OCS Study, MMS 2001-031

NISTIR6735

SEABIRD TISSUE ARCHIVAL AND MONITORING

PROJECT: Protocol for Collecting and Banking

Seabird Eggs

Geoff Weston York

Barbara J. Porter

Rebecca S. Pugh

Da vid G. Roseneau

Kristin Simac

Paul R. Becker

Lyman K. Thorsteinson

and Stephen A. Wise

••e

••

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NIST CENTENNIAL.

NlST

Nation<:d Institute of Standards and Techn.:>logy

Technoio9Y AdminIstration, U.S. Department of Commerce
,

DCS Study, MMS 2001-031

NISTIR6735

SEABIRD TISSUE ARCHIVAL AND MONITORING

PROJECT: Protocol for Collecting and Banking

Seabird Eggs

~USGS scilmce tor" challfJing womJ

Geoff Weston York'

Barbara J. Porter

Rebecca S. Pugh'

David G. Roseneau'

Kristin Simac I

Paul R. Becker'

Lyman K. Thorsteinson"

and Stephen A. Wise2

IU.S. Department of the Interior

U.S. Geological Survey

Alaska Biological Resources Center

2National Institute of Standards and Technology

Chemical Science and Technology Laboratory

3U.S. Department of the Interior

U.S. Fish and Wildlife Service

Alaska Maritime National Wildlife Refuge

4U.S. Department of the Interior

U.S. Geological Survey

Biological Resources Division - Western Office

April 2001

U.S. Department of Commerce

Donald L Evans, Secretary

National Institute of Standards and Technology

Karen H. Brown, Acting Director
CONTENTS

page

List of Tables .iii

List of Figures .iii

Acknowledgments iv

Disclaimer v

INTRODUCTION '" 1

Environmental Specimen Banking 1

Project Goal and Objectives................................................................................................. 2

Banking Seabird Eggs 2

MANAGEMENT SySTEM 6

MATERIALS 8

Materials Required for Collecting Eggs

and Shipping them to the Processing Laboratory... 8

Materials Required for Processing Eggs 8

Materials Required for Packaging Samples

and Shipping Them to the Specimen Bank. 9

Seabird Data Form 9

METHODS 15

Egg Collection Field Procedures 15

Egg Processing Laboratory Procedures. 17

Egg Sample Archival Procedures 21

REFERENCES 22

11
ACKNOWLEDGMENTS

The authors would like to acknowledge the following individuals for their work and support in

developing the Seabird Tissue Archival and Monitoring Project.

Egg collections were made by the following individuals of the U.S. Fish and Wildlife Service,

Alaska Maritime National Wildlife Refuge: Laura Greffenius (Little Diomede Island); Dean Kildaw

(St. George Island); Arthur B. Kettle (East Amatuli Island, Barren Islands); Leslie Slater, Alexis

Paul, and Susan Hatch (St. Lazaria Island). The authors would also like to thank Kevin Winker,

Curator of Birds at the University of Alaska Museum, for archiving the eggshells from the project.

In addition, the authors would like to acknowledge the help and support of the residents of Little

Diomede in collecting eggs at Little Diomede Island and the Minerals Management Service, Outer

Continental Shelf Environmental Studies Program, Anchorage, Alaska, for their support of the

Project, particularly Cleveland Cowles and Steve Treacy.

IV
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.

v
SEABIRD TISSUE ARCHIVAL AND MONITORING PROJECT:

Protocol for Collecting and Banking Seabird Eggs

INTRODUCTION

ENVIRONMENTAL SPECIMEN BANKING

Archiving biological and environmental samples for retrospective analysis is a major component of

systematic environmental monitoring. The long-term storage of carefully selected, representative

samples in an environmental specimen bank is an important complement to the real-time monitoring

of the environment. These archived samples permit:

1. The use of subsequently developed innovative analytical technology that was not available

at the time the samples were archived, for clear state-of-art identification an~ quantification

of analytes of interest,

2. The identification and quantification of analytes that are of subsequent interest but that were

not of interest at the time the samples were archived, and

3. The comparison of present and past analytical techniques and values, providing continued

credibility of past analytical values, and allowing flexibility in environmental monitoring

programs.

1

Seabirds, including albatrosses, pelicans, cormorants, terns, kittiwakes, murres, guillemots, and

puffins spend most of their lives at sea and have special adaptations for feeding in the marine

environment, including the ability to excrete the excess salt obtained from ingesting seawater. Many

species nest in dense groups (colonies) on steep, precipitous sea-cliffs and headlands.

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 (e.g., see Hatch et aI., 1993). Chemical analysis of seabird

tissues, including egg contents, can be particularly useful in determining whether contaminants (and

potential biological effects) associated with human industrial activities, such as offshore petroleum

and mineral exploration and development, are accumulating in marine environments. The collection

and archival of seabird tissues over a period of several years will be a resource for future analyses,

providing samples that can be used to determine historical baseline contaminant levels.

The specimen bank for the Seabird Tissue Archival and Monitoring Project (STAMP) is maintained

by the National Institute of Standards and Technology (NIST) in the satellite facility of the National

Biomonitoring Specimen Bank (NBSB) at the NIST Charleston Laboratory, Charleston, South

Carolina. The Charleston banking facility is designated for marine specimens as part of the National

Marine Analytical Quality Assurance Program. The NBSB design for long-term cryogenic storage,
which has been described by Wise and Koster (l9~5), is a result of 20 years of experience involving

cooperative efforts between NIST and the Environmental Protection Agency (EPA), and several

years of comparative studies with specimen banking programs in Germany, Japan, Sweden, and

Canada. Agencies using the NBSB include EPA, U.S. Department of Agriculture (USDA), Food

and Drug Administration (FDA), National Cancer Institute (NCI), National Oceanic and

Atmospheric Administration (NOAA), and U.S. Geological Survey, Biological Resources Division

(USGSIBRD). Both NOAA and USGSIBRD are primary participants in the use of the NBSB for

marine environmental research and monitoring. The two NOAA programs using the NBSB are the

National Status and Trends Program and the Marine Mammal Health and Stranding Response

Program. The USGSIBRD is collaborating with both NIST and NOAA in banking tissues from

Alaska marine mammals for the Alaska Marine Mammal Ti~sue Archival Project (AMMTAP).

': ~ I ~ • •

• 1[,

PROJECT GOAL AND OBJECTIVES

The goal of STAMP is to archive a representative collection of tissues from Alaskan colonial seabird

species for future contamin~nt 'analyses and documentation of long-term trends in environmental

quality. The Project has two objectives:' .

1. Collect specimens from representative Alaskan colonial seabird species suitable for

determining levels of organic and inorganic contaminants ..

Tissues collected forarchival are limited to freshly.killed birds taken by researchers or subsistence

hunters. Eggs arecollected as part of routine colony monitoring by USFWS personnel and processed

by USGS/BRD personnel according to NIST standard operating procedures (SOP). The reader is

referred to the Materials and Protocols Sections .

r: } ,

2. Transport, catalog, and archive samples in conditi~ms that are suitable for long-term storage

and eventual contaminant analyses.,

+ t- t~ \,( , r •

After collection, samples are packaged, transported, cataloged, and archived according to protocols

established by the NBSB. Storage is in liquid nitrogen vapor at -150 DC,which provides the best

conditions for, minimizing sample degradation. Samples will be available to project partners for

.future contaminant analyses. Requests for archived samples by other researchers and agencies will

< be considered by project partners ~J.1 a case-by-case basis.

, .

BANKING SEABIRD EGGS

, ~.I

'1.

Seabird tissues, particularly eggs, have played important roles in environmental monitoring in

Europe and Canada. The Canadian Wildlife Service (CWS) collects, banks, and analyzes eggs and

tissues from seabirds of the Atlantic and Pacific coasts as part of its Wildlife Toxicology Program.

(See Mineauet aI., 1984; Elliott, 1985; and Wakeford and Kasserra, 1997). Eggs are particularly

.1 .•...• :. '

" .

... ~ . - ,

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2
useful for the temporal and spatial monitoring of persistent organic pollutants (e.g., polychlorinated

biphenyls (PCBs), chlorinated pesticides, dioxins) and mercury. For example, the CWS successfully

documented temporal changes in PCBs and pesticides in the Great Lakes by analyzing banked

herring gull (Larus argentatus) eggs (Mineau et aI., 1984). Also, eggs from alcids (seabirds

belonging to the family Alcidae that includes murres, murrelets, auklets, guillemots, and puffins)

were identified as key indicators for circumpolar monitoring of persistent organic pollutants (POPs)

by all Arctic nations participating in the International Arctic Monitoring and Assessment Programme

(AMAP) Phase II - Years 1998 - 2003 (AMAP Scientific Experts Workshop, Girdwood, Alaska,

April 1998).

The AMAP report on the state of the Arctic environment summarizes knowledge on POPs in

seabirds living in arctic environments. However, the report is limited to the Canadian and

Scandinavian Arctic (AMAP, 1998). It presents data indicating that piscivorous seabirds feeding

at the top of the marine food web (e.g., cormorants, puffins, kitti wakes) have higher levels of PCBs

in their eggs than those feeding at lower levels (e.g., eiders). Levels in seabird eggs were higher in

the Scandinavian Arctic than in the Canadian Arctic and, within Canada, levels were higher in the

high eastern Arctic than in the lower western Arctic. Also, levels of PCBs approaching

concentrations known to affect hatching success were found in thick-billed and common murre (Uria

lomvia and U. aalge), puffin, black guillemot (Cepphus grylle), and black-legged kittiwake (Rissa

tridactyla) eggs from the Canadian and Norwegian regions of the Arctic (AMAP, 1998).

Presently, there are few data on the concentrations of POPs in colonial seabirds nesting in Alaska.

Kawano et aI. (1988) reported chlordane concentrations in thick-billed murres from the North Pacific

and Gulf of Alaska. However, the most comprehensive information on organochlorine residues in

Alaskan seabird eggs was obtained 25 years ago (Ohlendorf et aI., 1982). Extrapolating levels of

POPs from the western Canadian Arctic database is not appropriate, because atmospheric and

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

Alaska and Bering Sea, and the oceanic transport of other substances eastward along the northern

and eastern coasts of Siberia and into the Chukchi and Bering seas probably affect overall

contaminant patterns and levels in Alaskan seabirds. Local sources from existing and former

military installations also have to be considered.

More than 95 % of the seabirds breeding in the continental United States breed at Alaskan colonies

in the Gulf of Alaska and Bering and Chukchi seas (USFWS, 1992), and about 70 % of the Alaskan

birds nest on Alaska Maritime National Wildlife Refuge (AMNWR) lands (G. V. Byrd, personal

communication). In 1998 and 1999, the USGSIBRD, AMNWR, and NIST initiated ajoint program

to develop and test protocols for collecting, processing, transporting, and banking eggs collected

from selected colonies of common murres and thick-billed murres (Uria aalge and U. lomvia) on

AMNWR lands.

Murres spend about 80 % of their time at sea (USFWS, 1992). They are capable of diving to depths

of up to 200 m to capture prey, feeding on a wide variety of small fish, including capelin (Mallotus

villosus) and Pacific sand lance (Ammodytes hexapturus), other osmerids and gadids, sculpins,

3
pricklebacks, shannies, ronquils, and some larger invertebrates (e.g., shrimp, squid). Murres only

. come ashore to breed in large colonies on precipitous sea cliffs and headlands, where they lay single

eggs on bare rock ledges in' dense aggregations. The eggs are relatively large, yielding contents

ranging from 75 g to 95 g, which is sufficient for multiple analyses. Murres are also capable of

laying replacement eggs about 13 to 14 days after losing first eggs.

A protocol for collecting and banking seabird eggs was tested atmurre colonies in the AMNWR in

1'998 - 1999 (Yorket aI., 1999). Six colonies, ranging from the Arctic Ocean (Chukchi Sea) to the

North Pacific (Gulf of Alaska), were selected for routine sampling. These colonies are located at

Cape Lisburne in the Chukchi Sea, Little Diomede Island in Bering Strait, Bluff in Norton Sound

(northern Bering Sea), St. George Isl,andin the southern Bering Sea, Amatuli Island in the northern

Gulf of Alaska, and St. Lazaria Island in the southeastern Gulf of Alaska near Sitka (Fig. 1). During

this pilot phase, the draft collection, sample processing, transportation, and banking procedures were

evaluated for practicality and suitability for obtaining uncoptaminated samples and were modified

where necessary. The revised protocol is found in the Methods section of this report and serves as

i 'the SOP for obtaining future samples. ! i

Using the protocol developed in 1998, murre eggs were collected in 1999 from colonies on Little

'Diomede, St. George, Arnatuli, and St. Lazariaislands. 'The eggs were processed and transported

to the NBSB at NIST in Charleston, South Carolina, where they were archived at temperatures of

-150°C. The egg contents provide a good source of material for conducting preliminary chemical

analyses to establish baseline concentrations of POPs and other contaminants. The results will be

compared among colonies, and with published Alaskan and Canadian seabird data and data from

other species in Alaska. This work represents the first step toward developing a database that will

quantify and document the transfer of persistent organic compounds through seabird food webs in

N~a :

Protocols will-continue to be developed and refined throughout the life of the project as additional

species (e.g., kittiwakes, auklets, guillemots, and petrels) and tissues (e.g., liver, kidney, muscle,

feathers, and blood) are incorporated into it. The eggs of black-legged kittiwakes, Rissa tidactyla,

will probably be incorporated into the project in the near future.: This species is a surface feeder that

preys on small fish, particularly sand lance, capelin, and at higher northern Arctic latitudes, Arctic

-cod (Boreogadus saida). -They also occasionally take invertebrates (e.g., surface schooling

'. euphausiids). As in the case ofmurres, kittiwakes nest in large colonies on coastal and insular seacliffs and headlands. They build grass nests and lay up to 3 eggs (in Alaska, the typical clutch size

is 2 eggs). The eggs are smaller than murre eggs and provide less material for analyses. Work will

be conducted in the future if kittiwake clutches can be combined for banking and analyses and still

meet project objectives. Selected tissue samples may also be analyzed to determine the suitability

of the tissue types for determining levels of inorganic and organic contaminants.

, ,

4
( .... 40'

30'

Atlantic

Ocean .

'50" ..... ~ ~

q

60·

..

St. Lazaria I.

. &00

.... Jopan ~~

.~: ... \f\r.J.: ....•- ~ .-

-, eon •••

". Kur'l

Illondo

15O"E···· ..

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Pacific Ocean

180·

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150"W

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Figure 1. Locations of murre (Uria spp.) colonies selected for egg collections for STAMP

(indicated by .): Cape Lisburne (Chukchi Sea), Little Diomede Island (Bering Strait), Bluff

(northern Bering Sea), St. George Island (southern Bering Sea), Amatuli Island (central Gulf of

Alaska, and St. Lazaria Island (Eastern Gulf of Alaska).

5
MANAGEMENT SYSTEM

The STAMP specimens are collected, transported, processed, and banked by collaborating

researchers from the USGSIBRD, USFWS, and NIST. Eggs are collected by USFWS, Alaska

Maritime NWR personnel during routine monitoring studies at the colonies. The egg processing

laboratory is located at the USGSIBRD Alaska Biological Science Center, Anchorage, Alaska, where

USGSIBRD personnel are responsible.for removing the contents from the shells and shipping the

frozen processed samples to the NBSB at the NIST Charleston Laboratory in Charleston, South

Carolina. The remaining eggshells are shipped to the University of Alaska in Fairbanks, where they

are archived at the University of Alaska Museum.

, .

Eggshell Archiving University of Alaska Museum

Fairbanks, Alaska

University of Alaska - Fairbanks

K. Winker

After arrival at the NIST Laboratory, the samples are checked, cataloged, and banked by NBSB

personnel. The NBSB cataloging and archiving procedures are consistent with those employed by

NIST for the banking of marine mammaltissues for the Marine Mammal Health and Stranding

Response Program (MMHSRP) and Alaska Marine Mammal Tissue Archi val Project (AMMT AP)

(Becker et aI., 1997)

A list of seabird colonies, individuals collecting the eggs, and individuals responsible for specimen

processing and banking is shown in Table 1.

, ,

Table 1. Responsibilities for collecting, processing, and banking eggs from Alaskan seabird colonies for

STAMP.

Activity Location Responsibility ,

Egg Collecting Alaska Maritime NWR USFWS

Cape Lisburne, Chukchi Sea D. Roseneau, G.V. Byrd

Little Diomede 1.,Bering Strait D. Irons

. Bluff, Northern Bering Sea . ._- E. Murphy, G.V. Byrd

S1. George 1., Southern Bering Sea A. Sowls, G.V. Byrd

Amatuli I., Central Gulf of Alaska A. Kettle, G.V. Byrd

S1. Lazaria I., Eastern Gulf of Alaska L. Slater, G.V. Byrd

Egg Processing Alaska Biological Science Center

Anchorage, Alaska ,

USGS Biological Resources Division

. G. Weston York

K. Simac

Egg Content Banking NBSB. NIST

Charleston, South Carolina

NIST

R. Pugh

6
Requests for banked samples by researchers that are not associated with STAMP will be considered;

however, release of the tissues to outside investigators will be contingent upon the approval of

STAMP program managers from each of the collaborating agencies. Sample release will depend on

a determination that a surplus of requested sample material exists beyond anticipated sampling or

analytical needs of STAMP. Requests for samples must include a clear and concise statement ofthe

proposed work and be consistent with the goal and purposes of STAMP. The following specific

information should be included in the request for samples:

1. Name of principal investigator and affiliated research or academic organization

2. Specific sample and quantity desired

3. Explanation of proposed research to be conducted, including funding source

4. Justification for use of banked tissue

5. Research facility where analyses will be conducted

6. Analytical quality control procedures to be used and agreement to participate in NISTdirected interlaboratory comparison exercises for quality assurance (QA) purposes

7. Estimated date for completion of research, and schedule/date of subsequent reports

8. Agreement that all results and findings, including analytical data, be provided to the NBSB

and STAMP (this includes a data submission schedule)

9. Agreement that credit and acknowledgment will be given to USGS, USFWS, NIST, and

STAMP for use of banked tissues. The following acknowledgment will be inserted in all

publications, abstracts, or presentations:

The samples used in this study were provided by the National Biomonitoring Specimen Bank

at the National Institute of Standards and Technology and the Seabird Tissue Archival and

Monitoring Project (STAMP). STAMP is operated through the collaboration of the United

States Geological Survey, the United States Fish and Wildlife Service. and the National

Institute of Standards and Technology.

Shipping charges will be borne by the individual or institution requesting the samples. Additional

charges to cover costs associated with sample preparation and distribution may also be required.

STAMP will publish annual tissue inventory reports. These reports will provide information on the

status of seabird egg samples stored at the NBSB and the results of any chemical analyses that may

have been conducted on them.

7
8

" MATERIALS

The following materials are provided by the NBSB, unless noted otherwise.

I _

MA TERIALS REQUIRED FOR COLLECTING EGGS I .

AND SHIPPING THEM TO THE PROCESSING LABORATORY

I • " .

• Data recording forms

• Talc-free vinyl gloves .l.

,

• Teflon FEP (fluorinated ethylene propylene) bags for transporting individual eggs from field

• Tape for sealing Teflon bags . .

• Ziplock bags

• 'Water resistant cardboard egg 'labels ' c,

1

• 'Plastic containers (e.g., 5-gaIIon [18.9L] paint buckets); one container per 5 to 10 eggs

• 'Ovring sealing lids with two 12 mm holes per lid (for plastic container)

• 'Packing material (e.g., styrofoam peanuts) for plastic container

• 'Shipping labels .

MATERIALS 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, ziplock bags, and cardboard labels for storing egg shells

• Cardboard shipping tubes . i

• Labels for exterior of shipping tubes

• Tape for securing exterior labels '.J

• 'I L bottle containing high purity distilled (lIP) water or best available water for rinsing

samples

• 'I L bottle high grade ethanol (95 %) (EtOH) for rinsing instruments

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

• Calipers, accurate to 0.01 mm

• Titanium blade knife, serrated

• Teflon spatula for emptying egg contents

• Lab coats (disposable)

• 'Balance for weighing samples

• Surgical scissors and forceps

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

samples

• LN2 in container for freezing samples

• Dewar and lid

'Items provided by the processing laboratory or cooperating individuals.

-',
• Dry shippers (LN2

-cooled) with protective shipping covers

• Shipping labels

MATERIALS REQUIRED FOR PACKAGING SAMPLES

AND SHIPPING THEM TO THE SPECIMEN BANK

SEABIRD DATA FORM

An example of the data recording form is provided on pages 13 to 14. This 2-page form must be

filled out for each egg. Standard biological/environmental information is recorded on the first page

(page 13). The boxes at the top of this page provide space for the NIST identification codes. These

are assigned to the samples when they arrive at the NBSB. Other information to be recorded on this

page is listed below:

1. Sample ill Number: Assign a 14-digit alphanumeric code to the egg and enter this code here

using the following protocol: The first four letters (e.g., see below) should be used to identify

the colony and the first two numbers should be used to identify the egg (e.g., 01, 02, 03). The

next four letters should consist of the standard common name acronyms for the species the

egg was collected from (e.g, COMU = common murre, TBMU = thick-billed murre, BLKI

=black-legged kittiwake). The last four numbers should be used to identify the yearthat the

egg was collected (e.g., 1999). Examples of the alphanumeric codes that should be used for

the six study sites listed in Table 1 are shown here: Cape Lisburne (CLlS), e.g.,

"CLISOITBMUI999"; Little Diomede I. (LIDI), e.g., "LIDI02TBMU2000"; Bluff (BLUF),

e.g., "BLUF03COMU2001"; S1.George I. (STGE), e.g, "STGEOIBLKII999"; S1.Lazaria

I. (STLA), e.g, "STLA02COMU2000"; East Amatuli I. (EAAM), e.g,

"EAAMO1BLKI2002". All questions regarding proper labeling, including assigning

alphanumeric codes, should be directed to the NBSB contact listed on page 14.

2. Species: Enter the genus/species name.

3. Geographic Area: Enter the most common name of the general location where the egg is

collected. Egg collectors should be as specific as possible ( e.g., Cook Inlet, northeastern

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

4. La1. Long.: Record the latitude and longitude of the location where the egg is collected.

Latitudes and longitudes should be reported to the nearest tenth of a minute, if possible.

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

occurs (i.e, Cape Lisburne, S1.George Island, East Amatuli Island, S1.Lazaria Island).

6. Sample Source: Record any information pertinent to the agencies, persons, and research or

management programs responsible for egg collections (e.g., USFWS seabird monitoring

program, local subsistence harvest).

7. Site ill NamelNumber: Enter a name or number code to identify the sections or subsections

9
of the colonies where the egg was collected. If numbers or names do not exist, they should

be created and recorded in the fieldnotes (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. Date and Time of Collection: Record this information 'asthe 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-hourbasis (i.e.,

6:00 PM is reported as 1800).

9. Method of Colle~tion: D~scrib~. th~ method u~ed to coIlec'i the egg (e.g., collected by hand

using vinyl gloves, collected by local subsistence .harvesters without gloves; see Egg

Selection and Handling section). I' - -' ,

" ~. ·t

10. Date and Time of Death: If body tissues (e.g., flesh, bone, feathers) are collected from dead

seabirds for later analyses, record the time of death as day, month, year, and hour, if known,

• • l •

using the same format described in No.8, above. ,

, ";' e )

•... .' -j -.' , •

11. 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

.?ave used or are using large transformers, hydraulic fluids, an~ other chemicals).

-;, .., I' l

12. Field Storage Conditions: Report field storage conditions a~d the length oftime that the egg

was stored in the field before beingshipped to the processing facility.

~ ~ I ~ . , • ,.". l

I I

13. 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. This information should be recorded here

."' •• J

as day, month, year, and hour using the .same format described in No.8, above. I

14. Post-Processing 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 NBSB.

.' !• ~ ~... ~j " - .~. ••.•.. .< -

, I •. ,. ,

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

approved protocol was used, state this. If modifications were made, described them in detail.

'" r •. tl - ,

16. Com~ents: Enter any additional remarks pertinent to the collection, processing, or archiving

of the egg.- .' -.

17. Additional Samples: Note if other types of tissue samples wer~ obtained that were related

to the collected egg (e.g., body tissues from a dead member of the nesting pair). Information

should include types of samples, their purpose, and names and locations of individuals

receiving the samples. This space should be used to report if the egg was part of a multi-egg

clutch that was collected that year (e.g., in the case of kittiwakes): If it was, the identification

codes for the other eggs collected from the clutch should be noted here. This space should

also be used to report if the egg was collected from a nest site that was known to have been

10
used as a sampling site in previous years (i.e., a fact suggesting that the egg may have come

from the same breeding pair).

On the second page of the data recording form (page 14) enter data and information that are specific

to the egg:

18. Sample ID Number: The same 14-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.

19. Date and Time of Sample Preparation: Enter the date and time (start time) that the egg was

processed and placed in a container in preparation for LN2freezing. This information should

be reported here as day, month, year, and hour using the format described in No.8, above.

20. Processor: Enter the name of the individual(s) who processed the sample for LN2freezing

and packed and shipped it to the NBSB.

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

- 80 ·C or below in liquid nitrogen vapor. This information should be reported here as day,

month, year, and hour using the format described in No.8, above.

22. Date and Time of Shipping to NBSB: Enter the date and time that the sample was shipped

to the NBSB. This information should be reported here as day, month, year, and hour using

the format described in No.8, above, and should reflect the time that the sample left the

processing site.

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

processing site to the NBSB.

24. Date and Time Received at NBSB: Enter the date and time that the sample was received at

the NBSB. This information should be entered as day, month, year, and hour using the

format described in No.8, above.

25. Receiver: Enter the name of the individual receiving the sample at the NBSB.

26. Histological Samples: Record any information on any related samples that were collected for

histological slides. This information should include: the Individual/Organization making the

slides, the Final Destination of the slides, the kind of Tissues Sampled, and the method of

sample preservation (if appropriate).

27. Sample Weight: Record the weight of the sample (whole egg and eggshell contents).

28. Egg Measurements: Record the length and breadth of the egg, and the eggshell thickness as

per the laboratory protocol (pages 17 to 18).

29. Form Prepared by: Enter the name of the person that filled out the 2-page form.

11
.1

All of the appropriate information for express shipping of the samples is provided in the lower right

hand comer of the second page. This includes the full address Ofthe NBSB, as well as the name and

telephone number of the individual responsible for receiving the samples .

., ! . ,

..

"

, ,

I;.

"

. , ,

12
'---- __ I I I I~ __

SEABIRD TISSUE ARCHIVAL MONITORING PROGRAM

NATIONAL BIOMONITORING SPECIMEN BANK

SEABIRD DATA FORM

I

Sample ID Number: ---'- Species: _

Geographic Area: Lat.: Long.: ~ __

Colony Name: ---'-~Sample Source: _

Site ID Number/Name: _

II

Sample Type: Liver Kidney Muscle Feather Egg Blood Other _

Date and Time of Death (If Applicable):---------------- _

Date and Time of Collection: Method of Collection: _

Weather Conditions: _

Field Storage Conditions: _

Pre-shipment Storage Conditions: _

Date and Time Shipped from Study Site: _

Protocol: Standard Modified (Please note modification below)

Comments:

Additional Samples:

Collected by: _

13
III Sample ID Number: ""--- '___

Date and Time of Sample Preparation: Processor: _

Date' and Time of Freezing (~80° Cor belowje , _

,

Date and Time of Shipping to NBSB:_. .: Shipper: _

Date and Time Received at NBSB: Receiver: -'

IV Histological Samples:

IndividuaVOrganization:~'___..:.....;..__'_'_.:..__ __=_---------...:.-"------.:.---

.

Final Destination: __ -.,.. -'-- ___.:._ __

Tissues Sampled: _

V

Sample Weight: A

CJ

CJ

B .. 1' '; CJ""

CJ

Egg (whole) 0

Egg (contents) 0

Liver

Kidney

Egg Measurements: . Length :,'--'___'____c.m .. Breadthe j, c.m

Eggshell Thickness: mm (mean often measurements)

VI

Form Prepared by: -.,.. _

j- (print Name)

(Signature)

A copy of this form should be shipped with samples to: Rebecca Pugh

Research Biologist

NIST

219 Fort Johnson Road

Charleston, SC 29412

(843) 762-8647

14
METHODS

The seabird egg collection and banking protocol developed by STAMP for the NBSB is based on

protocols 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. This protocol is also consistent with those

used by the NBSB for the AMMTAP and MMHSRP (Becker et aI., 1988; 1991; 1999).

Taking into consideration field conditions during egg collections, the intent of the protocol is 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 NBSB, including storage under liquid

nitrogen vapor at -150°C. A generalized scheme for the protocol used by STAMP is presented in

Figure 2.

EGG COLLECTION FIELD PROCEDURES

Selecting and Handling Eggs

Ten eggs are collected from each target species at the designated nesting colonies. To avoid

obtaining eggs with large embryos, collections should be made as early as possible during the laying

period and preferably no more than 14 days after the first eggs are laid at the colonies. Collections

should be made under conditions that will minimize sample degradation prior to shipping and

storage. For species that lay single eggs (e.g., common and thick-billed murres), eggs suspected of

being replacement eggs should not be collected. For species that normally lay multiple eggs (e.g.,

kittiwakes), complete clutches will be collected whenever they contain more than one egg. The

following procedures are used to handle the eggs:

1. Remove eggs from the nest using talc-free vinyl gloves. Immediately, place each egg in

individual Teflon bags. Seal each Teflon bag with clean non-contaminating tape and place

the sealed bag containing the egg in two individual ziplock bags (i.e., one inside the other).

2. Identify and label eggs individually. Fourteen-digit alphanumeric identification codes (refer

to page 9) should be hand-printed on water resistant paper labels cut from "Rite in the Rain"

field notebook paper and placed inside the outer-most ziplock bag (i.e., the label should

reside between the two ziplock bags containing the Teflon-bagged egg). The labels should

be printed using soft to medium pencils (No.2 or 3) or indelible ink (pencil is preferred

because it is not as vulnerable to alcohol and other solvents as is "permanent" ink).

3. Enter the identification codes in the "Sample ID Number" space of the NBSB Seabird Data

Form (refer to pages 9 and 10). 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).

15
Field Collection:

10 eggs per species per colony

Refrigerate )

., t ~ ,

Transport Whole Eggs to Processing Laboratory ,

. Alaska Biological Science Center, Anchorage, Alaska r

I

.

. Whole Egg Measurement:

Length, Breadth, Mass

I ..»:

Egg Harvest:

Removal of Contents I ~

; Egg Contents: Eggshell:

Determine Mass Measure Eggshell Thickness and Mass

Place in Teflon Bag and Heat Seal . Place in Teflon Bag and Heat Seal

Place Teflon Bag in Cardboard Tube Maintain at < 20 ·C

I I

Freeze in Liquid Nitrogen I

Transport to Archive

Place in Liquid Nitrogen Dry Shipper University of Alaska Museum, Fairbanks, Alaska

I

Transport to the National Biomonitoring Specimen Bank

NIST Charleston, South Carolina

I

Archival:

> Log into ASTRO Database

Cryogenic Homogenization ' 1

.

Sample A SampleB ,

, .

50"10 50"10 ,

Store at ·150 ·C Store at ·150·C ,

I

"

'IAnalysis

, .

"

I

,

Figure 2. Generaliz~d scheme for the egg collection and banking protocol used by STAMP.

".1" r.,

"

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16
1. Carefully pack individually bagged eggs from the same colony in solid plastic containers

with enough shock- resistant packing material (e.g., styrofoam peanuts, crumpled paper, or

dry grass) to protect them during shipping. The plastic containers that are provided by the

processing laboratory or cooperating individuals (Table 1), consist of new, clean 5-gallon

(18.9 L) paint buckets with o-ring sealing lids or double-walled plastic cooler-type totes. If

buckets are used, they will have two approximately two 12 mm holes drilled in the lids to

allow for air pressure changes during shipment by aircraft. Two buckets (5 eggs per

container) or one tote (10 eggs per container) will be required to safely transport 10 eggs.

4. Refrigerate the eggs or keep them cool until they are shipped to the processing laboratory.

Transporting Eggs to the Processing Laboratory

2. Keep the packaged eggs as cool as possible (ideally at about 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 it ifit can not be frozen, but keep it in the shipment. 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.

USGSIBRD

Alaska Biological Science Center

1011 East Tudor Road

Anchorage, Alaska 99503

Attn: Geoff York

(907) 786-3928

3. Ship packaged eggs and Seabird Data Forms to:

EGG PROCESSING LABORATORY PROCEDURES

The seabird egg processing procedures are based on USFWS, Portland Field Office, SOP:F-003

protocol. Egg processing should take place' within a laboratory under the cleanest conditions

available. At a minimum, processing should be done in an enclosed covered area free of obvious

sources of potential contamination such as cigarette smoke, fuel oil fumes and smoke, and laboratory

chemicals such as formaldehyde. The work area should be thoroughly cleaned to remove dust and

other residues, and work surfaces covered with Teflon sheeting before processing begins. Processing

the eggs consists of six steps: measuring whole eggs, collecting egg contents ("egg harvest"),

measuring eggshell thickness, archiving eggshells, packaging and freezing egg contents, and

shipping frozen egg contents (egg sample) to the NBSB.

Measuring Whole Eggs

1. Wear talc-free vinyl gloves while handling the eggs and change gloves between each egg.

17
2. If possible, "candle" the eggs by placing each in front of a bright light source to determine

the approximate stage of embryo development and to determine if cracks are present in the

shell.

- .

3. Store the eggs in a refrigerator if they cannot be processed immediately after they arrive

at the laboratory. DO NOT FREEZE the eggs 'unless they are already cracked because

freezing whole unbroken eggs will crackthe shells. ' ,

\'

4. Clean each intact egg with a lint-free cloth and HPLC~grade distilled-deionized water that

is at or nearthe temperature of the egg. Change cloths between eggs.

_ .l',,. t : ."

~ ':~. ." ,r~ ~ ~'

5: Record any unusual features (e.g., cracked, dented, pitted, ordiscolor~d shells; unusual

sizes or shapes).

6. Weigh (g) each whole egg and record this value on the Seabird D~ta Form.

7. Measurethe length (ern) and breadth (ern)' of each egg <vith calipers at their greatest,

longest, and widest points and enter these measurements on the Seabird Data Form. To

obtain accurate measurements of length, make sure that the caliper jaws are parallel to the

longitudinal axis of the egg. For breadth, hoid'the jaws perpendicular to the"longitudinal

axis of the egg.

,'. ,,'

8. Carefully rinse each egg again with HPLC-grade water following the measurements and

allow them to air dry; , --' .

Collecting Egg Contents (Egg Harvest)

".t • . ',. /

1. Wear talc-free vinyl gloves while handling the eggs and change gloves between eggs.

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

a. Line a clean 250 mL beaker with a clean 76 mm x 203 mm Teflon bag. Place it on a

scale and tare (g). t

b. Place the egg lengthwise on a clean Teflon sheet. Using a knife with a titanium blade,

gently score around the egg (about its equator) using the serrated titanium blade knife.

Apply gentle, steady pressure and make several rotations around the egg. Once through

the shell, insert the tip of the k~iie blade to cut the membrane and begin separating the

two halves. Carefully cut 1/2 to 2/3 the distance around the egg. Invert the egg while

puIling apart the shell halves and pour the contents into the opened Teflon bag.

c. If necessary use a clean Te'flon spatula to scrape any remaining contents into the jar

(BE CAREFUL not to tear the shell membrane when using the spatula).

d.' Rinse the spatula with HPLC-grade water followed by 95 % EtOH after each use and

- '. , I •

allow to air dry. ,-

e. Weigh the beaker containing the Teflon bag with egg contents and record the tared

weight under EGG (contents) on the Seabird Data Form.

f. If the egg contains an embryo, carefully remove the embryo from the separated shell

" halves. Embryo and egg contents, if any, are stored separately. Length of the embryo

" 18
(crown - tail) is recorded.

g. Heat-seal the Teflon bag. Place it in another Teflon bag along with a "Rite in the Rain"

field notebook paper label containing the egg contents, mass, and identifying sample

number, and heat-seal the second bag.

Measuring Eggshell Thickness

1. Wear talc-free vinyl gloves while handling the eggshells and change gloves between

eggshells.

2. Rinse shell halves with HPLC-grade water, taking care not to tear the membrane. Shell

halves should be stored in a cool dry place for a minimum of 30 days to attain a constant

weight prior to taking additional measurements.

3. Weigh dried eggshells and record this measurement on the Seabird Data Form.

4. Measure eggshell thickness with a micrometer having rounded contacts. The micrometer

contacts should be rinsed with HPLC-grade water followed by a EtOH rinse between the

eggshells of the individual eggs. The measurements should be made at five separate

equidistant points around the circumference of both shell halves and 0.5 em from the cut

edge that separated each half.

5. Record the mean of the 10 measurements on the Seabird Data Form for each egg. If the

membrane is absent or has separated from the shell, note this fact in the Comments section

on the form.

Archiving Eggshells

1. . Place the eggshells of each egg (the two halves) in a small Teflon bag and heat-seal it.

Next, place the Teflon bag and a "Rite in the Rain" field notebook paper label containing

the sample number, eggshell thickness, and mass data inside a small ziplock bag. Maintain

the bagged shells at <20°C until they are ready to be shipped to the University of Alaska

Museum in Fairbanks, Alaska, where they will be permanently archived.

2. Package the eggshell samples and a copy of their respective Seabird Data Forms in a

durable container filled with appropriate shock-resistant material and ship to:

University of Alaska Museum

907 Yukon Drive

Fairbanks, AK 99775-6960

Attn: Kevin Winker

(907) 474-7027

19
Packaging and Freezing Egg Contents Samples '

1. Recordthe egg contents sample number and mass on the label designed for the cardboard

shipping tube. Place the double-bagged egg contents sample in an individual cardboard

tube and affix the sample label to the outer cardboard tube with the wide clear tape.

2. Freeze the labeled cardboard tubes containing the samples by immersing them in LN

z

for

10 minutes. IfLN2 is not immediatelY,available, freeze the samples in a laboratory freezer

capable of maintaining a temperature of -80°C or lower. (Special Note: LNzshould not be

stored in sealed containers. Personnel handling LNzshould wear boots, cuffless trousers,

non-absorbent aprons, loose insulating gloves, and safety glasses) .

.~;

Shipping Frozen Egg Samples

Frozen samples are transported in liquidnitrogen vapor dry shippers that are provided by the NBSB.

These' shipping containers will keep specimens 'frozen for 2 days to 5 days, depending on proper

. "charging" and proper handling of the containers. To properly charge a dry shipper, fill the container

% of the way with liquid nitrogen and let it stand for 24 hours. Refill the shipper 1f2 of the way and

let it stand for approximately 3 hours to 4 hours then pour. off the excess LN2• This is required to

fully saturate the absorbent material that is located in the lining of the container. The dry shipper

is now ready to receive samples.

1. •Place the frozen cardboard tubes containing the samples in the dry shipper.

2. Double check the Seabird Data Forms for completeness and accuracy. Any

deviations or modifications of the protocol must be noted on the form.

I' .

3. Place a copy of the completed forms in a ziplock bag or manila envelope.

Place this bag or envelope between the protecti ve shipping cover and the dry

,shipper; close and latch the shipping cover.

4. Ship the dry shipper(s) with the samples to the NBSBat NIST in Charleston,

South Carolina, within 48 hours or as soon as possible after sample collection

using 24-hour express package service to:

., . • !

• i .,'

NIST '.

219 Fort Johnson Road

.Charleston,.'

South, Carolina .:29412

Attn: Rebecca Pugh

(843) 762-8647 ,

The dry shipper(s) must not contain excess 'LNz when shipped, i.e., all LN

z

must be

absorbed in the container lining. Maximum holding time for the shippers is 10 days toI2

days. Shipping costs will be paid by NIST. Never ship samples late in the week, i.e.,

Thursday, or Friday, or before holidays, unless special arrangements have been made with

the shipping service and NBSBINIST laboratory personnel.

20
5. The NlSTINBSB personnel should be notified by telephone as soon as possible after the

frozen samples are shipped:

Rebecca Pugh (843) 762-8647 or

Paul Becker (843) 762-8503

EGG SAMPLE ARCHIVAL PROCEDURES

After the samples have been received at NIST, they are transported to the NBSB facility. The dry

shippers are unpacked and the egg contents samples are inspected for packaging problems and for

proper temperatures. 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 assigned an NBSB number, stored in liquid nitrogen vapor freezers, and logged into the

Archival Specimen Tracking Retrieval Operations (ASTRO) database and Paradox database.

Before a sample is moved to its permanent, long-term freezer storage location, it is cryogenically

homogenized using a cryogenic procedure designed to reduce the likelihood of changes in sample

composition due to thawing and refreezing (Zeisler et aI., 1983). The resulting homogenate is then

divided into several aliquots depending on the species (e.g., B001, B002, B003; contents of common

and thick-billed murre eggs average about 80 g and one egg from these species provides about 16

5-g aliquots). Some of the aliquots from each sample are stored in different freezers to provide

additional long-term security. Half of the aliquots from each sample are placed in long-term storage

and the other half are available for immediate and near-term analyses.

When a sample is moved to the long-term freezer storage location, a storage form is filled out

documenting the archiving process and the specific storage location ofthe sample. This information

is also entered into the computerized NBSB Paradox and ASTRO databases.

The samples will remain in the liquid nitrogen vapor storage freezers at approximately -150 ·C until

they are requested and released for future analysis. Detailed information describing each sample and

its source is recorded and maintained in the NBSB Paradox database. Hard copies of this

information will also be kept in the NBSB files, and upon request, will be made available to

interested parties.

21
·/ REFERENCES

AMAP. 1998. AMAP Assessment Report: Arctic Pollution Issues. Arctic Monitoring and

Assessment Programme (AMAP), Oslo, Norway.

Becker, P. R., S. A. Wise, R J. Koster and R. Zeisler. 1988. Alaskan Marine Mammal Tissue

Archival Project: A Project Description Including Collection Protocols, NBSIR 88-3750, National

Bureau of Standards, Gaithersburg, Maryland .

..

Becker, P.R., S. A. Wise, B. J. Koster and R. Zeisler. 1991. Alaskan Marine Mammal Tissue

Archival Project: Revised Collection Protocol, NISTIR 4529, National Institute of Standards and

Technology, Gaithersburg, Maryland.-, .•.

, . l .

Becker, P.R., S.A. Wise, L. Thorsteinson, RJ. Koster, & T. Rowles. 1997. Specimen banking of

marine organisms in the United States: current status and long-term prospective. Chemosphere.

34(9/10): 1889,.1906. . "I, - l • ,

.'

Becker, P. R., B. J. Porter, E. A. Mackey, M. M. Schantz, R. Demiralp,and S. A. Wise. 1999.

National Mammal \ Tissue Bank and Quality Assurance Program: Protocols, Inventory, and

Analytical Results, NISTIR 6279, National Institute of Standards and Technology, Gaithersburg,

Maryland.

Byrd, G. V., E. C. Murphy, G. W. Kaiser, A. Y. Kondratyev, and Y. V. Shibaev. 1993. Status and

ecology of offshore fish-feeding alcids (murres and puffins) in the North Pacific. In The Status,

"Ecology, and Conservation of Marine Birds of the North Pacijic,JK Vermeer, KT. Briggs, KH.

Morgan, and D. Siegel-Causey, eds. Canadian Wildlife Service, Ottawa .

. .

Elliott, J. E. 1985. Specimen banking in support of monitoring for toxic contaminants in Canadian

wildlife. In: International Review of Environmental Specimen Banking, S.A. Wise and R. Zeisler,

eds. U.S. Department of Commerce, National Bureau of Standards: Spec. Publ, 706.4-12.

, ,

Hatch, S.JCA.,B: D. Roberts, and B. S. Fadely. 1993. Adult survival of black-legged kittiwakes,

Rissa tridactyla, in a Pacific colony. Ibis 135:247-254.

Kawano, M., T. Inoue, T. Wada, H. Hidaka, and R. Tatsukawa. 1988. Bioconcentration and residue

patterns of chlordane compounds in marine mammals: invertebrates, fish, mammals and seabirds.

Environ. Sci. Technol.22:792-797.

Mineau.P.; G.A. Fox, RJ. Nordstrom, D.V. Weseloh, D.J. Hallett, andJ.A. Ellentonl. 1984. Using

the Herring Gull to monitor levels of organochlorine contaminants in the Canadian Great Lakes. In:

Toxic Contaminants in the Great Lakes, J. O. Nriagu andA. S. Simmon, eds. Chapter 19

Nordic Council of Ministers. 1~95. Nordic Environmental Specimen Banking - Methods in Use in

ESB. Manual for the Nordic Countries. Copenhagen, Denmark.

22
Ohlendorf, H. M., J. C. Bartonek, G. J. Divoky, E. E. Klaas, and A. J. Krynitsky. 1982.

Organochlorine Residues in Eggs of Alaskan Seabirds. U.S. Fish and Wildlife Service. Special

Scientific Report - Wildlife No. 245, Wash. D.C. 41 p.

I •

I

U.S. Fish and Wildlife Service. 1992. Alaska Seabird Management Plan. U.S. Fish and Wildlife

Service, Divsion of Migatory Birds. Anchorage, Alaska.

U.S. Fish and Wildlife Service, Portland Field Office. 1993. Bird egg collection, measurement,

preparation, and shipment for residue analysis. SOP: F-003

Wise, S. A. and B.J. Koster. 1995. Considerations in the Design of an Environmental Specimen

Bank: Experiences of the National Biomonitoring Specimen Bank Program. Environ. Health

Perspect. 103 (Suppl. 3): 61-67.

Wakeford, B. J., and M. T. Kasserra. 1997. The relationship between the Canadian Wildlife Service

Specimen Bank and the Wildlife Toxicology Program: the effect on specimen collection.

Chemosphere 34 (9/10): 1933-1938.

York, G. W., D. Roseneau, P. Becker, and B. Porter. 1999. Protocols and Procedures for Collecting

and Banking Seabird Eggs. Draft - March 15; 1999. In: National Marine Mammal Tissue Bank and

Quality Assurance Program. 1998 Report, Volume 1. Pp. 88 - 101. NIST, Gaithersburg, MD.

Zeisler, R., J. K. Langland, and S. H. Harrison. 1983. Cryogenic homogenization procedure for

biological tissues. Anal. Chern. 60: 2760-2765.

23