Arch Environ Contam Toxicol (2008) 55:670682 DOI 10.

1007/s00244-008-9199-1

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-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), organochlorine pesticides, 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 signicantly higher than those from a reference site (\1 pg/g) 25 km away. Geometric mean wood duck egg TEQs (6 pg/g) were one-fth 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 signicantly 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 proles, lack of contamination in reference site eggs, and
T. P. Augspurger (&) U.S. Fish and Wildlife Service, 551-F Pylon Drive, Raleigh, NC 27606, USA e-mail: tom_augspurger@fws.gov 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

decline in contaminant concentrations after process changes at the mill provide strong evidence that mill discharges inuenced contamination of local wood duck eggs. Collectively, 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 eld and lab investigations. Colonial 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 productivity and health of sibling embryos can be collected. The value of using colonial waterbird reproductive outcomes, deformity rates, and contaminant accumulation in integrated 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 articial structures can provide some of the same monitoring benets 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.

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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). Articial nest structures for raptors such as great horned owls (Bubo virginianus), osprey (Pandion haliaetus), and American kestrels (Falco sparverius) have also been used for contaminant assessments (Lincer and McDufe 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 pollutant 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 articial nest boxes, large clutch size, and well-documented breeding biology and foraging ecology (Hepp and Bellrose 1995). The rst use of wood ducks in polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) eld studies indicated that the species was sensitive to these pollutants (White and Hoffman 1995). That led others to use wood ducks in eld assessments of dioxin-like compounds (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-tetrachlorodibenzo-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 factors (TEFs) (Beeman and Augspurger 1996). Because those samples were taken prior to mill process changes, this contemporary assessment provides the rst 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 efuent from 1957 to 1987, and the Eastmost River, a distributary channel where slower ow favors pollutant deposition. In addition to PCDDs and PCDFs, mercury, organochlorine 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 sh 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 conrm the expected low concentrations.

Methods Sample Collection Ten to 17 nest boxes were installed at each of three locations 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 federal permits. 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

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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, and distilled water, was used to score each eggshell at its equator, and egg contents were collected into I-Chem Research glass jars with Teon-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 of 15 13C-labeled PCDDs and PCDFs surrogates was 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 certied 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 specic PCBs, extracts were subjected to acid- and base-treated silica gels and adsorbent chromatography on activated silica gel. Extracts were further puried by high-performance size-exclusion chromatography 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 quantied 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 column 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 the calibration procedure. Column performance was veried by analyzing standards of individual components and observing the chromatographic resolution of the TCDDs, HxCDDs, and hexachlorodibenzofurans (HxCDFs). Similarly, relative retention times for other congeners were evaluated with respect to labeled analogues. The efciency of the extraction and cleanup for PCDDs and PCDFs was measured by comparing the quantity of the labeled surrogates detected in the nal 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 nal extract were used to calculate the amounts 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 conrmation 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 HewlettPackard 5890 Series II GC with cool on-column capillary injection systems. Analytical columns were 60 m 9 0.25 mm 9 0.25 lm DB-5 (5% phenyl-, 95% methylsilicone; Agilent) and DB-17 (50% phenyl-, 50% methylsilicone; Agilent). The temperature program began at 60C, ramped to 150C at 15C/min, then ramped to 260C at 1C/min, and nally ramped to 300C at 10C/min for a 15-min hold. The electron capture detector (ECD) temperature was 330C. A mix of several Aroclors was used to produce secondary PCB congener calibration standards which were previously quantied based on pure primary PCB standards (AccuStandard, New Haven, CT, USA). The PCB congeners were matched and identied 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

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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 surrogates 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) were chosen to represent more volatile early-eluting PCBs (Cl13), midrange-eluting congeners (C146), and latereluting 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 columns and temperature program were the same as described above for PCB analyses. Six levels of standards (29 components) 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-pesticide-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 58mg 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 refrigeration (Stickel et al. 1973), regression equations specic to wood duck eggs (Hoyt 1979) were used to estimate egg fresh weight from length and breadth measurements. This allowed calculation of egg-specic 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 t 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 the PCDDs and PCDFs was largely due to values reported at less than MDLs at the reference site, resulting in a leftskewed distribution; log transformations of contaminant data for the three Roanoke basin study sites were generally normally distributed). Accordingly, pollutant concentrations were compared among sites with the nonparametric KruskalWallis rank sum test followed by Tukeys HSD test for mean separation (based on rankings of concentrations among all sites). Statistical signicance 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-heptachlorodibenzofuran (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 contemporary 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 correlation (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. Statistical 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 inuence 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 the majority of TEQs in these samples). Blanks indicated no signicant 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 triplicate combustion, amalgamation, and analysis of ve samples was B2.5. PCDDs, PCDFs, TEQs, and Mercury There were generally only ve 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 signicantly elevated 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 signicantly different from those from areas just downstream in the Roanoke River and Eastmost River. The TEQs from PCDDs and PCDFs were signicantly 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 contribution differed among sites (Table 1). The proportion of TEQs from TCDD was signicantly 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 signicantly 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 samples 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 congener and the TEQs have declined signicantly and by about vefold 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 signicantly 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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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) 0.8 Ab 0.51.3 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-HxCDD 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 TCDD/TEQ (%) TCDF/TEQ (%) Mercury 0.0056 0.0020 \0.0001 0.0440 \0.0001 \0.0001 \0.0001 0.2812 \0.0004 0.6704 \0.0001 0.0223 \0.0001 0.0033 0.3 A 0.20.5 0.3 A 0.20.4 0.5 A 0.30.8 0.3 A 0.20.5 2.4 A 1.53.8 10 A 5.517 4.1 A 2.56.7 0.2 0.10.3 0.3 A 0.20.4 0.2 0.10.2 6A 49 13 A 66 A 0.04 A 0.020.06 Roanoke River (n = 18) 0.5 A 0.30.9 0.3 A 0.20.4 0.3 A 0.20.4 0.6 A 0.50.8 0.3 AB 0.20.4 2.8 A 1.94.0 13 A 8.521 3.3 A 2.05.5 0.2 0.10.2 0.2 AB 0.20.3 0.2 0.10.2 5A 37 11 AB 68 A 0.04 A 0.020.05 Eastmost River (n = 14) 0.8 A 0.51.1 0.3 A 0.20.5 0.3 A 0.20.5 0.7 A 0.41.0 0.3 A 0.20.5 2.8 A 1.74.7 15 A 9.623 4.3 A 2.76.8 0.2 0.10.3 0.4 A 0.30.5 \0.2 8 (ND) 6A 49 12 AB 68 A 0.04 A 0.030.05 PLNWR (ref. site; n = 15) \0.2 B 11 (ND)c \0.2 B 10 (ND) \0.2 B 9 (ND) \0.2 B 9 (ND) \0.2 B 9 (ND) 0.3 B 0.20.5 1.4 B 0.9 2.2 0.4 B 0.30.4 \0.2 11 (ND) 0.2 B 0.10.3 \0.2 11 (ND) 1B 11 10 B 30 B 0.02 B 0.010.03

2,3,7,8-TCDD

\0.0001

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% condence 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, heptachlorodibenzo-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 b c

KruskalWallis test (df = 3, a = 0.05) Means followed by the same letter capital letter are not signicantly different Number of samples in which analyte was not detected

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 corresponding 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). Concentrations 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.

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1,2,3,4,7,8HxCDD -0.013 0.930 0.657 \0.001 0.100 0.707 \0.001 0.483 0.008 0.958 -0.044 0.765 0.014 0.925 -0.064 0.663 0.086 0.559 0.517 \0.001 -0.184 0.210 -0.196 0.183 -0.058 0.698 0.019 0.741 \0.001 0.668 \0.001 0.872 \0.001 \0.001 0.529 \0.001 0.502 \0.001 0.546 -0.104 0.337 0.454 0.642 0.843 \0.001 0.658 \0.001 \0.001 0.792 \0.001 \0.001 \0.001 0.601 0.696 0.240 0.110 0.069 0.688 0.653 0.504 0.521 0.860 \0.001 0.928 0.810 0.581 \0.001 0.372 0.009 0.211 0.150 0.395 0.005 0.002 0.989 0.127 0.389 -0.004 0.981 0.636 \0.001 0.066 0.099 0.095 0.026 0.953 0.013 0.036 1,2,3,6,7,8HxCDD 1,2,3,7,8,9HxCDD 1,2,3,4,6,7,8HpCDD OCDD 2,3,7,8TCDF 1,2,3,7,8PeCDF 2,3,4,7,8PeCDF 1,2,3,4,7,8HxCDF -0.038 0.797 0.655 \0.001 0.385 0.007 0.173 0.240 0.525 \0.001 -0.122 0.408 -0.188 0.200 -0.092 0.536 0.897 \0.001 0.600 \0.001 TEQs 0.970 \0.001 0.158 0.283 -0.038 0.798 0.056 0.706 0.035 0.814 0.041 0.780 -0.037 0.801 0.994 \0.001 0.019 0.899 0.072 0.628 -0.021 0.886

Table 2 Correlation matrix of dioxin and furan congeners and World Health Organization avian TCDD toxic equivalent concentrations in wood duck eggs from the lower Roanoke River, North Carolina, 20022005

Chemical

1,2,3,7,8PeCDD

2,3,7,8-TCDD

0.174

0.238

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-HxCDD

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

p 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

Arch Environ Contam Toxicol (2008) 55:670682

Note: TCDD, tetrachlorodibenzo-p-dioxin; PeCDD, pentachlorodibenzo-p-dioxin; HxCDD, hexachlorodibenzo-p-dioxin; HpCDD, heptachlorodibenzo-p-dioxin; OCDD, octachlorodibenzo-pdioxin; TCDF, tetrachlorodibenzofuran; PeCDF, pentachlorodibenzofuran; HxCDF, hexachlorodibenzofuran; TEQ, TCDD toxic equivalent concentrations, using avian toxic equivalency factors of Van den Berg et al. (1998). Values are correlation coefcients and p-values; all comparisons are for n = 48. Other 2,3,7,8-substituted congeners were not detected in more than half the samples

Arch Environ Contam Toxicol (2008) 55:670682

1992-93 2002-05

677 Table 3 Productivity of wood ducks nesting in boxes erected along the lower Roanoke River basin, 20022003 Dataseta All nests Clutch size (n) Hatching success (%) p-valueb Welch Creek (n = 8) 0.031 0.068 10 ABc 522 95 88100 Roanoke River (n = 18) 16 A 1418 79 5886 (n = 9) 13 1215 75 2593 Eastmost River (n = 14) 10 B 616 85 71100 (n = 10) 8 416 91 52100

100 90 80

pg / g fresh wet weight

70 60 50 40 30 20 10 0
TCDD p=0.0016 TCDF p=0.0005 TEQ p=0.0005

Nests with B16 eggs Clutch size (n) Hatching success (%) 0.273 0.250

(n = 7) 9 423 100 89100

Note: Clutch sizes are geometric mean and 95% condence intervals, and percentage hatch data are median and interquartile range. Only active nests (those with one or more eggs) were included in these calculations 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 c a

Fig. 2 Trends in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8tetrachlorodibenzofuran (TCDF), and TCDD-equivalent (TEQ) concentrations in wood duck eggs from nest boxes along the lower Roanoke River, near Plymouth, North Carolina (geometric mean and upper 95% condence interval; n = 10 in 19921993 and n = 48 in 20022005). Interyear comparisons by Wilcoxon rank sum test

KruskalWallis test (df = 2, a = 0.05)

Means followed by the same capital letter are not signicantly different

Productivity Wood duck productivity data indicate high hatching success at all sites (Table 3). Clutch sizes were signicantly 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 signicant association between hatching success and either TCDD, TCDF, or TEQs. When data for all nests at the three lower Roanoke River sites (n = 40) were evaluated, weak but signicant negative correlations were determined for the association between clutch size and each of these parameters (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

signicant (although the Spearman q of -0.373 between clutch size and TCDF in the culled dataset would be signicant 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 a Welch 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 ducks utility in coplanar halogenated aromatic hydrocarbon monitoring: (1) the signicantly greater PCDD, PCDF, and TEQ accumulation in eggs from the study areas compared to those from the reference site, (2) the signicantly lower PCDD and PCDF concentrations in eggs collected during 20022005 compared to 19921993, and (3) the congener proles from all sites and all years. Wood duck eggs from nests along Welch Creek, the lower Roanoke River, and the Eastmost River were

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contaminated with PCDDs and PCDFs. On a TEQ basis, concentrations in these areas signicantly 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 sufcient to reect 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 susceptibility 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 reect 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 rst 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 Roanoke 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 vefold 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 sh 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 catsh (Ictalurus punctatus) llets 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 llets averaged about 28 pg/g) to 20022004 (when TEQs in llets 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 (Sanderson 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 signicantly over the decade, from a geometric mean of 82% (95% condence interval, 73 92%) in 19921993 to 68% (95% condence interval, 63 74%) in 20022005. Congener proles 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 proles 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 component analyses of congener proles (Choi et al. 2001) and dioxin ngerprinting could be employed to the residues measured in wood duck eggs to aid in source identication. 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 ngerprinting applications. The congener proles, lack of contamination in reference 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 reects 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 Carolina 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 denitive 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

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679

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 Roanoke River, NC Range Roanoke River, NCa Range Pocosin Lakes National Wildlife Refuge, NC Range Bayou Meto, AR Area Ia Range Area IV Range Wisconsin River, WI Range Shiawassee National Wildlife Refuge, MI Range Rose Lake Game Area, MI Range 5 2003 5 2003 6 19951996 12 19881989 17 19881989 15 20022003 10 19921993 n 48 Year(s) 20022005 TCDD 0.7 0.14.9 2.9 0.431 \0.2 \0.20.3 36 2480 \1 NDb \2 ND c TCDF 3.8 0.525 24 1.1460 0.4 0.20.9 23 2240 1 \13 1.1 \16 TEQs 6 130 30 2500 1 0.84 59 4720 2 \14 18 343 140 57246 2 1.13.7 Williams (2004) Williams (2004) Custer et al. (2002) White and Hoffman (1995) White and Hoffman (1995) This study Beeman and Augspurger (1996) Reference This study

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 b c

TEQs recalculated from datasheets provided by study author Analyte not detected No data on individual analytes

median and average percentage hatch for these areas is normal (Hepp and Bellrose 1995). Mercury, TCDD, TCDF, and TEQs were not signicantly correlated with percentage hatch. There were signicant but weak inverse relationships 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 signicant 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 measured 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 but the 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 Wisconsin River pulp and paper mills. They estimated a no-

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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 success (Elliott et al. 1989, 2001). Field studies of tree swallows at a PCB- and dioxin-contaminated site indicated that 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 concentrations were signicantly 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 of 0.14 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 productivity 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 nonortho-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 signicant 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 tissues (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 avian assessments is noteworthy. Tree swallow eggs can accumulate 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 documented. 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 concurrent 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 uptake from local forage (Custer et al. 2003, 2005; Maul et al. 2006). These data facilitated development of PCB bioaccumulation models for tree swallows (Echols et al. 2004). Also, dioxin-like compounds have been implicated in adverse effects on tree swallows in the eld (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 reference site, the decline of PCDDs and PCDFs following cessation of molecular chlorine bleaching, and the dominance of TCDD and TCDF in congener proles provides strong evidence that contamination of lower Roanoke River wood duck eggs reects pulp and paper mill impacts.
Acknowledgments Assistance in the lab and eld was provided by Mike Tanner, Sara Ward, Jean Richter, Wendy Stanton, and Michelle Chappel. Partial funding was provided through USFWSs Environmental Contaminants Program (Study ID No. 200240001).

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