“Examination of the Relationship of Selenium Concentrations 

and Mallard Egg Hatchability in Field-Collected Eggs” 

Summary of 

U.S. Fish and Wildlife Service Region 8 document: 

“Toxic Thresholds of Selenium in Mallard Eggs: Data Report” 

DEPARTMENT OF THE INTERIOR 

U.S. FISH AND WILDLIFE SERVICE 

REGION 8 

USFWS ID # 200310005.2 

USEPA Interagency Agreement # DW-14-92253201-3 

U.S. Fish and Wildlife Service 

Environmental Contaminants Division 

Sacramento Field Office. August, 2011. 

Prepared for: 

Charles Delos 

USEPA Work Assignment Manager 

HECD Contract: EP-C-09-001; Work Assignment 5-04 

U.S. Environmental Protection Agency 

Health and Ecological Criteria Div. (4304T) 

1200 Pennsylvania Avenue, NW, 

Washington, DC 20460 

Ph: 202-566-1097 Fax: 202-566-1140 

E-mail: delos.charles@epa.gov

Prepared by: 

739 Hastings Street 

Traverse City, Michigan 49686 

Phone: (231) 941-2230 

Facsimile: (231) 941-2240 

Principal Contact Person: 

Keith Taulbee 

ktaulbee@glec.com

January 21, 2014
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page i

Great Lakes Environmental Center, Inc.

TABLE OF CONTENTS 

 

Page No. 

Examination of the Relationship of Selenium Concentrations and Mallard Egg Hatchability in 

Field-Collected Eggs ........................................................................................................................1 

Summary of Methods .......................................................................................................................1 

Summary of Results Reported in U.S.FWS (2011) .........................................................................2 

Summary of Additional Analyses Performed ..................................................................................3 

REFERENCES ................................................................................................................................5 

LIST OF TABLES 

 Page No. 

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

 Wastewater Treatment Facility. Taken directly from Table 1 on page 8 of U.S. 

 FWS (2011) ....................................................................................................................2 

LIST OF FIGURES 

 Page No. 

Figure 1. Relationship between selenium concentration in mallard eggs (shown as the natural 

 log of mg Se/kg dry weight) and proportional hatch of corresponding mallard 

clutches. Data shown are for the entire 2000-2005 study period ..................................6 

Figure 2. Relationship between selenium concentration in mallard eggs (shown as the natural 

 log of mg Se/kg dry weight) and proportional hatch of corresponding mallard 

 clutches. Each figure represents the relationship for a single year (shown in the lower 

 left of each plot) during the 2000-2005 study period ....................................................7 
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 1 of 7 

Great Lakes Environmental Center, Inc.

SUMMARY 

 OF 

U.S. FISH AND WILDLIFE SERVICE REGION 8 DOCUMENT: 

“TOXIC THRESHOLDS OF SELENIUM IN MALLARD EGGS: DATA REPORT” 

Examination of the Relationship of Selenium Concentrations 

and Mallard Egg Hatchability in Field-Collected Eggs 

This document provides a summary of the U.S. Fish and Wildlife Service Region 8 document 

"Toxic Thresholds of Selenium in Mallard Eggs: Data Report," and reports results of additional 

analyses regarding the relationship between selenium concentrations in mallard eggs and 

hatchability using data presented in the Appendices of the U.S. FWS (2011) report. The U.S. 

Fish and Wildlife Service conducted a study to examine the relationship between selenium 

concentrations and proportional hatch in mallard eggs collected in the field between 2000-2005 

by the U.S. Fish and Wildlife Service. Eggs were collected in Davis, CA from nesting sites in 

fields irrigated with treated sewage water with slightly elevated selenium levels discharged by 

the city (U.S. FWS 2011). City of Davis personnel routinely pass eggs collected from all nests 

found prior to mowing to the North Delta Conservancy, who then artificially incubate eggs, raise 

hatchlings, and return the birds to the wild (U.S. FWS 2011). Between 2000-2005, the U.S. 

FWS worked with these organizations to collect representative eggs from each clutch and then 

relate the chemical concentrations found within the eggs to hatchability of the corresponding 

clutches. The authors note that the objective of this study was to generate sufficient data across a 

range of selenium concentrations in the eggs above background levels such that they would be 

able to distinguish between two alternative hypothesized thresholds of selenium toxicity in 

mallards based on laboratory toxicity testing: a threshold of 6-10 μg/g dry weight estimated by 

the Services versus a threshold of 16-20 μg/g dry weight estimated by the regulated community 

(U.S. FWS 2011). The methods followed in this study are summarized in the following 

paragraphs. 

Summary of Methods 

For this study, eggs were collected from nests during the 2000-2005 nesting seasons (U.S. FWS 

2011). The majority of collected eggs were mallard (Anas platyrhynchos) eggs; however, 

gadwall (Anas strepera) and cinnamon teal (Anas cyanopter) eggs were also collected. Once 

nests were located, clutch size was determined, and one egg from each clutch was randomly 

selected for dissection and chemical analysis (U.S. FWS 2011). For large (8-12 eggs) clutches, 3 

additional eggs were selected for additional chemical analysis (U.S. FWS 2011). Eggs 

designated for chemical analysis were taken to the Sacramento Fish and Wildlife Office for 

processing, and remaining eggs were taken to the North Delta Conservancy for artificial 

incubation and hatching (U.S. FWS 2011). 

Eggs designated for chemical analysis and dissection were processed within six hours of 

collection. Egg length, breadth, and weight were measured. Egg stage and development were 

determined through direct observation following methods of Caldwell and Snart (1974). Egg 
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 2 of 7 

Great Lakes Environmental Center, Inc.

contents were stored and frozen at -20°C for chemical analysis. All frozen egg samples were 

analyzed for selenium by Laboratory and Environmental Testing, Inc., in Columbia, MO, using 

hydride generation atomic absorption spectroscopy (U.S. FWS 2011). A subset of 31 eggs was 

selected for additional inorganic analysis, and a subset of 5 eggs was selected for additional 

organic analysis, to determine whether additional contaminants besides selenium were present 

and affecting hatchability (U.S. FWS 2011). 

Summary of Results Reported in U.S.FWS (2011) 

Paired hatchability and egg selenium data are available for a total of 96 mallard clutches 

collected between 2000 and 2005 (U.S. FWS 2011). Only clutches that had three or more days 

of natural incubation were used (U.S. FWS 2011), to control for the fact that hatchability of 

artificially incubated eggs that had been initially incubated naturally for less than three days was 

lower than for eggs naturally incubated for three or more days (Henny et al. 2000). Selenium 

concentrations in eggs ranged from 1.7 – 14 μg/g Se dry weight, with a geometric mean of 4.2 

μg/g Se dry weight (U.S. FWS 2011). Hatchability of individual clutches ranged from 40-100%, 

and average yearly hatchability ranged from a low of 82% in 2003 (a year with four clutches 

collected, one of which had 40% hatchability) to a high of 100% in 2005 (U.S. FWS 2011). 

Average hatchability across all 96 clutches between 2000-2005 was 91% (U.S. FWS 2011). 

Ranges and average selenium concentration and hatchability results are shown in Table 1, which 

is reproduced directly from U.S. FWS (2011). 

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

Wastewater Treatment Facility. Taken directly from Table 1 on page 8 

of U.S. FWS (2011). 

Intra-clutch variability in egg selenium concentrations was assessed by measuring the coefficient 

of variation (standard deviation / mean) for 15 clutches (11 of which were for mallards) from 

which selenium was measured in four eggs. Coefficients of variation ranged from 3.8% to 

74.3%, with an average of 18.7%, or 14.1% if the clutch with a coefficient of variation of 74.3% 

is omitted (U.S. FWS 2011). The authors concluded that intra-clutch variability was comparable 

2000 2001 2002 2003 2004 2005 All Years

# Clutches 

Collected 23 17 13 4 28 11 96

M ean [Se]* 

(Range)

4.1 

(1.7 - 14)

3.4 

(2.4 - 5.2)

5.0 

(3.2 - 7.3)

4.1 

(2.1 - 5.7)

4.7 

(1.8 - 11)

3.6 

(1.9 - 12)

4.2 

(1.7 - 14.0)

M ean % 

Hatch 

(Range)

87% 

(50 - 100)

90% 

(66.67 - 100)

98% 

(83.33 - 100)

82% 

(40.00 - 100)

91% 

(50.00 - 100) 100% 91% 

(40.00 - 100)

* Geometric mean presented as μg/g dry weight.
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 3 of 7 

Great Lakes Environmental Center, Inc.

to other studies, and that the method of randomly selecting one egg to represent clutch-wise 

selenium exposure was appropriate (U.S. FWS 2011). 

Eleven of the 18 inorganic elements besides selenium that were measured were found in at least 

one egg, and barium, copper, iron, magnesium, manganese, strontium, and zinc were detected in 

all 31 eggs (U.S. FWS 2011). The National Irrigation Water Quality Program (NIWQP) 

identified seven contaminants among the 18 inorganic elements samples in the subset of 31 eggs 

(U.S. DOI 1998; Seiler et al. 2003). Among these, arsenic, boron, and mercury were detected 

above background levels, but well below NIWQP no observable effect levels (U.S. FWS 2011). 

Zinc was slightly above background, and assumed to not be affecting hatchability (U.S. FWS 

2011). Copper concentrations were below background levels, and neither cadmium nor 

molybdenum was detected in the 31 eggs (U.S. FWS 2011). 

Among all inorganic elements, concentrations of barium were elevated, with concentrations 

ranging from 8.2-77.4 μg/g, with a geometric mean of 27.6 μg/g dry weight (U.S. FWS 2011). 

These concentrations were much higher compared to other studies cited by the authors (U.S. 

FWS 2011). The authors determined the elevated concentrations were not the result of laboratory 

error. Finally, the authors examined the correlation between barium concentrations and 

hatchability, and found no significant relationship (P=0.95), concluding that barium did not 

impact hatchability in this study (U.S. FWS 2011). 

In the five eggs analyzed for organic compounds, DDE and PBDE congener concentrations were 

detected at levels below toxicity thresholds for similar species, and determined to not be 

negatively affecting hatchability (U.S. FWS 2011). Similarly, three phthalate esters were 

detected in eggs, but the authors noted these chemicals are recognized to have a low degree of 

acute and chronic toxicity, have low water solubility and bioaccumulative potential, and likely 

had no effect on hatchability (Autian 1970; Parkerton and Staples 2003; U.S. FWS 2011). 

The authors did not report results investigating correlations between selenium concentrations in 

mallard eggs and hatchability. They recommend that these data be incorporated into larger 

datasets to assess threshold selenium effect level; specifically, to add these data to the master 

database maintained by the Service to perform subsequent statistical analyses to determine the 

toxicity threshold in mallard eggs (U.S. FWS 2011). 

Summary of Additional Analyses Performed 

The correlations between selenium concentrations in mallard eggs and hatchability of 

corresponding clutches were analyzed by GLEC for the entire 2000-2005 dataset, as well as for 

each individual year between 2000-2005, using data reported in Appendix A of U.S. FWS 

(2011). Consistent with the original analysis, values highlighted in red in Appendix A were 

excluded from this analysis as a result of a lack of natural incubation (U.S. FWS 2011). For all 

correlation analysis, proportional hatch was logit transformed, and selenium concentrations were 

natural log transformed prior to analysis. Clutches with 100% hatchability were set to 97.5% 

prior to analysis to allow for the logit transformation (R Core Team 2013). There was no 

correlation between selenium concentrations and proportional hatch for the full 2000-2005 
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 4 of 7 

Great Lakes Environmental Center, Inc.

dataset (P=0.90, Figure 1). Similarly, there was no correlation between selenium concentrations 

and proportional hatch for individual years (Figure 2, P>0.05), with the exception of 2003, 

where there was a statistically significant positive correlation (P=0.04, r2=0.92) between 

selenium concentrations and hatch (Figure 2). The number of clutches found in 2003 was much 

lower than for all other years (4 of 96), and the anomalous positive correlation was driven by a 

clutch with 40% hatch at a selenium concentration of 2.1 mg/kg coupled with three clutches with 

selenium concentrations between 4.5-5.7 mg/kg and hatches between 88%-100% (Figure 2). 

The relationship appears to be merely the result of an outlier value with low hatchability 

combined with a small sample size, as clutches from other years during the study were found to 

have 100% hatch with lower Se concentrations. 

The authors of the report suggested that the range selenium levels expected to be found in this 

study would facilitate the testing of the hypothesis of whether the selenium toxicity threshold for 

mallard eggs occurs between 6-10 mg/kg dw, or between 16-20 mg/kg dw (U.S. FWS 2001). 

The range of selenium concentrations observed in this study was too low to investigate the 

higher (16-20 mg/kg) threshold, but there were sufficient data to at least perform a coarse 

investigation of whether the effects of selenium toxicity could be observed at the lower (6-10 

mg/kg) threshold. For this analysis, the 96 values for the entire 2000-2005 sampling period were 

divided into two bins, one with selenium concentration less than 6.0 mg/kg dw, and one with 

selenium concentration of 6.0 mg/kg dw and higher. The less than 6.0 mg/kg dw bin consisted 

of 76 clutches, and the 6.0 mg/kg dw and higher bin consisted of 20 clutches. The geometric 

mean of the selenium concentration in the less than 6.0 bin was 3.47 mg/kg dw, and was 8.32 

mg/kg dw in the 6.0 and greater bin. Next, a Kruskal-Wallis (non-parametric ANOVA) test was 

performed to determine whether there were any statistically significant differences in hatch 

between the two bins. Results of the Kruskal-Wallis test revealed no significant (P=0.59) 

differences between the two groups, suggesting that for these data, there was not a selenium 

toxicity threshold between 6-10 mg/kg dw. This result is not surprising, given that the average 

hatchability for the less than 6.0 mg/kg bin was 91.4%, and for the 6.0 mg/kg and higher bin, the 

average hatchability was 91.1%. 

Overall, the results of the correlation analysis indicate that for these data, there is no relationship 

between mallard hatchability and selenium concentrations in eggs across the range of field 

concentrations observed in this study. Furthermore, the results of the binning analysis suggest 

that for these data, the concentration range of 6-10 mg egg Se/kg dw does not represent a 

selenium toxicity threshold for mallard ducks in the field. 

 
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 5 of 7 

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REFERENCES 

Autian, J. 1973. Toxicity and Health Threats of Phthalate Esters: Review of the Literature. 

Environmental Health Perspectives 4:3-26. 

Caldwell, P.J. and Snart, A.E. 1974. A Photographic Index for Aging Mallard Embryos. Journal of 

Wildlife Management 38:298-301. 

Henny, C.J., Grove, R.A. and Bentley, V.R. 2000. Effects of Selenium, Mercury, and Boron on 

Waterbird Egg Hatchability at Stillwater, Malheur, Seedskadee, Ouray, and Benton Lake 

National Wildlife Refuges and Surrounding Vicinities. National Irrigation Water Quality 

Program Information Report No. 5. Bureau of Reclamation, Denver, Colorado. 

Parkerton, T.F. and Staples, C.A. 2003. An Assessment of the Potential Environmental Risks 

Posed by Phthalates in Soil and Sediment. The Handbook of Environmental Chemistry 3:317-

349. 

R Core Team. 2013. R: A language and environment for statistical computing. R Foundation for 

Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/. 

Seiler, R.L., Skorupa, J.P., Naftz, D.L. and Nolan, B.T. 2003. Irrigation-Induced Contamination 

of Water, Sediment, and Biota in the Western United States – Synthesis of Data from the 

National Irrigation Water Quality Program. Professional Paper, 1655. U.S. Geological Survey, 

Denver, Colorado. 

U.S. Department of the Interior (U.S. DOI). 1998. Guidelines for Interpretation of the 

Biological Effects of Selected Constituents in Biota, Water, and Sediment. National Irrigation 

Water Quality Program Informational Report No. 3. U.S. Department of the Interior, 

Washington D.C. 

U.S. Fish and Wildlife Service (U.S. FWS). 2011. Toxic thresholds of selenium in mallard eggs: 

Data Report. Prepared by Aceituna, Kevin N., and Maurer, Thomas C. USFWS ID# 

20031005.2. U.S. Fish and Wildlife Service, Environmental Contaminants Division. 

Sacramento, CA. 
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 6 of 7 

Great Lakes Environmental Center, Inc.

Figure 1. Relationship between selenium concentration in mallard eggs (shown as the 

 natural log of mg Se/kg dry weight) and proportional hatch of corresponding 

 mallard clutches. Data shown are for the entire 2000-2005 study period. 

ln (mg Se/kg egg dw)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0
Examination of the Relationship of Selenium Concentrations January 21, 2014 

and Mallard Egg Hatchability in Field-Collected Eggs: 

Summary of “Toxic Thresholds of Selenium in Mallard Eggs: Data Report” Page 7 of 7 

Great Lakes Environmental Center, Inc.

Figure 2. Relationship between selenium concentration in mallard eggs (shown as the 

 natural log of mg Se/kg dry weight) and proportional hatch of corresponding 

 mallard clutches. Each figure represents the relationship for a single year 

 (shown in the lower left of each plot) during the 2000-2005 study period.

ln (mg Se/kg egg dw)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0

ln (mg Se/kg egg dw)

0.8 1.0 1.2 1.4 1.6 1.8

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0

2000 2001

ln (mg Se/kg egg dw)

1.0 1.2 1.4 1.6 1.8 2.0 2.2

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0

2002

ln (mg Se/kg egg dw)

0.6 0.8 1.0 1.2 1.4 1.6 1.8

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0

2003

ln (mg Se/kg egg dw)

0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0

2004

ln (mg Se/kg egg dw)

0.5 1.0 1.5 2.0 2.5 3.0

Proportional Mallard Hatch

0.0

0.2

0.4

0.6

0.8

1.0

2005