DEPARTMENT OF THE INTERIOR

U.S. FISH AND WILDLIFE SERVICE

R EGION 8



## Toxic Thresholds of Selenium in Mallard Eggs : Data Report

FINAL REPORT

USFWS ID # 200310005.2

USEPA Interagency Agreement # DW

- 14

- 92253201

- 3

Prepared by:

Kevin M. Aceituno Environmental Contaminants Specialist

and

Thomas C. Maurer Environmental Contaminants Specialist Chief, Investigations and Prevention Branch

U.S. Fish and Wildlife Service

Environmental Contaminants Division

Sacramento Field OfficeToxic Thresholds of Selenium in Mallard Eggs: Data Report

2 August , 2011

BLANK PAGEToxic Thresholds of Selenium in Mallard Eggs: Data Report

3

INTRODUCTION

As part of t he Department of the Interior’s irrigation drain water research program of the mid

1980's to mid

- 1990's several experimental feeding studies were conducted with game

- farm

mallards ( Anas platyrhy n chos ) to determine toxic threshold concentrations of selenium in avian eggs (summarized in Heinz

, 1996). Coordinat ed field studies of shorebirds, primarily black

necked stilts ( Himantopus me xicanus ) and American avocets ( Recurvirostra americana )

, wer e also conducted in an attempt to document toxic threshold concentrations of selenium in avian eggs (summarized in Skoru pa
, 1998). Results of these efforts indicated that the selenium toxic threshold point, based on reduced egg viability, occurs somewhe re in the region of 6

- 10 μg/g (dry weight basis or dwb). Based on well documented bioaccumulation factors f or selenium,

o nly 2

- 4 ug/L of selenium in water can lead to 6

- 10 μg/g selenium (dwb) in avian eggs (Skorupa

, 1998). These results are one of the core scientific foundations for the U.S. Fish and Wildlife Service’s (Service) recommendation that the U.S. Environmental Pr otection Agency ( EPA ) revise the national chronic water quality criterion for selenium downwa rd from 5 ug/L to 2 ug/L ( USFWS

, 2000: California Toxics Rule Biological Opinion).

A debate in the professional literature (Fairbrother et al.

, 1999; Skorupa

, 1999; Fairbrother et al.

, 2000; CH2M HILL

, 2000) and at professional meetings (Adams et al.

, 2000) has taken place regarding the Service’s data interpretations. Fairbrother et al. (1999) and Adams et al. (2000) re

examined the Service’s experimental feeding studies with game

- farm mallards. Ba sed on new statistical analyses of existing data, Fairbrother et al. (1999) and Adams et a l. (2000) argued that the data from laboratory studies of mallard ducks support a critical threshold value of 16

- 20 μg/g

selenium (dwb) in avian eggs. The authors’ analysis forms one of the core scientific foundations for the Regulated Community ’ s recommendation that EPA should either retain the current water quality criteria for selenium, or revise it upward. Skorupa (1999) responded to those

recommendations by identifying additional data points not consid ered by Fairbrother et al. (1999) and Adam s et al. (2000)

, and by showing that the authors’ statistical methods, when applied to the complete data set, yield a critical threshold value of 7.9 μg/g (dwb); a number

consistent with the Service’s recommendation

. A third, and independent, analysis of the

Service’s laboratory data for mallards was conducted by CH2M HILL (2000) under contract to Chevron Oil Company. This analysis identified additional data points not included in any of the

earlier a nalyses and this larger data set yielded an estimated t hreshold value of 8.4 μg/g (dwb);

also a number consistent with the Service’s recommendation

.

Since the Service’s original laboratory feeding experiments were based on relatively small sample sizes, typically 10

- 15 ducks per treatment group, and because t here is a fair amount of individual variation in sensitivity to selenium poisoning among mallard ducks, all of the various analyses of the data have fairly wide statistical confidence boundaries. For example, CH2M

HILL ’ s analysis estimated the threshold p oint at 8.4 μg/g, but with 95% confidence intervals thatToxic Thresholds of Selenium in Mallard Eggs: Data Report

4

extended from 1.0 μg/g to 13.7 μg/g

. Thus, currently existing data, while supporting the Service position more than it supports other interpretations , does not conclusively eliminate either position statistically speaking. This lack of statistical power associ ated with small sample sizes has led to scientific uncertainty.

Th e Service had a uniquely inexpensive opportunity to conduct a large

- sample field study well

suited to conclusively resolving the debate over the precise location of the selenium toxicity threshold point for mallard eggs. The City of Davis, California (City) , a community 15 miles

west of Sacramento

, routinely disposes of treated sewage water by irrigating upland fields. The

treated sewage water contains a slightly elevated concentration of selenium (approximately 4 ug/L). To maximize evaporative transpiration of the applied sewage w ater, the fields are periodically mow ed to pro mote new growth. The fields have consistently attracted large numbers of nesting mallards

, and as a conser vation measure City personnel search for duc k nests before they mow a field. T hey salvage the eggs from all nests found and turn them over to the

No rth Delta Conservancy where the eggs are artificially incubated, and t he hatched ducklings are raised until they are old enough to be re

- introduced into the wild. Because the water has slightly elevated selenium levels, the duck eggs contain slight ly elev ated selenium levels as well . Past monitoring of mallard eggs conducted by the City of Davis (Mike Conners, City of Davis, pers. comm.) has revealed egg selenium concentrations as high as about 20 μg/g (dwb) and as low as normal background of about 1

- 3 μg /g (Skorupa and Ohlendorf

, 1991). This range of exposure is ideal for a large sample size determination of the critical threshold point for toxicity. Typically the City of Davis sends anywhere from 300 to 800 mallard eggs per nesting season to the North

D elta Conservancy. This report summarizes the methods and data generated by an investigation designed to provide statistical resolution of the selenium toxicity threshold point for mallard eggs with enough precision to distinguish between two alternative i nterpretations of laboratory toxicity testing results: (1) a lower threshold estimate d by Service of 6

- 10 μg/g (dwb ) versus (2) a higher threshold of 16

- 20 μg/g estimated by the Regulated Community.

METHODS

Egg Collection

Egg collection was done in coordination with City personnel, and took place during the 2000 to 2005 nesting seasons (April through July). During this time the Overland Flow site of the Davis Wastewater Treatment Facility (Figures 1 & 2) was monitored and searched for nests once every ten days. Although mallards were the primary target species, gadwall ( Anas strepera ) and cinnamon teal (

Anas cyanoptera ) nests were also loc ated and collected. Nest searches were conducted using a chain dragged between two A TVs. This method is commonly used to locate nests of cryptic species as discussed in Higgins et al. (1969). When a duck was observed flying off a nest a spotter identified the species while others continued to move to the sighting. Upon

location of the nest, clutch size was recorded and one egg was randomly selected for dissection and chemical analysis. Random selection was done by using a random number table to generateToxic Thresholds of Selenium in Mallard Eggs: Data Report

5

a number zero through nine. One person would hold a finger over the egg closest to the approach and begin scanning over the clutch, while another person would observe the second indicator on a digital clock and indicate when to stop scanning (when seconds match the randomly generated number). The randomly selected egg was designated as the “A” egg, placed inside an egg carton inside of a cooler, and taken back to the Sacramento Fish and Wildlife Office (SFWO) for processing. The remainder of the clutch was collected and stored in an egg carton at ambient temperature with the air cell f acing upward, and transported to the artificial incubation facility within the same day. When a large clutch (i.e., 8

- 12 eggs) was located, the “A” egg was collected as well as three additional eggs (also randomly selected using the methods

described abov e) . All four eggs were collected for chemical analysis to ensure that an individual egg from each clutch would provide an appropriate representation of clutch

- wide selenium exposure. To assess the variability of selenium among eggs within a single clutch we calculated the coefficient of variation for each large clutch sampled using the following formula:

Artificial Egg Incubation

Collected eggs were sent to the North Delta Conservancy (Conservancy) in Walnut Grove,

California for artificial incubation and hatching. E gg s were incubated in a GQF M odel 1202

incubator (GQF Manufacturing Company, Savannah, GA) at 99.5o F and 56% relative humidity until hatching, or sufficiently past term to indicate failure to hatch. T he Conservancy’s artificial

incuba tio n (hatchability) rate for mallard clutches was 92% (i.e. 92% equals the site

- specific

control ha tchability), this was based on observing hatching rates of clutches collected from other areas of the region that were delivered to the Conservancy for artifici al incubation. The fate of each egg was monitored to determine hatching success for each clutch. Hatching success was measured by determining the percent of eggs we delivered to the Conservancy that hatched.

Failed

- to

- hatch eggs were returned to the SFW O where they were dissected according to the protocol outlined above and the fate o f each embryo was determined.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

6 Figure 1. City of Davis Wastewater Treatment Facility

Figure 2.

The Davis Wastewater Treatment Facility Overland Flow Site. Woodland

Sacramento

DavisToxic Thresholds of Selenium in Mallard Eggs: Data Report

7

Egg Processing

Eggs collected for inspection and chemical analysis were held at ambient temperature and processed within six hours of collection. Egg length and breadth were measured to the nearest 0.01 millimeter using di gital calipers, and whole egg mass was measured to the nearest 0.01 gram using a digital scale. A small hole was then cut in the blunt end of the egg over the air sac using stainless steel scissors and the egg shell bisected so the contents could be inspe cted and removed. To ensure no cross contamination between eggs occurred, scissors were rinsed with dilute nitric acid and deionized water between samples. Egg stage and condition were determined through direct observation of egg contents using the metho ds outlined in Caldwell and Snart (1974). Egg contents were stored in certified clean I

- Chem® 200 series glass jars with

Teflon

- lined caps and immediately frozen at - 20o C.

Chemical Analysis

Samples selected fo r selenium analysis were sent to the Labor atory and Environmental Testing, Inc. (LET) facility in Columbia, Missouri and analyzed using hydride generation atomic absorption spectroscopy (HGAA). LET is a Service contract laboratory which utilizes Analytical Control Facility (ACF) approved analytic al techniques and QA/QC procedures (SOPs used by LET are available upon request).

In addition to seleniu m analysis a subset of eggs was randomly selected for full inorganic analysis including aluminum, arsenic, boron, barium, beryllium, cadmium, chromium, copper, iron, mercury, magnesium, manganese, molybdenum, nickel, lead, strontium, vanadium and zinc. These additional analyses were do ne to determine if any contaminants besides selenium were present and if they had the potential to influence hatchability. Eggs selected for full inorganic scan were processed in the same manner as eggs analyzed for selenium. Inorganic

scans were also co nducted by LET using either inductively

- coupled plasma emission spectrometry (Al, B, Ba, Be, Cd, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Sr, V), graphite furnace atomic absorption spectrometry (Pb), or cold vapor atomic absorption spectrometry (Hg). All inorganic

dat a presented in this report are in parts per million (μg/g) on a dry weight basis.

An additional subset of eggs was also selected for organic chemical analysis to ensure that organic contaminants were not influencing hatchability. Eggs selected for organi c analysis were processed using the same methods as eggs selected for inorganic analysis except dissecting tools

were rinsed with hexane between samp les to avoid cross contamination . Samples were then sent to the California Department of Fish and Game’s W ater Pollution Control Laboratory (WPCL) in Rancho Cordova, California. The organic analysis was done using gas chromatography

- mass spectrometry and included organochlorine pesticides scan (e.g., DDT , chlordane , dieldrin), congener specific polychlorinate d biphenyl (PCB) scan, congener specific polybrominated diphenyl ether (PBDE) scan, and phthalate scan. When randomly selecting eggs for organic analysis, only fresh and one day old eggs were considered to control for potential chemical

breakdown. This also assisted in reporting results, as organic contaminants are presented as parts per million (μg/g) on a fresh wet weight basis.

Following chemical analysis, selenium concentrations for sample eggs were matched to theToxic Thresholds of Selenium in Mallard Eggs: Data Report

8

hatchery performance data of sibli ng eggs, providing a complete data set suitable for creating a statistically precise estimate of the toxicity threshold point for mallard eggs exposed to relatively

low concentrations of selenium . Henny et al. (2000) found that hatching success in artific ially incubated eggs depended on egg stage at the time of collection. They found that mallard eggs incubated naturally for less than three days hatched at a lower rate than eggs incubated for three or more days. To control for this confounding effect, we only analyzed clutches that had three or more days of natural incubation at the time of collection.

RESULTS

Selenium and Hatchability Analysis Between 2000 and 2005 a total of 96 mallard clutches were found with 3 or more days of natural

incubation. Th ese clutches were used for selenium and hatching analysis. Selenium concentrations in the randomly collected “A” egg ranged from 1.7 to 14 μg/g (dry weight) with a mean of 4.2 μg/g (geometric mean). Hatching success of individual clutches ranged from 50 to 100% with an overall mean of 91%. Annual breakdown of selenium and hatching data is shown in Table 1 (See Appendix A for complete data set).

Table 1. Selenium and hatching data for mallard clutches collected from the Davis Wastewater Treatment Facility.

Intra

- Clutch Selenium Variation Clutches large enough to facilitate intra

- clutch analysis were not found during the 2000 field season, but a total of 15 large clutches were found dur ing the 2001 to 2005 seasons. Eleven clutches were mallard, three were gadwall and one was cinnamon teal. The mean coefficient of variation was 18.1% and ranged from a minimum of 3.8% to a maximum of 74.3%. The clutch

with 74.3% variation had on e egg with a selenium concentration substantially more elevated than the other two (13 μg/g compared to 3.8 and 4.2 μg/g). When this clutch was omitted the mean coefficient of variation dropped to 14.1%. Complete variation data for all clutches are prese nted in Table 2 (See Appendix B for complete data set).

Table 2. Intra

- clutch variation in selenium concentrations.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

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Complete Inorganic and Organic Contaminant Analysis A subset of 31 eggs was randomly selected for an inorganic scan. Of the 18 elemen ts included in the scan, 11 were detected in at least one egg; however, only barium, copper, iron, magnesium, manganese, strontium and zinc were detected in all 31 eggs. The results indicate extremely elevated concentrations of barium with a geometric mea n of 27.61 μg/g (see discussion) . A complete summary of detection frequency, range and means for each metal is presented in Table

3.

An additional subset of 5 fresh eggs was selected for organic analysis. All five eggs contained 4,

4’ dichlorodiphenyldichloroethylene (DDE), a metabolite of DDT, at concentrations ranging from 0.08 to 0.22 μg/g (fresh wet weight). Three phtha late esters were also detected: Di

- n

- butyl ph th alate (DBP) ranged from 0.06 to 0.18 μg/g, Bis

- 2

- ethyhexyl phthalate (DEHP) ranged from 0.08 to 0.21 μg/g, and Diphenylph th alate (DPP) ranged from 0.07 to 0.11 μg/g. Four PBDE congeners were detected in all f ive eggs as well. BDE 47 ranged from 0.001 to 0.17 μg/g, BDE 99 ranged from 0.003 μg/g to 0.23 μg/g, BDE 100 ranged from 0.003 μg/g to 0.10 μg/g, and BDE 155 ranged from 0.002 μg/g to 0.021 μg/g. The remaining organic contaminants were not consistently p resent in all eggs, were below detection limits, or were below quantifiable concentrations. A complete list of organic analytes and results is presented in Appendix C.

Table 3. Results summary of full metals scan . Concentrations are presented in μ g/g o n a dry weight basis.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

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DISCUSSION

A concern during this investigation was ensuring that observed decreases in hatchability were due to low level selenium exposure instead of exposure to additional contaminants known to impair avian reproduction. The National Irrigation Water Quality Program (NIWQP) identified several contaminants of concern on irrigated lands due to their potential adverse effects on wildlife. This includes seven inorganic contaminants: arsenic, boron, cadmium, copper, mercury, molyb denum and zinc (U.S. DOI, 1998; Seiler et al., 2003). For these seven elements we compared the data from our full scan to background concentrations, no observable effect levels (NOEL), and lowest observable effect levels (LOEL) compiled by NIWQP (Table 4)

. Cadmium and molybdenum were no t detected in any of the Davis d uck eggs, and copper was detected at concentrations below the established background level. Arsenic, boron and mercury were all detected at levels above background, but the mean concentratio n of all three were considerably lower than NOEL or LOEL concentrations. Zinc concentrations were above the established background level. However, it is unlikely that zinc was impairing hatchability since it is an essential element, and concentrati ons in the Davis duck eggs were not substantially higher than background. Additionally, birds are considered relatively tolerant to zinc and effects on avian offspring are only seen in laboratory experiments in which hens are fed extremely elevated

doses of zin c (Eisler, 1993).

Table 4. Comparison of Davis Ducks data to metals identified by the National Irriga tion WaterToxic Thresholds of Selenium in Mallard Eggs: Data Report

11 Quality Program as a potential concern on irrigated lands (US DOI, 1998; Seiler et al., 2003).

Concentration s are presented in μ g/g dry weight

.

The inorganic scan also revealed elevated concentrations of barium ranging from 8.2 to 77.4 μg/g with a geometric mean of 27.6 μg/g. We were unable to find any barium toxicity thresholds for avian eggs, but barium concentrations were much higher comp ared to eggs from other studies. The highest barium concentrations found in mallard eggs came from a study done in the Cook Inlet in Alaska where concentrations ranged from 2.3 to 5.5 μg/g dry weight (USFWS, 1994). The highest barium concentrations found in California came from great blue heron ( Ardea h erodias ) and great egret ( Ardea alba ) eggs from Stone Lakes National Wildl ife Refuge (Thomas, 1999). Great blue heron egg concentrations ranged from 2.05 to 6.91 μg/g, and great egret eggs ranged from 3.12 to 6.66 μg/g. In another study conducted in California, Schwarzbach et al (2006) documented barium concentrations in failed

- to

- hatch California clapper rail ( Rallus longirostris obsoletus ) eggs from San Francisco Bay . They found eggs with deformed embryos contained higher concentrations of barium (2.2

– 4.1 μg/g) than eggs without deformed embryos (0.20

– 0.59 μg/g). Although the difference in barium concentrations was significant, a similar trend was also found in strontium, aluminum and chromium concentrations and the authors were unable to definitively conclude that barium was responsible for the abnormal growth observed.

The Davis mallard eggs contained barium concentration at least on order of magnitude highe r than other eggs from through out the United States. Due to this discrepancy, we ensured that the results were not the product of laboratory error. ACF conducts a complete QA/QC check on all data before results are considered final. Further, LET conduct s matrix spikes and sample duplicates as a measure of analytical accuracy. We inspected the raw lab data and both matrix spike and duplicate results indicated that the elevated concentrations were not a result of laboratory error. Moreover, in an effort to determine the impacts of elevated barium, we examined the relationship between barium concentration in the egg and hatching success in the corresponding clutch, but found no correlation between barium concentration and hatching success (p = 0.95). Desp ite elevated barium levels, the overall hatching success of eggs collected for this study is considered high and it does not appear that barium was impairing hatchability.

In addition to inorganic contaminants, a number of organic chemicals were also dete cted in Davis Duck eggs. DDE is known to impair avian reproduction and was detected in mallard and gadwall eggs. The presence of DDE is not surprising given a long history of DDT use in

Calif ornia and its persistence in the environment. The impact of DD E on avian reproductiveToxic Thresholds of Selenium in Mallard Eggs: Data Report

12

performance is highly dependent on species. In white ibis ( Plegadis chihi ), a fresh wet weight concentration as low as 0.25 μg/g in eggs was associated with 4.5% egg shell thinning (King et al., 1980). A 15.4 % decrease in thickne ss in common goldeneye ( Bucephala clangula ) egg

shells occurred at egg concentrations of 0.52 μg/g (Zicus et al., 1988). The black duck ( Anas

rub ripes ), a close relative of the mallard, appears to be less sensitive to DDE exposure with eggshell thinning o ccurring at 46.3 μg/g (Longcore et al., 1971). DDE concentrations found in this investigation are below the lowest toxicity benchmarks, and are orders of magnitude lower than a toxicity benchmark for a closely related species. Therefore, DDE does not app ear to be adversely impacting hatchability.

Three phthalate esters were also detected in this investigation. These chemicals have been largely used as plasticizers in a broad range of applications. We were unable to find toxicological effect thresholds for phthalate esters in avian eggs, or any studies characterizing phthalate concentrations in eggs. It has been recognized that phthalate esters have a low degree of acute and chronic toxicity to aquatic wildlife given their lack of water solubility and

b ioaccumulative potential (Autian, 1970; Parkerton and Staples, 2003) thus we do not think these chemicals impaired hatching in this study.

The presence of PBDE congeners found in this study likely reflects the widespread use of these

chemicals as fire ret ardants. PBDE products are typically a mixture of different congeners and the leading commercial mixture, a pentaBDE product commonly referred to as DE

- 71, contains primarily BDEs 47, 99, 100 and 153 (EPA, 2006). These are the four congeners detected in this study, but determining the impact of these chemicals is difficult given a lack of toxicity thresholds for avian eggs. Many studies have investigated total PBDEs in eggs. Among the highest were peregrine falcon ( Falco peregrinus ) eggs with an average concentration of 6.6 μg/g (Chen et al., 2008). McKernan et al (2009) conducted a dosing study using DE

- 71, and found that injecting eggs with 10 and 20 μg/g DE

- 71 decreased pipping and hat ching success in

American kestre ls ( Falco sparverius ), but had no effect on chickens ( Gallus gallus ) or mallards. The PBDE concentrations detected in this study are well below those found in other eggs, and are considerably lower than dosing concentrations that had no toxicological impact. Given these comparisons, we d o not consider any decreases in hatchability to be caused by PBDEs.

Because the laying order of eggs within the clutches collected was not observed, we relied on random selection and analysis of one egg to provide a representation of selenium concentratio ns in all eggs. Many studies have shown that there is no relationship between selenium concentration and laying order in mallard eggs produced from selenium

- dosed hens (Heinz et al., 1987; Heinz et al., 1989; Stanley et al., 1994). Additionally, Bryan et al. (2003) found no intra

clutch variation in selenium concentrations of common grackle ( Quiscalus quiscala ) eggs and Smith et al. (1988) also found no variation in black

- crowned night

- herons ( Nycticorax

nicticorax ). Further, both of these studies docume nted similar coefficients of variation to what

we found in our intra

- clutch comparison (13.8% in common grackles, 16% in black

- crowned night

- herons). Our results and the findings of others indicate that the randomly selected “A” eggs provide a suitable re presentation of clutch

- wide selenium exposure.

This investigation was necessary because laboratory

- based selenium toxicity testing is limited to small samples sizes, largely due to the expense of housi ng and caring for captive ducks. TheseToxic Thresholds of Selenium in Mallard Eggs: Data Report

13

studies have yield ed results that are relatively imprecise and consequently open t o alternative interpretations. Additionally, many field studies examining selenium toxicity are focused on already contaminated sites, thus egg concentrations from these studies ten d to fall into the higher effect range. This still leaves the lower concentration range poorly populated with data, resulting in the lack of statistical power required to determine low level toxicity effects. We capitalized on a unique opportunity to gen erate data regarding the impact of selenium on reproductive performance in mallards. The goal of this investigation was to collect enough eggs from adult mallards exposed to slightly elevated levels of selenium to provide greater statistical resolution

of low level effect concentrations. In total, nearly 100 clutches were sampled and the low level of selenium exposure is reflected in an average egg concentration of 4.2 μg/g. This is above the expected background concentration of selenium in eggs (1 to 3 μg/g; Skorupa and Ohlendorf, 1991), but still well below egg concentrations ( > 60 μg/g) from areas considered highly impaired due to selenium contamination (Ohlendorf, 2003).

RECOMMENDATIONS

The Service used an opportunity to collect data suitable for integration w ith existing toxicity studies. The next step in the process is to take the data generated in this investigation and incorporate it into larger data sets utilized by researchers to assess threshold selenium effect

levels. The eventual result will be a statistically powerful, field

- based data set with the ability to accurately define the toxicity threshold of selenium to reproductive performance in mallards.

Selenium is a widespread and potent contaminant threat to trust resources and resolut ion of the uncertainty surrounding the toxic threshold point will have significant regulatory implications. The next step to take is to add these data to the master database maintained by the Service and perform the appropriate statistical analyses that wi ll provide a refined selenium toxicity threshold for mallard eggs.

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- 1876.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

17 

### Appendix A

Selenium and Hatching DataToxic Thresholds of Selenium in Mallard Eggs: Data Report

17 Appendix A: Table 1. Selenium and hatching data from 2000. Samples highlighted in red were omitted from analysis due to lack of natural incubatio n or insufficient hatching data.

Egg Condition Key: LN=live, normal; LA=live, abnormal; LU=live, unknown; DN =dead, normal; DA=dead, abnormal; DU=dead, unknown; UU=unknown, unknownToxic Thresholds of Selenium in Mallard Eggs: Data Report

18 Appendix A: Table 2. Selenium and hatching data from 2001. Samples highlighted in red were omitted from analysis due to lack of natural incubatio n.

Egg Condition Key: LN=live, normal; LA=live, abnormal; LU=live, unknown; DN=dead, normal; DA=dead, abnormal; DU=dead, unknown ; UU=unknown, unknownToxic Thresholds of Selenium in Mallard Eggs: Data Report

19 Appendix A: Table 3 . Selenium and hatching data from 2002.

Egg Condition Key: LN=live, normal; LA=live, abnormal; LU=live, unknown; DN=dead, normal; DA=dead, abnormal; DU=dead, unknown ; UU=unknown, unknown

Appendix A: Table 4. Selenium and hatching data from 2003.

Egg Condition Key: LN=live, normal; LA=live, abnormal; LU=live, unknown; DN=dead, normal; DA=dead, abnormal; DU=dead, unknown; UU=unknown, unknownToxic Thresholds of Selenium in Mallard Eggs: Data Report

20 Appendix A: Table 5. Selenium and hatching data from 2004. Samples highlighted in red were omitted from analysis due to lack of natural incubatio n.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

20 Egg Condition Key: LN=live, normal; LA=live, abnormal; LU=live, unknown; DN=dead, normal; DA=dead, abnorma l; DU=dead, unknown; UU=unknown, unknownToxic Thresholds of Selenium in Mallard Eggs: Data Report

21 Appendix A: Table 6. Selenium and hatching data from 2005.

Egg Condition Key: LN=live, normal; LA=live, abnormal; LU=live, unknown; DN=dead, normal; DA=dead, abnormal; DU=dead, unknown ; UU=unknown, unknownToxic Thresholds of Selenium in Mallard Eggs: Data Report

24 

### Appendix B

Intra

- Clutch Selenium DataToxic Thresholds of Selenium in Mallard Eggs: Data Report

25

Appendix B: Table 1. Intra

- clutch Selenium Data. Concentrations presented as μ g/g on a dry weight basis.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

26 

### Appendix C

Results of Inorganic and Organic ScansToxic Thresholds of Selenium in Mallard Eggs: Data Report

25 Appendix C: Table 1. Results of inorganics scan. Concentrations are presented as μ g/g on a dry weight basis.

*Non

- detects are reported as less than the method detection limit (in red)

.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

28

Appendix C: Table 1. Results of organochlorine scan. Concentrations presented as ng/g wet weight.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

29

Appendix C: Table 2. Congener specific PCB scan. Results presented as ng/g wet weight.Toxic Thresholds of Selenium in Mallard Eggs: Data Report

30

Appendix C: Table 3. Congener specific PBDE scan. Results presented as ng/g wet weight.

Appendix C: Table 4. Phthalate esters scan. Results presented as ng/g wet weight.