence site eggs, and decline in local concentrations

following a switch from molecular chlorine to chlorine

dioxide bleaching at the mill provide strong evidence that

lower Roanoke River wood duck egg contamination

reflects pulp and paper mill impacts. These observations

also support using wood duck eggs for monitoring PCDD

and PCDF status and trends.

PCDD and PCDF Data in Context

Table 4 provides additional context on eastern North Car-

olina wood duck egg PCDD and PCDF concentrations.

While concentrations in the 19921993 collections were

among the highest recorded for this species, concentrations

are now in the midrange of those reported by others.

There was no definitive evidence of adverse impacts

from current PCDD and PCDF contamination. One

unhatched embryo with exencephaly was observed in 2002.

The same malformation was noted in an embryo from the

19921993 assessment which was also a conjoined twin

(Beeman and Augspurger 1996). Wood duck productivity,

as measured by clutch size and percentage hatch, was high

compared to other sites (Hepp and Bellrose 1995); the

percentage hatch had a wide range in Welch Creek, the

Roanoke River, and Eastmost River (0 to 100), but the


123


10/14

median and average percentage hatch for these areas is

normal (Hepp and Bellrose 1995). Mercury, TCDD, TCDF,

and TEQs were not significantly correlated with percentage

hatch. There were significant but weak inverse relation-ships between clutch size and TCDD, TCDF, and TEQs

when all data (n = 40) were included. Smaller clutch size

has been shown to be an experimentally reproducible effect

of dioxin-like compounds in multigeneration American

kestrel studies (Fernie et al. 2001), but the relationships

observed here had a low explanatory power (Spearmans

q * -0.4) and were not significant when the analysis was

restricted to clutch sizes B16 eggs (an assumed normal

clutch size from one female). This is evidence that the

correlations may be an artifact of supranormal clutch sizes

(likely from more than one female). However, it is noted

that two nests with the greatest TEQs had among the lowest

clutch sizes (a clutch of 10 eggs with a corresponding TEQ

in a sibling embryo of 30 pg/g and a clutch of 8 eggs with a

corresponding TEQ of 28 pg/g).

The absent or weak relationships between pollutants and

productivity in wood duck eggs from this site in 2002

2005 are not unexpected, given the decline in contaminant

concentrations. The maximum TEQ concentrations mea-

sured in 20022005 are two orders of magnitude lower

than those which did not adversely affect wood duck

embryos following preincubation egg injection of TCDD

(Augspurger et al. 2008). The 6 pg/g geometric mean TEQ

concentration in wood duck eggs from nests downstream of

the mill is less than known adverse effect levels for all butthe most sensitive avian species, the domestic chicken, for

which adverse effects begin near 10 pg/g (USEPA 2003).

Further, only two of the eggs from our 20022005

assessment exceed a 2050 pg/g reproductive effects

threshold for TEQs and wood ducks (White and Hoffman

1995). Because White and Hoffmans (1995) assessment

used a TEF of 0.1 for TCDF (which was generally present

at concentrations similar to those of TCDD), their reported

effects threshold may be an underestimate. Even so, 46 of

48 eggs collected in 20022005 from the Roanoke River

basin had TEQ concentrations lower than this estimate.

Recent molecular characterization of the ligand binding

domain of the aryl hydrocarbon receptor in wood ducks

further indicates that the species would not be responsive to

the concentrations we encountered (Head 2006).

Field no-effect concentrations for other species are also

well above the geometric mean TEQ for wood duck eggs

from the Roanoke River basin in 20022005. Woodford

et al. (1998) found no effect of dioxins and furans on

hatching success of ospreys nesting downstream of Wis-

consin River pulp and paper mills. They estimated a no-

Table 4 Geometric mean and range of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and TCDD toxic

equivalents (TEQs) in wood duck eggs from this assessment and others

Site n Year(s) TCDD TCDF TEQs Reference

Roanoke River, NC 48 20022005 0.7 3.8 6 This study

Range 0.14.9 0.525 130

Roanoke River, NCa

10 19921993 2.9 24 30 Beeman and Augspurger (1996)

Range 0.431 1.1460 2500Pocosin Lakes National Wildlife Refuge, NC 15 20022003 \0.2 0.4 1 This study

Range \0.20.3 0.20.9 0.84

Bayou Meto, AR

Area Ia

17 19881989 36 23 59 White and Hoffman (1995)

Range 2480 2240 4720

Area IV 12 19881989 \1 1 2 White and Hoffman (1995)

Range NDb

\13 \14

Wisconsin River, WI 6 19951996 \2 1.1 18 Custer et al. (2002)

Range ND \16 343

Shiawassee National Wildlife Refuge, MI 5 2003 c

140 Williams (2004)

Range 57246

Rose Lake Game Area, MI 5 2003 2 Williams (2004)

Range 1.13.7

Note: All concentrations are picograms per gram wet weight. TEQs were calculated with toxic equivalency factors for birds from Van den Berg

et al. (1998)a TEQs recalculated from datasheets provided by study authorb

Analyte not detectedc No data on individual analytes


123


11/14

effect level in eggs of 136 pg/g TEQs (using the TEFs of

Safe 1990). In a retrospective analysis, Elliott et al. (2001)

reported negative correlations between great blue heron

chick growth and TEQs at about 360 pg/g. Concentrations

corresponding to observed health effects in chicks upon

hatching were thought not to have impaired hatching suc-

cess (Elliott et al. 1989, 2001). Field studies of tree

swallows at a PCB- and dioxin-contaminated site indicatedthat the concentration at which reproductive failure became

common was 180 pg/g TCDD (Custer et al. 2005).

Other pollutants were present at levels below those

associated with adverse effects to birds. Mercury concen-

trations were significantly higher in eggs from the nests of

the Roanoke River basin sites compared to the reference

site. Mercury is a contaminant of concern in this area due

to historic releases from a now closed chlor-alkali plant at

the mill, and the higher concentrations of mercury in wood

duck eggs downstream of the mill may be indicative of

local contamination. While higher than reference site

concentrations, the maximum mercury concentration of0.14 lg/g fresh wet weight from a nest box on Welch

Creek is well below adverse-effect thresholds. Heinz

(1979) reported abnormal egg-laying and lower produc-

tivity in female mallards fed 0.5 lg/g methylmercury; eggs

of these females contained 0.790.86 lg/g mercury. In

more recent dietary mercury studies with mallards, a wide

range of mercury burdens was associated with normal

hatching and embryo mortality with adversely affected

eggs estimated to contain as little as 0.74 lg/g mercury

(Heinz and Hoffman 2003). In a review of the mallard data

and other avian toxicological data, Thompson (1996)

indicated that mercury concentrations in eggs between 0.5

and 2.0 lg/g were associated with reproductive

impairment.

Only nine samples were analyzed for PCBs which are

low in other media for the lower Roanoke River. Based on

the low total and mono-ortho-PCBs, we infer that non-

ortho-PCBs were also low and not important contributors,

relative to PCDDs and PCDFs, to the TEQs at this mill site.

Organochlorine pesticide concentrations were also low.

Summary

We documented highly significant differences in spatial

and temporal PCDD and PCDF accumulation which

illustrate the utility of the wood duck in measuring PCDD

and PCDF status and trends. While useful for monitoring,

interpretation of contaminant residues in wood duck eggs

should be considered in context of their trophic status.

Omnivorous birds such as the wood duck typically have

PCDD, PCDF, and PCB concentrations intermediate to

those of herbivorous and piscivorous species, in adult tis-

sues (Senthilkumar et al. 2002; Braune and Malone 2006)

and eggs (Custer et al. 2002). Wood duck contaminant

burdens are likely only representative of other omnivorous

species at a contaminated site, with higher concentrations

expected in insectivorous and piscivorous species.

The strengths and weaknesses of using wood ducks in

avian PCDD/PCDF assessments should be considered

when selecting sentinel species at other sites. The growing

body of data on successful use of tree swallows in avianassessments is noteworthy. Tree swallow eggs can accu-

mulate marked concentrations of dioxin-like compounds;

TCDD concentrations exceeding 1000 pg/g (Custer et al.

2005) and TEQs (based on the TEFs of Van den Berg et al.

1998) up to 25,400 pg/g (Secord et al. 1999) are docu-

mented. Concentrations of PCBs and TEQs from PCBs,

PCDDs, and PCDFs were an order of magnitude higher in

eggs of tree swallows than those of wood ducks in a con-

current assessment of those species (Custer et al. 2002).

Site-to-site differences in accumulation and the ability to

measure concentrations in eggs and sibling chicks in this

altricial species allow estimates of contaminant uptakefrom local forage (Custer et al. 2003, 2005; Maul et al.

2006). These data facilitated development of PCB bioac-

cumulation models for tree swallows (Echols et al. 2004).

Also, dioxin-like compounds have been implicated in

adverse effects on tree swallows in the field (McCarty and

Secord 1999a, b; Custer et al. 2003, 2005; Martinovic et al.

2003). Wood ducks remain a useful option. In our study,

the greater PCDD and PCDF contamination of Roanoke

River basin wood duck eggs compared to a nearby refer-

ence site, the decline of PCDDs and PCDFs following

cessation of molecular chlorine bleaching, and the domi-

nance of TCDD and TCDF in congener profiles provides

strong evidence that contamination of lower Roanoke

River wood duck eggs reflects pulp and paper mill impacts.

Acknowledgments Assistance in the lab and field was provided by

Mike Tanner, Sara Ward, Jean Richter, Wendy Stanton, and Michelle

Chappel. Partial funding was provided through USFWSs Environ-

mental Contaminants Program (Study ID No. 200240001).

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