“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, 2014Examination 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 Co ncentrations and Mallard Egg Hatchability in Field-Collected Eggs .......................................................................................................... ..............1 Summary of Methods ............................................................................................................ ...........1 Summary of Results Report ed in U.S.FWS (2011) .........................................................................2 Summary of Additional An alyses 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 di rectly from Table 1 on page 8 of U.S. FWS (2011) ................................................................................................................... .2

LIST OF FIGURES

Page No.

Figure 1. Relationship between selenium concen tration in mallard eggs (shown as the natural log of mg Se/kg dry weight) and pr oportional hatch of corresponding mallard clutches. Data shown are for the entire 2000-2005 study period ..................................6

Figure 2. Relationship between selenium concentr ation in mallard eggs (shown as the natural log of mg Se/kg dry weight) and pro portional hatch of corresponding mallard clutches. Each figure represents the relati onship for a single year (shown in the lower left of each plot) duri ng the 2000-2005 study period ....................................................7Examination 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 SE RVICE 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 se lenium concentrations in mallard eggs and hatchability using data presente d 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 personn el routinely pass eggs co llected from all nests found prior to mowing to the North Delta Conservanc y, who then artificially incubate eggs, raise hatchlings, and return the birds to the w ild (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 concentra tions 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 ab ove 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 threshol d of 16-20 μg/g dry weight esti mated by the regulated community (U.S. FWS 2011). The methods followed in th is study are summarized in the following paragraphs.

Summary of Methods

For this study, eggs were collected from nest s 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 determine d, 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, an d weight were measured. Egg stage and development were determined through direct observation following methods of Caldwell and Snart (1974). EggExamination 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 Labor atory and Environmental Testing, Inc., in Columbia, MO, using hydride generation atomic absorption spectrosco py (U.S. FWS 2011). A subset of 31 eggs was selected for additional inorganic analysis, and a subset of 5 e ggs 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 ar e available for a total of 96 mallard clutches collected between 2000 and 2005 (U.S. FWS 2011). On ly 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 thr ee 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). Hatchabili ty of individual clut ches ranged from 40-100%, and average yearly hatchability ranged from a lo w 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 ha tchability 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 concen trations was assessed by measuring the coefficient of variation (standard de viation / mean) for 15 clutches (11 of which were for mallards) from which selenium was measured in four eggs. Coefficients of varia tion ranged from 3.8% to 74.3%, with an average of 18.7%, or 14.1% if the cl utch with a coefficien t 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 μ g/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 th e method of randomly selecting on e egg to represent clutch-wise selenium exposure was appr opriate (U.S. FWS 2011).

Eleven of the 18 inorganic elements besides seleni um that were measured were found in at least one egg, and barium, copper, iron, magnesium, mangane se, 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 thes e, 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 m ean 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 c oncentrations were not the result of laboratory error. Finally, the authors examined the co rrelation between barium concentrations and hatchability, and found no significant relationshi p (P=0.95), concluding th at 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 toxic ity 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 author s noted these chemicals are rec ognized to have a low degree of acute and chronic toxicity, have low water so lubility 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 result s investigating correla tions between selenium concentrations in mallard eggs and hatchability. They recommend that these data be incorporated into larger

datasets to assess threshold selenium effect leve l; specifically, to add these data to the master database maintained by the Service to perform su bsequent statistical analyses to determine the toxicity threshold in malla rd eggs (U.S. FWS 2011).

Summary of Additional Analyses Performed

The correlations between selenium concentratio ns in mallard eggs and hatchability of corresponding clutches were anal yzed by GLEC for the entire 2000-2005 dataset, as well as for each individual year between 2000-2005, using da ta 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 l ack 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 tr ansformation (R Core Team 2013). There was no

correlation between selenium concentrations and proportional hatch for the full 2000-2005Examination 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 si gnificant positive correlation (P=0.04, r 2 =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 sele nium concentration of 2.1 mg/kg c oupled 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 re sult 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 lowe r 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 se lenium 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 eff ects of selenium toxicity could be observed at the lower (6-10 mg/kg) threshold. For this analysis, the 96 va lues for the entire 2000-2005 sampling period were divided into two bins, one with selenium concentration less th an 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 th e 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 Kruska l-Wallis test revealed no significant (P=0.59) differences between the two groups, suggesting th at 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 wa s 91.4%, and for the 6.0 mg/kg and higher bin, the average hatchability was 91.1%.

Overall, the results of the correl ation analysis indicate that for th ese data, there is no relationship between mallard hatchability a nd 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 fo r 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

Great Lakes Environmental Center, Inc. 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 Photograp hic 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, Ma lheur, Seedskadee, Ouray, and Benton Lake National Wildlife Refuges and Surrounding Vicin ities. National Irrigation Water Quality Program Information Report No. 5. Bur eau of Reclamation, Denver, Colorado.

Parkerton, T.F. and Staples, C.A. 2003. An A ssessment 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 environmen t 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 No lan, 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. Prof essional 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 C onstituents in Biota, Water, a nd 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. T oxic thresholds of selenium in mallard eggs: Data Report. Prepared by Aceituna, Kevi n 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 con centration in mallard eggs (shown as the natural log of mg Se/kg dry weight) a nd proportional hatch of corresponding

mallard clutches. Data shown a re 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 3.0Proportional Mallard Hatch

0.0 0.2 0.4 0.6 0.8 1.0Examination 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 con centration in mallard eggs (shown as the natural log of mg Se/kg dry weight) a nd 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 3.0Proportional 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 1.8Proportional 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 2.2Proportional 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 1.8Proportional 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 2.6Proportional 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 3.0Proportional Mallard Hatch

0.0 0.2 0.4 0.6 0.8 1.0

2005