NISTIR 7678 

SEABIRD TISSUE ARCHIVAL AND MONITORING 

PROJECT (STAMP): Project Overview, and Updated 

Protocols for Collecting, Processing and Banking

Seabird Eggs

Lauren B. Rust1

Rebecca S. Pugh1

Amanda J. Moors1

Stacy S. Vander Pol1

Paul R. Becker1

And David G. Roseneau2

1

U.S. Department of Commerce 2U.S. Department of the Interior 

National Institute of Standards and Technology 2U.S. Fish and Wildlife Service 

Chemical Science and Technology Laboratory Alaska Maritime National Wildlife Refuge 

Analytical Chemistry Division 95 Sterling Hwy. #1 

Hollings Marine Laboratory Homer, AK 99603 

 331 Fort Johnson Road 

 Charleston, SC 29412 

July 29, 2010
ii

Table of Contents

LIST OF FIGURES ....................................................................................................................IV

LIST OF TABLES......................................................................................................................IV

LIST OF APPENDICES ............................................................................................................IV

ACKNOWLEDGEMENTS ........................................................................................................ V

DISCLAIMER.............................................................................................................................. V

ABSTRACT.................................................................................................................................VI

SEABIRD TISSUE ARCHIVAL AND MONITORING PROJECT: ..................................... 1

Introduction................................................................................................................................... 1

STAMP Project History ............................................................................................................... 1

Use of Seabird Eggs for Monitoring............................................................................................ 3

Specimen Banking for STAMP.................................................................................................... 4

Management System..................................................................................................................... 5

Access Policy.................................................................................................................................. 7

PROJECT GOALS AND OBJECTIVES................................................................................... 8

MATERIALS ................................................................................................................................ 9

Materials in Egg Collecting Kits.................................................................................................. 9

METHODS.................................................................................................................................. 12

Study Area and Sampling Sites ................................................................................................. 12

Egg Collection Field Procedures................................................................................................ 13

Collecting murre eggs................................................................................................................. 13

Labeling the eggs......................................................................................................................... 13

Bagging the eggs.......................................................................................................................... 14

Packing the eggs for shipping .................................................................................................... 15

Storing and shipping the eggs.................................................................................................... 15

Collecting gull eggs ..................................................................................................................... 16

Labeling the collected gull eggs ................................................................................................. 18
iii

Bagging the eggs.......................................................................................................................... 18

Packing the eggs for shipping .................................................................................................... 19

EGG PROCESSING PROCEDURES...................................................................................... 20

Material Required for Processing Eggs.................................................................................... 21

Seabird Data Form ..................................................................................................................... 21

Receiving Whole Eggs................................................................................................................. 25

Measuring Whole Eggs............................................................................................................... 25

Removing Egg Contents from Shells......................................................................................... 28

Homogenizing Egg Contents...................................................................................................... 30

Pipetting Egg Contents into Sample Jars and Vials................................................................ 31

Cleaning Glassware .................................................................................................................... 35

Drying and Shipping Eggshells.................................................................................................. 37

Shipping Frozen Egg Samples ................................................................................................... 38

Egg Sample Archival Procedures.............................................................................................. 40

SUMMARY ................................................................................................................................. 41

LITERATURE CITED .............................................................................................................. 41
iv

LIST OF FIGURES

Figure 1: Packing eggs for transport............................................................................................. 10

Figure 2: Murre identification photo guide................................................................................... 11

Figure 3: Gull identification photo guide...................................................................................... 11

Figure 4: The 1999-2009 Seabird Tissue Archival and Monitoring Project (STAMP) seabird egg 

collecting sites............................................................................................................................... 12

Figure 5: Pre-printed murre egg labels. ........................................................................................ 14

Figure 6: Pre-printed gull egg labels............................................................................................. 18

Figure 7: 12-digit Sample ID number........................................................................................... 22

Figure 8: Weighing an egg on a TexWipe on a digital scale. ....................................................... 27

Figure 9: Filling out Part IV on the Seabird Data Form. .............................................................. 27

Figure 10: Measuring a rinsed and dry egg with digital calipers.................................................. 28

Figure 11: Egg contents in a glass measuring cup ready to be weighed....................................... 30

Figure 12: Homogenizing egg contents in a pre-cleaned, UHV foil-covered 1-L glass beaker with 

the high-speed VirTis blender....................................................................................................... 34

Figure 13: Homogenized egg content aliquots in 5-15 mL Teflon® jars and 6 polypropylene 

cryovials ready to be frozen.......................................................................................................... 35

Figure 14: Storage ID Number. .................................................................................................... 40

LIST OF TABLES

Table 1: Organizations and individuals responsible for collecting, processing and banking for 

STAMP. .......................................................................................................................................... 6

LIST OF APPENDICES

Appendix 1: Seabird Data Form. .................................................................................................. 46

Appendix 2: Marine ESB Storage Data Form .............................................................................. 48

Appendix 3: Abbreviations for colony sites and bird species....................................................... 49
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ACKNOWLEDGEMENTS

The following villages, organizations, and agencies are acknowledged for their egg collection, 

processing and funding contributions to STAMP: Bureau of Indian Afairs, Alaska Regional 

Subsistance Branch, Anchorage; U.S. Fish and Wildlife Service, Alaska Maritime National 

Wildlife Refuge, Homer; U.S. Fish and Wildlife Service, Office of Migratory Bird Management, 

Anchorage; U.S. Fish and Wildlife Service, Marine Mammals Management, Anchorage; U.S. 

Geological Survey Biological Resources Division, Alaska Science Center, Anchorage; U.S. 

Geological Survey Biological Resources Division, Western Regional Office, Seattle; Minerals 

Management Service, Anchorag; Alaska Department of Fish and Game, Dillingham; University 

of Alaska; University of Washington, Seattle; Oregon State University; World Wildlife Fund and 

Perdue University; Alaska Sea Otter & Sea Lion Commission, Anchorage; Alaska Native 

Science Commission, Anchorage; Transboundary Ecologic LLC, Anchorage; State Nature 

Biosphere Preserve ‘Komandorskiy’, Commander Islands, Russian Far East; Village of Point 

Hope, Point Hope; Maniilaq Association, Kotzebue; Native Village of Deering, Deering; Natural 

Resources Division, Kawerak, Inc., Nome; Bering Straits Native Corporation, Nome; Native 

Village of Shaktoolik, Shaktoolik; Native Village of Savoonga, St. Lawrence Island; Paimiut 

Tribal Council, Village of Paimiut, Hooper Bay; Native Village of Mekoryuk, Mekoryuk; St. 

George Traditional Council, St. George Island; St. George Island; Togiak Traditional Council 

Environmental Program, Togiak; Native Village of Nanwalek, Nanwalek); Seldovia Village 

Tribe, Seldovia; Tatitlek Village IRA Council, Tatitlek; Sitka Tribe of Alaska, Sitka; and the 

residents of the Native Village of Diomede, Little Diomede Island. 

DISCLAIMER

Certain commercial equipment or instruments are identified in this paper to specify adequately 

the experimental procedures. Such identification does not imply recommendations or 

endorsement by the National Institute of Standards and Technology nor does it imply that the 

equipment or instruments are the best available for the purpose.
vi

ABSTRACT

The Seabird Tissue and Archival Monitoring Project (STAMP) is a collaborative effort by the 

U.S. Fish and Wildlife Service (USFWS), the Alaska Maritime National Wildlife Refuge 

(AMNWR), and the National Institute of Standards and Technology (NIST) to monitor longterm (decadal) trends in persistent, potentially harmful bioaccumulative contaminants in the 

Bearing and Chukchi seas and Gulf of Alaska using seabird tissues, i.e., primarily eggs (York et 

al. 2001). The cryogenic banking of eggs for retrospective analysis is a major component of 

STAMP. Before STAMP, data and information on contaminants in Alaskan seabirds were very 

limited. Analysis of banked eggs is helping to develop baseline data on contaminants in major 

Alaskan Marine Regions. To date, approximately 14,000 egg sample aliquots representing over 

1,453 eggs from 44 sampling sites have been banked at NIST’s Marine Environmental Specimen 

Bank in Charleston, SC. These data are also significant in providing information on 

contaminants in a human subsistence resource that is prominent in many rural Alaskan diets.

This report is to provide an up-to-date description of the STAMP protocols for collecting, 

processing and banking seabird eggs. 
1

SEABIRD TISSUE ARCHIVAL AND MONITORING PROJECT:

Project Overview, and Updated Protocols for Collecting, Processing and 

Banking Seabird Eggs

Introduction

The Seabird Tissue and Archival Monitoring Project (STAMP) is a collaborative effort by the 

U.S. Fish and Wildlife Service Alaska Maritime National Wildlife Refuge (USFWS-AMNWR),

the National Institute of Standards and Technology (NIST) and the Bureau of Indian Affairs

Alaska Region Subsistence Branch (BIA-ARSB) to monitor long-term (decadal) trends in 

persistent, potentially harmful bioaccumulative contaminants in the Bearing and Chukchi seas 

and Gulf of Alaska using seabird tissues, i.e., primarily eggs (York et al. 2001). This approach is 

similar to that of the Alaska Marine Mammal Tissue Archival Project (AMMTAP), started in 

1987 by NIST and the National Oceanic and Atmospheric Administration (NOAA) as part of the 

Outer Continental Shelf Environmental Assessment Program sponsored by the Minerals 

Management Service. It was developed to cryogenically bank tissues collected from marine 

mammals harvested by Alaskan subsistence hunters (Becker et al. 1993). Specimens from both 

AMMTAP and STAMP are banked in the Marine Environmental Specimen Bank (Marine ESB) 

at the Hollings Marine Laboratory, Charleston, SC. 

Northern seabirds feed near the top of the food chain and have the potential for accumulating 

anthropogenic contaminants. Before STAMP, data and information on contaminants in Alaskan 

seabirds were very limited. Analysis of banked eggs is helping to develop baseline data on 

contaminants in major Alaskan Marine Regions, monitor long-term trends in these potentially 

harmful substances, and conduct retrospective studies. These data are also significant in 

providing information on contaminants in a human subsistence resource that is prominent in 

many rural Alaskan diets.

This report provides up-to-date description of the STAMP protocols for collecting, processing

and banking seabird eggs (York et al. 2001). For recent findings on contaminant levels and the 

geographic differences that have been found in them see Roseneau et al. (2008) and Vander Pol 

et al. (2009).

STAMP Project History 

STAMP was initiated in 1998 when protocols for collecting, processing, and banking seabird 

eggs were first developed by USFWS, the U.S. Geological Survey Biological Resources 

Division (USGS-BRD), and NIST with funding support from the U.S. Department of Interior’s 

Minerals Management Service (MMS). The project was designed to track trends in 

environmental quality by 1) collecting Alaskan seabird eggs using standardized protocols so to 

minimize contamination, 2) processing and banking the contents under conditions that ensure 

chemical stability during long-term (decadal) storage, and 3) analyzing aliquots of the stored 
2

material to determine current baseline levels of persistent bioaccumulative contaminants (e.g., 

chlorinated pesticides, polychlorinated biphenyls [PCBs], brominated flame retardants [PBDEs],

butyltin compounds [organotins], and mercury concentrations and isotopes (Vander Pol et al. 

2009). Methods and protocols were developed for STAMP in 1998 (York et al. 2001), and 

specimen collections began in July 1999. The USGS-BRD stopped participating in the project in 

2007 and AMNWR took over all of the processing responsibilities in addition to their original 

duties of collecting eggs. Note: eggs were not obtained in 2007 because AMNWR needed to 

obtain additional equipment, supplies, and lab space, and lacked the time to process them.

Since its beginning, STAMP has involved many Federal and State of Alaska agencies, regional 

and local organizations, and individuals from local Alaskan villages that have an interest in the 

project. Funding support has come from MMS, USGS-BRD, USFWS AMNWR, Bureau of 

Indian Affairs Alaska Region Subsistence Branch (BIA-ARSB), and the North Pacific Research 

Board (NPRB), with in-kind contributions from USFWS, USGS-BRD, and NIST. A major 

expansion began in 2005 when the NPRB provided three years of funding to STAMP through 

NPRB Project 0543, “Expanding the Seabird Tissue Archival Project (STAMP) in the North 

Pacific: Geographic Patterns in Contaminant Residues in Seabird Eggs used in Rural Subsistence 

Diets.” The results of this work were published in Roseneau et al. 2008 and Vander Pol et al.

2009. In 2008, additional support was provided by the NPRB to STAMP for a joint study by 

USFWS AMNWR, NIST, and Kawerak Inc. in Nome, Alaska, to investigate “Mercury levels in 

murre and gull eggs harvested for food in the Norton Sound region and potential sources of 

contamination” (NPRB Project 0822).

STAMP has received recognition from the 2007-2008 International Polar Year (IPY) Program 

and as a contributor to both the Arctic Monitoring and Assessment Programme (AMAP) and the 

Conservation of Arctic Fauna and Flora (CAFF), both working groups of the International Arctic 

Council. In 2008, STAMP was designated as an AMAP/CAFF Coordinated Monitoring Effort 

(CME), the goals of which are to: (1) form a more complete picture of the overall state of the 

Arctic ecosystems, and their extent of structural integrity, resiliency, and sustainability; (2)

identify and/or quantify stressors affecting sustainability of Arctic ecosystems, and therefore the 

Arctic’s living resources; and (3) seek efficiencies of operation as directed by Senior Arctic 

Officials (AMAP/CAFF 2007). 

Numbers of seabird colonies, colony locations, and species targeted by STAMP has evolved 

since 1999. In 1999, STAMP began collecting common and thick-billed murre eggs, and in 

2001, the program was expanded to include black-legged kittiwake eggs. In 2004, glaucous gull

(Larus hyperboreus) and glaucous-winged gull (L. glaucescens) eggs were added to the project 

due to the request by and funding contributions from the BIA-ARSB. Gulls were included

because their eggs are an important subsistence food in many coastal regions of Alaska. Also, 

these two species of gulls feed on a wide variety of fish and invertebrates and also scavenge on 

marine mammal carcasses and refuse in community dumps and land-fills where they have 

opportunities to be exposed to relatively high levels of anthropogenic contaminants (Vander Pol 

et al. 2009). Currently, STAMP criteria for selecting study sites and species include the overall 

geographic distribution and regional importance of the nesting colonies; their locations relative 

to onshore, nearshore, and offshore environments; the logistical feasibility of collecting eggs at 

them; the trophic position and foraging strategies of the birds; the foraging habitats used by the 
3

birds; and the use of the birds’ eggs in rural subsistence diets. (Note: STAMP temporarily 

stopped collecting kittiwake eggs in 2005 to concentrate efforts on completing the murre and gull 

work).

Use of Seabird Eggs for Monitoring 

Little was known about contaminants in Alaskan seabirds before the advent of STAMP. More 

than 95% of the seabirds breeding in the continental United States nest at colonies in the Bering 

and Chukchi seas and Gulf of Alaska (USFWS 1992). The Bering and Chukchi seas are not only 

under the influence of airborne contaminants transported across the pole from Eastern Europe, 

but also from atmospheric and oceanic transport from Asia across the Pacific Ocean. Overall 

contaminant patterns and levels in Alaskan seabirds are probably influenced by atmospheric 

transport of contaminants from Asia eastward and northward into the Gulf of Alaska, oceanic 

transport from Asia via the eastward flowing North Pacific Current, and the transport of 

substances into the Bering and Chukchi seas from the Northern Gulf of Alaska via the westward 

moving Alaskan Stream and Alaskan Coastal Current (Stabeno et al. 1999; Li et al. 2002). 

Existing and former military installations may also contribute to local point source “hot spot” 

pollution patterns in Alaska (Vander Pol et al. 2003).

During its early planning stages, STAMP participants identified the seabird egg as the first tissue

of choice for study by the project. There is a relatively long history of using bird eggs for 

environmental monitoring and for investigating the health status of bird populations (Vander Pol 

et al. 2003). Seabirds are long-lived and slow to mature. They occupy high positions in the 

marine food web and are considered sensitive indicators for the marine environment (prey 

includes krill, small fish, and squid). Breeding success, timing of nesting, diets, and survival 

rates may provide early indications of changing environmental conditions (Hatch et al. 1993). 

Eggs are particularly useful for the temporal and spatial monitoring of persistent organic 

pollutants (e.g., polychlorinated biphenyls (PCBs), chlorinated pesticides, dioxins) and mercury 

(York et al. 2001). POPs are long-lived organic compounds that bioaccumulate and have 

potential harmful effects. Examples include polychlorinated biphenyls (PCBs), chlorinated 

pesticides, (i.e., DDT, chlordane, toxaphene, hexachlorobenzene [HCB], hexachlorocyclohexane 

[HCH], and dieldrin), dioxins, and furans. Although most of these substances are restricted or 

banned in most developed countries, they are still manufactured for export and remain in use in 

many developing countries. POPs can be found in the environment many thousands of 

kilometers from their points of release because they can be transported through the atmosphere 

as gases and aerosols (Vander Pol et al. 2003). Mercury is another contaminant that is 

atmospherically transported to remote regions of the globe. Bioavailability of mercury depends 

on microbial conversion of inorganic mercury to methylmercury, which is fat-soluble, 

bioaccumulates much like POPs, and is toxic. Although most of the mercury emission to the 

atmosphere is natural (e.g., volcanic emissions), the greatest source of anthropogenic emission is

fossil fuel combustion (particularly coal) and waste incineration (AMAP 2002).

Currently, STAMP is focused on collecting, banking, and analyzing murre and gull eggs because 

they are harvested in many rural Alaskan coastal communities where they play important roles in 

local diets (Iknokinok and Georgette 1997). Common and thick-billed murre eggs are of 

particularly value for monitoring long-term trends in environmental quality. Both of these 
4

species feed at upper trophic levels and have the potential to accumulate and store contaminants 

in relatively large amounts (Springer et al. 1984, 1986, 1987; Roseneau et al. 2000). However, 

some resource partitioning occurs between the two species that is probably reflected by species 

differences in contaminant loads. Both common and thick-billed murres feed on Pacific sand 

lance (Ammodytes hexapterus), capelin (Mallotus villosus), and small cod (e.g., walleye pollock,

(Theragra chalcogramma); Pacific cod, (Gadus macrocephalus); saffron cod, (Eleginus 

gracilis); and Arctic cod, (Boreogadus saida). However, thick-billed murres tend to forage 

farther from shore and at greater depths than common murres and they also feed on a variety of 

benthic species, including invertebrates that they catch on or near the bottom. In contrast, 

common murres usually forage closer to shore at shallower depths on small mid-water fishes 

(Swartz 1966, 1967; Springer et al. 1984, 1986, 1987; Roseneau et al. 2000; Vander Pol et al.

2009).

Initial data from murre eggs obtained at colonies associated with deep oceanic habitats in the 

Bering and Chukchi seas and Gulf of Alaska suggested that there were north-south and east-west 

geographic gradients in contaminant levels (Christopher et al. 2002; Vander Pol et al. 2003;

Vander Pol et al. 2004; Day et al. 2006). In 2005, the North Pacific Research Board (NPRB) 

provided funding support to STAMP to verify the presence of these patterns, by adding more 

coastal and mainland colonies to the project and analyzing subsets of these egg collections (see 

Figure 4 and Appendix 3). This work confirmed that there were north-south and east-west 

gradients in contaminant levels in the Bering and Chukchi seas and Gulf of Alaska, but it also 

found relatively high mercury levels in murre eggs from Norton Sound that were similar to those 

initially reported by STAMP for murre colonies in southeastern Alaska (Roseneau et al. 2008, 

Vander Pol et al. 2009).

In 2003, STAMP expanded the murre component of the project and created a new gull module 

because of the importance of their eggs in rural subsistence diets. The gull component was 

initiated in May 2004, when the BIA-ARSB began funding local village residents to collect eggs 

at several coastal gull colonies. The BIA had become interested in STAMP when a number of

rural organizations indicated that they wanted more information on contaminants in the eggs of 

these birds (e.g., Point Hope IRA Council Parks and Wildlife, Point Hope; Maniilaq Association 

Subsistence Division, Kotzebue; Natural Resources Division, Kawerak Inc., Nome; Native 

Village of Mekoryuk; St. George Traditional Council Island Sentinel Program; Togiak 

Traditional Council Environmental Program; Seldovia Village Tribe Environmental Program; 

Tatitlek IRA Council; and Sitka Tribe of Alaska) (Roseneau et al. 2008, Vander Pol et al. 2009)

Specimen Banking for STAMP

The contents of seabird eggs collected by STAMP are stored cryogenically (-150 °C) at the 

Marine Environmental Specimen Bank (Marine ESB) at the Hollings Marine Laboratory, in 

Charleston, South Carolina. The Marine ESB is a cryogenic facility that stores well-documented 

environmental specimens collected as part of ongoing research and monitoring programs 

conducted in marine and coastal environments. Aliquots of the STAMP egg specimens provide 

the samples needed to determine current baseline levels of contaminants. Remaining portions of 

these specimens plus unanalyzed egg specimens are maintained in the Marine ESB for future 

retrospective analyses. 
5

Cryogenically banking egg contents for retrospective analysis is an important component of 

STAMP. The long-term storage of carefully selected, representative samples in a specimen bank 

is an important complement to real-time contaminant analysis in an environmental monitoring 

program. For example, the Canadian Wildlife Service successfully documented temporal 

changes in PCBs and pesticides in the Great Lakes region by analyzing herring gull (L. 

argentatus) eggs that were collected and banked as part of its Wildlife Toxicology Program 

(Mineau et al. 1984; Elliott 1985; Wakeford and Kasserra 1997; Norstrom and Hebert 2006). 

Also, the decrease in PCBs and chlorinated pesticides and the increase in mercury shown by 

Braune et al. (2001) for the Prince Leopold Island area are based on a reanalysis of archived 

samples of seabird eggs collected between 1975 and 1998. Eggs collected, processed, and 

banked by STAMP using standardized protocols will allow researchers to identify and study new

analytes of interest in future years and they will also allow analytical techniques to be compared 

over time. These comparisons will provide valuable information on the accuracy and 

effectiveness of current techniques versus new, more sophisticated methods that may be 

developed in upcoming years.

To date, approximately 1,453 eggs representing over 14,000 egg sample aliquots have been 

banked in the Marine ESB as a part of the STAMP. 

Management System

Eggs are collected for STAMP by participating agencies (e.g., USFWS, USGS-BRD, BIAARSB), rural organizations (e.g., tribal and village councils) and rural residents (e.g., subsistence 

hunters), and air-freighted to Homer, Alaska where they are processed by AMNWR personnel at 

the AMNWR laboratory. Collection and processing procedures are described in detail in the 

Methods and Materials section of this report.

Processing procedures result in (1) frozen egg contents (egg specimens) that are expressed 

shipped frozen along with the corresponding seabird data forms to the Marine ESB for baseline 

analyses and banking, and (2) dried eggshells that are shipped to the University of Alaska 

Museum of the North in Fairbanks, Alaska, for long-term storage.

After the Marine ESB receives the frozen specimens and seabird data forms, they are checked for 

potential problems (lost labels, thawed specimens, lost or incomplete data forms, etc.) and then 

logged into the Marine ESB specimen tracking database. The samples are kept frozen until all 

cataloging procedures are complete and then they are put in Liquid Nitrogen (LN2) vapor 

freezers for long-term (decadal) storage at -150 o C. After the samples have been formally 

entered into long-term storage and the data base tracking system, aliquots are available for 

analyses. Cataloging and banking procedures for STAMP specimens are the same as those used 

by the Marine ESB for other programs, including the Marine Mammal Health and Stranding 

Response Program (MMHSRP), the Alaska Marine Mammal Tissue Archival Project 

(AMMTAP), the Mussel Watch Program, and the Peregrine Falcon Monitoring Program. Details 

on these procedures have been described elsewhere (Pugh et al. 2007).
6

Organizations and individuals responsible for collecting, processing, and banking eggs are listed 

in Table 1.

Table 1: Organizations and individuals responsible for collecting, processing and banking eggs for STAMP.

______________________________________________________________________________

Activity Location Responsible Party

______________________________________________________________________________

Egg Collecting US Fish and Wildlife Service Alaska Maritime NWR

D. Roseneau, G. Thomson

J. Williams, A. Kettle

L. Slater

US Fish and Wildlife Service Selawik NWR

Refuge Biologist

US Fish and Wildlife Service Togiak NWR

Refuge Biologist

US Fish and Wildlife Service Office of Migratory Birds

D. Irons

Alaska Biological Science Center USGS Biological Resources Division

S. Hatch

University of Alaska Institute of Arctic Biology

E. Murphy

University of Washington Aquatic & Fishery Sciences

 J. Parrish

Kawerak Inc. Subsistence Resources Program

 S. Tahbone, E. Trigg

Egg Processing Alaska Biological Science Center USGS Biological Resources Division

(1999-2006) Anchorage, Alaska K. Simac, G. York

Egg Processing Alaska Maritime NWR USFWS

(2006-2009) Homer, Alaska D. Roseneau

Eggshell Archiving University of Alaska Museum University of Alaska - Fairbanks

Fairbanks, Alaska K. Winker

Egg Content Banking Marine Environmental Specimen Bank NIST

Charleston, South Carolina R. Pugh, A. Moors, L. Rust
7

Access Policy

Researchers not associated with STAMP can request aliquots of banked specimens; however, 

release of these materials to outside investigators requires approval from the project managers of 

the collaborating agencies and is contingent upon the determination that enough material will be 

left in the Marine ESB to allow STAMP to conduct retrospective analyses and monitor long-term 

trends. Requests for samples must include clear, concise descriptions of the proposed work and 

coincide with STAMP’s long-term goals. The following information must be supplied with the 

sample requests:

1. The name(s) of the principal investigator(s) and affiliated research or academic 

institutions.

2. The specific sample material requested and quantity desired.

3. A brief description of the proposed research and its funding source.

4. A brief justification for use of the banked tissues.

5. The name of the research facility where the analytical work will be conducted.

6. A brief description of the analytical quality control procedures that will be used and a

signed agreement stating that the research facility will participate in NIST directed

interlaboratory comparison exercises for quality assurance (QA) purposes.

7. The estimated date for completion of research, and preparing/submitting subsequent 

reports and publications.

8. A signed agreement stating that copies of all results and findings, including analytical 

data, will be provided to the collaborators of STAMP, along with a data submission 

schedule.

9. A signed agreement stating that credit and acknowledgment will be given to USGS, 

USFWS, NIST’s Marine ESB, BIA-ARSB, AMNWR and STAMP for collecting and 

processing seabird eggs.

The requesting parties will be responsible for all shipping charges. Additional charges may also 

be required to cover the costs of retrieving, documenting, and packaging the samples for 

shipment.
8

PROJECT GOALS AND OBJECTIVES

STAMP’s current goals are to collect and bank tissues (primarily eggs) from Alaskan colonial 

seabird species to determine current baseline contaminant levels, track long-term trends in 

environmental quality, and provide material for retrospective analysis. At present, specific 

objectives are to:

1. Develop and use standardized protocols for collecting, processing, analyzing, and 

banking seabird eggs.

2. Collect eggs from a broad range of Alaskan thick-billed murre, common murre, 

glaucous gull, and glaucous-winged gull colonies scattered throughout the Bering and 

Chukchi seas and Gulf of Alaska for several nesting seasons to determine current 

baseline contaminant levels and verify the presence/absence of geographic patterns.

3. Process the eggs under controlled laboratory conditions to ensure the sample material 

does not become contaminated by outside sources.

4. Cryogenically bank the egg contents in the Marine ESB under controlled laboratory 

conditions to ensure chemical stability during long-term (decadal) storage.

5. Send the eggshells to the University of Alaska Museum of the North for long-term 

(decadal) storage.

6. Analyze subsamples (aliquots) of the egg contents under controlled laboratory 

conditions to determine current baseline contaminant levels.

7. Describe current geographic and temporal patterns in the Alaskan and northeastern 

Pacific marine environments.

8. Use the contaminant patterns observed in eggs to improve our understanding of the 

cycling and transport of these chemicals in the environment.
9

MATERIALS

STAMP developed the first protocol for collecting seabird eggs in 1999 (York et al. 2001). In 

2004, these protocols were revised and updated (Roseneau et al. 2008, Vander Pol et al. 2009). 

The following details represent the most current protocols that are being utilized to date and 

reflect new techniques for collecting, processing, packaging, and transporting samples.

Egg collecting kits are provided by AMNWR and mailed to participating partners free-of-charge.

AMNWR also pays the return shipping costs.

Materials in Egg Collecting Kits

• 50-quart coolers or Rubbermaid Action Packers with collection protocols

• Foam padding to protect the eggs (Figure 1)

• All necessary state and federal permits

• Laminated photographs to help differentiate between common and thick-billed murres 

and Glaucous-Winged and Glaucous Gulls (Figures 2 and 3 respectively)

• Data recording forms

• Pre-printed shipping labels

• Egg labels

• Several “keep upright-handle with care” and “keep cool-do not freeze” stickers

• Plastic bags

• Disposable talc-free gloves

• Permanent markers and pencils

• Duct tape

Permits: State and federal permits needed to collect eggs from Alaskan seabird colonies are 

obtained by STAMP’s AMNWR project manager. The permits are state-wide and cover almost 

all of the nesting locations in the Beaufort, Chukchi and Bering seas, Gulf of Alaska, and 

Aleutian Islands. They allow up to 15 common murre, 15 thick-billed murre, 36 glaucous gull, 

and 36 glaucous-winged gull eggs to be obtained from the listed colonies every year. The permits 

were reviewed by the USFWS permit committee and Alaska Department of Fish and Game 

biologists during January-March 2009. They were renewed on 1 April 2009 and are currently in 

effect until 31 March 2012. They can be renewed every three years until 2020, and new 

collecting locations can be added at any time. Annual reports, due by 15 January every year, are 

compiled and submitted by STAMP’s AMNWR project manager. Note: eggs obtained from 

locations outside Alaska (e.g., Tatoosh Island) are covered by permits held by the participating 

collectors.
10

Figure 1: Packing eggs for transport. Top left: eggs placed on the foam padding; Top right: layering the foam 

pads in the provided coolers; Middle right: Supplies provided in the kit, and Bottom: Cooler ready to be 

shipped. (Note: The eggs in the top left picture are not shown in their individual bags.)
11

Figure 2: Murre identification photo guide.

Figure 3: Gull identification photo guide.

Glaucous-Winged Gull 

Glaucous Gull 

 Thick-billed Murre Common Murre

Thick-billed Murre
12

CANADA

SIBERIA

CHUKCHI SEA

BEAUFORT SEA

BERING SEA

GULF OF ALASKA

Cape Lisburne

Cape Thompson

Noatak R. delta

Cape Denbigh

Little Diomede I.

St. Lawrence I.

Sledge I.

King I.

Triangle I.

Cape Pierce

Hooper Bay

St. George I.

Bogoslof I.

Buldir I.

St. Lazaria I.

Viesokoi Rock

E. Amatuli I.

Kodiak I. (Merry I.)

Middleton I. Gull I. 

Kikertalik Lk.

Duck I. 

Ualik Lk.

Shaiak I.

Tatitlek

Shoup Bay

Bluff

Sinuk R.

delta

Safety Sound

Cape Deceit (Rock I.)

Kukpuk R. delta

Tatoosh I. (Washington State; not shown here) 

Carolyn Island & Cape Darby

Shaktoolik

Shishmaref Inlet (4 separate colonies)

Unalakleet

Stuart I.

Brevig Mission

ALASKA

Aiktak I.

Map courtesy of Dave Roseneau.

METHODS

Study Area and Sampling Sites

The STAMP study area encompasses sites in the Bering and Chukchi seas, Aleutian Islands, 

Gulf of Alaska, and northeastern Pacific Ocean, and by the close of the 2009 breeding season, 

the project successfully sampled 44 colonies in these regions (Figure 4). 

Figure 4: The 1999-2009 Seabird Tissue Archival and Monitoring Project (STAMP) seabird egg collecting 

sites.
13

The seabird egg collecting and banking protocols developed by STAMP for the Marine ESB is 

based on methods used by the Canadian Wildlife Service Specimen Bank (Elliott, 1985) and 

Nordic Countries (Nordic Council of Ministers, 1995). However, some modifications have been 

made to reflect input from the USFWS Alaska Maritime NWR. The egg banking protocol is 

based directly on procedures used by Marine ESB staff to catalog and bank AMMTAP and 

MMHSRP tissue samples (Becker et al. 1988, 1991, and 1999).

The murre and gull egg collecting protocols were designed to obtain uncontaminated fresh eggs 

during the early incubation period to avoid obtaining replacement eggs. They have continued to 

evolve over the years to become less complex and minimize egg breakage during transport. The 

intent of these updated protocols is to make them more user-friendly for rural residents to use at 

remote locations under typical Alaskan field conditions and to obtain fresh, well-defined 

specimens uncontaminated by extraneous sources of trace elements and organic compounds, and 

to package and transport these samples as quickly as possible under conditions that eliminate or 

minimize specimen degradation prior to storage. Sample storage and inventory procedures 

follow those routinely performed at the Marine ESB, including storage under LN2 vapor at -

150°C.

Egg Collection Field Procedures

The procedures below have been reproduced from NISTIR 7562 with modifications and updates.

Collecting murre eggs

Each murre egg collection kit consists of 1 plastic Coleman cooler capable of holding 15 murre 

eggs. 

1) The permit allows 15 murre eggs to be collected. Use the egg labels to identify which 

species of eggs are being collected. 

2) Collect the eggs as soon as possible after the birds have started laying eggs. If not 

possible, indicate in the ‘Comments’ section on the datasheet when they were 

collected (for example, “about 2 weeks after egg laying began”).

3) Using talc-free vinyl gloves, remove the eggs from the nest. Always wear gloves and 

try not to touch the eggs with bare hands, particularly if there’s a chance that there 

might be any residual outboard fuel, oil, or insect repellant on them as this will 

contaminant the shell and contents. Note on the datasheet if no gloves were available 

and the eggs were picked up with bare hands. 

Labeling the eggs

The collection kit contains pre-printed labels for the eggs (Figure 5). Use the enclosed pencil to 

fill out one label for each murre egg that is collected (DO NOT use ball point pens). Assign a 

different number for each nest you collect eggs from (1-15). If more than 1 egg is present in a 
14

nest, use the same nest number for each egg collected from that particular nest, so it’s known that 

the eggs labeled with the same nest numbers came from the same clutch of eggs. 

1) Identify and label eggs individually. The species and location will be pre-printed on 

the labels. Fill in the missing information (name, egg collection date, assigned nest 

number, and any comments (e.g. “collected late in the season”, “did not use gloves”, 

“eggs almost ready to hatch”). The labels should be printed using soft to medium 

pencils (No. 2 or 3) and placed between the two plastic bags.

2) One Seabird Data Form should be filled out per clutch (see Appendix 1). In the 

comments section on page 2, make a note if more than one egg was found in that 

clutch. The assigned 12-digit sample ID number should be hand-printed on the

Seabird Data Form as soon as possible after the eggs have been collected (storage and 

shipping times should be added to the forms just before the samples are shipped to the 

processing laboratory). Refer to Figure 7 below for instructions on how to assign the 

identification codes.

Collected by:__Tom Jones______

Date:___July 1, 2009___________

Species:___Common Murre____

Location:___Duck Island______

Egg No.:__________6__________

Comments:_Collected late in season

Figure 5: Pre-printed murre egg labels.

Bagging the eggs

Place each egg in individual plastic bags and seal it with clean non-contaminating tape. 

1) Gently squeeze the air out of the plastic bag before closing it and gently wrap the 

closed bag around the egg.

2) Place the bagged egg into a second plastic bag along with a completed pre-printed 

label.
15

3) Gently squeeze the air out of the second plastic bag before closing it and gently wrap it 

around the egg. The double-bagged, labeled egg is now ready to be packed for 

shipping. Note: squeezing the air out of the bags before sealing them is important—it 

prevents the eggs from rolling around loose in bubbles of trapped air.

Packing the eggs for shipping

Use the Coleman coolers as shipping containers for the eggs. Each cooler is designed to safely 

hold up to 15 murre eggs (Figure 1). If collecting both common and thick-billed murre eggs, use 

the cooler marked “COMU Egg Collection Kit” for the common murre eggs and the cooler 

marked “TBMU Egg Collection Kit” for the thick-billed murre eggs (check the egg labels in the 

kits—they also indicate which species the kit is for).

Each cooler contains 3 large loose pieces of foam padding (about 10 cm thick) labeled as:

Layer #1, Layer #2, and Layer #3, and a foam block glued to the underside of the lid to fill the 

recessed cavity. Pack the bagged eggs in the coolers as described below.

1) Remove foam Layers #1 and #2 from the cooler and leave Layer #3 in the bottom of the 

container.

2) Lay 8 of the doubled-bagged eggs on their sides in the two grooves in layer #3 in the 8 

marked spots. Don’t let the eggs touch each other or the walls of the cooler—try to space 

them about 2.5 cm apart. Put small pieces of foam between the eggs and use some tape to 

secure them in place.

3) Put Layer #2 back in the cooler, grooved side up, and gently push it down onto the first 

layer of eggs to hold them firmly in place.

4) Lay 7 of the doubled-bagged eggs on their sides in the two grooves in Layer #2 in the 7 

marked spots. Again, don’t let the eggs touch each other or the walls of the cooler—try to 

space them at least 2.5 cm apart.

5) Put Layer #1 back in the cooler following the directions marked on it and gently push it 

down onto the second layer of eggs to hold them firmly in place. Layer #1 should stick up 

a little above the lip of the cooler (about 2.5 cm). If it sticks up more than that, remove it 

and try to re-adjust Layer #2 (in other words, gently push it down a little more firmly 

onto the eggs in Layer #3).

6) When the cooler is ready to ship, fill out the enclosed collection form and lay it on top of 

Layer #1 in the container.

7) Close the lid and tape the cooler shut by wrapping 4-5 layers of tape completely around it 

about 15 cm in from each end, just like it was originally taped shut.

8) Tape the pre-printed shipping label securely to the top of the lid, and stick the “Keep 

Cool” and “Handle with Care” labels on the top and sides of the containers. The cooler is 

now ready to ship.

Storing and shipping the eggs

Ship the eggs to Homer, see address below, as soon as possible after collecting them. If that is 

not possible, keep the eggs as cool as possible but do not freeze them. Keeping them stored in a 

refrigerator is best, otherwise, keep them in the cooler and place it outside in a cool shady place.
16

If an egg cracks during packaging and freezer facilities are available (e.g., Cape Lisburne), the 

egg can be salvaged by freezing it as soon as possible and shipping it frozen to the processing 

laboratory. The decision to retain such an egg in the collection will be made by the processing 

laboratory personnel based on the condition of the egg after shipment.

Contact Dave Roseneau at the Alaska Maritime National Wildlife Refuge in Homer, AK, before 

sending the eggs and specify the date the samples are to be shipped as well as the name of the air 

carrier. If the cooler cannot be sent directly to Homer, Dave or Vern will request it to be sent to a 

hub community like Kotzebue, Nome, Bethel, Fairbanks, Anchorage, or Kodiak where they can 

arrange to have it transferred to another air carrier that will bring it to Homer. After the samples 

are shipped, contact Dave again and provide him with the actual date of shipment as well as the 

air carrier’s airway bill number so the shipment can be tracked.

Dave Roseneau,

USFWS Alaska Maritime National Wildlife Refuge

95 Sterling Highway #1

Homer, Alaska 99603-7472

Phone (907-226-4613)

Fax (907-235-7783

E-mail: dave_roseneau@fws.gov

Vern Byrd can be contacted if Dave is not available (phone 907-235-6546, fax 907-235-7783, or 

e-mail <vernon_byrd@fws.gov>). Note: the government phone system will not accept collect 

calls. Please call direct, give them your number, and tell them to call you back. 

Use the air carrier servicing your community that is willing to ship the eggs freight collect if 

possible. Call or e-mail Dave at the number above if there are shipping problems.

Collecting gull eggs

The collection kit for gull eggs consists of 2 plastic Coleman coolers large enough to hold up to 

18 gull eggs each for a total of 36 eggs. 

Some kits also contain disposable cameras for taking pictures of nesting areas and birds. If there 

is a disposable camera in the collection kit, please use it to photograph the nesting area where the 

eggs are collected, as well as the collecting activities. Close up pictures of the birds will help 

confirm the species identity for the long-term database. Send the camera back with the egg 

shipment.

The permit allows the collection of up to 36 gull eggs from a total of 12 nests. Gulls typically lay 

2-4 eggs per clutch. Do NOT take eggs from more than 12 nests and do NOT collect more than a 

total of 36 eggs. Collect the eggs during the first 2 weeks of the laying season starting a few days 

after the birds have finished laying eggs and have completed their clutches. If that’s not possible, 
17

collect the eggs when possible and note how early or late it was compared to when the birds first 

started laying eggs (for example, “right when they first started laying eggs”, “about three weeks 

after they started laying eggs”, “just before the eggs hatched”, etc.). 

Here are some tips when deciding which nests to collect from:

First Choice: In the gull nesting area, look for nests that have 3 eggs in them. Try to find 12 

nests that have 3 eggs in each one of them. In this particular case, the total number of eggs 

collected will be 36 (36 eggs from 12 nests containing 3 eggs each) and the collection will be 

complete.

Second Choice: If 12 gull nests with 3 eggs in each are not observed, take all of the eggs 

from as many 3-egg nests as possible, and then look for nests that have 2 eggs in them. Take 

all of the eggs from enough of the 2-egg nests to meet the sampling quota of 12 nests. For 

example, if only 3 nests that contain 3 eggs are found, 9 more nests that contain 2 eggs each 

will need to be located. In this particular case, if 9 nests containing 2 eggs each are found, the 

total number of eggs collected will be 27 (9 eggs from 3 nests containing 3 eggs each and 18 

eggs from 9 nests containing 2 eggs each).

Third Choice: If it is not possible to find any nests with 3 eggs, try to find 12 nests that have 

2 eggs in each one of them, and collect all of these eggs. In this particular case, the total 

number of eggs collected will be 24 (24 eggs from 12 nests containing 2 eggs each).

Fourth Choice: If only nests with 2 eggs per nest are found, take all of the eggs from as many 

nests as possible and then look for nests that have 1 egg in them. Collect eggs from enough 

of the 1-egg nests to meet the sampling quota of 12 nests. For example, if 5 nests containing

2 eggs each are found (10 eggs), 7 more nests that contain 1 egg each to equal 12 nests will 

need to be collected. In this particular case, if 7 nests containing 1 egg each are collected, the 

total number of eggs collected will be 17 (10 eggs from 5 nests containing 2 eggs each and 7 

eggs from 7 nests containing 1 egg each).

Fifth Choice: If it happens to be a year when nesting conditions are poor and the gulls only 

manage to lay 1 egg per pair, try to collect 12 eggs from 12 nests that contain 1 egg each. In

this particular case, if 12 nests that have 1 egg each are found, the total number of eggs 

collected will be 12 (12 eggs from 12 nests containing 1 egg each). Note: if at least 6 nests 

that contain 1 egg each cannot be found, don’t collect any eggs and wait until the next year.

Also, contact Dave Roseneau and let him know if the birds failed to lay a normal number of 

eggs. 

Always wear a set of gloves provided in the collection kit when handling the eggs. Wearing 

gloves ensures that contamination from gasoline, oil, or insect repellant, possibly on your hands, 

does not come in contact with the eggs. All of these substances can penetrate the egg shells and 

contaminate the egg contents. As a general rule, always try to avoid touching the eggs with bare 

hands.
18

Labeling the collected gull eggs

The collection kit contains pre-printed labels for the eggs. Use the enclosed pencil to fill out 

one label for each egg collected. Do not use ball point pens. When filling out the labels, use a 

different number for each nest where an egg was collected. Number the nests from 1 to 12 in 

collection order and use these numbers to label the eggs. If more than 1 egg is present in a nest, 

use the same nest number for each egg collected from that particular nest. Eggs labeled with 

the same nest numbers were collected from the same clutch of eggs. For example, if nest no. 5 

at the Kukpuk River contained 3 eggs and all 3 were collected, each one of the three separately 

bagged eggs from that nest should have a label with the number “5” written on it like shown 

below in Figure 6.

1) Identify and label the collected eggs individually. The species and location will be 

pre-printed on the labels in the collection kit. Fill in the required information: name,

egg collection date, assigned nest number, and any comments. For example, 

“collected late in the season”, “did not use gloves”, “eggs almost ready to hatch”, 

etc. The labels should be printed using soft to medium pencils (No. 2 or 3) and 

placed between the two plastic bags.

2) Enter the identification codes in the “Sample ID Number” space on the Marine ESB 

Seabird Data Form. This form should be filled out completely as soon as possible 

after the eggs have been collected. Storage and shipping times should be added to 

the forms just before the samples are shipped to the processing laboratory.

Collected by: __Tom Jones______

Date: ___July 1, 2009_________

Species:__Glaucous Gull______

Location: ___Kukpuk River___

Nest No.: _______5___________

Comments: __did not use gloves__

Figure 6: Pre-printed gull egg labels.

Bagging the eggs 

Put each egg in individual plastic bags found in the collection kit (never put more than 1 egg 

in a single bag). After the egg is placed in a bag, please do the following:
19

1) Gently squeeze the air out of the plastic bag and seal the bag closed. Gently wrap the 

closed bag around the egg.

2) Put the bagged egg into a second plastic bag with a completed label.

3) Gently squeeze the air out of the second plastic bag and seal it closed. Gently wrap 

the bag around the egg. The double-bagged, labeled egg is now ready to be packed 

for shipping.

Packing the eggs for shipping

Use the Coleman coolers for shipping containers and carefully pack the bagged eggs in them 

following the directions listed below.

The coolers are designed to safely hold up to 18 gull eggs each. Each cooler contains 3 large 

pieces of foam padding about 10 cm thick labeled Layer #1, #2, and #3, and 1-2 smaller pieces 

of foam glued into the recessed cavity in the underside of the lid (Figure 1). Pack the eggs in the 

coolers as follows:

1) Remove foam Layers #1 and #2 from the cooler and leave Layer #3 in the bottom of the 

container.

2) Lay 10 of the doubled-bagged eggs on their sides in the two grooves in layer #3 in the 10 

designated spots. Do not let the eggs touch each other or the walls of the cooler. Space 

them about 2.5 cm apart. Use pieces of tape to secure the eggs to the foam to keep them 

in place.

3) Put Layer #2 back into the cooler, grooved side up, and gently press it down onto the first 

layer of eggs to hold them firmly in place.

4) Lay 8 of the doubled-bagged eggs on their sides in the two grooves in Layer #2 in the 8 

designated spots. Again, do not let the eggs touch each other or the walls of the cooler. 

Space them at least 2.5 cm apart and use a piece of tape to secure them to the foam.

5) Put Layer #1 back into the cooler following the directions marked on it and gently press it 

down onto the second layer of eggs to hold them firmly in place.

6) Put the shipping form on top of Layer #1.

7) If a disposable camera is sent in the collection kit, fit it into one of the gaps next to the 

foam glued to the underside of the lid and tape it in place.

8) Close the lid and tape it shut by wrapping 2-3 layers of duct tape completely around the 

cooler about 15 cm from each end.

9) Tape the pre-printed shipping label securely to the top of the lid, and stick the “Keep 

Cool” and “Handle with Care” labels on the top and sides of the container. The cooler is 

now ready for shipping (Figure 1).

Keep the packaged eggs as cool as possible (ideally ay 4°C) until they are shipped. If an egg 

cracks during packaging and freezer facilities are available (e.g., Cape Lisburne), it can be 

salvaged by freezing it as soon as possible and shipping it frozen to the processing laboratory. 

Do not discard the egg if it cannot be frozen, but keep it in the shipment. The decision to retain 
20

such an egg in the collection will be made by the processing laboratory personnel based on the 

condition of the egg after shipment.

Ship to Dave Roseneau. Please refer to the murre ‘egg storing’ and ‘shipping’ instructions above. 

PLEASE CONTACT DAVE ROSENEAU OR VERN BYRD BEFORE SHIPPING TO 

HOMER, AK.

EGG PROCESSING PROCEDURES

The updated processing protocol currently being used by AMNWR personnel is summarized 

below. Procedures used to process murre and gull eggs are the same—the only difference is that 

the contents of multi-egg gull clutches are pooled before they’re homogenized (murres lay 1-egg 

clutches).

All processing is done under controlled laboratory conditions by personnel wearing vinyl talcfree gloves using chemically cleaned equipment (e.g., titanium knives; stainless steel blender 

shafts, blades, forceps, and spatulas; glass measuring cups, glass beakers and pipettes) and an

AirClean® Systems positive-pressure laminar flow hood lined with UHV foil and equipped with 

a HEPA 0.3 micron filter to minimize chances of contamination. Egg contents are removed and 

weighed under the positive-pressure hood. Contents put in chemically cleaned measuring cups 

and beakers are covered with clean UHV foil before they’re transferred to a VirTis high-speed 

blender station to be homogenized. The foil covering is kept in place during blending operations 

(blender shafts and blades are inserted into the contents through small slits cut in the foil with 

titanium knives), and the covered containers are returned to the positive-pressure hood before the 

foil is removed and the homogenized contents are pipetted into clean Teflon® jars and cryovials. 

Chemical cleaning of glassware, knives, blender shafts and blades, forceps, spatulas, and other 

processing tools is done under a standard negative-pressure laboratory fume hood lined with 

UHV foil. All of the cleaned items are covered by foil or wrapped in protective layers of it 

before they are transferred back to the positive-pressure hood and used for processing samples. 

The AMNWR laboratory in Homer, Alaska, became fully functional in 2008. It contains both 

NIST and AMNWR supplied equipment. Items provided by NIST include an AirClean®

Systems positive-pressure laminar flow hood, a stainless-steel Interscience BagMixer 400, a 

high-speed VirTis blender with stainless steel shaft and blade capable of turning 30,000 RPM, a 

heat sealer for sealing Teflon bags®, a cordless pipette gun, two serrated titanium knives, a large 

10-L Nalgene container for storing a supply of Type 1 DI water. AMNWR-supplied items 

include a standard negative-pressure laboratory fume hood for working with volatile chemicals; a 

secure chemical storage locker; a Type 1 DI water making system; a drying oven; two batterypowered digital calipers capable of measuring eggs to thousandths of centimeters (e.g., three 

decimal places—0.000 cm); a digital Ohaus scale capable of weighing eggs and their contents to 

hundredths of grams (e.g., two decimal places—0.00 g); several glass 1-L beakers; glass funnels; 

stainless steel tongs, forceps, spatulas, and wire; a 18 cubic foot refrigerator/freezer for 

temporarily storing whole eggs and homogenized samples; and a computer and printer to enter 

and compile data and print data forms, protocols, and address labels.
21

Material Required for Processing Eggs

• Talc-free vinyl gloves

• 356 mm x 406 mm Teflon FEP bags or sheets to provide clean working surfaces

• Lint-free Tex-wipes

• 250 ml beaker to hold Teflon bags used to hold egg contents

• 76 mm x 203 mm Teflon FEP bags, plastic bags, and cardboard labels for storing egg 

shells

• Cardboard shipping tubes

• Labels for exterior of shipping tubes

• Tape for securing exterior labels

• High purity distilled (HP) water or best available water for rinsing samples

• High grade methanol (99 %) (MeOH) for rinsing instruments

• High grade acetone (99 %) for rinsing instruments

• High grade hexane (95 %) for rinsing instruments

• Micrometer, (0 to 25 mm) accurate to 0.01 mm

• Calipers, accurate to 0.01 mm

• Titanium blade knife, serrated

• BagMixer

• Stainless steel hand blender

• Hexane-rinsed aluminum foil

• Lab coats (disposable)

• Balance for weighing samples

• Surgical scissors and forceps

• Insulated gloves, safety glasses, and tongs for handling the liquid nitrogen (LN2) and 

frozen samples

• LN2 in container for freezing samples

• Dewar and lid 

Seabird Data Form

During processing, a 2-page Seabird Data Form must be filled out for each clutch of eggs 

(Appendix 1). Standard biological/environmental information at the time the egg is collected is 

recorded on the first page. The boxes at the top of the first page provide space for the 

NIST/Marine ESB identification numbers. These are assigned to the samples when they arrive at 

the Marine ESB. The additional information to be recorded on the first page is listed below:

1. Sample ID Number: Assign a 12-digit alphanumeric code to the egg and enter this 

code here using the following protocol: The first four characters (letters) identify the 

colony, the next two characters (numbers) identify the clutch (e.g., 01, 02, 03). The 

next four characters (letters) consist of the standard common name acronyms for the 

species the egg was collected from (Appendix 3), and the last two characters 

(numbers) specify the year the egg was collected (e.g., ‘99) (Figure 7). 
22

NOTE: From 1999-2006, the sample ID consisted of 14-digits. The year was written 

out (e.g.1999). In 2008, due to space constraints, the code was shortened to 12-digits 

and only the last two numbers of the sampling year is now written out.

Figure 7: 12-digit Sample ID number.

2. Species: Enter the genus/species name.

3. Geographic Area: Enter the general location where the egg is collected (e.g., Cook 

Inlet, northeastern Chukchi Sea, southeastern Bering Sea, Bering Strait).

4. Lat.&Long.: The latitude and longitude of the nesting colony where the eggs were 

obtained. In most cases, the person receiving the eggs at the processing facility 

determines these coordinates by looking the location up in the USGS Dictionary of 

Alaska Place Names by Donald Orth, or by using USGS place names and Google 

Earth to calculate them. In some cases, they are calculated by using Google Earth in 

conjunction with maps or written descriptions provided by the egg collectors.

5. Colony Name: Enter the common name of the seabird nesting colony where collection

occurs (e.g., Cape Lisburne, St. George Island, East Amatuli Island, St. Lazaria 

Island).

6. Sample Source: General information on who was responsible for obtaining the clutch 

of eggs (e.g., USFWS seabird monitoring program, local subsistence harvest).

7. Site ID Name/Number: Enter the study name or plot number to identify the sections or 

subsections of the colonies where the egg was collected. If numbers or names do not 

exist, they should be created and recorded in the field notes (e.g., West Arch, Spire 

Rock). This name/number code will help ensure that eggs will be obtained from the 

same parts of the colonies each sampling year.

8. Sample Type: The type of tissue collected (e.g., liver, kidney, muscle, feather, blood, 

egg, other).

 STLW 13 TMBU 09

Colony 

Name 

(ex: St. 

Lawerence)

nth clutch

number 

collected

species type 

(ex: thickbilled murre)

Calender 

year that the 

egg was 

collected
23

9. Date and Time of Collection: Record this information as the day, month, year, and 

hour. The month should not be numbered, but should be written in abbreviated form 

(Jan, Feb, Mar, Apr, May, Jun, Jul, Aug, Sep, Oct, Nov, Dec) and the hour should be 

on a 24-hour basis (i.e., 6:00 PM is reported as 1800).

10.Method of Collection: Describe the method used to collect the egg (e.g., “collected by 

hand using vinyl glove”, “collected by local subsistence harvesters without gloves”;

refer to “Collecting murre/gull eggs” section).

11.Date and Time of Death: Record the time of death as day, month, year, and hour, if 

known, using the same format described in No. 9 above.

12.Weather Conditions: Note the general weather conditions (e.g., wind speeds and 

directions, temperature, precipitation) that were present when the egg was collected, 

along with anything that might be pertinent to potential contamination sources (e.g., 

radar sites that may have used or are using large transformers, hydraulic fluids, and 

other chemicals).

13.Field Storage Conditions: Report field storage conditions and the length of time that 

the egg was stored in the field before being shipped to the processing facility.

14.Date and Time Shipped from Study Site: Record the date and time that the egg was 

shipped from the field study site to the processing facility (e.g., “shipped to Kotzebue 

at 1000 hrs on 07/07/2009 on Bering Air, shipped to Fairbanks at 1400 hrs on 

08/11/2009 on Alaska Airlines, shipped to Homer at 1700 hrs on 08/12/2009 on ERA 

Airlines”).

15.Pre-Shipment Storage Conditions: Report the storage conditions at the processing 

facility and the length of time that the egg was stored at the site before being shipped 

to the Marine ESB.

16.Protocol: Indicate the protocol that was used to collect or process the eggs. If the 

“Standard” approved protocol was used, state this. If the protocols were “Modified”

described them in detail.

17.Comments: Enter any additional remarks pertinent to the collection, processing, or 

archiving of the egg. If body tissues (e.g., flesh, bone, feathers) are collected from 

dead seabirds for later analyses, note the date and time they were collected.

18.Additional Samples: A brief comment about other samples that may have been 

obtained from the same sampling site in the same year (e.g., “10 other useable 

samples (clutches) were collected from the same location on the same date”). Also, 

any information that might indicate eggs were collected from the same nest site in 

previous years.
24

19.Collected by: The full name of the person that collected the eggs or a general 

statement about how they were obtained (e.g., “collected by unnamed subsistence 

hunters from Point Hope”.)

The second page of the Seabird Data Form pertains to biological data and information that are 

specific to the egg and the processing of the egg:

20. Sample ID Number: The same 12-digit alphanumeric code found on the first page of 

the form (Item No.1) is entered here in case the two pages become accidentally 

separated.

21. Date and Time of Sample Preparation: Enter the date and start time that the egg 

sample was processed and prepared for LN2 freezing. 

22. Processor: Enter the full name of the individual(s) who processed the sample.

23. Date and Time of Freezing: Enter the date and time at which the egg sample is frozen 

at – 80 °C or below in LN2 vapor (-150 °C) in preparation for shipping to the Marine 

ESB.

24. Date and Time of Shipping to Marine ESB: Enter the date and time that the sample 

was shipped to the Marine ESB. This information should be reported here as day, 

month, year, and hour using the format described in No. 9 above, and should reflect 

the time that the sample left the processing site.

25. Shipper: Enter the full name of the person responsible for shipping the sample from 

the processing site to the Marine ESB.

26. Date and Time Received at Marine ESB: This information is recorded by the receiver 

at the Marine ESB. Enter the date and time that the sample was received at the 

Marine ESB. 

27. Receiver: The full name of the individual that received the sample at the Marine ESB.

28. Egg Measurements: Whole egg measurements (weight, expressed as 000.00 g; length, 

expressed as 0.00 cm); breadth (expressed as 0.00 cm), egg content weights 

(expressed as 000.00 g), and eggshell weights (expressed as 000.00 g).

29. Sample Aliquots: Record the number of polypropylene vials, Teflon jars, and Teflon 

bag (s) that the sample has been aliquoted into (e.g., 6 polypropylene vials—B01P, 

B02P, B03P, BO4P, B05P, B06P; 5 Teflon jars—B01T, B02T, B03T, B04T, B05T; 

Teflon bags—1).

30. Form Prepared by: Enter the full name and signature of the person that filled out the 

2-page form.
25

31. Shipping Information: The shipping address for the form and processed samples are 

listed at the bottom of the second page of the form, including the name and phone 

number of the person responsible for receiving the samples.

Receiving Whole Eggs

When a cooler containing eggs arrives, open it and carefully put the bagged eggs and the 

shipping form in a USPS Priority Mail 12” x 12” cardboard box (2 boxes may be required for 

gull eggs), tape the box shut and write the collecting location, collector’s name, arrival date, and 

number of eggs on its top; place in the STAMP refrigerator.

Measuring Whole Eggs

1) When the eggs are ready to be processed, pick one of the sampling sites (e.g., the 

Sinuk River delta) and take the box(s) that contain the specific eggs out of the 

STAMP refrigerator and open it. Sort through all of the bagged eggs looking at the 

nest numbers written on the egg labels. Assemble a full clutch of eggs (some clutches 

only contain 1 egg and some may contain 2 or 3 eggs), take it out of the box(s). Close 

the box(s), put it back in the refrigerator, and take the clutch of eggs into the lab.

2) Turn the AirClean® Systems positive-pressure laminar flow hood on and leave it on 

during processing. Keep the folding doors closed until you are ready to use it and 

close them between clutches of eggs.

3) Check to make sure that the STAMP Ohaus digital scale is sitting level on a piece of 

UHV foil under the right front corner of the AirClean® Systems positive-pressure 

hood. If it’s not, wipe it off with an alcohol-moistened TexWipe, put it on a piece of 

foil under the right front corner of the hood, and level it by adjusting the legs until the 

level bubble is centered. Note: always check to make sure the bubble is centered and 

the scale is level before using it.

4) After the scale is in place and the hood is on and running properly, follow the 

directions listed below. Note: these directions assume you are working with a clutch 

of 3 eggs; however, in most cases, you’ll be working with clutches that only contain 1 

or 2 eggs, so be prepared to modify the steps accordingly.

A) Using a pencil, fill out the blank seabird data form with the species (glaucous gull, 

thick-billed murre, etc.), colony location (Safety Sound, Norton Sound, etc.), date 

collected, collector’s name, egg processor’s name, the date you froze the samples 

(when the samples were placed in the STAMP freezer), 12-digit alphanumeric ID 

code, shipper’s name, receiver’s name, and any unusual features about the egg 

(e.g. cracked, dented, pitted, discolored).

B) Place one TexWipe per egg on a piece of foil near a sink to help keep the eggs in 

place. 
26

C) Put on a pair of gloves and remove the egg(s) from the plastic bag(s) and place 

them on the TexWipes.

D) Remove the paper labels from the plastic bags and place them with the datasheet. 

Once you remove your gloves, staple them to the data form.

E) Rinse the eggs with DI water then wipe them dry with TexWipes (1 TexWipe per 

egg). Place them back on the TexWipes on the foil in a line, and designate them 

Eggs A, B, and C. Use the convention that the left-most egg is always Egg A, the 

right-most egg is always Egg C, and the egg in the middle is always Egg B.

F) Replace your gloves.

G) Put a piece of TexWipe on the scale and zero it by pushing “TARE”. The 

TexWipe will help keep eggs from rolling off.

H) Place Egg A on the scale (Figure 8), weigh it to the nearest tenth of a gram 

(i.e.,00.0 g), and record the weight on the data form in column A on the whole 

egg (g) line (Figure 9, Appendix 2).

I) Measure Egg A using calipers at its greatest (longest and widest) part (Figure 10).

Turn the calipers on, turn it to “millimeter” mode. The millimeter mode allows 

you to make measurements to 2 decimal places (e.g., 00.00 mm). After measuring 

each egg, convert the millimeter (mm) readings to centimeters (cm) by moving 

the decimal place 1 place to the left (record length and breadth to nearest 

thousandth of a centimeter–i.e., 0.000 cm) and record the information in column 

A on the length and breadth lines (Figure 9).

J) Repeat steps F through I for Eggs B and C and record the data on the appropriate 

lines in columns B and C, respectively (Figure 9).
27

Photo courtesy of Dave Roseneau

Figure 8: Weighing an egg on a TexWipe on a digital scale.

EXAMPLE

IV Egg Measurements: A B C

Whole egg (g) 94.6 g 95.2 g broken – thrown away

Length (cm) 7.391 cm 7.302 cm NA

Breadth (cm) 5.053 cm 5.124 cm NA

Egg contents (g) 80.0 g 85.0 g NA

Figure 9: Filling out Part IV on the Seabird Data Form.
28

Photo courtesy of Dave Roseneau

Figure 10: Measuring a rinsed and dry egg with digital calipers.

Removing Egg Contents from Shells

1) All of the tasks must be performed under the AirClean® Systems positive-pressure 

hood, clean room or equivalent. Wear talc-free vinyl gloves while handling the eggs 

and change gloves between clutches.

2) Remove the contents of each egg using the following procedure:

A) Turn on the master switch on the back of the AirClean® Systems unit, open the 

hood’s folding door to turn the light and filter fan on and let the unit run for about 

30 seconds. Be sure all the chemically clean glassware needed is available under 

the hood. If not, put on a pair of vinyl talc-free gloves, clean the items under the 

negative-pressure fume hood according to the ‘cleaning protocols’ below, wrap 

them in clean pieces of UHV foil, and put them against the back wall of the hood 

behind the working area. Be sure there are several pieces of UHV foil in the hood.

B) Go to the heat sealing station, take the plastic clamp off the heat sealer’s arm and 

turn it on by setting the temperature dial to 4 so it will be ready when needed. 

C) Put on a pair of vinyl talc-free gloves, pick up a clean glass measuring cup from 

the back of the hood, set it down on the left side of the hood about 10 inches back 

from the folding door, and remove the UHV foil covering. 
29

D) Fold a small piece of clean UHV foil firmly over the rim of the cup. It should 

cover about half of the rim and extend 4 cm down the side. 

E) Set the cup on the scale, press the “TARE” button to zero the readout, and return 

the cup to its spot on the foil several inches back from the door.

F) Pick up Egg A, large end up, in one hand; take it to the open hood; hold the cup 

steady with your free hand; and crack it open on the cup’s foil covered rim, just 

like you would crack a chicken egg open on the rim of a mixing bowl (2-3 firm 

taps should do it; rotate the egg slightly between taps to extend the crack about 

1/3 of the way around its widest point).

G) After the shell is cracked, quickly move the egg over the center of the cup, hold 

the large half in your free hand, and break the egg open by applying upward 

pressure and pulling them gently apart. Note—practice cracking open some 

chicken eggs on the foil-covered rim of a measuring cup. Gull egg shells are 

fairly thin and they’re not that much harder to break open than chicken eggs. 

However, murre egg shells are thicker and stronger and they’ll require firmer, 

more forceful taps on the rim of the measuring cup to crack them open cleanly. To 

practice breaking a murre egg open, use an egg from a colony where 15 eggs 

were collected—if contents are lost or pieces of shell fall in it, there will still be 

14 eggs left from the sampling site.

H) Hold the shell halves at steep angles over the cup until the contents have drained 

into it. If some of the albumen persists and clings to the insides of the shells, set 

them down on a clean piece of UHV foil, unwrap one of the clean stainless steel 

spatulas and gently scrape the remaining material into the cup. Try not to tear the 

shell membrane and get pieces of it in the contents.

I) When all of the contents from Egg A are in the cup, take the shell halves to the 

lab sink, rinse them out with Type 1 DI water, put them down on a clean piece of 

foil or TexWipe, and designate them “Egg A”.

J) Go back to the hood and put the measuring cup containing Egg A’s contents on 

the scale (Figure 11) and pencil in the weight on the appropriate line of the data 

form (Figure 9).

K) Push the “TARE” button on the scale to zero it. Place the cup in the open space on 

the left side of the hood.

L) Write the unique 12 character alphanumeric code assigned to the clutch and “Egg 

A” on a paper lunch bag, put the shell halves in it, and set it aside.

M) Repeat steps E through L with Eggs B and C, using the same measuring cup. 

Remember to “TARE” the scale after Egg B is weighed. Note: if the clutch only 

contains one egg, you can skip this step.
30

Photos courtesy of Dave Roseneau

N) After all of the egg contents are in the measuring cup, place the cup back in the 

hood and cover it tightly with UHV foil. Make sure the foil extends about 3 cm

below the rim and put a large rubber band around the cup just above the handle to 

hold the foil in place.

NOTE: If the egg contains an embryo >2cm, put the egg contents into a Teflon bag, heat seal the 

bag, place in a labeled cardboard tube and send to Charleston where it will be 

cryohomogenized.

Figure 11: Egg contents in a glass measuring cup ready to be weighed.

Homogenizing Egg Contents

1) Take the covered cup to the VirTis blender station and clamp it in with the handle 

facing out (make sure the clamps are tight and secure).

2) Remove a foil-wrapped blender shaft with attached blade and a foil wrapped titanium 

knife from the back of the AirClean® Systems hood and take them to the blender 

station.

3) Unwrap the blender shaft/blade and insert it into the blender and tighten the knob 

holding it in as much as you can. Make sure it is tight.
31

4) Lower the blender blade until it’s just above the foil covering the cup. Unwrap one of 

the titanium knives and cut a small slit in the foil slightly longer than the blade and 

widen it slightly with the tip of the knife.

5) Lay the knife back down on its foil wrapping and guide the blender blade through the 

slit in the foil. Lower the blades until it touches the bottom of the cup, and then raise

it up about 1.5 cm and secure it in place by tightening the knob that controls blade 

height. Re-tighten the knob.

6) Fold a small piece of clean UHV foil loosely around the blender shaft to cover the slit 

in the foil (i.e., “tent” the foil around the shaft).

7) Put a pair of safety glasses on, set the blender speed dial to 1, and turn it on (Figure 

12). After it runs for a few seconds, increase the speed to 3, let it run for a few more 

seconds, and then increase the speed to 5 and dial it back to 1. Blending is complete 

when the contents at the bottom are a uniform color and no albumen can be seen 

swirling around. Turn the blender off and reset the dial to 1. If pieces of albumen are 

still present, turn the blender back on and repeat the speed settings one more time, 

turn it off, and recheck the contents—any remaining pieces of albumen should be 

gone. Note: do not over-blend the contents. The longer you blend it, the frothier it 

will become and will make pipetting difficult.

8) Loosen the knob controlling blade height, gently pull the blade up through the slit in 

the foil cover, trying not to rip the foil, and place a small clean piece of foil over the 

hole. 

9) Remove the shaft/blade from the blender and take it and any other tools you used 

(e.g., titanium knives, forceps, spatulas, wire loops) to the sink and put them in a 

beaker of tap water to soak until you’re ready to clean them.

10) Unclamp the foil-covered measuring cup and move it to the work area under the 

positive-pressure hood. Let the homogenized contents stand for a few minutes to let 

any froth that formed dissipate.

Pipetting Egg Contents into Sample Jars and Vials 

1) Take out 5 Teflon® 15 mL jars and labels, neatly print the 12 character alphanumeric 

code assigned to the clutch and “B01T” on the first label, and then repeat this process 

with the 4 remaining labels, numbering them B02T, B03T, B04T, and B05T, 

respectively. Attach the labels to the jars by bending them slightly in the middle and 

sliding the ends under the rims on top of the lids. Note: homogenized samples from 

eggs obtained in 1999-2006 were labeled in a different order. Teflon® jars were 

labeled B01T-B05T followed sequentially by the polypropylene cryovials labeled 

B06P-B10P. This was originally thought to make banking easier, but it didn’t and the 

new numbering system described here was adopted in 2008.
32

2) Take out 5-6 15 mL polypropylene cryovials, depending on how much homogenized 

material there is, and neatly print the 12 character alphanumeric code assigned to the 

clutch and “B01P” in the frosted area of the first vial, and then repeat this process 

with the 4-5 remaining vials, numbering them B02P, B03P, B04P, B05P, and B06P, 

respectively. Put the cryovials in one of the vial holders.

3) Put a 16 cm-wide piece of clean UHV foil in of the hood beside the scale.

4) Put the cryovial holder containing the labeled vials on the foil. Remove the caps from 

the vials and set them open side down on the foil just in front of the holder.

5) Put the labeled Teflon® jars on the foil in a line next to the cryovial holder. Remove 

the lids and set them open side down on the foil by the jars.

6) Place the covered measuring cup containing the homogenized sample to the front of 

the hood near the vials and jars, remove the foil covering, and discard it (don’t reuse 

it).

7) Remove the pipette gun from its protective plastic bag and set it on or near the scale 

under the hood (make sure the scale is turned off).

8) Take a clean glass pipette from the storage area at the back of the hood and slide it 

into the pipette gun. Note: Always keep a supply of clean pipettes wrapped in a layer 

of clean UHV foil at the back of the hood and remember to keep the pipette gun 

charged by plugging it in after each processing session.

9) Set the pipette gun on the negative pressure setting, put the pipette tip about 1 inch 

below the surface of the homogenized sample in the measuring cup, press the trigger 

gently and suck up about 9-10 mL of the liquid, reset the gun to the positive pressure 

setting, put the pipette in the first Teflon® jar, press the trigger gently, and carefully 

fill it about half full. Note: when filling the pipette—press the trigger gently and let 

the liquid flow into it slowly and steadily. Never overfill the pipette because if liquid 

gets into the mechanism that holds the pipette in place, the gun will have to be 

disassembled and cleaned before it can be used again. Avoid sucking up any froth 

that might be floating on top of the sample for the same reason. 

10) Repeat the above process with jars B0T2-B0T5, but fill them to the 15 mL line just 

below their tops.

11) After the jars are full, begin filling the cryovials to the 15 mL line just below their 

tops. Always fill them in sequence (i.e., B01PB06P), in case you run out of sample 

material before you can fill the last vial.

12) After the jars and vials are filled, pull the used pipette from the gun and put it on a 

TexWipe outside the hood. Lay the pipette gun down on a clean piece of UHV foil 

near the hood.
33

13) Screw the lids back on the jars and carefully set them upright in a small tray beside 

the hood. Screw the caps back on the vials and carefully set the holder containing 

them in the tray with the Teflon® jars (Figure 13). Note: keep the jars and vials 

upright until the samples have been frozen.

14) Take the tray containing the aliquots to the freezer. Set the Teflon® jars upright 

along one edge of the freezer and transfer the cryovials to the cryovial holder that’s 

already in place.

15) Bring the tray and empty cryovial holder back to the lab and pencil in the date and 

time the aliquots were frozen on the seabird data form. Pencil in the number of 

aliquots of Teflon® jars and cryovials that were filled on the appropriate lines of the 

data form and list them by their numbers. See Appendix 1—e.g., 5 (B01T,

B02T,B03T,B04T,B05T); 6 (B01P,B02P,B03P,B04P,B05P,B06P). 

16) Take the used pipette to the sink, rinse it out with tap water, and put it in the used 

glass disposal receptacle.

17) Return to the positive-pressure hood. If 10 mL or more of the homogenized sample

remains after filling the jars, do the following: place a Teflon® bag in a 1000 mL 

glass beaker and prop the mouth open with one of the stainless steel wire loops. 

Carefully pour the contents of the cup into the center of the bag without letting it 

touch the loop or run down the sides of the bag. Note: contents on the sides of the bag 

will prevent the heat sealer from sealing them properly. More than 1 bag may be 

needed to contain the remaining material.

18) After the excess contents are in the Teflon® bag, remove the loop, lift the bag out of 

the beaker, squeeze the air out of it, hold it closed, and take it to the heat sealing 

station. Gently pull the top of the closed bag across the unit’s Teflon®-coated 

element until the homogenized liquid in the bottom is about half an inch below it, pull 

the unit’s arm down and hold it closed on the bag for about 3 seconds, release the 

arm, wait a few seconds, and then take the bag off of the element by gently pulling up 

one side. Note: at a temperature setting of 4, hold the unit’s arm down on the bag for 

about 2-3 seconds—any longer could melt the bag. Always separate sealed bags from 

the Teflon®-coated element slowly to avoid tearing the seams open. Practice on an 

empty Teflon® bag if needed.

19) After the bag is sealed, roll it up, put it in a white cardboard cryotube, and neatly print 

the 12 character alphanumeric code assigned to the clutch on the side with a cyropen. 

20) Place the tube(s) in the freezer. Record the number of Teflon® bags used on the 

appropriate line of the seabird data form. 

21) Check over the seabird data form for the clutch of eggs you just processed for any

obvious errors or omissions and file in the data form drawer. When all of the clutches 
34

Photo courtesy of Dave Roseneau

from the same collecting site are processed, use the penciled in versions to make 

complete electronic copies in MS Word.

22) Take the measuring cup and any beakers used to the sink and fill them up with tap 

water until you’re ready to begin cleaning them.

23) Replace the UHV foil under the AirClean® Systems positive-pressure hood and the 

negative-pressure laboratory fume hood with fresh ones.

24) Be sure all of the chemicals, Type 1 DI water, tools, glassware, and pieces of UHV 

foil are replenished to process another clutch of eggs.

Figure 12: Homogenizing egg contents in a pre-cleaned, UHV foil-covered 1-L glass beaker with the highspeed VirTis blender.
35

Photo courtesy of Dave Roseneau

Figure 13: Homogenized egg content aliquots in 5-15 mL Teflon® jars and 6 polypropylene cryovials ready to 

be frozen.

Cleaning Glassware

All glassware and stainless steel tools used to process the eggs must be chemically cleaned under 

the negative-pressure fume hood using the procedures listed below between each new clutch of 

eggs. Always wear safety goggles and talc free vinyl gloves when working with chemicals under 

the hood. 

1) Before cleaning, prepare the negative-pressure fume hood. Turn it on, wipe the work 

surface down with methanol-dampened TexWipes and cover the work surface with a 

layer of clean UHA foil. Wipe down one of the medium-sized Rubbermaid® containers, 

line it with UHV foil, and set it on the foil at the back of the hood. Retrieve the 4-L glass 

waste bottle, methyl alcohol, acetone, and hexane squeeze bottles from the chemical 

storage locker. Fill the labeled squeeze bottles about half full, and return the large bottles 

to the locker. Open the waste bottle and set it aside in the back of the hood. Take a shortstemmed glass funnel and put it in the open mouth of the waste bottle. Tear off about 20 

inches of UHV foil and lay it down near the front of the hood to provide a work area 

(replace this piece of foil with a fresh piece after it has been used a few times). Tear off 

several pieces of foil about 8 inches wide, tear them in thirds, and set them at back of the 

hood. Tear off several more pieces of foil about 12 inches wide and put them in the back 

of the hood. Close the hood and leave it running until cleaning is ready to begin. Note: 

the larger pieces of foil can be torn into smaller pieces, as needed, to wrap clean tools in, 

and the smaller pieces will be used to cover the mouths of the measuring cups after you 

clean them. 
36

2) Using a TexWipe, wash the measuring cups, blades, spatulas, wire loops, and titanium 

knives with tap water and a drop or two of non-phosphate soap, and then give them a 

good rinse under the tap.

3) Rinse the tools and the inside of the measuring cups and beakers with Type I DI water

using one of the squirt bottles.

4) Open the fume hood and place the rinsed items on a piece of clean aluminum foil under 

it.

5) When cleaning stainless steel tools (blender, knife blades, wire loops, shafts, spatulas, 

titanium knives) hold them over a spare glass measuring cup, one at a time, and using the 

squeeze-bottles rinse them with three squirts of methanol, three squirts of acetone, and 

then three squirts of hexane and let them dry on a piece of foil for a few minutes. Pour 

excess liquid into the waste container. Once dry, wrap each in a clean piece of UHV foil 

and place them at the back of the positive-pressure AirClean® Systems hood until they’re 

needed again.

6) To clean the glass measuring cups and glass or polypropylene beakers, hold them upright 

in one hand and use the squeeze-bottles to rinse them out three separate times with 

methanol, acetone, and hexane by squeezing each of the chemicals around them just 

below their rims and letting the chemicals run down inside them. After each threechemical rinse, empty the excess liquid into the waste bottle. Once rinsed, set them 

upright on the foil in the work area, close the hood, and let them dry for a few minutes. 

Once dry, open the hood, cover their mouths with pieces of UHV foil, and take them 

back to the AirClean® Systems hood and store them against the back wall.

7) Back under laboratory hood, pour the waste chemicals into it using the glass funnel and 

close the bottle. Place the STAMP squeeze-bottles and funnel in one of the STAMP trays

and close the hood window down leaving it open a few inches. Let the fan run for about 

10-15 minutes and then turn it off and close the hood all the way.

8) To clean pipettes, hold several of them at a time over a spare glass measuring cup and 

squirt them inside and out with three squirts of the three chemical mentioned above and 

place them in the foil-lined Rubbermaid® container (lean them against the side of the 

container so they’re standing at an angle). Close the hood and let them dry for several 

minutes, then wrap them in UHV foil and take them to the AirClean® Systems hood and 

store them against the back wall.

9) Back under to the laboratory hood, pour the waste chemicals into it using the glass funnel 

and close the bottle. Remove the glass funnel from the waste bottle, hold it over a spare 

measuring cup, rinse it several times with hexane, and set it down on a clean piece of 

UHV foil. Retrieve the 4-L bottle of hexane from the chemical storage locker, put it 

under the hood, remove the cap, insert the funnel, pour the contents of the hexane 

squeeze bottle into it, remove the funnel, replace the cap, and take it back to the storage 

locker. Note: never leave hexane in the squeeze bottle over night because it will 
37

eventually ruin the cap—always leave the cap off of the squeeze bottle after you empty it 

so it can dry out.

Drying and Shipping Eggshells

Once the egg contents are removed, rinse the shell halves out with Type 1 DI water; set them 

open side down on a clean TexWipe and let them drain dry for several minutes. Place the 

eggshells in individual paper lunch bags that has the alphanumeric identification number of the 

clutch and “Egg A”, “Egg B”, or “Egg C” (as appropriate) written on it; close the bag by folding 

the top over and creasing it; and set it aside on the counter top near the drying oven (see 

“Removing contents from shells” section above). Then, as time allows over the next few days, do 

the following:

1) Open the drying oven; Take 12 small TexWipes out of their packet and put 6 of them on 

the top rack and 6 of them on the bottom rack, spacing them about 1 inch apart. Assign 

them the numbers 1-12, using a left-to-right, back-to-front, and top-to-bottom scheme as 

shown below.

Top Rack

Bottom Rack

2) Pick up one of the paper bags containing the shells from one of the eggs, write #1 on it, 

remove the shell-halves, and put them on TexWipe square #1 in the back left-hand corner 

of the top rack (see above).

3) Choose 11 more bags and repeat the above process until there are shells on all of the 

TexWipe squares on both oven racks.

4) Close the drying oven’s door, turn it on, and set the temperature dial to 3.

5) Leave the shells in the oven for 48 hrs. Once dry, refer to the empty paper bags and write 

the eggs’ 12 character alphanumeric identification numbers on 12 clean plastic bags.

6) Remove the shells from the oven, put each in a plastic bag, and put the sealed bags in a 

12” x 12” x 12” USPS Priority Mail box padded with bubble wrap and tape it shut.

7) Repeat steps 1 through 6 with the rest of the clutches.

1 2 3

4 5 6

7 8 9

10 11 12
38

8) Temporarily store the boxes containing the shells in one of the lab cabinets under the 

counter until you are ready to ship them to Dr. Kevin Winker at the University of Alaska 

Museum in Fairbanks, Alaska.

Dr. Kevin Winker

University of Alaska Museum

907 Yukon Drive

Fairbanks, AK 99775-6960

Attn: Kevin Winker

(907) 474-7027

9) When all the shells are ready to be shipped to the museum in Fairbanks, pack the boxes in 

the larger cardboard cartons stored in the lab (four boxes per carton), address them to 

Kevin, put several “Fragile—Handle with Care” and “Priority Mail” stickers on them, 

weigh them, print out the postage on the postage meter, and take the metered cartons to 

the Post Office.

10) Call or e-mail Dr. Kevin Winker to let him know a shipment of eggshells is on the way 

via Priority Mail.

Shipping Frozen Egg Samples

The Marine ESB provides LN2 vapor dry shippers in protective fiberglass containers to transport 

frozen samples to Charleston. When properly charged, these “dry-shippers” will keep samples 

frozen for several days. 

To prepare the samples for shipping, put all of the Teflon® jars and polypropylene vials from 

each clutch into individual Teflon® bags (e.g. one bag should contain 5 Teflon® jars and 5-6 

cryovials) and seal them shut two or three times with the heat sealer. Bag the clutches one at a 

time and run them through the heat sealer as soon as you can to avoid moisture condensing on 

the sides of the bags. If moisture is present, the seams will not seal properly. If this happens, seal 

the bag again 1-2 more times just above the original seam to help keep it from popping open 

when it’s shipped. 

When you’re ready to send samples to the specimen bank, contact Rebecca Pugh in Charleston at

(843)762-8952 (rebecca.pugh@noaa.gov) and ask her to send you a fully charged dry-shipper 

via FedEx. If Rebecca is unavailable, contact Amanda Moors at (843)762-8953 

(amanda.moors@noaa.gov) or Lauren Rust at (843)762-8951 (lauren.rust@noaa.gov). Always 

try to contact Rebecca on Wednesday, so she’ll have plenty of time to charge the shipper over 

the weekend and send it out via FedEx on Monday morning. When it arrives, put the Teflon®

bags containing the samples in it and then arrange to have FedEx pick it up the following 

morning (Wednesday; it must be ready by 9 AM). If it leaves Homer Wednesday morning, 

Rebecca will receive it Thursday afternoon. Do not send shippers to the Marine ESB late in the 

week. If the samples cannot be sent out by Wednesday, call a Marine ESB personnel to discuss 

further options. 
39

The dry-shippers consist of heavy cylindrical fiberglass containers that have LN2 vapor-charged 

flasks secured inside them. The flasks are charged with nitrogen vapor by filling them threequarters of the way up with LN2, letting them stand for 24 hours, refilling them to the half way 

mark, letting them stand for another 3-4 hours, and then pouring off the excess LN2. The 

fiberglass containers containing the charged flasks have hinged lids that latch closed with 

stainless steel clamps. The LN2 flasks are sealed with large foam stoppers (plugs). When you’re 

ready to put samples in a flask, be sure to put on a heavy long-sleeved shirt, sweater, or light 

jacket, have a pair of insulated gloves handy, and follow the directions below (the vapor in the 

flasks is about -70 oC and it will freeze bare skin fast!).

1) Bring the dry shipper into the laboratory and open it, removing the foam stopper. 

2) Wearing cryogloves, gently place the frozen bags containing aliquoted eggs and the 

cardboard tubes containing remaining sample into the dry shipper(s). Leave enough 

room to return the foam stopper.

3) Double check the seabird data forms for completeness and accuracy. Any deviations or 

modifications of the protocol must be noted on the form. Note: please also send the 

datasheets by e-mail/disk.

4) Place a copy of the completed and signed forms in a plastic bag or manila envelope.

Place this bag or envelope between the protective shipping cover and the dry shipper; 

close and latch and secure with cable ties.

5) Fill out a FedEx label and attach it to the top of the shipper. Ship the dry shipper(s) with 

the samples to the NIST Marine ESB in Charleston, South Carolina, within 48 hours or 

as soon as possible using 24-hour express package service to:

NIST Charleston Lab

Hollings Marine Laboratory

331 Fort Johnson Road

Charleston, South Carolina 29412

Attn: Rebecca Pugh

(843) 762-8952

6) Notify the NIST Marine ESB personnel by telephone as soon as possible after the frozen 

samples are shipped:

Rebecca Pugh (843) 762-8952 or

Amanda Moors (843) 762-8953 or

Lauren Rust (843) 762-8951 or

Paul Becker (843) 725-4815

Maximum holding time for the shippers is 10 to12 days. Shipping costs will be paid by NIST. 

Never ship samples late in the week, or before holidays, unless special arrangements have been 

made with the shipping service and the NIST Marine ESB laboratory personnel.
40

Egg Sample Archival Procedures

When the dry-shippers arrive at the Marine ESB, the egg samples are unpacked, checked for 

temperature and packaging problems, and put in temporary cold storage. The seabird data forms 

and samples are compared to ensure that they are complete and correspond with one another. 

After the samples and forms have been checked the samples are ready to be stored in a long-term 

location in the LN2 vapor-phase freezers (-150°C).

When a sample is moved to the long-term freezer storage location, a Marine ESB Storage Form 

(Appendix 2) is completed documenting the archiving process and the specific storage location 

of the sample. Each egg sample is given a unique Marine ESB Storage ID Number (Figure 14), 

according to the format described in Pugh et al. (2007). This number is entered onto the seabird 

data form as well as the storage datasheet to act as a cross reference. In general, the first two 

characters (letters) represent the project (e.g. “ST” for STAMP), the next two characters 

(numbers) represents the year of the project the eggs were collected (e.g. “01” is the first year of 

the project and the eggs were collected in 1999, “02” is the second year of the project and the 

eggs were collected in 2000, etc.). The next character (letter) indicates the tissue type (e.g. “E” 

for egg, “M” for embryo), the following character(s) (number(s)) identifies the next sequential 

number of egg or embryo that has been collected since the project started (e.g. “001” is the first 

egg banked, “250” is the 250th egg banked), and the last character (letter) is always a “C”, 

indicating the eggs are banked in Charleston.

 ST 09 E 1289 C 

Figure 14: Storage ID Number.

The storage datasheet also includes the number of eggs in a clutch (e.g. A, B, and C), their 

corresponding weights, the date they were banked, the date they were homogenized, the storage 

locations of the aliquots and the personnel’s initials. When a subsample is requested, the date the 

subsample is removed is also recorded here. The information above is entered into the 

computerized Marine ESB’s Paradox and Freezerworks databases, and hard copies are filed 

away. After a request for samples is received and approved, Marine ESB personnel will remove 

one 5g egg subsample (Teflon jar or polypropylene cryovial) for each requested sample from its 

LN2 storage location and give them to the requesting parties. When this occurs, the 

corresponding storage forms and computerized databases are updated to include the dates the 

samples were pulled from the freezers, the name of the person receiving them, and a description 

of the analyses that will be run on them. 

STAMP 

Project

nth year of the 

project the egg 

was collected

(2007)

“Egg” 

sample

Egg is 

banked in 

Charleston

nth of egg 

collected 

since project 

started
41

The seabird field datasheet (Appendix 1) containing detailed information from the time the egg 

was collected to the time it is received at the Marine ESB is also entered into Paradox, 

Freezerworks and Excel databases. The field datasheet is scanned, given the file name 

corresponding with the Storage ID number and saved in a secure STAMP folder. The storage ID 

numbers are added to the field datasheet to act as a cross reference; the hardcopy is filed away at 

the Marine ESB, and upon request, will be made available to interested parties. The samples will 

remain in the LN2 vapor-phase storage freezers at approximately -150° C until they are requested 

and released for future analysis.

SUMMARY

This report provides detailed information on the most current updated versions of the protocols 

used to collect, process, and bank seabird egg samples for STAMP. Current plans are to store all 

samples collected to date and in upcoming years in the Marine ESB indefinitely so that they can 

be used by NIST personnel and other researchers to track long-term trends in contaminates in 

Alaska’s marine environments and serve as a source of material for retrospective analyses.

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46

Appendix 1: Seabird Data Form.

SEABIRD TISSUE ARCHIVAL MONITORING PROGRAM

SEABIRD DATA FORM

I Sample ID Number: KUKP01GLGU08 Species: Glaucous Gull

Geographic Area: Eastern Chukchi Sea, Alaska Lat.: 68o 22’ 52.25” N Long.: 166o 19’ 32.20”

W 

Colony Name: Kukpuk River Delta Sample Source: ______________________________ 

Site ID Number/Name:_________________________________________________________

II Sample Type: Liver Kidney Muscle Feather Egg Blood Other: Egg

Date and Time of Death (IfApplicable):________________________________ 

Date and Time of Collection: 07/05/2008 (time unknown)Method of Collection: Gloved

hand

Weather Conditions: Unknown

Field Storage Conditions: Stored outside in cool place until 07/06/2008 and then 

shipped to Anchorage via Kotzebue at about 1100 hrs on 07/07/2008 (Frontier Air). 

Arrived in Anchorage on 07/08/2008 and kept in a cool place in the Frontier hanger until 

they were picked up by ERA Airlines and shipped to Homer about 0700 hrs on 

07/09/2008. They arrived in Homer at about 0750 hrs and were picked up and delivered 

to FWS headquarters and put in the STAMP refrigerator at about 0900 hrs that same 

morning. 

Pre-shipment Storage Conditions: (see above).

Date and Time Shipped from Study Site: Taken to Point Hope via ATV on 

07/05/2008, shipped to Anchorage on 07/07/2008, and then shipped to Homer on 

07/09/2008 (see above).

Protocol: Standard.

Comments: Latitude and longitude calculated using Google Earth (approximate 

center of the river delta—see Google image)

Additional Samples: 14 other useable samples (clutches) were collected from the 

same location on the same date.

Collected by: Elijah Lane
47

III Sample ID Number: KUKP01GLGU08 Storage ID Number: __

Date and Time of Sample Preparation: 07/09/2008 at 1335 hrs. Processor: Karen Corbell

Date and Time of Freezing (-80° C or below): 07/09/2008 at 1520 hrs (about -30o C)

Date and Time of Shipping to MESB: 16 July 2008 at 1000 hrs Shipper: Karen Corbell

Date and Time Received at MESB: ___ Receiver: Rebecca Pugh

IV Egg Measurements: A B C

Whole egg (g) 89.40 g 

Length (cm) 6.951 cm 

Breadth (cm) 4.967 cm

Egg contents (g) 79.40 g 

Egg shell weight (g) 9.70 g 

Comments: 1-egg clutch. The difference between the whole egg weight and the sum of the 

egg contents and shell weight reflects the amount of contents that were lost during processing.

V Sample Aliquots:

Number of Polypropylene Vials: 5 (B01P, B02P, B03P, B04P, B05P)

Number of Teflon Jars: 5 (B01T, B02T, B03T, B04T, B05T)

Number of Teflon Bags: 0

VI 

 Form Prepared by: __________David G. Roseneau ______ ___ (Print Name)

 

 _____________________________________ _ (Signature)

A copy of this form should be shipped with samples to: Rebecca Pugh

Research Biologist

NIST, 331 Fort 

Johnson Road

Charleston, SC 2941
48

Appendix 2: Marine ESB Storage Data Form
49

Appendix 3: Abbreviations for colony sites and bird species.

STAMP Egg Collecting Sites Abbreviation

Cape Lisburne, eastern Chukchi Sea CLIS

Kukpuk River delta, eastern Chukchi Sea KUKP

Cape Thompson, eastern Chukchi Sea CTOM

Noatak River delta, Kotzebue Sound NOAT

St. Lawrence I. northern Bering Sea STLW

Sledge Island, Norton Sound SLED

Safety Sound, Norton Sound SAFE

Cape Denbigh, Norton Sound CDEN

Hooper Bay, eastern Bering Sea HOOP

Triangle Island, eastern Bering Sea NUNI

Shaiak Island, Bristol Bay TOGI

St. George I., southeastern Bering Sea STGE

St. Paul I., southeastern Bering Sea STPA

Bogoslof Island , southeastern Bering Sea BOGO

Aiktak I., eastern Aleutian Is. AIKT

Buldir Island, western Aleutian Is. BULD

East Amatuli I., northern Gulf of Alaska EAAM

Gull I., Kachemak Bay GULL

Duck I., western Cook Inlet DUCK

Kodiak I., northwestern Gulf of Alaska CHBY

Tatitlek vicinity, northeastern Prince William Sound TATI

St. Lazaria I., southeastern Gulf of Alaska STLA

Viesokoi Rock, southeastern Gulf of Alaska SITK

Cape Deceit, Kotzebue Sound DEER

Cape Pierce, Bristol Bay CPEI

Ualik Lake, Bristol Bay UALK

Kikertalik Lake, Bristol Bay KILK

Little Diomede I., Bering Strait DIOM

Shoup Bay, northern Prince William Sound SHBY

Middleton I., northern Gulf of Alaska MIDD

Bluff, Norton Sound BLUF 

Tatoosh I., northeastern Pacific Ocean TATO 

Shishmaref Inlet (2nd East Channel), eastern Chukchi Sea SHFA 

Shishmaref Inlet (Egg I. and Arctic Lagoon), eastern Chukchi Sea SHFB

Shishmaref Inlet (Tin Cr.), eastern Chukchi Sea TINC 

Brevig Mission, northern Bering Sea BREV 

King Island, northern Bering Sea KING 

Sinuk River delta, Norton Sound SINU 

Carolyn I., Golovin Bay, Norton Sound GOBA 

Cape Darby, Norton Sound CDAR

Shaktoolik, Norton Sound SHAK

Unalakleet River delta, Norton Sound UNAL

Stuart I., Norton Sound STUA

Seabird Species Acronyms

Black-legged Kittiwake (Rissa tridactyla) BLKI

Glaucous Gull (Larus hyperboreus) GLGU

Glaucous-winged Gull (L. glaucescens) GLGW

Common Murre (Uria aalge) COMU
50

Thick-billed Murre (U. lomvia) TBMU

Unidentified Murre species (U. spp.) UNMU