United States
                                  Environmental Protection
                                  Agency
                                  Health Effects Research
                                  Laboratory
                                  Research Triangle Park NC 27711
vvEPA
                                  Research and Development
                                  EPA-600/S1 -81 -025  May 1 981
Project Summary
                                  Pilot  Program  for the
                                  National  Environmental
                                  Specimen  Bank—Phase  I

                                 Sally H. Harrison, Rolf Zeisler, and Stephen A. Wise
                                   This work was performed under a
                                 joint NBS/EPA research program to
                                 develop state-of-the-art protocols for
                                 the sampling, storage, and analysis of
                                 biological and environmental-type
                                 matrices. This report summarizes the
                                 procedures used in the initial phase of
                                 a pilot program for determining the
                                 feasibility of the National Environmental
                                 Specimen Bank. A special clean-labo-
                                 ratory/storage  facility  has  been
                                 designed and completed for use in this
                                 program. Detailed protocols for sam-
                                 pling, storage, and analysis of human
                                 liver samples have been designed for
                                 this study. The implementation of
                                 these protocols for human liver sam-
                                 ples is described in this report.
                                   This Project Summary was developed
                                 by EPA's Health Effects Research Lab-
                                 oratory, Research Triangle Park, NC,
                                 to announce key findings of the re-
                                 search project that is fully documented
                                 in a separate report of the same title
                                 (see Project Report ordering informa-
                                 tion at back).

                                 Introduction
                                   In response to the growing concern
                                 for the potential dangers to human
                                 health and the environment by the
                                 increasing influx of man-made sub-
                                 stances in our ecosystem, the U.S.
                                 Environmental Protection Agency (EPA)
                                 and the National Bureau  of Standards
                                 (NBS) are currently studying the feasi-
                                 bility of establishing a National Environ-
                                 mental Specimen Bank  (NESB).  The
                                  program for such a bank would incorpo-
                                  rate a well-defined system of collection,
                                  analysis, and long-term storage of
                                  selected  environmental samples to
                                  provide (1) real-time monitoring data for
                                  pollutant trend analysis and (2) properly
                                  collected and stored samples for retro-
                                  spective analysis.
                                   The National Bureau of Standards is
                                  currently involved in a Pilot Environ-
                                  mental Specimen Bank Program which
                                  will provide actual working experience
                                  in all  stages of the  banking effort:
                                  specimen collection, processing, storage,
                                  analysis, and data management. The
                                  purposes of this pilot study are: (1) to
                                  develop analytical protocols for sampling
                                  and storage of four types of environ-
                                  mental samples, (2) to improve analytical
                                  methodologies for both trace elements
                                  and trace organics, (3) to evaluate the
                                  feasibility of long-term specimen storage
                                  at various conditions (-25°C, -80°C,
                                  -120°C, and freeze-dried at room tem-
                                  perature), and (4) to provide a "bank" of
                                  samples for retrospective  analysis in
                                  future years as analytical methodologies
                                  improve. The experience gained in this
                                  pilot study will be used to evaluate the
                                  feasibility of the NESB Program. The
                                  rationale for this specimen banking
                                  program, a review of the NBS activities
                                  relating to this program, and an outline
                                  of the proposed pilot specimen bank
                                  study at NBS have been described
                                  previously. In these previous reports, the
                                  preliminary plans for sample collection
                                  and storage were reviewed. During this

-------
      Donors 100/Y
       Baltimore,
        Maryland
Donors
Donors
Donors
1OO/Y
Minnesota
                                                     Donors 100/Y
                                                        Seattle,
                                                      Washington
       Teflon Bag
    LN2 Dry Shipper
                             Teflon Bag
                           LN2 Dry Shipper
   Teflon Bag
LN2 Dry Shipper
                                  N B S
                                Receiving Lab
                                                          Section A
                                                      Long Term Storage
                                                       -120°Cto-196°C
    Section B
Storage Evaluation
            Figure 1.    Sample collection scheme for human livers.
initial year of the proposed pilot study,
the collection, processing, storage
evaluation, and trace element analysis
of the first environmental specimens,
human livers, were implemented. These
activities are described in detail in this
report. In addition,  the design of the
clean  laboratory/storage facility is
discussed.

Collection of Liver Samples
  Contracts for procurement of liver
samples were negotiated  and awarded
to three medical schools located in
Baltimore, Maryland; Minneapolis,
Minnesota; and Seattle, Washington.
Each  contractor is providing 100 liver
samples this  year for a  total  of 300
samples (see Figure 1).
  Due to the extremely low levels of
trace elements and organic pollutants in
human liver tissue, the analytical chemist
must  exercise extreme caution to avoid
possible contamination of the sample.
(The lack of contamination control has
made existing banks of samples useless
for general trace constituent analyses.)
A detailed sampling protocol, designed
from the viewpoint of analytical chemists,
is utilized to minimize contamination.
The written sampling protocol outlines
in detail the case selection criteria and
the steps to be followed by the contractor
in obtaining a "valid" sample. The
implementation of  this protocol  is a
                                    result of extensive interaction of NBS
                                    personnel with the contractors to insure
                                    the quality of the samples.
                                      The individual collecting the liver
                                    samples uses (1) non-talced gloves, (2) a
                                    special titanium-bladed knife for bisect-
ing the liver (to avoid contamination
from other elements, i.e., Cr, Ni, etc.), (3)
Teflon sheets upon which the sample is
placed, and (4) high-purity water to
rinse the sample. The samples are then
sealed in Teflon  bags, frozen at liquid
nitrogen temperature, and shipped to
NBS. A data form, sent with each liver
sample, contains information about the
donor and specimen,  e.g., date of birth,
sex, residence, ethnic group, height,
weight, smoker, occupation (if known),
diagnosis of autopsy, date  and time of
death and autopsy, and weight of sample.
Approximately 100 samples have been
received and stored  in the pilot bank
facility at NBS.
  The schemes for the collection and
allocation of subsamples  for storage
evaluation are shown in Figures 1 and
2. The liver specimens are received as
duplicate sections of the left lobe identi-
fied as sections "A"  and "B." All "A"
sections are placed in liquid nitrogen
freezers for long-term storage, and the
"B" sections are used for storage
evaluation, i.e., real-time analysis and
long-term storage evaluation at different
conditions. Approximately 10 percent of
the samples will be homogenized using
a cryogenic brittle  fracture technique.
This homogenization provides about 20
aliquots of 5-8 g  which are stored in
Teflon jars. To solve  the question of
                                                                  Sample Storage
                                                                     Evaluation
                                                                     Cryogenic
                                                                  Homogenization
                                                                     Transfer to
                                                                  20 Teflon PFA jars,
                                                                      6-8g/iar
                                              Figure 2.    Allocation of subsamples for storage evaluation.

-------
•8
•§
    0.2
                 20
                                                                100
                                                                   *- N
                       =N umber of Livers Analyzed
Figure 3.
Probability of detecting change in the concentration due to storage
condition.
appropriate temperature for sample
storage, these sample aliquots will be
stored under four different conditions:
freeze-dried at room temperature, frozen
at -25°C, at -80°C, and in liquid nitrogen
vapor at -120°C to -190°C. The concen-
trations of trace constituents found
after storage under those conditions
will be compared to the data found in the
"real-time" analysis of each specimen
(Figure 2). The "A" sections are reference
samples which  may be used to re-
evaluate results on a particular "B"
section. At the end of the pilot program,
the "A" sections stored at  liquid nitrogen
temperatures will represent a valuable
bank of validated samples available to
the scientific community. In addition, a
large quantity of data, from the analyses
of the "B" sections, will be available on
many of these samples.
  The success of the NESB will be
determined in a large part by the ability
to preserve the  integrity of the trace
substances (i.e., organic,  inorganic, and
organometallic  species) in  samples
during long-term  storage. Changes in
the forms and concentrations of the
numerous environmentally important
substances in specimens stored for
extended periods may occur in several
ways. Processes such as surface ad-
sorption and  sample degradation may
reduce the concentrations of various
components. In addition, continued bio-
chemical and enzymatic activity may
                           produce species which may not have
                           been present in the original sample. On
                           the other hand, contamination of the
                           specimen fluids could lead to apparent
                           increases in trace substances' concen-
                           trations. Superimposed on these
                           processes are the factors which will
                           affect the process  rates, such as con-
                           tainer material, contact time and area,
                           storage temperature, pH, and  initial
                           species concentration. All of these
         factors are important considerations in
         evaluating the suitability of long-term
         storage.
           The probability for the discovery of
         changes in the concentrations of trace
         constituents during storage has been
         assessed by statistical treatment of the
         expected experimental parameters,
         e.g., number of  specimens analyzed,
         homogeneity of subsamples, and ana-
         lytical error (K. R. Eberhardt and L.
         DeRobertis,  Center for Applied Mathe-
         matics, Statistical Engineering Division,
         NBS). Figure 3 consists of a series of
         curves generated from the experimental
         parameters of 5 percent inhomogeneity
         and 5 percent analytical error. These
         curves determine the probability of
         detecting a particular percent change in
         concentration (e.g., 6 and 4 percent for
         curves 1.0 and 0.7, respectively) for a
         given number of liver samples analyzed.
         For example, we would have a 98 per-
         cent probability of detecting a 6 percent
         change (Point A) and 78 percent proba-
         bility of detecting a 4 percent change in
         concentration (Point B) if 30 livers are
         analyzed. If the sample inhomogeneity
         or analytical  error increased  to 10
         instead of 5 percent, the percent change
         detectable at Points A and B would be
         12 and 8 percent, respectively. Based on
         these curves, the number of samples to
         be analyzed was set at 30. Analyses of a
         smaller number of samples would sig-
         nificantly reduce the  probabilities of
         detecting small changes, whereas in-
         creasing the number of samples ana-
                          Table 1.    Number of Livers to be Analyzed for Storage Evaluation Study

                                     Year                80        81      82        83        84
Real Time Analysis 36
1 Year Storage LNZ
-85°C Samples
-25°C from '80
RT
2 Years Storage LNz
-85°C
-25°C
RT
3 Years Storage LN'.
-85°C
-25°C
RT
4 Years Storage LNZ
-85°C
-25°C
RT
30 30 30
12
12 Samples
12 from '83
12

Samples
from '82


Samples
from '81


Samples
from '80

30
30
30
30
30
30
30
30
30
30
30
30
24
24
24
24
                           Livers Analyzed/Year
36
78
30
30
456

-------

1
II
Voltammetry
2 Staff Weeks
For 3 Samples
5 Grams
1
;^^^^^
CU, CD, PB, \
Nl, ZN, (AL) J
^^.^

<


1
IV
Instrumental
2 Staff Weeks
For 3 Samples
1 Gram
I
2 Irradiations
2 Counts
I
/WV, SE, FE, ZN, CO}
\SB, RBJAG, HG, CR>

Human Liver
lection of Half Left Lobe
10 to 15 Grams
Homogenized




Neutron
__i_ J\fli\'at'"n
Analysis
)
(
Che
rSepai

V
Radiochemical
2 Staff Weeks
For 3 Samples
1 Gram
1
2 Irradiations
4 Fractions
4 Counts
1
CU, CD, AS^\
SE, SB )


1
mica/
rations ~™|
|
VI
CHE LEX- 100, INAA
2 Staff Weeks
For 5 Samples
4 Grams
I
2 Irradiations
3 or 4 Counts
I
(V, MN, CU, CD7\
-E, CO, CFt, AC, \
MO, SN, U, (ZN]/
I
III
Atomic Absorption
2 Staff Weeks
For 3 Samples
2 Grams
I
( HG and BE J

                                                                                                            I
                                                CZD
                Figure 4.    Analytical scheme for the determination of trace elements in human livers.
lyzed would not increase greatly the
probability of detecting changes.
  The 30 specimens used for the storage
evaluation represent approximately 10
percent of the specimens collected per
year. A major limitation to this number
is the present analytical capacity. In
order not  to exceed the currently avail-
able analytical capability, most of the
analytical work is deferred to the year
1984, when the program should have a
larger analytical capacity. For the evalu-
ation of the storage of human livers,
Table 1 gives the  analytical  needs in
terms of numbers of livers to be analyzed
each year.
Trace Element and Trace
Organic Analysis
  The trace element analysis activities
of the specimen bank project have
concentrated on three main tasks this
year: (1) development of a comprehen-
sive analytical scheme, (2) evaluation of
the sample collection protocols, and (3)
initiation  of  the analysis of the first
year's collected samples.
  The current analytical protocol is
directed toward the analysis of most of
the first-priority elements  proposed by
the International Workshop on Monitor-
ing Environmental Materials and Speci-
men Banking. Additional elements
occurring as biological major elements
and trace elements in the samples are
included in the protocol because their
levels might supply  information about
the "normal" state of the specimen. The
elements under consideration, their
priority, and the analytical techniques to
be used appear in Table 2. The analytical
scheme (Figure 4) incorporates the NBS
Center for Analytical Chemistry approach
of multi-technique analysis for quality
control. The first three techniques
listed, i.e., atomic absorption spectros-
copy (AAS), anodic stripping voltammetry
(ASV) or linear sweep voltammetry

-------
(LSV), and instrumental neutron activa-
tion analysis (INAA), comprise the
current routine  analytical scheme.
Radiochemical neutron activation anal-
ysis (RNAA) will be performed on a
limited  number of samples to provide
multi-technique quality control. Accord-
ing to the proposed storage evaluation
scheme, 36 livers are being  analyzed
during the first year of sample collection.
  Analytical methods for the analysis of
two classes of organic compounds, i.e.,
organochlorine pesticides and polycyclic
aromatic hydrocarbons (PAH) are cur-
rently being developed. Preliminary
results  have been obtained using the
following analytical procedures: (1) ex-
traction with acetonitrile, (2) liquid-
liquid partition into hexane, (3) concen-
tration by evaporation, (4) high-perform-
ance  liquid  chromatography (HPLC) to
isolate the compounds of interest, and
(5) gas chromatography (GC) analysis on
capillary columns with selective electron
capture  detection.  An HPLC method,
using a chemically bonded amine column
has been developed to  isolate the
organochlorine pesticides and PAH
from  interfering organic constituents.
GC methods have been developed for
the determination of the 16 pesticides
on the priority pollutant list. Research is
continuing on the methodology for
quantitation of these compounds in liver
samples in order to initiate real-time
analyses for organics in 1981. Future
plans include  (1) the evaluation of
homogeneity for organics, (2) the devel-
opment of procedures for comparison of
organochlorine  content determined by
GC and determined by neutron activa-
tion analysis, and (3) expansion of the
pilot program to include development of
analytical protocols for the mussel/oyster
samples.

Safety Requirements
  The handling of human liver samples
carries the potential risk of exposure to
infectious diseases, particularly hepatitis
B virus or "serum hepatitis." Asa result,
certain  precautions are  necessary to
insure protection of the analysts involved
in sample processing and analysis.
  To eliminate potentially infectious
liver specimens from the specimen bank
samples, a blood sample  is removed
from the donor at the time of autopsy to
be used for hepatitis B screening. In
addition, liver specimens from the right
lobe are removed for preparation  of
listological slides. These slides are
examined by the pathologists for evi-
 Table 2.    Analysis of Trace Elements in Human Liver

  Element     Concentration (vg/gj*       Analytical Methodb
Category^
Be
F
Al
V
Cr
Mn
Fe
Co
Ni
Cu
In
As
Se
Rb
Sr
Mo
Pd
Ag
Cd
Sn
Sb
Ba
Pt
Hg
71
Pb
ND
0.06 - 1.4
1.6 - 2.6
< 0.007- 0.09
0.005- 0.27
0.5 - 1.9
70 -210
0.017- 0.16
0.009- 0.32
3.2 - 14.7
31 - 80
0.006- 0.46
0.097- 0.68
7 - 12
0.01
0.4 - 1.6
ND
0.006- 0.07
0.5 - 4.9
0.08 - 0.65
0.01
0.01
ND
0.005- 0.25
0.001- 0.009
0.8 - 2.3
AAS

ASV/LSV
R
INAA
INAA
INAA
INAA
ASV/LSV
ASV/LSV, RNAA
ASV/LSV, INAA
RNAA
INAA, RNAA
INAA

RNAA, INAA

INAA
ASV/LSV. RNAA
R
INAA. RNAA

R
AAS
R
ASV/LSV
1
1
2
1
1
1
2
1
1
3
1
1
1
2
3
1
1
2
1
1
2
3
3
1
1
1
 "Fresh weight, range of concentrations from reference 4.
 ''Techniques used at NBS for Pilot Specimen Bank Program:
   AAS - atomic absorption spectroscopy,
   ASV/LSV - anodic skipping voltammetry/linear sweep voltammetry,
   INAA = instrumental neutron activation analysis,
   RNAA = radiochemical neutron activation analysis.
 C1) First priority element (3);
 2) Trace elements available  with applied methods to monitor "normal" body
   concentrations for non-pollutants.
 3) Trace elements of environmental importance not included in first priority list.
                R - Research initiated    ND = No data available

dence  of  infectious  diseases.  Liver
samples are placed in temporary storage
at liquid nitrogen temperatures  at NBS
until the results of the screening are
received from the hospitals supplying
the livers.
  A Safety Advisory Committee has
been established to advise the  Center
for Analytical Chemistry concerning
suitable safety  precautions for the
handling and processing of these liver
samples at NBS. Recommendations
were made by the committee regarding
procedures to safeguard personnel. The
Safety Advisory Committee will be
requested to review annually the pro-
cedures of the pilot specimen bank
program.

-------
Sally H. Harrison, Rolf Zeisler, and Stephen A. Wise are editors with the Center
  for Analytical Chemistry, National Bureau of Standards, Washington, DC
  20234.
George M. Goldstein is the EPA Project Officer (see below).
The complete report, entitled "Pilot Program for the National Environmental
  Specimen Bank—Phase I." (Order No. PB81 -173 320; Cost: $8.00. subject to
  change) will be available only from:
        National Technical Information Service
        5285 Port Royal Road
        Springfield, V'A 22161
        Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
        Health Effects Research Laboratory
        U.S. Environmental Protection Agency
        Research Triangle Park,  NC 27711
                                                                                t, US GOVERNMENT PfllNTINO OFFICE-1»1 -757-012/7088

-------