-------
Table 61
Final Set of Multiple Regression Analyses
Dependent Variable = In (blood lead)
Backward Stepwise
MAXR Stepwise
6. (3)+soil, 234
dust
0.270
0.0556 0.0452
soil
0.0952 0.0208
0.270
soil
0.0952
Model*
n
r-square
b**
u-value
r-square
b
o-value
1. Soil
358
0.170
0.2058
0.0001
0.170
0.2058
0.0001
2. Dust
246
0.247
0.2888
0.0001
0.247
0.2888
0.0001
3. Air+@
161
0.184
0.1420
0.0001
0.184
0.1420
0.0001
4. (3)#+soil
356
0.198
soil
0.198
soil
0.1430
0.0001
0.1430
0.0001
air
air
0.0649
0.0083
0.0649
0.0083
5. (3)+dust
246
0.276
dust
0.276
dust
0.2353
0.0001
0.2353
0.0001
air
air
0.0556 0.0452
0.0208
dust
0.2015 0.0001
dust
0.2015
0.0001
7. (3)+soil, 234
dust,
areal
area2
0.282 dust
0.2232 0.0001
air
0.3304 0.0040
0.282
dust
0.2232
areal
0.3067
0.0001
0.0064
areal
-.8398 0.0110
area2
0.2478
0.0040
^independent variables used in model construction
**the unstandardized regression coefficient for air lead, for the
natural log transformations of soil lead or dust lead, or for area, as
identified in the table.
0PBHOBBY, ST0RMWIN, AGES, AGES_SQ, INCOME, M0UTH1*, MOUTH2*, CPH0R,
C0RIFL, SMOKET, VITAMINT, and WASH. (* Variables used to construct
these principal component vectors were: EATSNOWT, ORAL, CPUFDT,
CORIFU, CORIOT, CORIPA, and CORISW.)
//the variables in the most predictive model from the preceding step,
i.e., SMOKET and AIR.
77
-------
Table 62
Definitions of Variables Used in Final Set of
Multiple Regression Analyses (Table 61)
Variable Definition Type
Soil
Soil lead level
Continuous
Dust
Dust lead level
Continuous
Air
Air lead level
Continuous
Areal
Home not in Area 2 or in Area 3
Categorical
Area2
Home not in Area 1 or in Area 3
Categorical
PBHOBBY
Absence or presence of lead hobby in home
Categorical
STORMWIN
Absence or presence of storm windows
Categorical
AGES
Child's age
Continuous
AGES-SQ
Child's age squared
Continuous
INCOME
Family Income
Categorical
M0UTH1
Eigenvector (see Table 60)
Continuous
MOUTH2
M •• tl *«
Continuous
CPOHR
Daily number of hours of outdoor play
in the neighborhood
Continuous
CORIFL
Daily number of hours of indoor play
on the floor
Continuous
SMOKET
Absence or presence of smoker in home
Categorical
VITAMINT
Use or nonuse of vitamins, minerals,
or other dietary supplements
Categorical
78
-------
Table 63
Testing for the Influence of Hand-to-Mouth Activity
Dependent Variable = In (blood lead)
Backward Stepwise
MAXR Stepwise
Mode13 n r-square b p-value r-square b p-value
234 0.282
0.0001
0.282
0.0001
LN(DUST)
AIR
SMOKET
AREA1
AGEMARK*
0.2232 0.0001
0.3304 0.0040
0.1184 0.0439
-.8398 0.0110
0.2232 0.0001
0.3304 0.0040
0.1184 0.0439
-.8398 0.0110
246 0.293
0.0001
0.293
0.0001
LN(DUST)
AIR
SMOKET
AREA1
0.2296 0.0001
0.3178 0.0047
0.1194 0.0377
-.7812 0.0158
0.2296 0.0001
0.3178 0.0047
0.1194 0.0377
-.7812 0.0158
AGEMARK**
^Independent variables used in model construction.
*AGEMARK is an interaction term between (1) a categorical variable whose
value 1 denotes age less than or equal to 2 years and whose value 2 denotes
age greater than 2 years and (2) the natural log transformations of soil
lead levels. Although included in the model construction, AGEMARK did not
appear as a significant contributor to the dependent variable, i.e., the
natural log transformations of blood lead.
**AGEMARK is an interaction term between (1) a categorical variable whose
value 1 denotes age less than or equal to 2 years and whose value 2 denotes
age greater than 2 years and (2) the natural log transformations of dust
lead levels. Although included in the model construction, AGEMARK did not
appear as a significant contributor to the dependent variable, i.e., as the
natural log transformation of blood lead.
79
-------
Table 64
Final Multiple Regression Results For Predicting Children's
Blood Lead Levels
Ln Blood Lead (n =
234; r2 = 0.28)
Parameter
P#
Variable*
Estimate
F statistic
ALPHA
0.3328
LN (DUST)
0.2232
27.62
0.0001
AIR
0.3304
8.46
0.004
AREA 1
-0.8398
6.57
0.011
SMOKET
0.1184
4.11
0.044
*ALPHA = Y-axis intercept.
LN(DUST) a Natural logarithm of lead level found in grab sample for household vacuum
cleaner.
AIR = Area-specific ambient air lead level.
AREA 1 = Home is in Area 1.
SMOKET = Someone in household smokes.
^Significance level. Variables are considered to be statistically significant
contributors to log-transformed blood lead when p is less than 0.05.
80
-------
Table 65
Mean Levels of Metals in Blood, Urine, and Hair Samples From Children by Area*
Blood
Urine
Urine
Hair
Hair
Hair
Cadmium
Lead
Arsenic
Lead
Cadmium
Arsenic
Area
(ng/ml)
(ng/ml)**
(ng/ml)**
(ug/g)
(ug/g)
(ug/g)
1
0.50 (16)
2.6 (17)
9.8 (17)
15.5 (13)
0.83 (13)
772 (12)
3
0.42 (9)
1.0 (16)
7.0 (16)
6.5 (16)
0.62 (16)
258 (8)
T test
0.44
0.17®
0.60®
0.02®
0.28
0.09®
Probability"*"
*Sample sizes are in parentheses.
**Standardized to 10 mg creatinine/dl.
"•"Probability that the mean levels are essentially the same in both areas.
Statistically significant differences are considered to exist when p is less
than 0.05.
^Corrections have been made for unequal variances.
-------
Table 66
Mean Levels of Metals in Children's Blood, Urine, and Hair Samples
According To Household Storm-window Use*
Children Living in Children Living
Households Without in Households With
Area Sample** Storm-Windows Storm-Windows p#
BCD
0.46
(7)
0.53
(9)
0.55
UPB
2.16
(6)
.88
(11)
0.74
UAS
1.5 2
(6)
14.3
(11)
0.11®
HPB
19.2
(4)
13.9
(9)
0.62
HCD
0.74
(4)
0.87
(9)
0.71
HAS
688
(4)
813
(8)
0.82
BCD
0.0
(1)
0.48
(8)
A
UPB
2.49
(4)
0.55
(12)
0.35®
UAS
11.3
(4)
5.6
(12)
0.18
HPB
4.2
(3)
7.0
(13)
0.27
HCD
0.31
(3)
0.70
(13)
0.2O
HAS
0.0
(1)
295
(7)
A
*Sample sizes are in parentheses.
**BCD = Blood cadmium (ng/ml).
UPB = Urine lead (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (mg/ml), standardized to 10 mg.
HPB = Hair lead (ug/g).
HCD = Hair cadmium (ug/g).
HAS = Hair arsenic (ug/g).
^Probability that the mean levels are essentially the same in both as analyzed by the
Student's t test. Statistically significant differences are considered to exist when
is less than 0.05.
^Corrections have been made for unequal variances.
A All values are the same for one class level; no t test performed.
82
-------
Table 67
Mean Levels of Metals in Children's Blood, Urine, and Hair Samples
According To Play Surface Type*
Children Who Children Who
Played Mainly Played Mainly on
Area Sample** on Grassy Surfaces Nongrassy Surfaces P#
BCD
0.63
(3)
0.48
(13)
0.45
UPB
0.69
(3)
3.04
(14)
0.08®
UAS
11.1
(3)
9.6
(14)
0.91
HPB
21.2
(3)
13.8
(10)
0.60®
HCD
0.88
(3)
0.82
(10)
0.88
HAS
512
(3)
858
(9)
0.58
BCD
0.60
(1)
0.40
(8)
A
HPB
0.22
(3)
1.22
(13)
0.42
UAS
4.49
(3)
7.61
(13)
0.52
HPB
6.36
(3)
6.56
(13)
0.94
HCD
0.49
(3)
0.67
(13)
0.46
HAS
399
(1)
238
(7)
A
*Sample sizes are in parentheses.
**BCD = Blood cadmium (ng/ml).
UPB = Urine lead (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
HPB » Hair lead (ug/g).
HCD = Hair cadmium (ug/g).
HAS = Hair arsenic (ug/g).
^Probability that the mean levels are essentially the same as analyzed by the
Student's t test. Statistically significant differences are considered to exist when p
is less than 0.05.
^Corrections have been made for unequal variances.
A All values are the same for one class level; no t test performed.
83
-------
Table 68
Mean Levels of Metals in Children's Blood, Urine, and Hair Samples
According To Absence and Presence of Household Member Who Smoked*
Children Who Children Who
Lived With Lived With One
Area Samples** No Smoker or More Smokers
BCD
0.41 (9)
0.61
(7)
0.10
UPB
1.85 (10)
3.73
(7)
0.38
UAS
11.8 (10)
7.0
(7)
0.65
HPB
13.2 (6)
17.5
(7)
0.52
HCD
0.65 (6)
0.99
(7)
0.29
HAS
384 (6)
1159
(9)
0.15®
BCD
0.37 (7)
0.60
(2)
0.047®
UPB
1.15 (12)
0.70
(4)
0.69
UAS
6.67 (12)
8.09
(4)
0.74
HPB
7.25 (13)
3.36
(3)
0.12
HCD
0.70 (13)
0.28
(3)
0.15
HAS
122 (6)
665
(2)
0.03
*Sample sizes are in parentheses.
**BCD = Blood Cadmium (ng/ml).
UPB = Urine lead (mg/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
HPB =» Hair lead (ug/g).
HCD = Hair cadmium (ug/g).
HAS = Hair arsenic (ug/g).
^Probability that the mean levels are essentially the same as analyzed by the
Student's t test. Statistically significant differences are considered to exist when p
is less than 0.05.
^Corrections have been made for unequal variances.
84
-------
Table 69
Mean Levels of Metals in Blood and Urine
Samples from Adults by Area*
Blood Urine Urine
Cadmium Cadmium Arsenic
Area (ng/ml) (ng/ml)** (ng/ml)**
I 0.93 (15) 0.56 (16) 4.44 (16)
3 1.05 ( 6) 0.32 (16) 5.60 (16)
T Test 0.74 0.26® 0.65
Probability#
*Sample sizes are in parentheses.
**Standardized to 10 mg creatinine/dl.
//Probability that the mean levels are essentially the same
in both areas. Statistically significant differences are
considered to exist when p is less than 0.05.
^Corrections have been made for unequal variances.
Table 70
Area-Specific Mean Adult Ages and Lengths of Continuous
Residence in Homes Where Interviews Were Conducted*
Mean
Age
Mean Length of
Residence
Area
(Years)
(Years)
1
61.6 (17)
31.6 (17)
3
62.3 (16)
24.0 (16)
T Test
Probability #
0.90
0.31
~Sample sizes are in parentheses
//Probability that the means are essentially the same in both areas.
Statistically significant differences are considered to exist when p
is less than 0.05.
85
-------
Table 71
Mean Levels of Metals in Adult Blood and Urine Samples According To
Their or Their Spouse's History of Having Worked in a Lead-Related Industry*
Area
Sample**
Adults
With
Lead-Job
History
Adults
With No
Lead-Job
History
p#
1
BCD
0.77 (9)
1.17 (6)
0.34@
UCD
0.51 (9)
0.63 (7)
0.77
UAS
6.26 (9)
2.09 (7)
0.24
3
BCD
— (1)
1.05 (6)
A
UCD
0.23 (1)
0.32 (15)
A
UAS
0.0 (1)
5.97 (15)
A
*Sample si2es are in parentheses.
**BCD a Blood cadmium (ng/ml).
UCD = Urine cadmium (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
//Probability that the mean levels are essentially the same in both
areas, as analyzed by the Student's t test. Statistically
significant differences are considered to exist when p is less than
0.05.
(^Corrections have been made for unequal variances.
A All values are the same for one class level; no t test performed.
86
-------
Table 72
Mean Levels of Metals in Adult Blood and Urine Samples According To
Yard-Working Habits*
Adults Who
Worked in Adults Who
Their Yards and/ Did No
or Their Neighbor's Yard
Area Sample** Yards Work p#
1
BCD
0.94
(14)
0.70
(1)
A
UCD
0.59
(15)
0.15
(1)
A
UAS
4.57
(15)
2.45
(1)
A
3
BCD
1.47
(3)
0.63
(3)
0.37
UCD
0.37
(11)
0.22
(5)
0.51
UAS
6.04
(11)
4.62
(5)
0.74
*Sample sizes are in parentheses.
**BCD = Blood cadmium (ng/ml).
UCD = Urine cadmium (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
#Probability that the mean levels are essentially the same in both areas,
as analyzed by the Student's t test. Statistically significant
differences are considered to exist when p is less than 0.05.
A All values are the same for one class level; no t test performed.
87
-------
Table 73
Mean Levels of Metals in Adult Blood and Urine Samples According To
Habits of Eating Neighborhood-Grown Fruits and Vegetables*
Area
Sample**
Adults
Who
Ate
Neighborhood
Produce
Adults
Who Did
Mot Eat
Neighborhood
Produce
P#
1
BCD
0.93
(15)
(0)
A
UCD
0.60
(15)
0.06
(1)
A
UAS
4.66
(15)
1.11
(1)
A
3
BCD
1.27
(3)
0.83
(3)
0.66
UCD
0.38
(9)
0.24
(7)
0.49
UAS
4.66
(9)
8.01
(7)
0.28
*Sample sizes are in parentheses.
**BCD = Blood cadmium (ng/ml).
UCD = Urine cadmium (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
^Probability that the mean levels are essentially the same in both areas,
as analyzed by the Student's t test. Statistically significant
differences are considered to exist when p is less than 0.05.
-------
Table 74
Mean Levels of Metals in Adult Blood and Urine Samples
According To Habits of Eating Fish Caught Locally*
Adults Adults
Who Who Did
Ate Locally Not Eat Locally
Area Sample** Caught Fish Caught Fish
1
BCD
0.83
(7)
1.01
(8)
0.57®
UCD
0.38
(7)
0.71
(9)
0.39
UAS
6.65
(7)
2.71
(9)
0.32®
3
BCD
0.70
(2)
1.22
(A)
0.61
UCD
0.09
(2)
0.37
(13)
0. AO
UAS
6.99
(2)
5.81
(13)
0.85
*Santple sizes are in parentheses.
**BCD = Blood cadmium (ng/ml).
UCD = Urine cadmium (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
//Probability that the mean levels are essentially the same in both areas,
as analyzed by the Student's t test. Statistically significant
differences are considered to exist when p is less than 0.05.
^Corrections have been made for unequal variances.
89
-------
Table 75
Mean Levels of Metals in Adult Blood and Urine Samples According
To Current Cigarette-Smoking Habits*
Area
Sample**
Adults
Who
Currently
Smoked
Adults
Who Did
Not Currently
Smoke
P it
1
BCD
1.53
(3)
0.92
(5)
0.49®
UCD
1.30
(3)
0.29
(5)
0.32®
UAS
0.82
(3)
1.98
(5)
0.61
3
BCD
1.50
(3)
0.70
(2)
0.50
UCD
0.63
(7)
0.09
(5)
0.02®
UAS
7.76
(7)
2.98
(5)
0.34
*Sample sizes are in parentheses.
**BCD = Blood cadmium (ng/ml).
UCD = Urine cadmium (ng/ml), standardized to 10 mg creatinine/dl.
UAS = Urine arsenic (ng/ml), standardized to 10 mg creatinine/dl.
//Probability that the mean levels are essentially the same in both areas,
as analyzed by the Student's t test. Statistically significant
differences are considered to exist when p is less than 0.05.
(^Corrections have been made for unequal variances.
90
-------
Ł
APPENDIX 1
STATE OF MONTANA
Department of Health and Environmental Sciences
HELENA—A public meeting to explain the scope and aims of the "Superfund" study
of East Helena children's exposure to lead will be held at 7 p.m. Tuesday, August 2
in the East Helena fire hall.
A canvass began last week to identify households in East Helena, and the control
area in northeast Helena, that contain children from one to five years of age. All
families in such households will then be asked to participate in the study.
Qualified medical and technical personnel from the Lewis and Clark City/County
Health Department will be doing the biologic and environmental sampling necessary to
obtain data necessary to complete the study.
Blood samples of children will be taken, as well as random samples of urine, hair
and stools from a select number of children and urine samples from some adults.
Children's hands will be dipped into a weak, vinegar-like solution of nitric acid.
Lead content will also be checked in paint samples from household walls and
baseboards, household dust, yard and garden soil,"and some garden vegetables.
Analysis of samples will begin immediately, and should be completed by November 1.
Homeowners will be notified of any indications that samples contain lead in excess of
established safety standards.
According to Hal Robbins, State Department of Health and Environmental Sciences,
participation in the census and study is strictly voluntary, and any information given
to interviewers will be held in strict confidence.
Robbins emphasized that the department considers this an extremely important
health-related study, as the last one undertaken in the area was done seven years ago.
The project has been reviewed and approved by the Centers for Disease Control,
and by the Environmental Protection Agency. The $385,000 cost of the study comes
from the EPA's Superfund program, which was set up to clean up existing hazardous and
toxic wastp* fnr «npria1 research oroiects sir.h as ctrf ^lena studv.
NEWS RELEASE
DATE: July 29, 1983
CONTACT: Hal Robbins
Air Quality Bureau
449-3454
-------
APPENDIX 2
FROM THE DIRECTOR
MONTANA DEPARTMENT OF HEALTH AND ENVIRONMENTAL SCIENCES
You may have read in the newspaper—or heard on the radio or television—
about a study planned this summer which will determine children's exposure to
lead for families living in the Helena Valley. All families in the East Helena
area and families in the northeast part of Helena will be contacted to locate
preschool children during the next few weeks.
A Department of Health census worker will call on you to ask whether you
have young children, their ages, and when you and your children will be home
this summer. They may also ask whether you have a garden or not, your
employment, and length of East Helena area residency. Your information will be
used to locate families who will again be visited in July and August, 1983, to
gather blood, possibly urine, and environmental samples. All information you
give to the interviewer will be held in strict confidence. Any information
released by the State Department of Health, and Envirormental Sciences will be
used only for statistical purposes in a manner in which no information about you
as an individual can be identified.
The Department considers this to be an extremely Important health-related
study and desires to sample all children in the 1 through 5 year old group.
Though your participation is voluntary, the real benefits may come to your
children. Your cooperation will be appreciated both in answering the census
questions and later with the collection of samples.
On the other side of this letter are the answers to questions you may have
about this study.
Thank you for your cooperation.
Sincerely,
John Drynan, M.O.
,w/upttier Information my be obtained from: The A1r QuaHtv
-------
What is this study all about? The Department will conduct this study of all
children in selected areas to locate those who might have more lead levels in
their bodies than those established by the Centers for Disease Control (CDC) of
Atlanta, GA. If such children are identified, they will be encouraged to seek
medical assistance, and the sources of lead in their envirorments will be
determined, so clean-up measures can follow.
Who is involved? Montana State Health Department, COC, the Environmental
Protection Agency (EPA), and the City-County Health Department will all be
involved in this same manner with this project. ASARCO and the East Helena
school system are also helping with the study.
How was my family selected? You live in one of the three geographical study
areas. The first area includes those who live within one-mile radius of the
ASARCO lead smelter and essentially includes the City of East Helena. Families
between 1 and 2.25 miles radius of the smelter are in the-.-second area. Those
living in the northeast part of Helena, north of Prospect, east of Harris, and
west of the Interstate, constitute the third (control) area. Yours is one of
about 375 families participating in the study.
Who is paying for the project? CDC and EPA are providing funds for this work.
How will samples be collected? East Helena area children will be asked to visit
a clinic where medical people will draw blood samples. Thereafter teams will
visit homes where preschool children live to collect blood, fran those who
didn't participate in the clinics. In some instances urine may also be
collected. House dusts, yard and garden soils and some-vegetables will be
sampled to characterize the nearby environment. A questionnaire will be filled
out for each child.
How often will I be visited? Most families will be visited twice--once during
the census interview and again when a team visits your home. Additional visits
may be requested of some families.
-------
What about the test results of my children and my residence? You will be given
the results either by letter or during a visit by a Department representative.
Explanation of the results will come from your physician and or Department
personnel.
-------
APPENDIX 3
Block #
I. D.
Letter
Area
File
Eligible C._
Eligible A."
Map
Vacant-
Montana Department of Health
and Environmental Sciences
Date 1
Date 2
Date 3
(T)
TIT
TTT
QUESTIONNAIRE -83(1)
I. IDENTIFICATION (All residences)
1. Name
No Contact/ III
/ /
Person Interviewed
2. Address
Phone
3. Mailing address (If different)
Head of Household
4. Years at this Address
5. Years in East Helena Study Area
6. Age
7. Work in E. Helena industry Y/N _
8. Farm/ranch near E.H. Y/N
Spouse
Yrs:
Y/N
Yrs.
9. Do you have a garden Y/N
Yrs.
Yrs.
Y/N
Y/N
Yrs.
Yrs.
10. How many families live at this address?
II. QUESTIONS FOR ALL FAMILIES WITH CHILDREN AGES 1 THROUCH 5
1. Names of all children
Normally at residence
A .
B .
C .
D .
E.
Birthdate
M D Y
/_/_/_/
/_/_/__/
/_/_/__/
/_/_/__/
I I I I
Sex
M F
Househol d
Member*
Y N
//////
/ /
Relationship
/_/_/ /__/_/
/_/_/ /_/__/
/ / / / / /
///ill
2. W111 you be on vacation between July 18 and August 19? Y/N
If yes, when will you be away?
3. Is 1t best to visit you and your children -morning
(during the period July 18-August 19) -afternoon
-evening
Date
Date
III. CONTACTS:
Personal
Telephone
*Is this usual place or residence
Date
Comments
-------
RIGGS
mm STREET
/
11
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o
70
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~xJ
M
X
P~
m m cxi ~x>
-I n>
o ro
uia n in •
_j. A~
cr cr
(Begin)
SE.
-------
APPENDIX 5
Participant Consent - General
I understand that the Montana Department of Health and Environnental Sciences,
with the assistance of the Center for Disease Control is conducting a study to
determine the possible health effects of lead exposure on pre-school age
children living in East Helena, its vicinity, and in a control area.
I understand that there will be three parts to the study;
A. Interview — Collection of information concerning:
(1) Health history, habits and activities of children in my home fran 1
through 5 years of age;
(2) Occupations of adults in my home,
(3) Types of cooking utensils used in my home, and
(4) Hobbies of any family member involving lead.
B. Environmental Testing
(1) Collection of dust samples within my home,
(2) Collection of soil and vegetable samples in the yard of my home
(leaving small holes),
(3) Examination of walls at my home for presence of lead in paint. (This
will be done by placing an x-ray fluorescent instrument against the
wall. The procedure will not damage nor alter the appearance of the
wal1.)
(4) Washing of child's hands to measure lead which may enter the mouth.
The wash solution, a solution similar to vinegar, will be washed from'
the hands after the sampling.
C. Examination of Blood samples
(1) A blood sample, approximately 2-3 ml. will be taken from a vein in the
arm of each child in my household ages 1 through 5 years to be tested
for indicators of lead toxicity. (There should be no problems
-------
associated with collecting the blood sample, other than slight, tem-
porary discomfort and the possibility of a small bruise at the site
where the needle enters the skin, which will disappear in a few days.)
I voluntarily agree to take part in this study and consent to having my-
child/children participate.I understand t'-at my/my childrens' participation
involves: (1) being interviewed regarding the topics described above, (2)
contributing a sample of blood, urine and hand dust, (3) allowing soil, dust and
vegetable samples to be taken from my residence, and (4) allowing examination of
the paint in my home. I have been assured that personal identifying information
will be kept in confidence by DHES and neither I nor any member of my family
will be identified by name in published reports of the results of this study, j
also understand that I may decline to answer specific questions as I see fit and
that I am free to withdraw my/our child's participation in the study at any
time. I understand that I will be informed in writing of the results of these
tests at the completion of the-study, unless additional follcwup is indicated,
in which case, I will be notified immediately. I understand that if I have
further questions' concerning the study, Information can be obtained by con-
tacting the Air Quality Bureau at 449-3454.
Participant
Signed:
Parent/Guaroi an ~~~~
Street
City/State
Interviewer
Signed
Date
1 copy to participant
1 copy to investigator
-------
Participant Consent - Adults
I understand that the Montana Department of Health and Environmental
Sciences, (DHES) with the assistance of the Centers for Disease Control (CDC) is
conducting a study to determine the possible health effects of lead and cadmium
exposure on people living in Esat Helena, its vicinity and in a control area. I
understand that there will be two parts to the study:
A. Environmental Testing
(1) Collection of dust samples within my home
(2) Collection of soil and vegetable samples in the yard of my home (leaving
small holes)
B. Examination of blood and urine samples
(1) A blood sample, approximately 2-3 ml. will be taken from a vein in my
arm to be tested for indicators of lead toxicity. (There should be no
problems associated with collecting the blood sample, other than slight,
temporary discomfort and the possibility of a small bruise at the site
where the needle enters the skin, which will disappearing few days).
(2) A urine specimen will be collected far cadmium analysis.
I voluntarily agree to take part in this study. I understand that my par-
ticipation involves: (1) Contributing a sample of blood and urine, and (2)
allowing soil, dust and vegetable samples to be taken from my residence. Any
leftover biologic specimens may be retained by CDC. I have been assured that
personal identifyng information will be kept in confidence by OHES and CDC and
neither I nor any member of my family will be identified by name in published
reports of the results of this study. The information shared with CDC will be
protected under the Federal Privacy Act.
-------
I also understand that I may decline to answer specific questions as I see fit
and that I .am free to withdraw my participation in the study at any time. I
understand that I will be informed in writing of the results of these tests at
the completion of the study, unless additional follcv/-up is indicated, in which
case I will be notified immediately. I understand that "f I have further
questions concerning the study, information can be obtained by contacting the
Air Quality Bureau at 449-3454.
Participant
Signed:
Street
City/State
Intervi ewer
Signed
Date
1 Copy to participant
1 copy to investigator
-------
APPENDIX 6
INTERVIEWING PROCEDURES
1. Complete necessary Information before the interview. Label consent form
and Interview form. Label each page with ID #.
2. Introduce yourself and your team members. Briefly explain everyone's
function in the study.
3. Read over the Participant Consent form with the volunteer, including a
brief explanation of all three parts of the study.
A. Interview
B. Environmental Testing
C. Biological Samples
4. Discuss any questions and ensure confidentiality.
5. Obtain volunteer signature on consent form and witness signature in writi
6. Proceed with questioning until finished.
7. Complete necessary information at end of Interview.
-------
Form approved
OMB No. 0920-0003
( 1-7)1.D. Number /
(SlocK) ~T"House)
EAST HELENA, MONTANA
CHILD QUESTIONNAIRE
CHILDHOOD LEAD EXPOSURE
1. Complete before interview:
A. Address:
No. Street Apt. if
(8-13) B. Date of interview / /
Mo. Day Yr.
(14-15) C. Interviewer No.
(16-19) D. Tine interview began: (Convert to military time)
MILITARY TIMETABLE
0100 AM
0700 AM
1300
PM
1900 PM
0200 AM
0800 AM
1400
PM
2000 PM
0300 AM
0900 AM
1500
PM
2100 PM
0400 AM
1000 AM
1600
PM
2200 PM
0500 AM
1100 AM
170U
PM
2300 PM
0600 AM
1200 N
1800
PM
2400 M
(20) 2. I would like to talk to the parent or legal guardian of the
Children who live in this house, preferably the one who can
tell us about how the other family members, especially the
child spends its time or younger children spend their time.
Is that person you?
1 - Yes 2 - No
(If answer is "no", ask who that person is and if you can
come back later to talk to him/her. Discontinue interview
until you are talking to who can tell you how the child/
children spend their time.)
This report is authorized by law (PL 96-510, Sect. 104 (b)). While your
response is voluntary, your cooperation is appreciated.
-------
I.D. 0
/
Page 2
3. What is your name?
First Middle Last
(21) 4. How long has this family been living at this address:
1 ¦ Less than 1 month
2 « 1 month or more but less than 2 months
3 ¦ 2 to 3 months
4 - More than 3 months but less than 6 months
5-6 months to 1 year
6 - More than 1 year but less than 5 years
7 • 5 years or more
9 =" Don't know or Unknown
IP THREE MONTHS OR LESS, TERMINATE INTERVIEW
5. Total number of persons living in household including any baby,
small children, and persons who usually live here but who are away
now, traveling, on vacation, in a hospital, or somewhere else.
(Include yourself)
(ENTER ANSWER ONLY WHEN YOU ARE SURE OF THE TOTAL NUMBER)
(22-23) Total Number:
RESPONSES IN TABLE 1 ON NEXT PAGE
6. A. What is the full name of youngest person living in household?
B. Circle code for sex; ask if necessary
C. On what date was he/she born?
Who is the next youngest? (Proceed up in age.)
(Repeat A,B,C for all members of the household.)
D. Ask as appropriate for all persons 16 and older:
What is (his/her/your) occupation—that is, what
does (he/she, do you) do?
-------
I.D. It
/
Page 3
HOUSEHOLD ROSTER
TABLE 1
(24-25)
Person
01
(35-36)
Person
02
(A6-47)
Person
03 —
(57-58)
Person
04
(68-69)
Person
05
F
M
L
A
Name
F
M
L
A
' Name
F
M
L
A
Name
F
M
L
A
Name
F
M
L
A
Name
B
Sex
M 1
<
F 2
(26)
B
Sex
M 1
<
F 2
<37)
B
Sex
H 1
<
F 2
(48)
B
Sex
M 1
<
F 2
(59)
B
Sex
H 1
(
F 2
(70)
C
Dace of
Birth.
MO.
DAY
YR.
AGE
C
Dace of
Birth
Mo.
Day
Yr.
Age
C
Date of
Birth
Mo.
Day
Yr.
Age
C
Date of
Birch
Mo.
Day
Yr.
Age
(27-28)
(29-30)
(31-32)
(33-34)
C
Date of
Birch
Mo.
Day
Yr.
Age
Occupation.
(38-39)
(40-41)
(42-43)
(44-45)
Occupacioa
(49-50)
(51-52)
(53-54)
(55-56)
Occupation
(60-61)
(62-63)
(64-65)
(66-67)
(71-72)
(73-74)
(75-76)
(77-78)
Occupation
-------
I.D. Q
Page 4
(79-80)
Person
06
A
Name
F
M
L
B
Sex
M 1
<
F 2
(81)
C.
Dace c
Birth
Mo.
Day
Yr.
Age
Occupation
(82-83)
(84-85)
(86-87)
(88-89)
(90-91)
Person
07
A
Name
F
M
L
B
Sex
M 1
(
F 2
(92)
C
Dace of
Birth
Mo.
Day
Yr.
Age
Occupation
(93-94)
" (95-96)
" (97-98)
"(99-100)
Person
(101-102) 08
A
Name
F
M
•L
B
Sex
M 1
<
F 2
(103)
C
Date of
Birth
Mo.
Day
Yr.
Age
Occupation
(104-105)
(106-107)
(108-109)
(110-111)
Person
(112-113) 09
Person
(123-124) 10
A
Name
M
L
A
Name
F
M
L
B
Sex
M 1
<
F 2
(114)
B
Sex
M 1
<
F 2
(125)
C
Date of
Birth
Mo.
Day
Yr.
Age
C
Date of
Birth
Mo.
Day
Yr.
Age
Occupation
(115-116)
(117-118)
(119-120)
(121-122)
Occupation
(126-127)
(128-129)
(130-131)
(132-133)
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i.d. a
/
Page 5
(134) 7. Are you the head of the household?
1 - Yes (Head)
2 - Yea (Co-Head)
3 ¦ No
(135-136)8. A. Who Is the head of the household?
(Put person "NUMBER" according to table 1.)
(137)
B. What is the highest grade or year of regular school that
(NAME OF HEAD) finished and got credit for?
1 - Graduate work 5
2 - 4 - year college degree 6
3 - Some college 7
4 - High School graduate 9
Some High School
7th or 8th grade
Less than seventh grade
Don't know or unknown
(138) C. Which of the statements below comes closest to the
total family income for this family before taxes in 1982?
1 ¦ Under $5,000 6 «¦ $25,000 or more
2 - $5,000 or more but less than $10,000 7 ~ Refused
3 ¦ $10,000 or more but less than $15,000 9 - Don't know or unknown
4 ¦ $15,000 or more but less than $20,000
5 - $20,000 or more but less than $25,000
I'm going to read you a list of different kinds of jobs that
expose people to lead dust or vapors. Have you or any member of
this household worked in one or more of these jobs during the
last 3 months?
1 ¦ Yes 2 ¦ No 9 ¦ Unknown
If yea, circle which ones —
LEAD-ZINC RELATED OCCUPATION CODES
(140-141) 01 - Lead smelter worker (152-153)
(142-143) 02 ¦ Foundry Worker
(144-145) 03 - Oil Refinery Worker (154-155)
(146-147) 04 - Painter (156-157)
(148-149) 05 - Battery Mfg. Plant Worker (158-159)
(150-151) 06 - Chemical Plant Worker
9.
(139)
07 - Paint-pigment, *iuc
copper Worker
08 " Plumber
09 - Glass Worker
10 - Other Lead-Related
Industry Worker
-------
I.D. 0
/
Page 6
Answer following questions in- Table 2 for each person who has
worked In a lead-zinc related occupation
A. What Is the name of the place where (you/he/she) work(s)?
B. How long have you (has he/she) worked there?
C. Do(es) (you/he/she) change out of (your/his/her) work clothes
and leave them at work?
D. Do(es) (you/he/she) shower at work before coming home?
E. What is (your/his/her) job title?
LEAD-RELATED JOBS
TABLE 2
Person No. A.
(160-161)
(Name of Workplace)
B. Time at workplace
Months:
(162-164)
C. Change Clothes
l.» Yes
(165) 2 - No
9 a Don't know
Occupation
Code
(166-167)
D. Shower
1 " Yes
(168) 2 - No
E.
.(Job Title)
9 " Don't know or Unknown
Person No.
(169-170)
A.
(Name of Workplace)
B. Time at workplace
Months:
(171-173)
C. Change Clothes
(174) 2 - No*
9 ¦ Don't know
(175-176)
Occupation
Code
D. Shower
1 - Yes
(177) 2 - No
E.
(Job Title)
9 ~ Don't know or Unknown
-------
I.D. #
/
Page 7
Person No.
(178-179)
A.
(Name of Workplace)
B. Time ac workplace
Months:
(180-182)
C. Change Clothes
1 " Yes
(183) 2 - No
9 ¦ Don'tt kaow
Occupation
Code
(184-185)
D. Shower
1 - Yes
(186) 2 - No
E.
(Job Title)
9 ¦ Don11 know or Unknown
Person No.
(187-188)
A.
(Name of Workplace)
B. Time at workplace
Months:
(189-191)
C. Change clothes
1 - Yes
(192) 2 - No
9 ¦ Don't know
Occupation
Code
(193-194)
D. Shower
1 » Yes
(195) 2 - No
E.
(Job Title
9 " Don11 know or Unknown
Person No.
(196-197)
A.
(Name ox Workplace)
B. Time at workplace
Honths:
(198-200)
C. Change clothes
1 - Yes
(201) 2 - No
9 - Don't knQw
E.
Occupation D. Shower
Code 1 ¦ Yes
(202-203) (204) 2 - No
9 " Don't know or Unknown
(Job Title)"
-------
I.D. If
/
Page 8
For questions 10-31 ask. abouC each child under age 6.
Scare with the youngest.
ENTER ANSWERS TO QUESTION 10 IN TABLE 3 BELOW
10. Now I would like to talk to you about (Child No. - under age 6)
A. Where does (Child No. ) spend his/her daytime hour?
(ACCEPT MULTIPLE ANSWERS FOR 4 OR MORE HOURS AT ANY LOCATION)
If not only at home, Ask:
B. About how many hours each day, on the average, does
(he/she) spend away from home?
C. What is the address where (he/she) spends
most sime away from home?
1 - At home 3 - At Day Care Center
2 ¦ At babysitters 4 ¦ At relatives
5 ¦ At some other place (Specify)
(Environmental sample will be taken later)
CHILD'S DAILY ROUTINE
TABLE 3
10A 10B 10C
Child Where Spends Hours
No. Time During Day Away Address away from home
(205) 1 12 3 4 5
(206-208) (209-210)
Number Street
City State Zip Code
10A 10B IOC
-Child Where Spends Hours
No. Time During Day Away Address away from'home
(211) 2 12 3 4 5
T212-214) (215-216)
Number Street
City State Zip Code
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I.D. 0
/
Page 9
(217)
10A 10B
Child Where Spends Hours
No. Time During Day Away
3 1 2 3 4 5
(T18-22QT~ (221-222)
IOC
Address away from hone
Number
Street
City
State Zip Cade
(223)
Child
No.
A
1QA
Where Spends
Tine During Day
1 2 3 4 5
~722Łr22Tr
10B
Hours
Away
(227-228)
IOC
Address away from hoze
Number Street
City State Zi? Code-
(229)
10A
Child Where Spends
No* Time During Day
" 1 2 3 4 5
T23ŁF232)~
10B
Hours
Away
(233-234)
IOC
Address away from home
Number
Street
City
State Zip Code
(235)
10A 10B
Child Where Spends Hours
No. Time During Day Away
6 1 2 3 4 5
"T236-238T" (239-240)
IOC
Address away from home
Number Street ~
City
State Zip CodT
-------
I.D. 0
/
Page 10
11. Where does.(he/she) usually play when outdoors around, this home?
1 ¦ In back, yard
2 - In front yard
3 ¦ Does not play outdoors
8 ¦ Other (Specify)
TABLE 4
ACCEPT MULTIPLE ANSWERS
Child
USUALLY
PLAYS
No.
(241)
1
(242 - 245)
1
2
3 8
(246)
2
(247 - 250)
1
2
3 8
(251)
3
(252 - 255)
1
2
3 8
(256)
4
(257 - 260)
1
2
3 8
(261)
5
(262 - 265)
1
2
3 8
(266)
6
(267 - 270)
1
2
3 8
SPECIFY OTHER
12. Is the ground there mainly grassy, concrete/asphalt, plain dirt
or soil, just a sandbox, or what?
1 ¦ Grassy
2 - Concrete/Asphalt
3 - Dirt/Soil
4 - Sandbox
7 - Other (Specify)
8 » Not applicable
CHILD
NO.
TABLE 5
ACCEPT MULTIPLE ANSWERS
(Circle answers)
GROUND MAINLY
Specify Other
(271)
1
(272 - 277)
2
3 4
7 8
(278)
2
(279 - 284)
2
3 4
7 8
(285)
3
(286 - 291)
2
3 4
7 8
(292)
4
(293 - 298)
2
3 4
7 8
(299)
5
(300 - 305)
2
3 4
7 8
(306)
6
(307 - 312)
2
3 4
7 8
-------
I.D. #
/
Page 11
13. AbouC how many hours each day does (he/she) usually spend playing
outdoors In this neighborhood?
14. Does (he/she) often take some food or a bottle with (hia/her)
out3ide to play?
1 » Yes 8 ¦ Not applicable
2 - No 9 » Don't know or Unknown
TABLE 6
CHILD
No.
14
HOURS
15
F00D/B0TTLZ
(313)
1
(314-315)
-
(316)
12 8
9
(317)
2
(318-319)
(320)
1 2 8
9
(321)
3
(322-323)
(324)
1 2 8
9
(325)
•4
(326-327)
(323)
1 2 8
9
(329)
5
(330-331)
MM*
(332)
12 8
9
(333)
6
(334-335)
(336)
12 8
9
15. Does (CHILD No. ) usually play alone, with other children
or mostly with adults? '
1 " Alone
2 - Other children
3 ¦ Adults
-------
I.D. ii / Page 12
TABLE 7
Child
No.
(337)
1
(338 - 340)
1
2
3
(3A1)
2
(342 - 344)
1
2
3
(345)
3
(346 - 348)
1
2
3
(349)
4
(350 - 352)
1
2
3
(353)
5
(354 - 356)
1
2
3
(357)
6
(358 - 360)
1
2
3
ENTER ANSWERS TO QUESTIONS 16 - 18 IN TABLE 8 BELOW
16. Are (his/her) hand or face usually washed before eating?
17. Are (his/her) hands or face usually washed before going to sleep?
18. Are (his/her) hands or face usually washed after making mud pies,
or playing with dirt or sand?
TABLE 8
1 « Yes, 2 - No, 8 ¦ Not applicable, 9 « Don't know or Unknown
Child
No.
BEFORE
EATS
BEFORE
SLEEP
AFTER
PLAY
(361)
1
(362 - 364)
1
2 8 9
1
2 8 9
12 8 9
(365)
2
(366 - 368)
1
2 8 9
1
2 8 9
12 8 9
(369)
3
(370 - 372)
1
2 8 9
1
2 8 9
12 8 9
(373)
4
(374 - 376)
1
2 8 9
1
2 8 9
12 8 9
(377)
5
(378 - 380)
1
2 8 9
1
2 8 9
12 8 9
(381)
6
(382 - 384)
1
2 8 9
1
2 8 9
12 8 9
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I.D. 0
/
Page 13
ENTER ANSWERS TO QUESTIONS 19-22 IN TABLE 9 BELOW
Has (CHILD NO. ) used a pacifier often in the last 3 months?
Does (he/she) often suck (his/her) thumb or fingers?
Does (he/she) sometimes chew on (his/her) fingernails?
A. Does (he/she) have a favorite blanket or stuffed toy?
IF YES ASK B AND G. OTHERWISE SKIP TO QUESTION 23.
B. Does (he/she) carry this around during the day?
C. Does (he/she) often put this in (his/her) mouth?
TABLE 9
1 * Yes, 2 - No, 8s Not applicable, 9 - Don't know or Unknown
(Circle correct number)
Child
No.
19
PACIFIER
20
SUCK
THUMB
21
CHEW
NAILS
22A
FAVORITE
22B
CARRY
22C
TOY IN
mouth
(385-391)
1
1
2
9
1
2 8 9
1
2 8 9
1
2 8
9
12 8 9
1
2 8 9
(392-398)
2
1
2
9 -
1
2 8 9
1
2 8 9
1
2 8
9
12 8 9
1
239
(399-405)
3
1
2
9
1
2 8 9
1
2 8 9
1
2 8
9
12 8 9
1
2 8 9
(406-412)
4
1
2
9
1
2 8 9
1
2 8 9
1
2 8
9
12 8 9
1
2 8 9
(413-419)
5
1
2
9
1
2 8 9
1
2 8 9
1
2 8
9
12 8 9
1-
2 8 9
(420-426)
6
1
2
9
1
2 8 9
1
2 8 9
1
2 8
9
12 8 9
1
289
19.
20.
21.
22.
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I.D. 0
/
Page 14
ENTER ANSWERS TO QUESTIONS 23 - 27 IN TABLE 10 BELOW^
23. How many hours during the day do you think (he/she) usually spend
playing on the floor when indoors in this home.
24. Does (he/she) often put (his/her) mouth on furniture or on the
window sill?
25. Many children put some things other than food into their mouths.
Would you say that (CHILD NO. ) does this a lot, just once in
a while or almost never?
26. Have you ever seen (CHILD NO. ) put paint chips in
his/her mouth?
27. Sometimes children swallow things other than food. Would you say
that (Child No. ) swallows things other than food a lot, just
once in a while, or almost never?
TABLE 10
1 ¦ A lot, 2 » Just once in a while,
3 ¦ Almost never, 9 ™ Don't know or Unknown
23
24
25
26
27
CHILD
HOURS
FURNITURE
THINGS
PAINT
SWALLOW
NO.
IN
MOUTH
CHIPS
(427-433)
1
^m
1
2
9
1
2 3 9
12 3
1
2 3 9
(434-440)
2
1
2
9
1
2 3 9
12 3
1
2 3 9
(441-447)
3
1
2
9
1
2 3 9
12 3
1
2 3 9
(448-454)
4
1
2
9
1
2 3 9
12 3
1
2 3 9
(455-461)
5
•WW
1
2
9
1
2 3 9
12 3
1
2 3 9
(462-468)
6
1
2
9
1
2 3 9
12 3
1
2 3 9
*27a. Specify things swallowed:
Child No. Item(s) swallowed
-------
I.D. 9
f
Page 15
RECORD ANSWERS TO QUESTION 28 IN TABLE 11 BELOW
28. A. During che last: 3 months has (he/she) been Caking any
vitamins?
B. During the last 3 months has (he/she) been taking any
minerals?
C. Was (he/she) taken any other kind of dietary supplement
in the last 3 months?
CHILD
NO.
1 - Yes, 2
28A
VITAMINS
TABLE
No, 9 ¦
11
• Don't know or Unknown
28B
MINERALS
(469)
1
(470)
1 2 9
(471)
1
2
9
NAME
NAME
NAME
NAME
(473)
2
(474)
12 9
(475)
1
2
9
NAME
NAME
NAME
NAME
(477)
3
(478)
12 9
(479)
1
2
9
NAME
NAME
NAME.
NAME
(481)
4
(482)
12 9
(483)
1
2
9
NAME
NAME
NAME
NAME
(485)
5
(486)
12 9
(487)
1
2
9
NAME
NAME
NAME
NAME
(489)
6
(490)
12 9
(491)
1
2
9
NAME
NAME
NAME
NAME
28C
SUPPLEMENT
1 2 9
(472)
NAME
NAME "
(476) 1 2 9
NAME
NAME
(480) 1 2 9
NAME
NAME
(484) 12 9
NAME
NAME
(488) 1 2 9
NAME
NAME
(492) 1 2 9
NAME
NAME
-------
I.D. #
/
Page 16
ENTER ANSWERS TO QUESTIONS 29 AND 30 IN TABLE 12 BELOW
29. A. Has (Child No. ) participated ia any kind of lead poisoning
screening program within the last 12 months?
1 ~ Yes, 2 ¦ No, 9 - Don't know or Unknown
IF "NO" SKIP TO QUESTION 30.
B. What were the results of that screening; were they norrsal or
high?
1 « Normal, 2 ¦ High, 9 " Don't Know or Unknown
30. Circle code for race of child. IF NECESSARY, ASK: What race is
(Child No. )?
1 - White, Non-Hispanic 5 ¦ Asian or Pacific Islander
2 ¦ White, Hispanic 6 *¦ American Indian or
3 ¦> Black, Non-Hispanic Native
4 ¦ Black, Hispanic 7 * Refused
9 ™ Unknown
TABLE 12
(Circle correct number)
Child
3 OA
30B
31
No.
SCREENED
RESULTS
RACE
(493-496)
1
.12 9
12 9
1
2
3 4 5
6
7
9
(497-500)
2
12 9
12 9
1
2
3 4 5
6
7
9
(501-504)
3
1 2 9
12 9
1
2
3 4 5
6
7
9
(505-508)
4
1 2 9
12 9
1
2
3 4 5
6
7
9
(509-512)
5
1 2 9
12 9
1
2
3 4 5
6
7
9
(513-516)
6
1 2 9
12 9
1
2
3 4 5
6
7
9
BE SURE QUESTIONS 10-30 HAVE BEEN ASKED FOR EACH CHILD UNDER 6 IN
THE HOUSEHOLD
-------
I.D. t
/
Page 17
31. Thinking about Che kinds of hobbles- that people might have, or*
work or activities that people might do around the home,
within the past 3 months has any member of this household
often:
HOBBIE CODES
1 - Yes, No - 2, 9 " Unknown
A. Painted pictures with artists paint?
B. Painted parts of the house or furniture In the house?
C. Worked with stained glass?
D. Cast lead Into fishing sinkers, bullets or anything
else?
E. Worked with soldering in electronics?
F. Worked on soldering pipes?
G. Made pottery at home?
(517)
1
2
9
(518)
1
2
9
(519)
1
2
9
(520)
1
2
9
(521)
1
2
9
(522)
1
2
9
(523)
1
2
9
(524)
1
V
"9 '
32.
(525)
1
2
9
33.
(526)
1
2
9
34.
(527)
1
2
9
35.
(528)
1
2
9
36.
(529)
1
2
9
37.
(530)
1
2
9
38.
(531)
1
2
9
39.
40.
(532)
1
2
9
1 ¦ Yes, 2 - No, 9 - Don't know or Unknown
Does/do the child/children frequently eat fruits or
vegetables grown anywhere close by in this neighborhood
When food is served, is it ever served in homemade or
imported clay pottery or ceramic dishes?
sometimes stored In the original can after being opened?
Give example if necessary: e.g. juice can.
Does/do Che child/children sometimes eat snow in the
wintertime?
Does anyone in this household smoke?
Does your home have storm windows?
What is Che name of the medical doctor who generally
examines or treats your child/children?
1.
2. Have no family' doctor
9. Don't know
-------
1*0. #
/
Page 18
(533) 12 3 49. A. Thank, you very much for answering these questions.
There may be a few things we talked about that I need
to clarify, hay I telephone you if 1 need to?
1 ¦ Yes, 2 " No, 3 - No Phone
IF YES, ASK:
B. What is your telephone number? (or that of nearby
neighbor or relative?)
/ /
(534) 12 9 50. Year house built
1 - Before 1955
2 ™ After 1955
9 » Don't know
II. ^COMPLETE AFTER INTERVIEW:
A. Time Interview Ended
(Military Time)
(535 - 538)
0100
AM
0700 AM
1300
PM
1900
PM
0200
AM
0800 AM
1400
PM
2000
PM
0300
AM
-0900 AM
1500
PM
2100
PM
0400
AM
1000 AM
1600
PM
2200
PM
0500
AM
1100 AM
1700
PM
2300
PM
0600
AM
1200 N
1800
PM
2400
M
B. Type of Housing Structure
(539) _______ 1. Constructed of:
1 - Frame 3 » Concrete Block
2 - Brick 4 - Combination
5 - Other (Describe)
(540) ______ 2. Structure is:
1 ¦ Sgl., 2 ¦ 2-Family, 3 » 3-or more Family
(541-542) ________ Number of Units
revised as of 7/28/83
-------
(1-7) I.D.0 /
(Slock) (House)
DEMOGRAPHIC
(16-19)
(20-23)
(24-27)
(28-31)
(32-35)
(36-39)
(40-43)
(44-47)
(48-51)
ADULT QUESTIONNAIRE
CHILDHOOD LEAD EXPOSURE
EAST HELENA, MONTANA
1. Subjects's name
(Lasc First Middle)
(8-13) 2. Subject's date of birth: Mo. Day Yr.
(14) 3. Subject's sex: _____ 1 » Male 2 ¦ Female
(15) 4. Subject's race: ____ 1 ¦ White (not hispanic)
2 ¦ Black (not hlspanlc)
3 ™ Hispanic
4 - American Indian or Alaskan native
5 - Asian or Pacific Islander
9 ~ Unknown
5. Subject's telephone: / /
(Area)
6. RESIDENCE:
A. List all places you have lived in the past 30 years,
starting with the most recent -
(Note to interviewer: Fill in last two
digits of year; e.g. 82 for 1982.)
FROM TO ADDRESS
-------
B. ADULTS: List all places you and jour spouse have worked
in Che past 20 years -
(52) Do you (have you) work(ed) in lead-zinc related industry?
1 " Yes 2 ¦ No 9 ¦ Unknown
LEAD-ZINC RELATED OCCUPATIONAL CODES
1 Lead smelter worker 7 Paint-pigment, zinc
2 Foundry worker copper worker
3 Oil refinder worker' 8 Plumber
4 Painter 9 Glass worker
5 Battery Mfg. plant worker 10 Other lead-related
6 Chemical .plant worker industry worker
HUSBAND
WHEN WORKED JOB JOB
FROM TO COMPANY DESCRIPTION CODE CHEMICALS
(2) (2) (2) (2)
( 53- 60)
( 61- 68)
( 69- 76)
( 77- 84)
( 85- 92)
( 93-100)
(101-108)
(109-116)
(117-124)
(125-132)
(133-140)
(141-148)
WIFE
WHEN WORKED JOB JOB
FROM TO COMPANY DESCRIPTION CODE CHEMICALS
(2) (2) (2) (2)
8. OUTDOOR ACTIVITIES:
1 ¦ Yes 2 ¦ No 9 ¦ Unknown or not stated
(Note to interviewer: Express each
as number of times per year
(average) even if activity is done
only in some seasons.)
-------
(149) A. Do you engage in sports that bring you in contact with soil
(e.g., baseball, football, jogging, etc.)?
(150-151) If yes, how often per year?
(152 ) B. Do you garden, weed or mow around your home?
(153 ) C. Do you garden, weed or mow someplace else in the
neighborhood?
If so, where?
9. DIETARY EXPOSUBES: 1 - Yes 2 - No 9 - Unknown
(154 ) A. Do you eat vegetables and fruits raised by yourself
or in the neighborhood?
(155 ) If yes: Are they root vegetables?
(e.g., carrots, potatoes)
Address where raised if not by (your)self
(156) Non-root vegetables and fruits?
Address where raised if not by (your)self
Fruit or Grown by In season during Average It times
vegetable Self or Neighbor which months eaten/week in season
(1) (2)
(157-159)
(160-162)
(163-165)
(166-168)
(169-171)
(172-174) muz zzmzzzzz znn
(175) _____ B. Do you eaC wild game or fish caught locally?
If yes:(Code: 1 ¦ Mammal 2 ¦ Bird 3 ™ Fish
Animal or Fish Location Average # times
where caught per Year Eaten
(1) (2)
(176-173)
-------
(188)
C. Do you eat meat or eggs from farm animals raised
in Che Ease Helena area?
(189-192)
(193-196)
•(197-200)
(201-204)
(205-208)
If yes:
(1 ¦ beef 2 ¦ pork 3 ¦ poultry 4 - eggs 5 « other)
Types of meat Location/ Average 0
(or eggs) address of times/year
farm
(1) (3)
(209) D. Do you drink milk from cows on farm3
in the East Helena area?
Address of farm
(210) E. Do you drink water from a private well or spring?
If yes:
(211-216)
(217-222)
(223-228)
Location of No. yrs When discontinued
well/spring Used (Year)
(2) (2) (2)
(229) 10. Have you ever sprayed oil around your property to keep
down dust?
1 • Yes 2 ¦ No 9 ¦ Unknown
(230-231) If yes, where did you get the oil? ¦
(Make list)
(232) 11A. Did you ever smoke or use tobacco products?
1 " Yes 2 - No 9 ¦ Unknown
If 'no', go to question 12.
(233) B. Do you now smoke or use tobacco products?
1 ** Yes 2 ¦ No 9 ~ Unknown
(234-235) If no, how long ago did you quit? (Years)
(236-237) C. At what age did you start smoking? (Years)
(238-239) D. On the average, how many cigarettes do/did you smoke a day?
-------
(240) E. Do/did you usually inhale the smoke when you smoke
cigarettes?
1 ¦ Ye3 2 - No 9 ~ Unknown
F. What brand of cigarettes do/did you generally smoke?
12. MEDICAL HISTORY
(241) Have you been treated or diagnosed by a doctor
in the past 2 years for any chronic or serious illness?
1 " Yes 2 - No ' 9 ~ Unknown
99 - Unknown year
Yr diagnosed Hospital or physician & address
(242-243)
(244-245)
(246-247)
(248-249)
(250-251)
(252-253)
(254-255)
Comments:
-------
APPENDIX 7
VENAPUNCTURE
1. Make sure consent form is signed.
2. Educate patient according to their level of comprehension with parent
present.
3. Assure patient of minimal discomfort.
4. Inspect patient's arms and hands for best venapuncture site.
5. Determine best method of venapuncture for the patient (butterfly 18 g or
conventional needle 21 g assembly).
6. Clean venapuncture site using B-D alcohol preps until alcohol prep shows
clean. Let air dry or dry with clean gauze.
7. Be sure patient is properly restrained by parent.
8. Apply tourniquet.
9. Palpate for vein.
10. Insert needle assembly.
11. Draw 2 B-D vacutainer evacuated tubes with EDTA preservative. Mix well;
invert 3-5 times.
12. Loosen tourniquet before last tube is full or before withdrawing needle.
13. Withdraw needle.
14. Apply pressure to venapuncture site until bleeding is stopped, then apply
band-aid.
15. Write in patient's name and sign labels. Attach labels to tubes.
16. Put tubes 1n cooler.
Processing Equipment:
1. Consent form
2. Butterfly 10 ga. or 21 ga. by 3/4", 12" tubing Infusion set, vacutainer
multiple sample Luer-Adapter, BeCton Dickinson vacutainer with EDTA pre-
servative, and vacutainer holder.
-------
3. Becton Dickinson (B-0) alcohol swab
4. Tourniquet
5. Cooler with "Blue Ice" packs to keep sample cool
6. Trained and qualified person to obtain blood samples (i.e., medical
technician, nurse, etc.)
-------
APPENDIX 8
Hair Collection (Children Only)
1. Instructions for Collecting Hair Samples
a. Use a new comb and pair of scissors for each subject*
b. Use disposable, powder-free plastic gloves to handle the hair
specimen*
c. Collect the hair samples:
1) Collect the hair samples from the nape area*
2) With a clean nylon comb, partition the hair between the
ears as shown in the diagram.
3) Fasten the hair above the ears out of the way with
aluminum clips*
4) At 8-10 sites on the nape area, gather 15-20 strands of
hair. Hold the end of the hair and cut the hair as
close to the scalp as possible with stainless steel
surgical scissors. A minimum of 400 mg is needed for
analysis *
5) From each cutting of hair from the scalp, cut off the
two inches of hair which were closest to the scalp and
store in a Ziploc plastic bag.
6) Discard the remaining length of hair.
7) Seal the Ziploc bag.
8) Affix the appropriate examinee ID number to the Ziploc
bag.
d* Hair samples may be shipped with the other specimens to CDC.
Place the hair samples on top of the shipper so that the hair
does not get wet.
e. Disinfect the scissors, clips and combs.
2. Equipment:
a. Clean nylon combs.
b. Stainless steel scissors.
c. Disposable, powder-free plastic gloves.
d. Aluminum Clips.
e. Ziploc bags.
f* Becton Dickinson alcohol swabs*
-------
(1-7) I. 0. No.
/
EAST HELENA, MONTANA
HAIR COLLECTION QUESTIONNAIRE
AGES 1 THROUGH 5
( 8 - 9) 1.
Child No. •
(10 - 11) 2.
Age
(12) 3.
Sex
(1 « Male 2 » Female)
(13 - 18) 4.
Date of Collection: Month ____ _____
Day
Year
(19 - 22) 5.
Sample No.
(23) 6.
When was the last time (Child's naae)'s hair was washed?
<___ 1 » Today or yesterday
2 - Two through six days ago
3 - Seven days ago or longer
(24) 7.
The last tine his/her hair was washed, was it washed at
' 1 - Home
2 ¦ Elsewhere?
(Specify)
(25 - 26) 8.
The last time his/her hair was washed, what brand of shampoo was used?
Specify brand _______
99 ¦ Don't know
(27) 9a.
Is this the regular brand of shampoo you use?
1 - Yes 2 - No
b. If no, what is7 ________________________
(28) 10a.
When washing his/her hair, do you ever use a conditioner or cream
rinse?
_____ (1 ¦ Yes 2 ¦ No)
If no, slcip to Question 12a.
(29-30) b. If ye«» which brand? _
99 - Don't know
-------
I. D. // /
(31) 11. How often do you use a conditioner or creme rinse?
(1 ¦ Occasionally 2 ¦ AlmoBt always)
(32) 12a» When washing his/her hair, do you ever use a
dandruff shampoo?
(1 - Yes 2 - No)
If no, skip to Question 14a.
(33-34) b. If yes, which brand?
99 ¦ Don't know
(35) 13* How often do you use a dandruff shampoo?
(1 " Occassionally 2 - Almost always)
(36) 14. Do you ever use any special treatment on his/her
hair, such as hair coloring?
(1 - Yes 2 - No)
If yes, specify
-------
APPENDIX 9
Urine Collection (Children and Adulcs)
I- Instructions for Obtaining Urine Samples
a. Instruct adults and children (or parents of child) to wash
hands with soap and water*
b. Instruct adults and children (or parents of child) to use a
moist towelette (Diaparene) to wash genitalia prior to
voiding:
1) Use the towelette once, wiping the labia from front to
back or the urethral opening on the penis,
2) Fold the towelette, and
3) Repeat the wiping as in step 1).
c. Instruct the participants how to collect a clean-catch urine
sample:
1) After wiping the genitalia, begin to urinate into the
toilet (the first small amount of urine may contain
small particles from the.opening of the urethra, and we
wish to discard this),
2) Collect the rest of the urine in the 132 ml (small cup)
specimen container, and
3) Leave the specimen in the bathroom.
d. Divide the urine specimen into the appropriate tubes as
follows:
1) Work over the bathroom sink.
2) Wear the powder-free lab gloves provided in the team kit.
3) For adult samples:
a) Place approximately 4 ml into the 6 ml white vial.
This is the plain urine sample (no preservatives
are in this vial).
b) Tap the top of the 50 ml blue-top tube before
opening. Once opened, pour up to 25 ml of urine
into the tube. The black line on the side of the
tube marks the 25 ml level. Cap, label, and Invert
the tube gently 4-5 times to mix the urine with the
small amount (250 ul) of nitric acid present in
this tube.
c) Tap the top of the 30 ml clear plastic tube before
opening. Pour up to 25 ml of urine into the tube.
A black line marks the maximum volume for filling
on this tube also (i.e., 25 ml). Cap, label as the
carbonate sample, and invert the tube 8-10 times
until the carbonate is dissolved. (As little as 5
ml of urine is an acceptable volume for this
sample.)
-------
4) For child samples: collecc plain and acid cubes (seeps
a and b only).
e. Pour any unused urine into the toilet, rinse the specimen
cup, and dispose of the cup in the team's garbage bag.
Ł• Collection of field blanks for the acid tubes: For every 25
participants, 3 acid tube field blanks will be collected. If
you are to collecc such a blank, a second sheet of labels
will be in the subject's file and the Urine (Acid) label will
be circled. Collection of this blank will be done as follows:
At the time of dividing the urine specimen into the appropri-
ate tubes, open an extra blue-topped tube and fill it with
deionlzed water to a level less Chan the black line mark.
Cap and invert as if lc were a normal urine sample. Label
and score wich Che other urine samples.
g. Collection of duplicate samples:
Every fifth participant will have two sets of urine tubes
collected. To facilitate this, Che following cable lists the
minimum amounts of urine to be collected:
BesC Volume Minimum Volume
Tube Type "Co Use (ml) Necessary (ml)
White vial (plain urine) 4 2
Blue-copped tube (acid urine) 25 5
Clear plascic tube (carbonate 25 5
urine, ADULTS ONLY)
Thus, for a child participate for whom duplicate samples are
needed, a minimum of 14-15 ml musC be collected to divide
among 4 tubes.
h. Place the Cubes and vials in the shipping containers in the
team's styrofoam cooler. Use refrigerator packs in the
cooler. Keep all Cubes and vials uprighc. Do noc lec the
Cubes touch Che ice packs directly.
1. After the samples have been returned Co the operating center
and logged in, chey muse be stored in a freezer at -20
degrees centigrade*
-------
APPENDIX 10
Handwash Sampling
Instructions for Collecting Handwash Samples
a. Be sure consent form is signed.
b. Educate parent as to the strength of the acid. Assure parent
that weak acid will not harm child provided hand is rinsed
well in tap water immediately after dipping.
c. Educate child according to their level of comprehension.
d. Inspect child's hands for open wounds or cuts, and if
present, do not dip that hand.
e. Dip one hand into wide mouth plastic container containing
approximately 500 ml of 0.1 H nitric acid with fingers
slightly moving for 20 seconds (use stopwatch). Remove hand
from container and immediately rinse well with running tap
water. Dry hand. Repeat above procedure on other hand.
f. Recap sample as quickly as possible to avoid any
contamination.
g. Label appropriately - note any nail polish and its color on
the sample sheets.
h. Handwash Blanks.
1) Take one extra handwash jar with you when you do the
child's hand dip.
2) Either before or after child's hands are dipped, open
blank hand dip jar for 40 seconds. Recap jar and label
accordingly.
Processing Equipment
a. Wide mouthed quart polyethylene mason jars (Bel Arts)
b. 500 ml of 0.1 normal nitric acid
c. Stopwatch
d. Paper towels
-------
APPENDIX 11
Soil Collection
1. Soil Sampling Procedure
a. Instructions for Selecting Soil Sample Locations
1) Front of House
The first soil sample will' be taken in the front yard.
Estimate the approximate center of the yard when facing
the house, move one and one-half meters (1.5) from the
curb and draw a circle one meter in diameter from chat
point* Samples will be taken at four equally spaced
locations in the circle, one at the northern most point
in the circle, one at the southern most point, one at
the eastern most point and one at the western most point.
If it is not possible to take a sample at the center of
the yard because of a walkway, driveway, etc., while
facing the home, move one meter to the right of the
obstruction and proceed. In the event it is not
possible to draw a circle, measure and collect four
subsamples from a rectangular configuration one-half
meter (.5) by two (2) meters. Soil collected at each
corner of the rectangle will be composited indentical to
those collected from the sampling circle. All samples
are obtained by compositing the four subsamples from the
circle or from the four corners of the rectangle.
If there is no well defined curb, as is the case in most
of the East Helena area or of an unpaved street, the
edge of the pavement or gravel will be used as the
curb. If there is some obstruction one meter from the
curb such as a sidewalk or drainage ditch, move toward
the house and to the right until the circle or rectangle
can be established.
2) Rear of House
The second soil sample will be taken in the rear of the
house in the back yard. If there is no back yard, then
the soil sample should be obtained in whatever open
space is available, but at least six meters from painted
surfaces such as walls of house, shed, or painted fence.
Samples from the front and rear of the home or residence
will composited for sample handling and analyses.
-------
3) Side of House
Estimate the approximate center of the house, front to
rear on one side and draw the one-meter circle 1/2 meter
from the foundation of the house. This should yield one
subsample immediately adjacent to the foundation, two
subsamples half a meter from the foundation and the
fourth subsample one full meter from the foundation*
This "side of house" procedure will be used to collect
one sample from significant structures which are
adjacent to the residence 9uch as garages, shops, etc.,
and one sample (using the one meter circle method) from
a major bare soil play area, if lead in paint is evident.
The environmental technician will be instructed to
sketch the house, grounds, and soil sampling sites on
the Environmental Survey form in order that the soil
sampling sites may be identified.
4) Other Public Areas
In multiple dwelling housing units it will not be
possible to obtain three composite soil samples for each
eligible child. In these instances, the areas in front
and rear of the apartments should be sampled,
composited, and recorded. One "side of building" sample
will be collected. Playgrounds and other areas held in
common by the residents and available to the children
must be sampled, using methods described above.
5) Play Areas
If a playground or vacant lot adjacent to the home is a
favorite play area, it will also be sampled. The
sampling will be based on whether a child plays more
than 50 percent of the time in the adjacent play area,
as determined by the parents. On these adjacent
playgrounds, lots, or other areas not related to a
dwelling, the sampling scheme to be followed is a
modification of that previously discussed. Draw an
Imaginary line to the opposite diagonal corner of the
playground or lot. Along this line every ten (10)
meters take samples in the manner prescribed. Follow
Che same procedure for the other two corners, making
sure to take a sample in the center where the lines
intersect.
It is necessary to clearly record the playground sites
since addresses will not Identify them adequately. The
distinction between bare earth samples and those in
grass or sod covered areas must be made in order to
determine the relative contribution each type might make
to blood lead levels. Any bare areas will be sampled
separately.
-------
If children play In a neighbor's yard, soil samples will
be collected in a manner as above afcer permission is
obtained.
b. Instructions for Collecting Soil Samples
1) Determine the appropriate area for sampling as described
above (Sample Locations).
2) .Place 0.7 m^ vooden template on the ground and orient
arrow (on template) to point north with the aid of a
compass.
3) Insert acid washed acetate liner in JMC "0"
Contamination Tube, and affix "0" Contamination Tube to
the JMC Backsaver N-2 Handle.
4) Sink "0" Contamination Tube vertically into the soil at
one corner of the 0.7 m* wood template. Withdraw in a
vertical manner after achieving desired core depth
(approximately 2 inches). Proceed to the next sample
corner (until all corners have been sampled) and repeat
this step. Thus, the sample cores will be contained
within one or two acetate tubes, depending on depth of
individual cores.
5) Remove acetate liner containing the sample from the "0"
Contamination Tube. Put acid washed caps on both ends
of the acetate liners are required to obtain the sample
and/or duplicate, they should be taped together with
proper identification labels on each acetate tube.
6) Store in an upright position to preserve soil core
stratification and to minimize mixing.
7) Clean "0" Contamination Tube by placing in a tap water
bath and using a large test tube brush to scrub the
inside surfaces free of organic soil and clay. Visually
inspect inner surfaces of "0" Contamination Tube (and
nozzle, particularly) to ensure soil has been removed.
If soil remains, repeat this step.
8) Rinse with distilled de-ionized water and then with
reagent grade acetone to aid in drying the inside of the
"0" Contamination Tube.
9) Use clean paper towels to wipe the exterior surface of
the "O" Contamination Tube clean and dry. Periodically
check the nozzle by wiping with a B-D alcohol swab to
Insure cleanliness at that critical interface.
10) Place dried "0" Contamination Tube in heavy duty Zlploc
bag and proceed to the next sample area.
11) Prior to in-field sampling, computer-aided random number
generation will be used to ascribe duplicate and blank
control procedures for 18.52 of the sample area.
-------
12) Duplicates: After obtaining cores as per the procedural
steps 1-11 above, rotate the 0.7 m^ template 90° and
extract duplicate cores (approximately 0 to 3.5 Inches
in depth).
13) Blanks: After the in-Łield cleaning procedures rinse
the "0" Contamination Tube with distilled-deionized
water. Collect the DI rinse water in an acid-washed
polyethylene container with a water-tight screw cap.
Label the containers appropriately and store in an
upright manner.
14) For sandy soils, and sand boxes in particular, use the
acetate liner and place a clean gloved hand at the
bottom of the acetate sleeve such that the contents
remain in the liner upon extraction. Composite these
samples in wtorl-palc bags, label and store in a separate
box.
Soil
Sampling Equipment
a.
JMC Backsaver M-2 Handle.
b.
"0" Contamination Tube.
c.
Acetate liners and caps.
d.
Wood template measuring 0.7 m2.
e>
Polyethylene gloves.
f.
Tap water bath.
g*
Large test tube brush.
h.
Distilled - deionized water.
i.
Reagent grade acetone (Baker analyzed)
j.
Becton Dickinson alcohol swabs.
-------
APPENDIX 12
Dust 'Collection
I* Instructions for Collecting Household Vacuum Bag Samples
a. Depending on vacuum type, reach in or rip open vacuum bag to
obtain sample*
b. Using plastic gloved hand, obtain a large handful of vacuum
bag contents.
c. Transfer to Ziploc plastic bag.
d. Label appropriately.
2. Instructions for Collecting Special Vacuum Cleaner Samples
a. Description of Sampling Apparatus.
A high-volume airborne particulate sampling device (Sierra or
General Metals) with an intake flow of about 55 cubic feet
per minute has been modified so a plexiglas hood can be
attached to the horizontally positioned stainless steel
filter holder. Two micron pore size Zeflour S by 10 inch
teflon filters will be used on the device to insure
collection of the very fine inhalable particles (down to
about 0.01 micron size) which are not retained by a typical
household vacuum cleaner. Smooth plastic dust attachments
along with clear plastic (tygon) 1-inch, thick-walled, 8-foot
Cubing lengths will transfer floor dusts to the hood of
deposit onto the teflon filter.
b. Sampling Procedure
1) Be sure hood-hose-wand attachments are clean. Check to
see that all parts have the same equipment number.
2) Be sure that sample transport box is clean and filled
with prenumbered and bagged teflon 8 by 10 inch filters
with back-up glass fiber filters.
3) Be sure sufficient cleaning items are available: 5
gallons of deionized-distilled water, brushes, paper
towels, ethanol, clean bags, gloves.
4) Allow 20-25 minutes per home for sampling.
5) Upon entering the home, identify a high-traffic carpeted
area between the living area and the kitchen which is
away from an outside entrance if possible.
6) Wipe the wooden 1-square meter template with an ethanol
soaked towel and dry, then position it on the floor with
enough room for the worker to gain access to two sides
of the template.
7) Clean the stainless steel filter holder with an ethanol
towel, dry and load the vacuum device with a
teflon/glass-backed filter pair, retaining the labeled
filter-holder bag for the about-to-be loaded filter.
Log the ID numbers, residence address, etc., on the data
sheet.
-------
Position the vacuum device adjacent to the template:
kneel on the plexiglaa template so it remains
stationary; turn the vacuum blower motor on;
simultaneously begin dusc collection when the stopwatch
is started; vacuum vigorously for 30 seconds; stop the
time without resetting it; reposition yourself on the
template perpendicular to the original position and
repeat the collection of dust for 30 seconds.
Remove the sample hood. With gloved hand3 gently raise
the edges of the filter paper to prevent dust from
falling off the filter and carefully reinsert the loaded
filter into the original plastic bag before sliding ic
horizontally into a slot in tha transport box.
Disassemble the vacuum apparatus placing the soiled
hood, hose, adaptors, etc., into the original bag for
return to the laboratory for cleaning. Outside the
residence, attach a hose on the vacuum blower motor
exhaust to blow the filter holder device clean, then
wipe the stainless steel filter holder with an ethanol
towel and dry.
-------
APPENDIX 13
XRF Analyses of Painted Surfaces
Instructions for Conducting Sampling
a. Explain basic purpose and function of the XRF to the property
owner* Answer any questions concerning the XRF and obtain
verbal consent for use of XRF.
b. Ask parents if there are any painted surfaces that the
children put their mouths on that should be checked by XRF.
Label and fill in all data necessary on XRF log sheet.
c. Check XRF for accuracy using the lead standard supplied with
each instrument. Run standards at each house before and
after taking household readings.
1) To run the standard - place lead standard on floor;
place XRF on standard; press trigger. Take three
readings and record the middle value, being careful to
note that the readings are within specified standard
deviations stated on the lead standard. Record results.
2) If standards are not within recommended standard
deviations, calibrate according to manufacturer's
recommendations.
d. Readings should be taken in all areas where the children
spend a lot of time and where the children can reach -
approximately 3 feet from the floor. Take three readings at
each station and record the middle value.
1) Take readings on exterior of house, 1 foot from the
right doorframe, and 3 feet from the surface on which
you are standing (ground or porch floor). Take readings
on back, front, and side doors, according to amount of
use. If surface is unpainted brick, or other surface
without a coating, take a reading on the door.
2) Take readings on one site in each room on a painted wall
surface. Readings should be taken throughout the house:
in the kitchen, all bedrooms, bathrooms, hallways,
dining room and living room. If walls are not painted,
a reading should be taken on the trim.
3) Other places readings should be taken are on painted
children's furniture and toys, fences, sandboxes,
exterior buildings, and fences.
e. Note chipping and peeling on trio and/or wall at each station
site, and record results.
f. Take reading on lead standard and record value.
g* Turn off XRF.
Processing Equipment:
a. Portable XRF with lead standard for calibration and precision
purposes* Princeton Gamma Tech portable XRFs (Model XK3)
will be used, with U. S. Department of Housing and Urban
Development lead standards.
b. Battery packs for operation of the XRF in the field.
-------
EAST HELENA, MONTANA
XRF READINGS
(1-7) I.D. NUMBER /
'(Block (House
(8 ) Area __________ Logged
PAINT
INSTRUMENT NUMBER XRF STANDARD XRF READING XEF READING
AT START AT END
( 9- 11) (12 - 15) (16 - 19) (20 - 23)
(Circle
:orrec
t numbers)
WALL
AREA CHIPPING
PEELING
Yes No
OR
NA
TRIM
CHIPPING OR
PEELING
Yes No NA
XRF SITE
1 - Wall
2 ¦ Trim
XRF
READING
( 24
-
31)
1.Living Room
1 2
8
1 2
8
1 2
•
( 32
-
39)
2.Bedroom 1
1 2
8
1 2
8
1 2
*
( 40
-
47)
3.Bedroom 2
1 2
8
1 2
8
1 1
•
( 48
-
55)
4.Bedroom 3
1 2
8
1 2
8
1 1
•
( 56
-
63)
5.Bedroom 4
1 2
8
1 2
8
1 2
•
( 64
-
71)
6.Bathroom
1 2
8
1 2
8
1 2
*
( 72
-
79)
7.Kitchen
1 2
8
1 2
8
1 1
( 80
-
87)
8.Dining Room
1 2
8
1 2
8
1 1
*
( 88
-
95)
9.Hallway
1 2
8
1 2
8
1 2
( 96
-
104)
10.Exterior
1 2
8
1 2
8
1 2
(105
(114
—
113)
122)
11.Other
(Specify)
12.
1 2
1 2
8
8
1 2
1 2
8
8
1 1
1 1
*
(123
-
131)
13.
1 2
8
1 2
8
1 2
%
(132
-
140)
14.
1 2
8
1 2
8
1 2
—. *-
DATE:
INSPECTOR:
rev 7/27/83
-------
APPENDIX 14
Floor Wipe Samples
1. Insturctions for Collecting Floor Wipe Samples
a* Determine sample site. Take dust sample only on a smooth
surface (no rugs or carpeting) on floor below the site used
to take the kitchen XRF reading.
b. Remove template from plastic bag* Handle template by the
outside so as not to contaminate the inside edges*
c. Lay template over sample site*
d. Open two alcohol swabs as follows:
1) Before tearing open alcohol swab, press the package from
top to bottom forcing the swab toward the bottom away
from the dotted line.
2) Tear along the dotted line and discard the smaller
portion of the package*
3) Invert package and squeeze out all the alcohol from the
package.
4) Carefully tear the two sides of the package off, being
careful not to touch or tear the swab*
5) Lay completely opened package on a convenient chair or
table.
6) Open a second alcohol swab in the same manner.
e* Fold swab into a triangle. Grasp one corner of the swab,
being careful to touch as little of the swab as possible,
then grasp the opposite corner until you have the tips of
both corners between your thumb and index finger,
f* Begin floor wipe. Place the triangular pad in inside upper
right corner and move to the left, moving in one direction
only. When you come to the left side of the template, lift
the pad, go back to the right side, place pad directly
underneath first pad path and repeat procedure until the
entire area within the template has been covered.
g. Place the pad with the dust covered side up on the inside of
the used package.
h. Using second alcohol swab as described above, make a series
of pad wipes from top to bottom until entire surface has been
covered.
1. Place second dust covered pad on top of the first with the
soiled surfaces facing each other,
j. Close the top of the package and roll toward the closed side,
k. Place sample in Zlplock bag.
1* Label appropriately.
2* Collection Equipment:
a* Plexiglass template framing a 400 cm2 area.
b« Becton-Dickinson low metal alcohol swabs.
c. Ziploc bags.
d. Gauze.
e. Travenol vinyl gloves.
-------
APPENDIX 15
Garden Vegetable Collection
1. Garden Selection
Select one garden plot (if available) per city block for
sampling. Select a well-ventilated garden plot in the southern
portion of each block* In residential areas without city blocks,
such as in certain subdivisions and on farms, select gardens which
are representative of the area.
2. Instruction for Collecting Garden Samples
a. Wear a pair of previously unused plastic gloves.
b. Collect approximately 400 grams each of 3 types of vegetables:
1) Type 1 - Leafy vegetable, such as lettuce and cabbage
leaves
2) Type 2 - Above ground vegetables, such as peas and beans
3) Type 3 - Underground vegetables, such as carrots and
radishes
c. Collect samples of leafy garden vegetables by clipping the
plant above any visible splash line or at least four cm from
the ground. Take only the edible portion of the plant.
d. Wear new plastic gloves for each vegetable type. Grasp the
plane with one hand and clip it with the other hand. Collect
samples from the middle and about 0.5 meters from each end of
a patch or row.
e. Place samples in brown paper kraft bags to prevent sweating,
date and label with preprinted labels for delivery to the
operations control center.
f. For each garden sampled, collect five 10 cm deep soil samples
for compositing. Sample according to the diagram shown below:
1 meter
/
1 meter
X
— X
g. Place the composite sample in a Ziploc plastic bag, date,
label, and deliver to the operations center.
-------
APPENDIX 16
EAST HELENA, MONTANA
SOIL AND VEGETATION SAMPLE
( 1 - 7) I.D. # /
PLACE LABEL
HEBE
SOIL AND VEGETATION SAMPLE
(Circle one)
Yes
(8) 1. Front Yard
(9) 2. Back Yard
(10) 3. Side of House
(11) 4. Play area
(12) 5. Other Location
If other, specify
(13) 6. Adjacent play area
(Vacant lot, etc.)
(14) 7. Garden soil composite
(15) 8. Adjacent structure soil
(16) 9. Kitchen dust sample
(17) 10. Vacuum carpet sample
(18) 11. Household vacuum bag dust
(19) 12. House cleaning:
No
2
2
2
2
2
Not
Applicable
8
8
8
8
8
8
2
2
2
2
2
Poor
Fair
Unknown
9
9
9
9
9.
8 9
8 9
8 9
8 9
8 9
3 ¦ Average
4 ¦ Excellent
-------
Page 2
I.D. Q /
13. Percentage
of grass cover:
OZ-25%
262-50%
512-75%
76S-
(20)
Front yard
1
2
3
4
(21)
Back yard
1
2
3
4
(22)
Play area
1
2
3
4
Date: Inspector:
Sketch the placement of the house on the property. Identify as accurately
as possible the location of each soil sample.
-------
APPENDIX 17
117
Letter to parents of children ages 1 though 5 living in Area III (Helena)
Address
Dear Mr. and Mrs.
You may be aware that the Montana Department of Health and Environmental
Sciences is conducting a study to investigate the possible lead exposure of pre-j
children living in the East Helena area.
A study of this nature requires a control group which means a separate,
nonexposed population to compare with those having probable lead exposure. A
section of Helena has been chosen as the control area. This letter assumes that
you live in this area.
A previous house to house survey Indicates that you have one or more childre
in the age range of 12 months through 5 years.
The study would consist of an interview, obtaining samples of blood, urine
and hand dust, measurement of lead in paint, and collection of dust v--'w.hin the
house and soil and from the yard.
It 1s not expected that significant amounts of lead will be found in your
child/children or 1n your house and yard. In any event, you will be given the
results of the tests.
If you have any questions, please call the Air Quality Bureau at 449-3454.
You will soon be receiving a telephone call requesting an appointment to
visit your home.
Your cooperation is appreciated.
Sincerely,
John J. Drynan, M.D., Director
DHES
-------
APPENDIX 18
Laboratory Methods - - Environmental Samples
1. Instructions for Soil Samples
a* Sample Preparation
1) Place clean white paper on work table.
2) Orient acetate liner lengthwise on clean paper* Remove
..both caps from liner*
3) Use PVC plunger (PVC pipe with silicone stopper) to push
sample out of acetate sleeve. Apply just enough
pressure with PVC plunger to extract sample. There will
be variation between samples as to the force necessary
for core removal. Make sure to grip acetate sleeve
firmly during this step.
4) Dissect sod layer, if present, from core. Remove the
sod layer at that zone where "crumbly soil" ir.eecs
"compacted soil." Thus, remove the portion from the top
of the compacted soil zone up through the surface
vegetation (1 to .25 inches), as follows:
a) Determine the depth of the sod layer by examining
soil structure.
b) Use clean heavy duty serrated plastic knives for
the separation process.
c) Use plastic knives for one separation and then
compile them in a box such that they may be washed.
(i) Wash plastic knives with soap and tap water,
then give a final rinse with
distilled-deionized water.
(ii) Let air dry and re-use.
5) After removing sod for all cores of a given sample
(four), composite the sod tufts in a Ziploc bag and
label appropriately. Store in a box. If there is no
sod layer present, one-inch segments may be measured
from the core without prior surface soil removal.
6) Place a plastic ruler along the length of the core.
Remove one inch of soil beginning at the compacted soil
zone just below the sod layer and composite these
one-inch segments in a labeled paper Kraft bag for a
given sample (not duplicates)*
7) Discard the rest of the soil.
8) Break up composited one-inch sample segments in labeled
paper Kraft bags by squeezing segments while in the bag
(using paper bag as a buffer between hands).
9) Place Kraft bags containing composite sample in oven at
80°C for 24 hours. This will yield data on a
dry-weight basis as processing to the analysis stage
proceeds.
10) Duplicates should be treated just like samples, with the
following exception. After removal of the sod layer (If
apparent), Instead of measuring off a one-inch segment,
measure off a three-inch segment. Composite these
-------
three-inch units in a labeled Kraft bag (with additional
"duplicate" identification). Break, up the segments as
per step if8, and place in oven to dry as described in
step 09.
Pass the 0-1 inch samples and 0-3 inch duplicates
through a 10, 2 millimeter (Tyler equivalent of a y
mesh) sieve by mechanical dry shaking on a Gilson Sieve
Tester for 5 minutes.
Transfer sieved soil from the sieve catch pan to
Whirl-pak polypropylene bags and affix proper
identification labels.
Clean the Gilson Sieve Tester as follows:
a) Run the putty knife on both surfaces of the two
sieves. Blow out with breathable compressed air.
b) Brush with plastic nylon dish brush on boch sides
of the two sieves. Blow out with breathable
compressed air.
c) Use a moist ethanol paper towel to gently rub the
bottom of the two sieves. Blow out with breathable
compressed air.
d) For Che catch pan, first blow it out with the
compressed breathable air, Chen wipe clean with a
moist ethanol paper towel. Use the compressed air
once again to dry the pan.
e) Wipe top of Gilson shaker with a dry paper towel,
and a quick blast of breathable compressed air.
b. . Instructions for Using SPEX Shatterbox Swing Mill Procedure
• 1) Dry samples prior to grinding by placing the Whirl-pack
bag in a drier where the temperature is between
35-40° C with the bag opened to allow moisture to
escape. Samples are to be dried a minimum of 6 hours.
2) Weigh 100 milligrams _+ 10 mg of Ivory Snow (sodium
stearate) into a plastic weighing dish for aid in
grinding.
3) When extracting sample material to grind, lightly shake
Che Ziploc bag such that the sample moves to one corner
of the bag. Run the pre-cleaned (ethanol-and paper
towel) stainless steel spatula down the inside seam to
the corner of the bag for a representative sample.
Gently shake the spatula while still within the sample
bag if too much sample is on the spatula, in order to
minimize sample loss.
4) To the grinding aid in the weighing dish, add 5.000 _+
0.0010 grams of sample* Record the residence
Identification number (the three digits following the
prefix 312- ) both on the side of the plastic
vial and on the vial cap. Following the sample 10,
Indicate original sampling location by the code:
Front * A
Side ¦ B
Play - C
Adjacent play area » D
Adjacent structure - E
Garden - F
Other #1 " G
Other 02 » S
11)
12)
13)
-------
An example; 312-167A represents a front yard sample
collected from residence 167.
5) Care must be exercised in handling large numbers of
samples to prevent mislabeling or sampling the same
Whirl-pack bag twice. Establishing sample handling
routines and work bench holding locations will prevent
•such problems.
6) Pour the 5 gram sample plus 0.1 gram grinding aid into a
.previously cleaned SPEX shatterbox tungsten-chromium
swing-mill grinding container. Place sample between the
center puck and the four inch ring to prevent spilling
the sample. Once most of the sample is placed in the
grinding container, invert the plastic weighing dish
over the puck and tap the bottom to insure the total
sample is transferred to the grinder. Make sure the
rubber 0-rlng is properly seated before placing the lid
on the grinding container. Carefully place the grinding
container into the shatterbox (onto the rubber mat) and
clamp the 12 inch pivotting arm over the container such
that the clamp centers on the grinding container lid.
Clockwise tighten the clamp knob until it can no longer
be tightened with two hands and lift the locking device
to prevent the knob from working loose during the
grinding process. Close the soundproofing shatterbox
lid, turn the toggle switch to on, rotate the timer to 5
minutes and press the white button on the timer knob.
Grinding will proceed and stop automatically.
7) Once the grinder stops, press the locking device to
loosen the tightening knob. Lift the unopened grinding
container from the shatterbox with two hands and place
it on a clean sheet of 8 1/2 x 11 inch paper in the
hood. Wear latex gloves and a face mask during this
process.
8) Slide and carefully lift the lid off the container and
hold it vertically on a second clean sheet of 8 1/2 x 11
inch paper. In this position gently brush the fine dust
on the lid down with single strokes, using a 1" nylon
paint brush, onto the center of the sheet of paper.
During this operation, Che hood vent fan must be turned
off to prevent loss of the sample. Gently set the heavy
grinding container lid aside. Brush the ring and puck
with increasingly smaller concentric circles in order to
remove sample material from the top of the puck and 4"
ring. Additionally, brush the exposed sides of both the
ring and puck while in the grinding bowl. Brushing in
this manner will result in concentrating the sample in
the bowl, and facilitate sample recovery and the
cleaning process. With two hands, tilt either the puck
or the ring from the grinding container so it can be
lifted onto the sheet of paper. Gently brush all sides
down, collecting the sample on the center of the sheet
of paper.
-------
9) Fold the middle of one side of Che sheet of paper which
holds the sample and carefully funnel the sample into a
previously cleaned and labeled vial. Cap the vial to
secure the sample and check it off from the printout as
being completed. Discard the plastic weighing dish and
the sheets of paper.
10) Use a vacuum (or breathable compressed air) to move dust
from Che grinding container, then moisten a paper towel
.with ethanol and wipe all interior grinder surfaces.
Repeat this process until the alcohol towel shows no
further soiling. The rubber 0-ring should be removed
and cleaned without undue stretching so as to extend its
lifetime. Re-assemble Che set-up for the nexc sample.
11) If covel wiping Che inner grinding parts does noc prove
sufficient, scrape Che surfaces with the stainless steel
Chicago Cuclery spatula and repeat towel wipes. If this
8till does not visually accomplish the cleaning, pour
abouC 5 grams of sand (Sargent-Welch 99.82 pure) into
the grinder and sec it up Co run for 2 minutes. Upon
completion clean Che device as per 9. above.
InscrucCions for Using SPEX X-Press (Pelletizer) Procedure
1) Assemble press componencs in preparaclon for soil
samples.
2) Pour ground sample inco aluminum sleeve. Ic may help co
rotate the plastic sample container and gently tap it
while pouring into the aluminum sleeve.
3) Slowly Insert aluminum plunger into aluminum sleeve.
4) Stabilize press componencs and apply pressure Co
aluminum plunger while firmly holding down the aluminum
sleeve.
5) Firmly grip the aluminum sleeve with one hand and the
aluminum plunger with the other hand.
6) Begin co extract the aluminum sleeve holding the
aluminum plunger in place. This will free the pellet
from the sleeve. The sleeve should be withdrawn about
1/4 of an Inch prior to excraccing Che aluminum plunger
in unison wlch the aluminum sleeve* This can be a
continuous mocion.
7) After extracting aluminum sleeve and plunger, see that
the sample is incacc in pellac form.
8) Pour a level Cablespoon full of boric acid in Che pellet
' chamber, carefully, so as noc Co discurb Che lncegrlcy
of che pellec form.
9) Inserc machined cylinder press; secure press chamber
base and lightly press down on machined cylinder.
-------
10) Place press chamber components in press, holding secure
Che bottom plate, mid-chamber and machined cylinder.
11) Screw down securing threads tightly on machined
cylinder, holding the press components securely.
12) Tighten hydraulic fluid grip (clockwise) and turn press
on. Watch the pressure gauge, and when it reaches 15
tons, count for 2 seconds and turn press off.
13) Let pressure decrease moderately, then slowly turn
¦ hydraulic fluid grip counterdoclcwise. Observe pressure
gauge as you turn the hydraulic grip, as the pressure
decrease should not be drastic.
14) Raise the securing threads via the wheel on top of the
press and remove press chamber components in the same
manner as they were placed into the press. That is,
holding the bottom plate, mid-chamber and machined
cylinder securely*
15) Carefully stand press components on machined cylinder
and remove the bottom plate (which is now the top). Put
PVC tubing where bottom plate was, and place once again
into the press. Align the washer with the securing
threads and continue to tighten. Hold the mid-chamber
of the press components securely until pellet is
extracted from the chamber.
16) Remove press components carefully, holding the machined
cylinder at the bottom so it will not fall out.
17) Remove PVC tube and grab pellet with gloved fingers. Be
careful not to touch soil surface of pellet. On the
"top" of the pellet, boric acid side, write the sample
three digit 0 and alpha coding (as was on plastic
vial). Record the date on the pellet as well and then
place in foam "nest", soil side facing down.
18) Use air hose on all pellet chamber components under the
hood, aiming at the exhaust duct.
19) Then use ethanol and paper towel (Hi Ori) to wipe
components clean of sample dust. The aluminum sleeve,
aluminum plunger, mid-chamber and the bottom plate
should be given special attention*
20) Blow out components and assemble as they are air
cleaned. The following order may be used: bottom
plate, mid-chamber, aluminum sleeve, aluminum plunger,
machined cylinder*
d. Sample Analysis Methodology
Pelleted soils were analyzed by energy dispersive X-ray
fluorescence (XRF)* This method involves the excitation of
characteristic X-rays in the sample by an external source of
radiation* Calibrated measurements of the intensities of
these X-rays quantify the sample's elemental concentrations.
The XRF system used was EG&E ORTEC TEFA III. Calibration
consisted of (1) entering standard concentrations with
associated uncertainties Into computer files; (2) selecting
metal filter types to increase background to peak ratios; (3)
setting regions of interest for all desired elemental peaks
including several regions for interfering elemental
-------
corrections; (4) running the standards to obtain spectra: (5)
performing absorption/enhancement corrections and stripping
peak overlaps caused by interferring elements; and (6)
adjusting the computer for an appropriate report format.
Once the X&F system was calibrated, samples were run in
batches of 24 by entering identification numbers and
initiating the computer-controlled analytical run. The
duration of each run vas 6 hours*
The first position sample in each batch was a standard used
for normalization to the previous calibration. The last
sample in each batch was a reference sample used for
comparisons. Samples were subjected Co 300 second live-cine
counts to optimize the statistics of the counting process.
The area of the pellet surface impacted by the X-ray beam was
approximately 1 square centimeter* The small soil particle
size (less than SO micron diameter) within the pellets
eliminated the particle size corrections ordinarily needed to
determine the concentrations of low atomic number elements.
Wet-chemical analyses were performed using (1) a Perkin-Elmer
sequential ICP-5500 system for inductively-coupled argon
plasma analyses; and (2) a Varian AA6 equipped with a hydride
generator for obtaining arsenic values,
e. Equipment
1) Sample Preparation
d •
Plastic serrated knives.
b.
Nasco Whirl-pack bags*
c.
Kraft bags (lunch bag size)*
d.
Gilson Sieve Tester, Model SS-15.
e.
No. 10, 2 millimeter mesh sieve (Tyler equivalent
to a 9 mesh).
f.
Sieve catch pan.
g-
Clean white paper.
h.
952 ethanol and paper towels.
Grinding
a.
SPEX Shatterbox cat. no. 8510*
b.
SPEX Shatterbox tungstens-chromium grinding dish
with ring and puck, cat. no* 8504.
c*
Whirl-pack polypropylene bags.
d.
Latex gloves*
e*
1" nylon brushes.
f.
13 dram plastic vials*
g.
Breathable compressed air.
h.
8 1/2" x 11" paper*
1.
Paper towels*
J.
95Z ethanol*
k*
Sargent-Welch 99.82 pure sand*
1.
Ivory Snow (stearic acid 99%).
Pelletizing Equipment
A •
SPEX X-Press cat. no. 36248*
b.
SPEX X-Press machined components.
c.
Machined aluminum sleeve and dowel.
d.
Latex gloves*
e.
Boric acid.
-------
12) The catch pan samples are then processed in a similar
fashion as soil samples. The SFEX swing mill and SPEX
X-Press procedures outlined in the soil subsection
should be followed. Grinding time for vacuum samples,
hovever, is 2 minutes, instead of 5 minutes.
13) For the vacuum samples less than 5 grams, it is
.important to maintain the ratio of 5 grams of sample to
0*10 grams of stearic acid (for matrix similarity in XS_F
analyses). If sieved vacuum samples are equal to or
less than 2.5 grams, they need not be processed further.
14) Clean the sieve network, as per Soil Samples a.14.,
above.
15) Assemble sieve network and ready for the next sample,
b. Processing Equipment:
1) Gilson sieve tester SS-15.
2) 2.0 millimeter sieve.
3) 850.0 micrometer sieve.
4) Sieving catch pan.
5) Stainless steel spatula.
6) 13 dram plastic snap cap prescription vials.
7) Breathable compressed air.
8) Plastic nylon brush.
9) 2 inch putty knife.
10) 95Z ethanol.
11>- Hi-Dri paper towels.
12) Ziploc bags.
3. Garden Vegetable Samples
a. Sample Preparation Methodology - Drying
In the laboratory, vegetables were individually removed from
kraft bags (with plastic gloves), dipped into a stainless
steel bowl containing six litters of cold tap water, stirred
lightly while intermittently submerging for 30 seconds each
composite sample, then rinsed once in running tap water.
This step was intended to be similar to the vegetable
cleaning done in the home. Vegetable samples were placed in
individual beakers for drying at 80°C for a minimum of 48
hours. Samples were then ground in a Wiley mill to pass a
40-mesh screen and ashed.
b. Sample Preparation - Methodology Digestion
The University of Montana, Missoula, MT, used the following
digestion procedure for preparing garden for vegetation rinse
samples. The procedure is appropriate for preservation of
potentially volatile metals, as adapted from Van Meter
(1974), and uses the following instructions:
1) Weigh out 0.25 g of sample.
2) Place in 2.5 cm by 25 cm acid washed Pyrex test tube.
3) Add ten milliliters of instra-analyzed, concentrated
nitric acid.
4) Seal tube with an oxygen-methane torch.
5) Place sealed Pyrex tube in sections of steel pipe, and
cap both ends.
6) Put capped steel pipe sections into 150°C oven for
three hours.
-------
7) After cooling, the Pyrex tubes are removed from the
ateel pipe sections and depressurized by opening a small
vent hole with the oxygen-methane torch. This should be
done under a hood with all the precautions of working
with concentrated acids (i.e., apron, protective
glasses, gloves, etc.).
8) Score the Pyrex tube, wipe with trace metal free B-D
alcohol swabs and remove the top of the Pyrex tubed
along scored groove.
9) Transfer solution into 100 ml beakers using a
concentrated nitric acid squirt bottle to aid in sample
solution recovery from the Pyrex tube.
10) Evaporate sample solutions to dryness on a heat plate
(under an acid hood) with gentle heat to remove the -
concentrated nitric acid.
11) Re-dissolve dried samples with 0.3 Molar lnstra-analyzed
nitric acid to a final volume of 10 ml in volumetric
flasks.
12) The 10 ml of final solutions were then transferred to
plastic screw-top vials.
c. Laboratory Analyses of Garden Vegetable Samples
MDHES analyzed the vegetable rinse samples by AA and ICAP to
determine the sample concentrations of lead, cadmium,
arsenic, zinc, and copper.
d. Equipment
1) Sample Processing
a. 2.5 cm by 25 cm acid washed Pyrex test tube
b. Instra-analyzed concentrated nitric acid
c. Oxygen-methane torch
d. Steel pipe sections with threaded caps
e. Oven capable of 150°C
f. Glass scorer (a zinc plated one was used in this
procedure)
g. Trace metal free B-D (Becton Dickinson) alcohol
swabs
h. 100 ml Pyrex beakers
1. Hotplate
j. Plastic screw top storage vials (10 ml)
Reference:
Van Meter, W. Heavy Metal Concentration in Fish Tissue of the Upper
Clark Fork River. Montana University Joint Water Resources Research
Center, Bozeman, MT (1974).
-------
4. S'"""ary of Environmental Specimen Analyses
Table A lists the sizes of the samples analyzed for different
environmental specimens. The type of sample treatment, the standard
reference materials used, and the method of handling the data from these
analyses. Table B lists the minimum laboratory detection limits along
with ICP/AAS wavelengths for the elements described within this report.
Instrumental X-ray calibrations were difficult because standards were
difficult to find which fell within the high range of expected soil
concentrations. For this East Helena study, we expected that elemental
soil lead concentrations would almost reach 10,000 ppm. National Bureau
of Standards soil and bulk reference materials contain only low lead
concentrations except for river sediment (at 714 ^ 28 ppm lead) and urban
particulate matter (at 6550 + 80 ppm lead), neither of which presented a
composition or matrix entirely similar to smelter soil materials around
Ease Helena, Montana. Although the U.S. Geological Survey has documented
elemental concentrations for numerous soil reference samples, none contain
more than about 30 ppm of lead. Consequently, MDHES obtained Canadian
(CANMET) soil and ore reference samples, the latter of which ranged from
about 4 to 64Z in lead. Several CANMET soils and a low-lead ore were
carefully mixed, blended and ground to provide "soil" lead values within
the expected range. Additional soil samples from the vicinity of the
Anaconda, Montana, copper smelter, previously analyzed by EPA16 NEIC
laboratory in Denver by X-ray and ICP methods, were also used as
calibration standards. While calibrations were nearly ideal for lead,
ranging from 4 to 8310 ppm, other elements such as copper and zinc
presented problems. Copper ranged from 7 to 15,800 ppm and zinc from 34
to 49,790 ppm, far greater ranges than desired for this study. For
wet-chemistry analytical methods, standard solutions for elemental
concentrations can easily be mixed as needed, but short of sample mixing
and blending, bulk XRJ? standards come with fixed compositions, often with
ideal concentration ranges for some elements and not for others.
Standards for the garden vegetables analyzed by MDHES in this study were
NBS reference materials which were both available and mostly suitable with
regard to concentration ranges, although high heavy metal concentrations
are lacking in NBS reference biological materials.
All ICP/AAS calibrations were set up using the EPA multiple element water
standard 478-11 followed by appropriate dilutions of samples, as needed,,
Co obcain concentration values. On the other hand, X-ray calibracions
utilized a suite of standards selected from reference materials where
possible. A minimum of six "standards" are needed before any X-ray
calibration corrections (for incer-elemental absorption and enhancements)
can be made. For this study, at least a dozen "standards" were used for
each calibration type to allow for needed corrections during initial
instrumental calibrations.
-------
4. Summary of Environmental Specimen Analyses
Table A lists the sizes of the samples analyzed for different
environmental specimens." The type of sample treatment, the standard
reference materials used, and the method of handling the data from these
analyses. Table B lists the minimum laboratory detection limits along
with ICP/AAS wavelengths for the elements described within this report.
Instrumental X-ray calibrations were difficult because standards were
difficult to find which fell within the high range of expected soil
concentrations. For this East Helena study, we expected that elemental
soil lead concentrations would almost reach 10,000 ppm. National Bureau
of Standards soil and bulk reference materials contain only low lead
concentrations except for river sediment (at 714+28 ppm lead) and urban
particulate matter (at 6550 + 80 ppm lead), neither of which presented a
composition or matrix entireXy similar to smelter soil materials around
East Helena, Montana. Although the U.S. Geological Survey has documented
elemental concentrations for numerous soil reference samples, none contain
more than about 30 ppm of lead. Consequently, MDHES obtained Canadian
(CANMET) soil and ore reference samples, the latter of which ranged from
about 4 to 64Z in lead. Several CANMET soils and a low—lead ore were
carefully mixed, blended and ground to provide "soil" lead values within
the expected range. Additional soil samples from the vicinity of the
Anaconda, Montana, copper smelter, previously analyzed.by EPA's NEIC
laboratory in Denver by X-ray and ICP methods, were also used as
calibration standards. While calibrations were nearly ideal for lead,
ranging from 4 to 8310 ppm, other elements such as copper and zinc
presented problems. Copper ranged from 7 to 15,800 ppm and zinc from 84
to 49,790 ppm, far greater ranges than desired for this study. For
wet-chemistry analytical methods, standard solutions for elemental
concentrations can easily be mixed as needed, but short of sample mixing
and blending, bulk XRF standards come with fixed compositions, often with
ideal concentration ranges for some elements and not for others.
Standards for the garden vegetables analyzed by MDHES in this study were
NBS reference materials which were both available and mostly suitable with
regard to concentration ranges, although high heavy metal concentrations
are lacking in NBS reference biological materials.
All ICP/AAS calibrations were set up using the EPA multiple element water
standard 478-11 followed by appropriate dilutions of samples, as needed,,
to obtain concentration values. On the other hand, X-ray calibrations
utilized a suite of standards selected from reference materials where
possible. A minimum of six "standards" are needed before any X-ray
calibration corrections (for inter-elemental absorption and enhancements)
can be made. For this study, at least a dozen "standards" were used for
each calibration type to allow for needed corrections during initial
instrumental calibrations.
-------
TABLE A
MDHES LABORATORY METHODS
Hand Wash
Nominal
Sample
Size
500 ml
Floor Wipe entire
sample
Sample
Treatment
None
Extracted with
Dil.Aq.Reg. in
hot sonic bath
Vegetation
0.25 g HNO3 press,
glass bomb
Soils 0.200 g
(Wet-chem.)
Microwave with
Aq.Reg. + HF
Soils and
Vacuum Dusts
(XRF)
5.000 g Sieved @ 1 ma
mesh, 0.100 g
Na-sterate +
5.000 g sample
to grinder;
press at 16
tons/sq.in.
pressure, boric
acid capsuled
Reference
None
DUES Hi-vol
method
U of Mont.
Van Meter
method
Matthes et al.
U.S. Bureau
of Mines
Modified EG&G
0RTEC method
Reporting'
ICP diskette
arsenic by hand
ICP diskette,
arsenic by hand
Hand calcula-
tions
Hand calcula-
tions
Printout to
keypunch
Standard Reference Materials used for the above analyses were:
NBS: River Sediment (1645), Fly Ash (1633), Urban Particulate
(1648), Spinach (1570), Orchard Leaves (1571), Wheat
(1567), Tomato (1573), Pine Needles (1575), and Bovine
Liver (1577).
USGS: AGV1, G-2 and BHVO.
CANMET: S01, S02, PD1 (non-ferrous dust) and CZN-1 (zinc
concentrate)
USEPA: 478-11 (multiple water standard), smelter soils: 4772,
4774, 4776, 4778.
-------
Sample Type
Soils
(ug/g - dry wt.)
Vegetation
(ug/g - dry wt.)
Floor Wipe
(ug/wipe)
Hand Wash
(mg/1)
Wavelength
Soils/Vac
(ug/g - dry wt.)
TABLE B
MDHES LABORATORY MINIMUM DETECTION LIMITS
Method
ICP
ICP
ICP
ICP
Elements
A1 As* Cd Cu Pb
5.0 0.25 2.5 2.5 12.5
0.04 0.40 0.40 2.0
0.80 0.04 0.40 0.40 2.0
0.02 0.001 0.01 0.01 0.05
XRF 50
Si Ti Zn
25.0 2.5 2.5
0.40
0.40 0.04
0.01 0.01
308.2 193.7 226.5 324.7 220.4 288.2 334.9 213.9
1.4 4.0 2.3 5.1 40
12
5.1
*Arsenlc concentrations were obtained using an atomic absorption instrument equipped with
hydride generation.
-------
APPENDIX 19
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-------
APPENDIX 20
ANALYTICAL METHODS AND QUALITY CONTROL ASSURANCE
FOR
EAST HELENA, MONTANA, CHILD HEALTH STUDY
Division of Environmental Health Laboratory Sciences
Center for Environmental Health
Centers for Disease Control
Atlanta, Georgia
-------
East Helena, Montana, Child Health Study
Division of Environmental Health Laboratory Sciences
Case No. 312
-------
Contents
Sections Prepared on October 3. 1983
Page
Brief History of Event 4
Toxicants Present 4
Proposed Study 4
Laboratory Analyses Requested 5
Intended Use of Data 5
Analytical Goals 5
Graphite Furnace Procedure for Blood Lead 6
Erythrocyte Protoporphyrin 14
Graphite Furnace Procedure for
Blood Cadmium 22
Graphite Furnace Procedure for
Urinary Arsenic 30
Graphite Furnace Procedure for
Urinary Lead/Cadmium 37
Urine B2 Microglobulin 47
Urinary Creatinine/Protein 56
Hair Arsenic/Cadmium/Lead 76
Sections Reported on April 30. 1984
Method Modifications 81
Quality Control Data - for Analytical Runs 83
Ranges of Values from Normal Individuals 88
-------
4
BRIEF HISTORY OF EVENT
A primary lead smelter located in East Helena, Montana, and operated by ASARCO
has been in operation since 1888. A smaller, related industry, American
Chemet, lies between the smelter and the city. The entire population of East
Helena (1,650 persons) lies within a 1-mile radius of the smelter stack.
Another 2,000 persons live within a 2-mile radius, but outside the city
limits. About 5.5 square miles of the area surrounding the stack has lead
exceeding 1,000 ppm in the upper 6 inches of soil.
In 1975, a random sample survey of children in East Helena found 34% of the
children with blood lead levels exceeding 30 ug/dl of whole blood. 12 inches
of snow was on the ground (temperature: 14°F). At the time of the study,
this is significant, since the highest blood lead levels are generally found
in the hottest part of the year. The testing must begin on schedule in the
middle of July, if children at greatest risk for lead poisoning are to be
identified.
TOXICANTS PRESENT
The principal toxicant is lead, which is probably in the form of lead oxide,
due to the smelter operation. The major routes of inorganic lead absorption
are the gastrointestinal tract and respiratory system. Many organs are
adversely affected by lead and, often, in very complex mechanisms, such as the
effects of lead on heme metabolism. Anemia is one of the early manifestations
of lead exposure as the result of the shortened life span of erythrocytes.
Other target organs of lead poisoning are the central nervous system,
peripheral nervous system, and the kidney. Additional toxicants of interest
are arsenic and cadmium that are being analyzed in environmental samples for
comparative data.
PROPOSED STUDY
The Montana Department of Health and Environmental Services (DHES), in
collaboration with the Division of Environmental Health Laboratory Sciences
(DEHLS), is conducting a study of children in East Helena, Montana, to
determine potential health risk due to lead poisoning. The DEHLS will perform
analytical services to meet the goals of the study and assure the validity of
biological quantitations for the study. This document presents the analytical
melthods/procedures and necessary quality assurances that the DEHLS will
provide DHES for the Child Health Study.
-------
5
LABORATORY ANALYSES REQUESTED
1. Whole Blood Lead
2. Erythrocyte Protoporphyrin
3. Whole Blood Cadmium
4. Urinary Arsenic
5. Urinary Lead/Cadmium
6. Urine B2~Microglobulin
7. Urinary Creatinine/Protein
8. Hair Arsenic/Cadmium/Lead
INTENDED USE OF DATA
1. Validation/Confirmation
The whole blood lead and erythrocyte protoporphyrin data generated by the
Division of Environmental Health Laboratory Sciences will be used to
confirm and validate the respective data from ESA laboratories. A
statistical comparative analysis will be performed with the data sets to'
determine the equivalency of the analytical results for the two
laboratories.
2. Epidemiologic
All other analytes assayed by the Division of Environmental Health
Laboratory Sciences are required for epidemiologic investigation of health
risk related to high concentrations of heavy metals in the environment.
Analytical data for these analytes will be used as specified in the study
protocol.
ANALYTICAL GOALS
The analytical goals for the study set the expected minimum performance for
the methods used and provide a mechanism to determine the actual laboratory
performance. First, internal quality control procedures provide the past
history (expected) performance of the methods and day-to-day performance by
the laboratory. Second, external quality control efforts independently
validate the analytical performance.
1. Internal Quality Control
A summary of the analytical goals for each analyte on the basis of the
internal quality control is presented in Table 1.
2. External Quality Control
A. A random selection of 10% of the study samples are submitted to the
laboratory as "blinded" duplicate samples to determine the
repeatability (sensitivity) of the analytical measurement.
B. Blind external quality control samples are included with study
specimens to independently evaluate the analytical systems for both
accuracy and sensitivity.
-------
6
GRAPHITE FURNACE PROCEDURE FOR BLOOD LEAD
(Revised August 18, 1983)
Determination of lead in whole blood is accomplished by flameless
(electrothermal) atomic absorption. The specimen is diluted with a 0.10% v/v
solution of Triton X-100, the lead homogeneously distributed throughtout by
lysing of the RBCs, and the absorbance of the resulting solution measured.
The method is based on a published procedure by Fernandez (1,2).
EQUIPMENT
Atomic Absorption Spectrophotometer: Perkin-Elmer Model 372.
Graphite Furnace: Perkin-Elmer Model 500.
Autosanvpler: Perkin-Elmer Model AS-1.
Parameter
Wavelength
Lamp Current
EDL Power
Slit
Signal Mode
Read Time
Inert Gas
Furnace Type
Background Corrector
Setting
283.3 nm
8 ma
9.5-10 W
0.7 (ALT)
ABS
5.0 sec
Argon
Pyrolytic
ON
Temperature Program
DRY 110 C 20 sec
(20 sec RAMP)
CHAR1 550 C 30 sec*
(10 sec RAMP)
ATOMIZE 2000 C 5 sec
(1 sec RAMP)
Inert Gas Flow 300 mL/min
20 mL/min@
ATOMIZE
~During the CHAR portion of the temperature program, the baseline is reset to
"0" with the BASELINE function of the Model 500.
lit may be necessary to reoptimize with new contact rings or change in lots
of graphite furnaces. Optimization should be checked with base ( 10 ug/dL)
and 1.00 ppm spike (84 ug/dL) to establish:
A. Peak height at maximum value.
B. "Correct" value to allow calculation of base blood or other specimen
tfor 84 ug/dL = 0.160A ± 15%]
Recorder: Perkin-Elmer Model 56, set at 5 mV chart speed at 20 mm/min.
Automatic Pipet: Model 25000 Micromedic Automatic Pipette, equipped with
a 1-mL dispensing pump (set at 100%) and a 200-uL sampling pump (set at
50%).
NOTE: A 1-mL sampling pump set at 10% has also been found satisfactory.
-------
7
REAGENTS
Water: Ultrapure water, prepared by a Milli-Q polishing system, is used
throughout.
Nitric Acid: Redistilled nitric acid (G. Frederick Smith) is used to
prepare a 1.0% v/v dilution by volumetric dilution with ultrapure water.
Triton X-100: Scintillation Grade (Eastman Kodak) is used to prepare
0.10% v/v solution by volumetric dilution with ultrapure water.
Stock and Working Lead Standards: A 1,000 mg/L stock solution of lead
nitrate is prepared from 1.5985 g of NBS SRM 928 lead nitrate, diluted to
1L with 1.0% v/v nitric acid. This solution is prepared every 6 months.
Intermediate standard of 10 mg/L and working standards of 0.10, 0.25,
0.50, 0.75 and 1.00 mg/L are prepared daily by volumetric dilution with
ultrapure water.
PLASTICWARE AND GLASSWARE
All plasticware and glassware used is cleaned by soaking 2A h in
detergent, followed by soaking for 3 days in 25% v/v nitric acid. The cleaned
items are then rinsed thoroughly with ultrapure water, and stored in a
dust-free environment.
Venous blood specimens are collected in either 5-mL Vacutainers (Beckton
and Dickinson) or 5-mL "Monoject" tubes (Sherwood Manuf). Both of these
containers employ aqueous dipotassium EDTA as anticoagulant.
Eppendorf micropipets (Brinkmann Instruments) are used to prepare spiked
aliquots for calibration; disposable polyethylene tips are used as received.
Beckmann Instruments' "Bio-Vials" are used for dilution of blood
specimens; these 4-mL containers are cleaned as above.
SPECIMEN COLLECTION
Collection of an uncontaminated whole blood specimen is a critical part of
many toxicological investigations. The following guidelines will provide
directions which, if carefully followed, will minimize the contamination of
whole blood by the many sources from which it may come. It cannot be
overemphasized that skin, clothing, dust, and many other sources of
contamination contain many times the levels of lead, arsenic, cadmium, and
other metals that will be determined in the collected specimen.
1. Clean the antecubital area thoroughly with: a) soap and water (Phisohex
has been shown to be free from significant metal contamination), followed
by b) alcohol (isopropanol or ethanol).
2. Puncture the skin/vein with a sterile, disposable needle capable of
multiple sampling. A suggested product is the B + D catalog #5749,
20-gauge needle. In some applications, the first blood specimen collected
will be used for other determinations; its use is to "rinse" the
collection needle with blood.
3. Collect one or more tubes of whole blood, using an appropriate
anticoagulant for the metal of interest. Anticoagulant/metal combinations
that have been shown to be compatible are:
Hercury-Heparin or Citrate
Lead-BDTA. Heparin, or Oxalate
Cadmium-EDTA. Heparin or Oxalate
(preferred anticoagulant underlined)
It is critical that a few "spares" of the lot of tubes used for collection
be sent to the laboratory along with the collected specimens. This will
-------
8
allow the laboratory to determine the metal content of the anticoagulant
used in that tube lot and to make appropriate blank corrections. Of
course, all lots of tubes should be screened before use in surveys.
4. It is critical that the collected specimen be thoroughly mixed after
collection, to insure the anticoagulant/blood mixture is uniform and that
clotting therefore will be prevented. Clotted specimens are nearly
useless!
5. Refrigerate the collected specimens, and ship them refrigerated by the
most expeditious means available. Heparin is by far the least "permanent"
of the anticoagulants listed, but it will prevent clotting for 2 weeks if
well-mixed during collection and if the specimens are refrigerated after
collection and during shipment.
6. Ship the collected specimens in well-padded, insulated containers (freeze
safe or the equivalent).
ANALYTICAL PROCEDURE
1. Aspirate 100 uL of blood into the delivery tip of the automatic pipet
and dispense sample and 1.00 mL of 0.10% v/v Triton X-100 into a
precleaned 4mL plastic vial.
2. Aspirate air into the delivery tip and dispense 1.00 mL of 0.10% v/v
Triton X-100 into the same vial. Cap the diluted specimen with a
plastic top.
3. Vortex thoroughly, and allow the diluted specimen to stand 15-20
minutes.
4. Re-vortex, and transfer a portion of the diluted specimen to a
precleaned polyethylene or polypropylene autosampler cup.
5. Measure the absorbance in triplicate, using the AS-1 autosampler to
dispense 20 uL of diluted specimen into the graphite furnace.
STANDARDIZATION AND CALCULATIONS
Standardization is accomplished by the use of a modification of the method
of standard additions. A bovine "base blood," typically containing less than
10 ug/dL of endogeneous lead, is diluted as in the procedure, and aliquots are
spiked with microliter additions of lead nitrate standards.
Standard Addition Procedure:
1. Base blood is diluted in the procedure, using twice the prescribed
volume (perform a "double" dilution).
2. Into five precleaned plastic autosampler cups, pipet 20 uL of 0.10,
0.25, 0.50, 0.75, and 1.00 mg/L lead nitrate, using a 20-uL Eppendord
micropipet with disposable plastic tips.
3. Add 500 uL of diluted base blood to each of the five cups, using a
500-uL Eppendorf with plastic tips. Allow the diluted specimen to
drain in the plastic tip, and apply several more displacement strokes
to assure a quantitative transfer. Nix by gentle swirling.
NOTE: Careful measurement by gravimetric methods have indicated that the
transfer of diluted specimens is quantitative, provided careful technique
is used.
4. Transfer the remaining diluted base blood to a sixth autosampler cup.
5. Measure the absorbances in triplicate as in the procedure.
-------
9
Calculations:
Two methods of calculation have been used with the described procedure,
each of which gives essentially identical results. Each individual
specimen may be analyzed by the procedure outlined above for standard
additions. Although potentially highly accurate, this method has an
extremely low throughput. Any differences in observed slope from spiked
aliquots of different specimens will be automatically compensated for by
this method. Since no measurable difference in slope was observed (within
experimental error) between spiked bovine and human blood, either a
regression analysis or an "average slope" method are normally used.
Method 1
Prepare spiked aliquots of all specimens, measuring absorbances in
triplicate. The average of three absorbances of the six aliquots are
taken to be:
A 3 average absorbance of unspiked specimen
B = average absorbance of first spike ( + 2 ng)
C = average absorbance of second spike ( + 5 ng)
D » average absorbance of third spike ( + 10 ng)
E a average absorbance of fourth spike ( + 15 ng)
F * average absorbance of fifth spike ( + 20 ng)
All spiked average absorbances are corrected for dilution, multiplying by
a factor df, where:
df = dilution factor a orig. volume + spike volume
orig. volume
In this case, with a 0.500-mL original volume and a 0.020-mL spike volume,
the factor is:
(0.500 + 0.020) mL =¦ 0.520 mL = 1.04
0.500 mL 0.500 mL
Calculations
1. Linear regression
Calculate mean of 2 or 3 measured absorbances for base and 5 standards.
Correct "standard" absorbances by the dilution factor 1.04.
Construct the following table:
Added
mg/dL Blood ^average absorbance A corrected (A corr-Abase)=N(1_5)
0 Base ^base
8.4 0.10 ppm spike Ai
21.0 0.25 ppm spike A2
42.0 0.50 ppm spike A3
63.0 0.75 ppm spike A4
84.0 1.00 ppm spike A5
0
Ai(l.04) Ni
A2(1.04) »2
A3(1.04) H3
A4(1.04) n4
A5(1.04) N5
-------
10
Using a calculator, enter the following:
ug/dL, A corr -Abase
(0
0
)
(8.4
Nl
)
(21.0
n2
)
(42.0
)
(63.0
n4
)
(84.0
n5
)
Calculate r2 slope, intercept. Typical values for these parameters are:
slope 1.8 - 2.2
intercept -1.0 - +1.0
r2 0.98
Using corrected (A - blank) specimen values, calculate ug/dL for unknowns.
2. Average slope
Using values in table above, calculate the following:
M2 N3, N4, N5
2.5 5.0 7.5 10.0
Average these values and calculate a conversion factor, cf:
cf = 8.4 ug/dL
n(1-5)
Where N(i_5)= average of Nj and four calculated values above.
Take corrected A values (- blank) for specimens, and calculate ug/dL by
multiplying by cf.
NOTE: [cf should be 0.45 - 0.55]
QUALITY CONTROL SYSTEM
Quality Control Statistics
The statistical format used for evaluation of quality control will be that of
two-way analysis of variance, ANOVA, with the construction of quality control
charts based on 95% and 99% confidence limits of the mean of duplicate
measurements, as well as range charts (3).
Precision and accuracy of the analytical system will be monitored as follows:
1) Ten analytical runs will be performed to characterize all control
materials used, with duplicate measurements performed per run.
2) Analysis of variance calculations will be performed on these 20 data
points, and quality control charts will be generated by computer for X and
range R.
3) A minimum of two control materials will be incorporated into each
analytical run of 20 unknown specimens, and data obtained for these
controls will be evaluated with the X and R charts from 2).
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11
- Blind Quality Control
Two types of blind quality control specimens will be incorporated into the
system:
1) blind duplicate specimens will be prepared, and inserted at an interval
determined by the supervisor, usually one blind duplicate per 20 unknown
specimens.
2) blinded control or reference material samples will be inserted into each
analytical run of 20 specimens, at the minimum rate of one blind control
per 20 specimens.
In both cases, the blinds should be identical in appearance to the specimens,
with the same containers, specimen volumes, and labelling. If desired, blind
quality control specimens can be evaluated with the same statistical methods
used for the control materials.
Run Format
In all cases, the following format will be used for specimen determination:
SAMPLE // Sample ID
1
Blank
2-5 (or greater)
Calibration Curve
6
Control I
7-27
Specimens
28
Control II
29-33
Calibration Curve
34
Blank
Action Limits
The analytical system will be declared "out of control" if one or more of the
following events occur (3):
X chart
1) A single X value falls above
limit.
2) Two successive X values fall
below the lower 95% limit.
3) Eight X values in succession
below the center line.
R chart
1) A single R value falls above the upper 99% limit.
2) Two successive R values fall above the 95% upper limit.
3) Eight R values in succession fall above the center line.
the upper 99% limit or below the lower 99%
either both above the upper 95% limit or both
fall either all above the center line or all
-------
If the system should be declared out-of-control, the following remedial action
should be taken:
1) Check for errors in recording levels of control samples, and if none are
found,
2) Check and calibrate instruments before performing further analyses on
analytical samples,
3) Reanalyze patient samples performed during the out-of-control run.
Method Performance
Limits of Detection
Limit of detection is in a practical sense determined by two factors: 1) the
slope sensitivity of the method, i.e., the response (in the case of atomic
absorption, the absorbance or absorbance-second measurement) for a given
concentration or amount of analyte, and 2) the random noise of the
instrumental system used in measurement, especially that noise at the measured
response for the blank for the determination (4).
According to the recommendations of the above reference, the limit of
detection will be defined as that concentration of analyte corresponding to an
absorbance or absorbance-second measurement equivalent to three times the
standard deviation of this signal measured at an analyte concentration at a
"low" level. In symbolic terms, this becomes:
CL = l^B
m
where cl is the concentration calculated to be the limit of detection, sg
is the standard deviation of the measurement of a blank or low concentration
sample; and m is the slope of the calibration curve (change in absorbance or
absorbance-seconds/change in concentration or amount of analyte). For a set
of measurements at the 12-ug/dL level, the standard deviation of the
measurement was 1.33 x 10~^A, which yields a limit of detection of
2.04 ug/dL (N = 6).
Accuracy and Precision
Accuracy and precision of the blood lead method have been estimated from the
determination of whole blood pools whose target values have been determined by
either a definitive method (5) or by a series of highly proficient reference
laboratories. The accuracy and precision of this method have been presented
in a previous publication.
Recent evaluation of the revised method was performed with the use of whole
blood pools whose target or "true" values were determined by the National
Bureau of Standards by stable isotope dilution-mass spectroscopy. Precision
at the "medical decision" level of 30 ug/dL was measured to be 6% or less (%
total CV) with accuracy of + 2 ug/dL or less versus ID-MS target values. The
accuracy and precision shown by these data are comparable to that obtained in
previously published work (6).
-------
13
References
1. Fernandez FJ. Microraethod for lead determination in whole blood by atomic
absorption, with use of the graphite furnace, Clin Chem 21 (4): 558-561
(1975).
2. Fernandez FJ. Automated micro determination of lead in blood. Atomic
Absorp Newslett 17 (5): 115-116 (1978).
3. A statistical quality control system for the clinical laboratory. In
Selection and Implementation of Methods in Clinical Chemistry, Commission
on Continuing Education, Council on Clinical Chemistry. American Soceity
of Clinical Pathology, Chicago, Illinois (March 1975).
4. "Winefordner J., and Long 6. Limit of Detection—A Closer Look at the
IUPAC Definition, Anal Chem, 55, (7): 713A-719A (1983).
5. "A national understanding for development of reference methods and
materials for clinical chemistry. J.H. Boutwell, ed., American Association
of Clinical Chemistry, Washington, D.C., 1978.
6. Paschal D and Bell C Improved accuracy in determination of blood lead by
electrothermal atomic absorption. Atomic Spectroscopy, 2: 146-50 (1981).
-------
ERYTHROCYTE PROTOPORPHYRIN
I. Principle
Free erythrocyte protoporphyrin (FEP) is measured by a modification of
the method of Sassa and Granick et al.(l) Protoporphyrin is extracted
from EDTA-whole blood into a 2:1 (v/v) mixture of ethyl acetate-acetic
acid, then back-extracted into dilute hydrochloric acid. The
protoporphyrin in the aqueous phase is measured fluorometrically at
excitation and emission wavelengths of 404 and 655 nm, respectively.
Calculations are based on a processed protoporphyrin IX (free acid)
standard curve. The final concentration of protoporphyrin in a
specimen is expressed as micrograms per deciliter of packed red blood
cells (ug/dL) RBCs; a correction for the individual hematocrit is made.
II. Instrumentation
A. Perkin-Elmer Model 650-10 spectrofluorometer, with R938
photomultiplier tube, xenon lamp, and custom-made microcell (10- x
75-mm) holder positioned to allow the passage of light through the
aqueous phase only.
(Perkin-Elmer Corp., Norwalk, CT)
B. Model 56 recorder
(Perkin-Elmer Corp.)
C. Cary Model 119 double-beam spectrophotometer
(Varian Associates, Palo Alto, CA)
D. Vortex mixer
(Fisher Scientific Co., Fairlawn, NJ)
E. Micromedic APS-2 pipetting station with 50-uL sampling and 200-uL
dispensing pumps and reagent dispenser, with inserts in racks
modified to accept 10- X 75-mm tubes.
(Micromedic Systems, Division of Rohm and Haas, Horsham, PA)
F. Mettler Model H18 analytical balance
(Mettler Instrument Corp., Hightstown, NJ)
G. IEC Centra-7 centrifuge
(International Centrifuge Co., Needham Heights, MA)
H. Hamilton Dispenser, with 2.5-mL syringe
(Hamilton Co., Reno, NE)
I. Micromedic High-Speed Automatic Diluter with 1.0-mL dispensing pump
(Micromedic Systems)
III. Materials
A. Protoporphyrin IX, dimethyl ester, 99.3* purity, grade 1 (Sigma
Chemical Co., St. Louis, MO)
NOTE: Store at -20°C over a desiccant. Purchase of one lot is
recommended.
B. Ethyl acetate, spectrophotometry quality
(J. T. Baker Co., Phillipsburg, NJ)
C. Acetic acid, glacial, "Baker Analyzed"
(J. T. Baker Co.)
D. Hydrochloric acid, concentrated, "Baker Analyzed"
(J. T. Baker Co.)
E. Kimble 10- X 75-mm disposable glass culture tubes
(Kimble Div., Owens-Illinois Co., Toledo, OH)
F. Parafilm M
(American Can Co., Greenwich, CT)
-------
G. Actinic glass volumetric flasks
(Corning Glassworks, Coming, NY)
NOTE: All nondisposable glassware used in this assay should be
washed in 10% (v/v) nitric acid and rinsed six times with
deionized water.
H. Formic acid, 88%, reagent grade
(J. T. Baker Co.)
I. Deionized water, greater than or equal to 1.0 megaOhm-cm at 25°C
(Continental Water Co., Atlanta, GA)
Reaaent Preparation
A. 7.0 mol/L Hydrochloric acid (HC1) (for hydrolysis)
Dilute 551 mL concentrated HC1 to volume with deionized water in a
1-liter volumetric flask.
B. 1.79 mol/L HC1 (for daily absorbance readings)
Dilute 1A1 mL concentrated HC1 to volume with deionized water in a
1-liter volumetric flask.
C. 0.43 mol/L HC1 (for analysis-extraction)
Dilute 68 mL concentrated HC1 to volume with deionized water in a
2-liter volumetric flask.
D. 1.5 mol/L HCl (for blanking spectrophotometer)
Dilute 118 mL concentrated HCl to volume with deionized water in a
1-liter volumetric flask.
NOTE: These dilutions assume concentrated HCl to be 12.7 mol/L.
The molar concentration of different lots of HCl should be
calculated by using the following formula:
relative density X % HCl
mol/L »
35.453
E. 2:1 (v/v) Ethyl acetate - acetic acid
Working under a hood, combine 200 mL ethyl acetate and 100 mL
glacial acetic acid. Mix the solution well; this volume is
sufficient for the standards, controls, and 80 specimens. (Prepare
this reagent daily, immediately before sampling the whole blood.)
Standard Preparation
NOTE: Prepare all standard solutions in actinic glass volumetric
flasks.
A. Protoporphyrin IX Standards (Concentrations are expressed in terms
of protoporphyrin IX free acid after the dimethyl ester has been
hydrolyzed. The millimolar absorptivity of protoporphyrin IX free
acid has conventionally been determined in 1.5 mol/L HCl; thus, the
daily absorbance reading of the hydrolysate is determined at this
acid concentration.(2))
1. 20 mg/dL Protoporphyrin IX Free Acid Hydrolysate (Stock
Standard)
Measure 42.0 mg protoporphyrin IX dimethyl ester (PPIX DME).
-------
16
Dilute to volume in a 200-raL actinic volumetric flask with 7
mol/L HC1, washing PPIX off weighing paper with a few drops of
formic acid. Add a small stirring bar, cover the flask with
aluminum foil, and mix contents at 20-25°C for 3 h, using a
magnetic stirrer. (Prepare weekly.)
2. 1,000 ug/dL Intermediate Stock
After 3 h, dilute 25.0 mL of 20 mg/dL solution with deionized
water to volume in a 500-mL actinic volumetric flask to yield a
1000 ug/dL solution, which is 0.35 mol/L with respect to HC1.
(Prepare weekly.)
3. 100 ug/dL Standard for Daily Absorbance Readings
Dilute 10.0 mL of 1,000 ug/dL intermediate stock to volume in a
100-mL actinic volumetric flask with 1.79 mol/L HC1 to yield a
100 ug/dL protoporphyrin IX standard, which is 1.5 mol/L with
respect to HC1. Use an aliquot of this standard for absorbance
readings, as in Section VI.B.
NOTE: The theoretical concentration of this solution with
respect to protoporphyrin IX free acid (PPIX FA) is calculated
as follows:
42 me PPIX DME 562.27 mg PPIX FA .1999 mg PPIX FA
200 mL 590.72 mg PPIX DME = mL
.1999 me PPIX FA 25 mL 10 mL = .0009975 mg/mL PPIX FA (99.75 ug/dL)
mL 500 mL 100 mL
99.95 ug 1 mmol x 10 dL 1 mg _ .00178 mmol/L
1 dL 562.27 1 L 1000 ug ~ PPIX FA
4. 100 ug/dL Standard for Dilutions
Dilute 5.0 mL of 1,000 ug/dL intermediate stock to volume with
0.43 mol/L HC1 in a 50-mL actinic volumetric flask.
5. 0-80 ug/dL Working Standards
Prepare the following working standards daily by diluting the
100 ug/dL standard with 0.43 mol/L HC1 according to the
following dilution scheme, using a Hicromedic APS-2 equipped
with 50-uL sampling and 200-uL dispensing pumps, and the
reagent dispenser.
Working
Volume
Volume
Standard
1,000 ug/dL
0.43 mol/L
Final
Concentration
Standard
HCl Diluent
Volume
80 ug/dL
400 uL
4,600 uL
5,000
uL
70 ug/dL
350 uL
4,650 uL
5,000
uL
60 ug/dL
300 uL
4,700 uL
5,000
UL
50 ug/dL
250 uL
4,750 uL
5,000
uL
40 ug/dL
200 uL
4,800 uL
5,000
uL
30 ug/dL
150 uL
4,850 uL
5,000
uL
20 ug/dL
100 uL
4,900 uL
5,000
uL
10 ug/dL
50 uL
4,950 uL
5,000
uL
0 ug/dL
0 uL
5,000 uL
5,000
uL
NOTE: It is especially important to work under subdued lights
when diluting and extracting the standard materials, which are
photo-labile.
-------
Procedure
NOTE: To protect hands against acids and solvents during sampling,
wear latex gloves. To avoid evaporation or degradation of specimens,
process samples as rapidly as possible. After centrifugation, samples
are stable for 1-3 h.
A. Thaw specimens and quality control materials of frozen EDTA-whole
blood at room temperature.
NOTE: Control pools with elevated levels of FEP are prepared from
blood (EDTA-anticoagulated) collected from cows that have been
administered lead acetate.
B. Using the spectrophotometer and quartz cuvettes, measure absorbance
at wavelength-maximum of the 100 ug/dL in 1.5 mol/L HC1 standard
solution against a blank of 1.5 mol/L HC1, scanning from 380-420
nm. (Wavelength-maximum is about 407-408 nm.) This measurement
will be used to determine standard concentrations. Clean cuvettes
after use with 5% Contrad solution, rinse thoroughly with deionized
water, followed by ethanol to remove water droplets.
C. Prepare the working standard dilutions from 100 ug/dL standard in
0.43 mol/L HC1, using 0.43 mol/L HC1 as a diluent. These dilutions
are unstable; therefore, prepare them as rapidly as possible.
D. Prepare the 2:1 ethyl acetate-acetic acid mixture and fill the
dispenser bottle of the Micromedic high-speed dilutor for
delivering 1.0 mL of reagent. Fill the dispenser bottle of the
Hamilton Dilutor with 0.43 mol/L HC1 for delivery of 1.0 mL.
(Place dilutors under hood to minimize fumes during usage.)
E. Before sampling, vortex thoroughly each standard dilution, quality
control pool, or whole blood specimen. Using the APS-2, transfer
10 uL of sample to a 10- X 75-mm disposable glass tube, in
duplicate.
F. Add 1.0 mL of the 2:1 ethyl acetate-acetic acid mixture to each
sample. Vortex thoroughly for 10 sec.
G. Add 1.0 mL of the 0.43 mol/L HCl to each sample. Wrap tube with
Parafilm and vortex thoroughly for 10 sec.
H. Sample in this order: standards, quality control pools, and whole
blood specimens in duplicate.
I. Prepare two blank tubes (0 standards) with 1.0 mL each of ethyl
acetate-acetic acid and 0.43 mol/L HCL, with 10 ul 0.43 HCL as
sample.
J. When all sampling is completed, centrifuge all tubes for 4 min at
1,400 rpm.
K. For samples outside the range of the standard curve, use a smaller
sample size or dilute sample with saline. For example,
5 uL ¦ 1:2 dilution
2 uL * 1:5 dilution
100 uL sample and 900 uL saline * 1:10 dilution, 10 uL sample used
-------
18
L. Perkin-Elmer 650-10 spectrofluorometer settings
slit(s) width
photomultiplier tube
cuvettes
range
PH Gain
response
mode
scan
wavelength
10 nm
R938 Hamamatsu
10- X 75-mm in microcell adapter
1
normal
normal
normal
off
404 nm excitation
655 nm emission
M. Allow 30-45 min for the 650-10 to warm up and stabilize after the
xenon lamp has been ignited.
N. Following the instruction manual, zero the Model 56 recorder with
"Recorder Zero" and "MEAS."
O. With shutter closed and sensitivity set on "1," zero the 650-10
spectrofluorometer using "Zero Adjust" with "Zero Suppression" OFF.
P. Open shutter. Turn "Zero Suppression" on. Put tube with blank
solution in sample compartment, and zero the digital readout
carefully using the zero suppression knob.
NOTE: Because of tube-to-tube variance, it is important to check
several different blank tubes and take the average amount of the
blank to be zeroed out.
Q. Place 80 ug/dL PPIX standard tube in sample compartment and set
digital readout to 80.0, using "sensitivity fine" knob. Check with
another 80 ug/dL standard to verify.
R. Proceed to read the standard curve, quality control pools, and
samples.
VII. Calculations
The millimolar absorptivity of protoporphyrin IX free acid in 1.5 mol/L
HC1 has been determined in our laboratory to be 296.87 + .45 (400
observations). Calculate the actual concentration of the 100 ug/dL
(.00178 mmol/L) working standard, using the following equation:
A > absorbance reading
b a cuvette pathlength, 1 cm
c s concentration, in mmol/L
e = millimolar absorptivity of protoporphyrin IX free acid in 1.5
mol/L HC1, 296.87
For example, if the daily absorbance reading of the 100 ug/dL standard
at wavelength maximum is 0.520, then:
A = ebc, and c ¦ A
eb
Where:
0.520
C *
=* .00175 mmol/L
(297 L/mmol-cm) (1 cm)
-------
19
Then: (.00175 mmol/L) (562.27 mg/mmol) (1000 ug/mg) (1L/10 dL) = 98.AO ug/dL
PPIX FA
Consider 98.40 as a percentage of 100 ug/dL and correct the standard
curve accordingly:
10 ug/dL X .9840 = 9.84
20 ug/dL X .9840 = 19.68, etc.
Perform a linear regression, with x = corrected standard concentration
and y = fluorescent intensity reading. Using the slope of the standard
curve and assuming zero intercept, calculate the concentration of
protoporphyrin IX per deciliter of whole blood for each specimen. To
correct for hematocrit and express results as ug/dL of RBC, use this
formula:
ug/dL whole blood
hematocrit X 100 = ug/dL RBC
VIII. CDC Modifications
The following modifications of the original methods are based on CDC
optimization experiments: (a) sample size increased from 2 uL to 10
uL; (b) ethyl acetate-acetic acid and 0.43 mol/L HC1 volumes increased
from 0.3 mL to 1.0 mL; (c) processed protoporphyrin IX standards used;
(d) hydrolysis time for the dimethyl ester decreased from 48 h to 3 h,
on the basis of the work of Culbreth et al.(3); and (e) 0.43 mol/L HC1
chosen for maximum fluorescent intensity of the extracted
QUALITY CONTROL SYSTEM
Quality Control Statistics
The statistical format used for evaluation of quality control will be that of
two-way analysis of variance, ANOVA, with the construction of quality control
charts based on 95% and 99% confidence limits of the mean of duplicate
measurements, as well as range charts (1).
Precision and accuracy of the analytical system will be monitored as follows:
1) Twenty analytical runs will be performed to characterize all control
materials used, with quadruplicate measurements performed per run.
2) Analysis of variance calculations will be performed on these 20 runs,
and quality control charts will be generated by computer for X and
range R.
3) Three levels of quality control materials will be incorporated into
each day's analytical run, and data obtained for these controls will be
evaluated with the X and R charts from 2).
-------
Blind Quality Control
Two levels of blind quality control pools will be incorporated into the system:
Blind duplicate specimens will be prepared and inserted at an interval
determined by the supervisor, usually one blind duplicate per 20
unknown specimens.
The blinds should be identical in appearance to the specimens, with the same
containers, specimen volumes, and labelling. If desired, blind quality
control specimens can be evaluated with the same statistical methods used for
the control materials.
Action Limits
The analytical system will be declared "out of control" if one or more of the
following events occur (1):
X chart
1) A single X value falls above the upper 99% limit or below the lower 99%
limit.
2) Two successive X values fall either both above the upper 95% limit or
both below the lower 95% limit.
3) Eight X values in succession fall either all above the center line or
all below the center line.
R chart
1) A single R value falls above the upper 99% limit.
2) Two successive R values fall above the 95% upper limit.
3) Eight R values in succession fall above the center line.
If the system should be declared out-of-control, the following remedial action
should be taken:
1) Check for errors in recording levels of control samples, and if none
are found,
2) Check and calibrate instruments before performing further analyses on
analytical samples,
3) Reanalyze patient samples performed during the out-of-control run.
-------
References
1. Sassa S, Granick JL, Granick S, Kappas A, and Levere RD:
Microanalyses of erythrocyte protoporphyrin levels by
spectrophotometry in the detection of chronic lead intoxication in
the subclinical range. Biochem Med 8:135-148 (1973).
2. Committee on Specifications and Criteria for Biochemical Compounds,
National Research Council Specifications and Criteria for
Biochemical Compounds, 3d ed. Washington, DC, National Academy of
Science (1972).
3. Culbreth P, Walter G, Carter R, and Burtis C. Separation of
protoporphyrins and related compounds by reversed-phase liquid
chromatography. Clin Chem 25:605-610 (1979).
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22
GRAPHITE FURNACE PROCEDURE FOR BLOOD CADMIUM
(Revised August 26, 1983)
Determination of blood cadmium is accomplished by flameless
(electrothermal) atomic absorption. The specimen is deproteinized with the
addition of nitric acid after dilution with water, and cadmium is determined
in the acid supernatant. A L'vov platform is used to decrease matrix effects
(1) and to increase precision and sensitivity (2). The procedure is based on
published work by Stoeppler et al. (3).
EQUIPMENT
Atomic Absorption Spectrophotometer: Perkin Elmer Model 372, with Model 500
graphite furnace, Model AS-1 autosampler, and Model 56 recorder. Pyrolytic
graphite furnaces and L'vov platforms are used.
Instrument Parameters:
Parameter Setting
Wavelength 228.8 nm
Lamp Current 6 ma
EDL Power 6 W
Slit 0.7 (alt)
Signal Model ABS
Read Time 4.0 s
Inert Gas Argon
Furnace Type Pyrolytic/L'vov
Background Corrector ON
Temperature Program
DRY
CHAR
ATOMIZE
COOL
Inert Gas Flow
150 C 25 s (5 s ramp)
450 C 25 s (5 s ramp)*
2000 C 5 s (1 s ramp)
20 C 10 s (Is ramp)
300 mL/min; 20 mL/min <§l ATOMIZE
*During the latter part of the CHAR, the baseline is reset to "0" by using the
B0C function of the 372; the settings at step 2 (char) are: READ 20s; BASELINE
28s: REC 29s. READ and REC are set "0" during ATOMIZE.
Recorder: Model 56 set at 5 mV; 20 mm/min speed.
Automatic Pipet: Micromedic Model 25000, with 1-mL sampling and dispensing
pumps; sampling pump set at "20%" (200 uL), dispensing at "50%" (500 uL).
REAGENTS
Water: Ultrapure water, polished by a Milli-Q system to 18 megaohm/cm purity
is used throughout.
Nitric Acid: Redistilled grade nitric (GF Smith) or Ultrex (JT Baker) is used.
Stock and Working Cadmium Standards: A 1,000 mg/L stock solution of reagent
grade cadmium acetate dihydrate is prepared from 237 mg of the cadmium salt,
dissolved and diluted to 100 mL with ultrapure water. To a 100-mL volumetric
flask, add 237 mg of the salt and 500 uL ultrapure nitric acid, diluting to
-------
23
the mark with ultrapure water. This stock solution should be prepared every 6
months. Intermediate stock of 10 mg/L is prepared by 1:100 volumetric
dilution of the 1,000 mg/L solution weekly; working and spiking stocks of 1.0
mg/L, 10, 25, 50, and 75 ng/mL are prepared daily from the 10 mg/L
intermediate stock.
PLASTICWARE AND GLASSWARE
All plasticware and glassware used is cleaned by soaking 24 h in
detergent, followed by soaking for 3 days in 25% v/v nitric acid. The cleaned
items are then rinsed thoroughly with ultrapure water, dried under class 100
conditions, and stored in a dust-free environment.
Venous blood specimens are collected in either 5-mL vacutainers (Beckton
and Dickinson) or 5-mL "Monoject" tubes (Sherwood Manuf.). Both containers
employ aqueous dipotassium EDTA as anticoagulant.
Eppendorf micropipets (Brinkmann Instruments) are used to prepare spiked
aliquots for calibration; disposable polyethylene tips are used as received.
Beckmann Instruments' "Bio-Vials" are used for dilution of blood
specimens; these 4-mL containers are cleaned as above.
SPECIMEN COLLECTION
Collection of an uncontaminated whole blood specimen is a critical part of
many toxicological investigations. The following guidelines will provide
directions which, if carefully followed, will minimize the contamination of
whole blood by the many sources from which it may come. It cannot be
overemphasized that skin, clothing, dust, and many other sources of
contamination contain many times the levels of lead, arsenic, cadmium, and
other metals that will be determined in the collected specimen.
1. Clean the antecubital area thoroughly with: a) soap and water (Phisohex
has been shown to be free from significant metal contamination); followed
by b) alcohol (isopropanol or ethanol).
2. Puncture the skin/vein with a sterile, disposable needle capable of
multiple sampling. A suggested product is the B + D catalog #5749,
20-gauge needle. In some applications, the first blood specimen collected
will be used for other determinations; its use is to "rinse" the
collection needle with blood.
3. Collect one or more tubes of whole blood, using an appropriate
anticoagulant for the metal of interest. Anticoagulant/metal combinations
that have been shown to be compatible are:
Mercury-Heparin or Citrate
Lead-EDTA. Heparin, or Oxalate
Cadmium-EDTA. Heparin, or Oxalate
(preferred anticoagulant underlined)
It is important that a few "spares" of the lot of tubes used for
collection be sent to the laboratory along with collected specimens. This
will allow the laboratory to determine the metal content of the
anticoagulant used in that tube lot and make appropriate blank
corrections. Of course, all lots of tubes used should be screened before
use in surveys.
4. It is critical that the specimen be thoroughly mixed after it has been
collected to insure that the anticoagulant/blood mixture is uniform and
that clotting will therefore be prevented. Clotted specimens are nearly
useless!
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24
5. Refrigerate the collected specimens, arid ship refrigerated by the most
expeditious means available. Heparin is by far the least "permanent" of
the anticoagulants listed, but it will prevent clotting for 2 weeks if the
specimen and anticoagulant are well-mixed at the time of collection and
refrigerated after collection and during shipment.
6. Ship the collected specimens in well-padded, insulated containers (freeze
safe or the equivalent).
ANALYTICAL PROCEDURE
1. Aspirate 200 uL of blood into the delivery tip of the automatic pipet;
dispense blood and 500 uL water into a precleaned 4-mL plastic vial.
2. Aspirate air into the delivery tip, and dispense an additional 500 uL
water into the same vial. This "double rinse" should give a quantitative
transfer of blood to the plastic vial.
3. Add 50 uL of ultrapure nitric acid to the diluted specimen, cap and mix
thoroughly on a vortex-type mixer until protein precipitation is complete,
as evidenced by the color of the solution changing to a dark brownish red
and the dispersion of precipitated protein/RBC*s throughout the diluted
specimen.
4. Centrifuge at 2,000 RPM for 5 minutes.
5. Decant the acid supernatant with a 250- or 500-uL Eppendorf pipet.
6. Measure the absorbance of the resulting supernatant in duplicate or
triplicate, using the AS-1 autosampler to dispense 20 uL of solution into
the graphite furnace.
STANDARDIZATION AND CALCULATIONS
Standardization is accomplished by the use of a modification of the method
of standard additions. A bovine "base blood," typically containing less than
1 ng/mL cadmium, is diluted per the procedure, and aliquots are spiked with
microliter additions of cadmium acetate standards.
Standard Addition Procedure:
1. Base (low cadmium) blood is prepared per analytical procedure, step 1-5,
using twice the prescribed volumes.
2. Into four precleaned autosampler cups, pipet 10 uL of 10, 25, 50, and 75
ng/mL cadmium standard, using a 10-uL Eppendorf pipet with disposable
plastic tips.
3. Add 250 uL of acid supernatant prepared from "base" blood to each of the
four cups.
4. Transfer 250 uL of the remaining supernatant to a fifth autosampler cup.
5. Measure the absorbance of the resulting solutions in duplicate or
triplicate.
Calculations:
Two methods of calculation have been used with the described procedure,
each of Which give essentially identical results. Each individual specimen
may be analyzed by the procedure outlined above for standard additions.
Although potentially highly accurate, this method suffers from extremely low
-------
25
throughput. Any differences in observed slope from spiked aliquots of
different specimens will be automatically compensated for by this method.
Since no measurable difference in slope was observed (within experimental
error) between spiked bovine and human blood, either regression analysis,
Method 2, or an "average slope" method, Method 1, are normally used.
Method 1 Average Slope
1. Calculate the average (mean) absorbance values for the solutions measured.
2. Correct the mean absorbances of the cadmium-spiked solutions by a dilution
factor, df, calculated as follows:
df = original volume + spiking volume
original volume
In this procedure, the df will be 1.04, which is
250 uL + 10 uL + 260 uL = 1.04
250 uL 250 uL
3. Construct the following table:
Specimen Corrected Absorbance ng/mL added Corrected Absorbance-At,ase
Base Abase ® ®
Spike! A (1.04) 2.5 A (1.04) - Abase
spike 1 spike 1
etc.
4. Calculate the factor, (ng/mL added)/(corrected absorbance - Afcase) for
each of the four spiked calibrators. The ng/mL additions are calculated
as additions to the original blood specimen and have the values 2.5, 6.25,
12.5, and 18.75 ng/mL.
5. Average the four factors from step 4 and multiply specimen absorbances
(controls or unknowns) by this average factor to calculate the cadmium
content in ng/mL. Make sure that all specimen absorbances are corrected
for blanks (dilute nitric acid) before calculation.
NOTE: The value for the average of (ng/mL)/(corrected absorbance -A^ase)
for this procedure usually falls in the range of 0.05-0.07 for the 2.5-ng/mL
addition. The calculated factors should be examined for consistency; this is
one indication of linearity of the calibration curve.
-------
Method 2-Linear Regression
1. Construct the following table:
X (ng/mL added)
Y (^corrected -Abase)
0
0
2.5
A (1.04) —A^ase
spike 1
6.25
etc. with spike 2
12.50
etc. with spike 3
18.75
etc. with spike 4
2. Using a calculator with linear regression curve fitting ability, and with
Y-X calculation capability, enter the X, Y pairs of data as in the above
table.
3. Calculate the r2, slope, and intercept for the data.
4. To calculate specimen values (controls or unknowns), enter the
blank-corrected A (Y) values into the calculator, and calculate X (ng/mL).
NOTE: These two approaches will give essentially equivalent results if:
1) the r2 value for the regression equation is high (0.98 or higher), and
2) the calculated intercept for the equation is near zero (0-0.05).
QUALITY CONTROL SYSTEM
Quality Control Statistics
The statistical format used for evaluation of quality control will be that of
two-way analysis of variance, ANOVA, with the construction of quality control
charts based on 95% and 99% confidence limits of the mean of duplicate
measurements, as well as range charts (4).
Precision and accuracy of the analytical system will be monitored as follows:
1) Ten analytical runs will be performed to characterize all control
materials used, with duplicate measurements performed per run.
2) Analysis of variance calculations will be performed on these 20 data
points, and quality control charts will be generated by computer for X and
3) A minimum of two control materials will be incorporated into each
analytical run of 20 unknown specimens, and data obtained for these
controls will be evaluated with the X and R charts from 2).
range R.
-------
Blind Quality Control
Two types of blind quality control specimens will be incorporated into the
system:
1) blind duplicate specimens will be prepared and inserted at an interval
determined by the supervisor, usually one blind duplicate per 20 unknown
specimens.
2) blinded control or reference material samples will be inserted into each
analytical run of 20 specimens, at the minimum rate of one blind control
per 20 specimens.
In both cases, the blinds should be identical in appearance to the specimens,
with the same containers, specimen volumes, and labelling. If desired, blind
quality control specimens can be evaluated with the same statistical methods
used for the control materials.
Run Format
In all cases, the following format will be used for specimen determination:
SAMPLE it Sample ID
1
Blank
2-5 (or greater)
Calibration Curve
6
Control I
7-27
Specimens
28
Control II
29-33.
Calibration Curve
34
Blank
Action Limits
The analytical system will be declared "out of control" if one or more of the
following events occur (4):
X chart
1) A single X value falls above
limit.
2) Two successive X values fall
below the lower 95* limit.
3) Eight X values in succession
below the center line.
R chart
1) A single R value falls above the upper 99% limit.
2) Two successive R values fall above the 95% upper limit.
3) Eight R values in succession fall above the center line.
the upper 99% limit or below the lower 99%
either both above the upper 95% limit or both
fall either all above the center line or all
-------
28
If the system should be declared out-of-control, the following remedial action
should be taken:
1) Check for errors in recording levels of control samples, and if none are
f ound,
2) Check and calibrate instruments before performing further analyses on
analytical samples,
3) Reanalyze patient samples performed during the out-of-control run.
METHOD PERFORMANCE
Limits of Detection
Limit of detection is in a practical sense determined by two factors: 1) the
slope sensitivity of the method, i.e., the response (in the case of atomic
absorption the absorbance or absorbance-second measurement) for a given
concentration or amount of analyte, and 2) the random noise of the
instrumental system used in measurement, especially that noise at the measured
response for the blank for the determination (5).
According to the recommendations in reference (5), the limit of detection will
be defined as that concentration of analyte corresponding to an absorbance or
absorbance-second measurement equivalent to three times the standard deviation
of this signal measured at an analyte concentration at a "low" level. In
symbolic terms, this becomes:
CL ~ 3sr
m
where cl is the concentration calculated to be the limit of detection, sg
is the standard deviation of the measurement of a blank or low concentration
sample; and m is the slope of the calibration curve (change in absorbance or
absorbance-seconds/change in concentration or amount of analyte). For a set
of measurements at the 0.6-ng/mL level, the standard deviation was
2.08 x 10-3A, which yields a detection limit of 0.28 ng/mL (N = 6).
Accuracy and Precision
At the present moment, there is only one commercial source of blood with
certified target values (6). Since this material is not available to the
laboratory, evaluation of precision and accuracy was performed by the
determination of cadmium in a bovine blood pool spiked with cadmium. Both the
unspiked or "base" material and the spiked material were analyzed for cadmium
with the described procedure. Precision at the "medical decision" level of
approximately 5 ng/mL was 10% CV (total CV, including within- and among-day
components). Performance of the proposed method for EPA quality control water
samples, prepared as dilute aqueous nitric acid control materials, has
consistently given + 10% accuracy; similar results have been obtained for
National Bureau of Standards' SRM 1643a, Trace Elements in Water.
-------
29
References
1. Atomic Spectroscopy, 4(3): 69-86 (1983).
2. Anal Chemistry, 84: 1515 (1982).
3. Fresenius Z. Anal Chem, 300: 372-80 (1980).
4. A Statistical Quality Control System for the Clinical Laboratory. In
Selection and Implementation of Methods in Clinical Chemistry, Commission
on Continuing Education, Council on Clinical Chemistry, American Society
of Clinical Pathology, Chicago, Illinois (March 1975).
5. Winefordner J and Long G. Limit of detection—a closer look at the IUPAC
definition," Anal Chem, 55 (7): 713A-719A (1983).
6. Behringwerke AG, Product Bulletin, Frankfurt, FRG, (June 1981).
-------
GRAPHITE FURNACE PROCEDURE FOR URINARY ARSENIC
(Revised August 30, 1983)
INTRODUCTION
Determination of arsenic in urine is a valuable part of many
epidemiological and environmental health surveys, since urine arsenic reflects
body burden, particularly recent undue absorption. Since the estimated half
life of arsenic in the body is comparatively short, it is imperative that
acute exposure cases be "sampled" as soon as possible after exposure in order
to accurately evaluate potential toxic risk.
This method is based in principle on the use of nickel as matrix modifier
(1-3) in order to thermally stabilize arsenic in the graphite furnace,
presumably by the formation of a stable nickel arsenide. Much of the
awkwardness and tedium of manual hydride methods is avoided in this automated
method. The Zeeman background correction system offers the increased
correction capacity needed at the primary resonance line of arsenic, 193.7 nm,
well into the "short" UV range where salts and other matrix residues absorb
strongly, causing high background absorbances.
EQUIPMENT
AA Spectrophotometer. P-E Zeeman/5000 with AS-40 Autosampler;
• Data Station 10
L'vov Platform. PE catalog # B-0109-324.
REAGENTS
Water: Ultrapure water, prepared by a Milli-Q polishing system* is used
throughout.
Triton X-100: Scintillation Grade (Eastman Kodak) is used to prepare
0.10% v/v solution by volumetric dilution with ultrapure water.
Matrix Modifier
Nickel nitrate, 1000 mg/L, made by dissolving 4.953 g
Ni(N0 ) 6Q § ij 200 mL ultrapure water, diluting to 1 L with
ultrapure water.
Stock and Working Arsenic Solutions. Arsenic, 1000 mg/L is used as received
from Fisher (Cat. //So-A-450) or J.T. Baker (Cat. //6919-1) . A 10-mg/L
intermediate stock is prepared weekly by volumetric dilution of 1.00 mL stock
to 100 mL; spiking solutions are prepared daily at 0.25, 0.50, 1.00, and 1.50
mg/L arsenic, also by volumetric dilution.
SPECIMEN COLLECTION
The judicious adherance to this protocol ensures urine specimens and
samples taken therefrom to be of high quality in terms of minimal
contamination from the ambient dust of air/clothes/skin.
1. Instruct subject to wash genitalia and hands prior to voiding and to be
either naked or to wear clean underclothes (NO OUTERCLOTHING) when voiding
into the collection bottle. A complete body washing (shower) with soap
and water is recommended.
-------
31
2. Collect 100+ mL of a first-morning void urine specimen in a 130-mL
collection container (B+D 133 mL containers Cat. #4013 have been
satisfactory).
3. Mix specimen thoroughly and decant 50-mL volume into a 50-mL plastic
container (Falcon Cat. #2098 has been satisfactory).
4. Add 500 uL of ultrapure (ultrex grade or equivalent) nitric acid, and mix
specimen thoroughly.
5. Refrigerate collected specimen, and ship refrigerated to laboratory.
6. Prepare a "blank" with 50 mL ultrapure water, acidified with 500 uL of the
nitric acid used in acidifying urine specimens. Return to laboratory with
acidified urine specimens as a "blank."
ANALYTICAL PROCEDURE
1. Pipet 1.00 mL urine specimen or control into a precleaned 4-mL plastic
vial.
2. Add 1.0 mL 4% v/v ultrapure nitric acid, prepared from J.T. Baker "ULTREX"
grade or 6F Smith "Redistilled" grade nitric acid.
3. Add 0.20 mL 0.10% v/v Triton X-100 and mix resulting solution thoroughly
by vortexing 10-20 sec.
4. For the matrix-matched standard addition procedure, add 10 uL of the four
standards above to four precleaned autosantpler cups.
5. Pipet 250 uL of diluted urine into four of the four cups, pipetting the
remaining diluted*urine into a fifth cup.
6. With the AS-40 set for 10-uL sample volume and 10-uL alternate (matrix
modifier) volume, pipet 10 uL of diluted urine, followed by 10 uL of
matrix modifier into the graphite furnace.
7. Measure the absorbances of the prepared solutions in triplicate.
CALCULATIONS
Since the slopes of the curves prepared from random urine have been shown to
vary up to + 100%, the method of additions is used for determination of the
unknown specimen concentrations. In the format above, the standard additions
correspond to addition of 22, 44, 88, and 132 ng/mL arsenic to the original
urine specimen.
In order to calculate the unknown concentrations, the following Table is
constructed:
Specimen ng/mL as added Average A A( 1.04)-dilution A(1 •04)-AunSpij{e(j
unspiked 0 A A 0
+ 10 uL 0.25 PPM 22 ng/mL Ai Aid.04) AxU.04) -A
etc.
With the entries in the last column to the right A(1.04) -A, entered as "Y,"
and ng/mL added as "X," perform a linear regression analysis on the data.
Calculate slope, intercept, and r-squared terms.
Typical values for these parameters are:
slope 0.40 - 0.60
intercept 0 - 0.10
r squared 0.98
-------
32
QUALITY CONTROL SYSTEM
Quality Control Statistics
The statistical format used for evaluation of quality control will be that of
two-way analysis of variance, ANOVA, with the construction of quality control
charts based on 95% and 99% confidence limits of the mean of duplicate
measurements, as well as range charts (4).
Precision and accuracy of the analytical system will be monitored as follows:
1) Ten analytical runs will be performed to characterize all control
materials used, with duplicate measurements performed per run.
2) Analysis of variance calculations will be performed on these 20 data
points, and quality control charts will be generated by computer for X and
3) A minimum of two control materials will be incorporated into each
analytical run of 20 unknown specimens, and data obtained for these
controls will be evaluated with the X and R charts from 2).
Blind Quality Control
Two types of blind quality control specimens will be incorporated into the
system:
1) blind duplicate specimens will be prepared and inserted at an interval
determined by the supervisor, usually one blind duplicate per 20 unknown
specimens.
2) blinded control or reference material samples will be inserted into each
analytical run of 20 specimens, at the minimum rate of one blind control
per .20 specimens.
In both cases, the blinds should be identical in appearance to the specimens,
with the same containers, specimen volumes, and labelling. If desired, blind
quality control specimens can be evaluated with the same statistical methods
used for the control materials.
Run Format
In all cases, the following format will be used for specimen determination:
range R.
SAMPLE #
Sample ID
6
7-27
28
29-33
34
1
2-5 (or greater)
Blank
Calibration curve
Control I
Specimens
Control II
Calibration Curve
Blank
-------
33
Action Limits
The analytical system will be declared "out of control" if one or more of the
following events occur (4):
X chart
1) A single X value falls above the upper 99% limit or below the lower 99%
limit.
2) Two successive X values fall either both above the upper 95% limit or both
below the lower 95% limit.
3) Eight X values in succession fall either all above the center line or all
below the center line.
R chart
1) A single R value falls above the upper 99% limit.
2) Two successive R values fall above the 95% upper limit.
3) Eight R values in succession fall above the center line.
If the system should be declared out-of-control, the following remedial action
should be taken:
1) Check for errors in recording levels' of control samples, and if none are
found,
2) Check and calibrate instruments before performing further analyses on
analytical samples,
3) Reanalyze patient samples performed during the out-of-control run.
METHOD PERFORMANCE
Limits of Detection
Limit of detection is, in a practical sense, determined by two factors: 1)
the slope sensitivity of the method, i.e., the response (in the case of atomic
absorption, the absorbance or absorbance-second measurement) for a given
concentration or amount of analyte, and 2) the random noise of the
instrumental system used in measurement, especially that noise at the measured
response for the blank for the determination (5).
According to the recommendations in reference (5), the limit of detection will
be defined as that concentration of analyte corresponding to an absorbance or
absorbance-second measurement equivalent to three times the standard deviation
of this signal measured at an analyte concentration at a "low" level. In
symbolic terms, this becomes:
Cl ¦ 3sp
m
where c^ is the concentration calculated to be the limit of detection; Sg
is the standard deviation of the measurement of a blank or low concentration
-------
34
sample; and m is the slope of the calibration curve (change in absorbance or
absorbance-seconds/change in concentration or amount of analyte). For a set
of measurements at the 8.5-ng/mL level, the standard deviation was
1.96 x 10~^A.sec, which yields a limit of detection of 4.0 ng/mL (N=4).
Accuracy and Precision
Accuracy and precision of the urinary arsenic method have been estimated from
determination of two different urine materials: a) a pooled urine from an
epidemiological study of arsenic in Fairbanks, Alaska, well water; and b) a
commercially available urine material (Fisher Urichem Level II). Target value
for the Alaska material was established with a published hydride evolution
method (6); the Level II by referee laboratories. Precision at the "medical
decision" level of approximately 100 ng/mL was 18% (total CV, including within
and among-day components), with agreement of + 15 ng/mL with hydride values
(6).
-------
35
References
1. Benard H and Pinta M, Atomic Spectroscopy, 3: 8-12 (1982).
2. Freeman H et al., Atomic Absorption Newsletter, 15: 49-50 (1976).
3. HGA Recommended Conditions for Arsenic, Version 1.0, Perkin-Elmer Corp.,
Norwalk, CT, 1982.
4. A statistical quality control system for the clinical laboratory. In
Selection and Implementation of Methods in Clinical Chemistry, Commission
on Continuing Education, Council on Clinical Chemistry, American Society
of Clinical Pathology, Chicago, Illinois (March 1975).
5. Winefordner J and Long G. Limit of detection—a closer look at the IUPAC
definition. Anal Chem, 55(7): 713A-719A (1983).
6. Cox DH, Jo Anal Tox, 4: 207-11 (1980).
-------
INSTRUMENTAL PARAMETERS FOR DETERMINATION OF URINARY ARSENIC
Wavelength
EDL Power
Slit 0.7 LOW
Signal Mode
Data Station 10
Read Time
Inert Gas
Inert Gas Flow
ATOMIZE
Zeeman Background
Corrector
Furnace Type
193.7 nm
8W
ABS
ON-PRINT ON
5 sec
Argon
300 mL/min; 20 mL/min@
ON
Pyrolytic
Temperature Program
DRY 180 C 20sec Ramp/25 sec Hold
CHAR 1150 C 5sec Ramp/15 sec Hold
ATOMIZE 2400 C Osec Ramp*/5 sec Hold
*Maximum Power Heating Used
-------
GRAPHITE FURNACE PROCEDURE FOR URINARY LEAD/CADMIUM
(Revised August 26, 1983)
EQUIPMENT
AA Spectrophotometer-Perkin Elmer Model 372 with Model 500 furnace, Model
AS-1 autosampler, and Model 56 Recorder or Perkin Elmer Zeeman/5000 with
Model AS-40 autosampler, and Model 10 Data Station.
L'vov Platforms - Perkin Elmer catalog it B-0109-324.
REAGENTS
Water: Ultrapure water, prepared by a Milli-Q polishing system is used
throughout.
Triton X-100: Scintillation Grade (Eastman Kodak) is used to prepare
0.10% v/v solution by volumetric dilution with ultrapure water.
Matrix Modifier:
A. Cadmium - make up a solution 4% by volume ultrapure nitric acid (GF
Smith redistilled or Baker ULTREX) and 0.001% by volume Triton X-100,
prepared by diluting 4.0 mL nitric acid and 1.0 mL 0.10% by volume
Triton to 100 mL with ultrapure water. *
B. Lead - make up a solution 4% by volume ultrapure nitric acid and 1%
weight by volume ammonium phosphate.
NOTE: These solutions should be checked daily for analyte content,
since they readily contaminate with airborne dust, dirt, and/or by-
handling.
Stock and Working Lead/Cadmium Standards: A 1,000-mg/L stock solution of
lead nitrate is prepared from 1.5985 g of NBS SRM 928 lead nitrate,
diluted to 1L with 1.0% v/v nitric acid. A 1,000-mg/L stock solution of
cadmium chloride is prepared from 1.6309 g of anhydrous reagent grade
cadmium chloride, diluted to 1L with ultrapure water. These solutions are
prepared every 6 months. With an intermediate lead standard of 10 mg/L
(10 ppm) and an intermediate cadmium standard of 1 mg/L (1 ppm), combined
working standards are made fresh daily as follows:
A. 0.100 ml of 10 ppm Pb
0.100 ml of 1 ppm Cd qs to 10.0 ml with ultrapure water
Final Cone. > 0.10 ppm Pb
10 ppb Cd
B. 0.25 ml of 10 ppm Pb
0.25 ml of 1 ppm Cd qs to 10.0 ml with ultrapure water
Final Cone. >0.25 ppm Pb
25 ppb Cd
-------
38
C. 0.50 ml of 10 ppm Pb
0.50 ml of 1 ppm Gd qs to 10.0 ml with ultrapure water
Final Cone. = 0.50 ppm Pb
50 ppb Cd
D. 0.75 mL of 10 ppm Pb
0.75 mL of 1 ppm Cd qs to 10. ml with ultrapure water
Final Cone. =0.75 ppm Pb
75 ppb Cd
SPECIMEN COLLECTION
The judicious adherance to this protocol ensures urine specimens and
samples to be of high quality in terms of minimal contamination from the
ambient dust of air/clothes/skin.
1. Instruct subject to wash genitalia and hands prior to voiding and to be
either naked or to wear clean underclothes (NO OUTERCLOTHING) when voiding
into the collection bottle. A complete body washing (shower) with soap
and water is recommended. *
2. Collect 100+ mL of a first-morning void urine specimen in a 130-mL
collection container (B+D 133-mL containers Cat.#4013 have been
satisfactory).
3. Mix specimen thoroughly and decant 50-mL volume into a 50-mL plastic •
container (Falcon Cat. #2098 has been satisfactory).
4. Add 500 uL of ultrapure (ultrex grade or equivalent) nitric acid, and mix
specimen thoroughly.
5. Refrigerate collected specimen, and ship refrigerated to laboratory.
6. Prepare a "blank" with 50 mL ultrapure water, acidified with 500 uL of the
nitric acid used in acidifying urine specimens. Return to laboratory with
acidified urine specimens as a "blank."
OVERALL HELPFUL HINTS AND REMINDERS:
1. Subject must be clean.
2. Handle all materials with clean hands or use plastic gloves.
3. Do not leave caps off containers for any long periods of time and handle
caps carefully to avoid contamination of cap's inside.
4. Tighten caps on shipping bottles to prevent leakage.
5. Do not make ink, etc., markings on containers. Use labels.
PLEASE RETURN ALL CONTAINERS AND OTHER MATERIALS. SINCE THE INITIAL COST
AND THE TIME EXPENDED FOR CLEANING PROHIBITS REPEATED REPLACEMENTS FOR
FUTURE ENDEAVORS.
ANALYTICAL PROCEUDRE
1. In a 4-ml precleaned plastic vial, pipet 1.0 ml urine into 1.0 ml matrix
modifier.
2. Vortex 30 sec.
3. Into four precleaned plastic autosampler cups, pipet 10 ul of the A, B, C,
and D standards, using a 10-ul Eppendorf micropipet with disposable tips.
4. Add 250 ul of diluted urine to each of the three cups, using a 250-ul
Eppendorf.
-------
5. Transfer the remaining diluted urine to a
6. Add 500 ul of each of the remaining urine
number of precleaned cups.
7. Measure the absorbance of a 20-uL aliquot
attached instrumental parameters.
CALCULATIONS
1. Average absorbance change.
In this method the differences in corrected absorbances between A, B
(df), C (df), D (df), and G (df) are calculated.
df g V + v = (0.250 + 0.010) mL = 0.26 = 1.04
v 0.250 = L 0.25
where V = added volume and v = original volume.
fifth autosampler cup.
samples to a corresponding
of solution in triplicate, using
-------
40
Construct the following table:
Specimen Soike (ng) ng/mL Added* Average Corrected Corrected
Absorbance Absorbance Absorbance
A A (1.04) Minus Unspikec
Unspiked
None
0 ng/mL
A
A
0
+ 10 uL O.lOppm Pb
1 ng
8 ng/mL
AxPb
Axd.04)
N
or 10 ppb Cd
0.1 ng
0.8 ng/mL
AxCd
Aid.04)
*1
+ 10 uL 0.25 ppm Pb
2.5 ng
20 ng/mL
A2Pb
A2(1.04)
n2
or 25 ppb Cd
0.25 ng
2.0 ng/mL
A2Cd
A2(1.04)
n2
+ 10 mL 0.50 ppm Pb
5.0 ng
40 ng/mL
A3Pb
A3 (1.04)
n3
or 50 ppb Cd
0.50 ng
4.0 ng/mL
A3Cd
A3(1.04)
n3
+10 mL 0.75 ppm Pb
7.5 ng
60 ng/mL
A4Pb
A4(1.04)
or 75 ppb Cd
0.75 ng
6 .0 ng/mL
A4Cd
A4(1.04)
n4
Calculate the following values:
N2. N3. n4
2.5 5.0 7.5
and average these values with . This average is then divided by a)
8ng/raL (for lead) or b) 0.8 ng/mL (for cadmium) and set equal to cf.
Calculate unknown concentrations by multiplying the average absorbance of each
unknown (after subjecting the blank) times the cf value for the appropriate
metal.
*To illustrate the calculation, assume a 10-uL lead spike of 0.10 ppm to a
250-uL volume of 1:1 diluted urine:
ng lead added = (0.010 mL) (0.10 ug/mL) (1000 ug/ng)
¦ (0.01) (0.1) (1000) = 1 ng
ng/mL lead in = 1 ng => 4 ng/mL
analyzed solution 0.250 mL
ng/mL in original urine = (4 ng/mL) (dilution factor) = 8 ng/mL
-------
2. Linear Regression
Construct the following table:
X (ng/mnL added) Y (N1-N5) - from previous calculation
0 0
8.0 (Pb) NX
0.8 (Cd) NX
20.0 (Pb) N2
2.0 (Cd) N2
etc.
Enter the X, Y pairs into a calculator and perform linear regression
analysis on the data points. Calculate slope, intercept, and r2 for
these data.
Typical Values:
Pb Cd
Slope 2.5-3.0 50-60
Intercept 0-5 0-5
r2 0.98 0.98
Using the Y - X function, calculate unknown concentrations from the
average absorbance of unknowns (after subtracting blank).
QUALITY CONTROL SYSTEM
Quality Control Statistics
The statistical format used for evaluation of quality control will be that of
two-way analysis of variance, AN0VA, with the construction of quality control
charts based on 95% and 99% confidence limits of the mean of duplicate
measurements, as well as range charts (1).
Precision and accuracy of the analytical system will be monitored as follows:
1) Ten analytical runs will be performed to characterize all control
materials used, with duplicate measurements performed per run.
2) Analysis of variance calculations will be performed on these 20 data
points, and quality control charts will be generated by computer for X and
range R.
3) A minimum of two control materials will be incorporated into each
analytical run of 20 unknown specimens, and data obtained for these
controls will be evaluated with the X and R charts from 2).
-------
42
Blind Quality Control
Two types of blind quality control specimens will be incorporated into the
system:
1) blind duplicate specimens will be prepared, and inserted at an interval
determined by the supervisor, usually one blind duplicate per 20 unknown
specimens.
2) blinded control or reference material samples will be inserted into each
analytical run of 20 specimens, at the minimum rate of one blind control
per 20 specimens.
In both cases, the blinds should be identical in appearance to the specimens,
with the same containers, specimen volumes, and labelling. If desired, blind
quality control specimens can be evaluated with the same statistical methods
used for the control materials.
Run Format
In all cases, the following format
_ SAMPLE it
1
2-5 (or greater)
6
7-27
28
29-33
34
will be used for specimen determination:
Sample ID
Blank
Calibration curve
Control I
Specimens
Control II
Calibration Curve
Blank
Action Limits
The analytical system will be declared "out of control" if one or more of the
following events occur (1):
X chart
1) A single X value falls above the upper 99% limit or below the lower 99%
limit.
2) Two successive X values fall either both above the upper 95% limit or both
below the lower 95% limit.
3) Eight X values in succession fall either all above the center line or all
below the center line.
R chart
1) A single R value falls above the upper 99% limit.
2) Two successive R values fall above the 95% upper limit.
3) Eight R values in succession fall above the center line.
-------
If the system should be declared out-of-control, the following remedial action
should be taken:
1) Check for errors in recording levels of control samples, and if none are
found,
2) Check and calibrate instruments before performing further analyses on
analytical samples,
3) Reanalyze patient samples performed during the out-of-control run.
METHOD PERFORMANCE
Limits of Detection
Limit of detection is, in a practical, sense determined by two factors: 1)
the slope uenuitivity of the method, i.e., the response (in the case of atomic
absorption the absorbance or absorbance-second measurement) for a given
concentration or amount of analyte, and 2) the random noise of the
instrumental system used in measurement, especially that noise at the measured
response for the blank for the determination (2).
According to the recommendations in the reference (2), the limit of detection
will be defined as that concentration of analyte corresponding to an
absorbance or absorbance-second measurement equivalent to three times the
standard deviation of this signal measured at an analyte concentration at a
"low" level. In symbolic terms, this becomes:
CL = Mb
m
where cl is the concentration calculated to be the limit of detection; sjj
is the standard deviation of the measurement of a blank or low concentration
sample; and m is the slope of the calibration curve (change in absorbance or
absorbance-seconds/change in concentration or amount of analyte). For a set
of urinary lead measurements at the 8.1 ng/mL level, the standard deviation of
the measurement was 2.88 x 10~3A, which yields a limit of detection of
2.9 ng/mL (N=5). For a set of urinary cadmium measurements at the 0.2 ng/mL
level, the standard deviation was 1.89 x 10~^A, which yields a limit of
detection of 0.1 ng/mL.
Accuracy and Precision
Urinary Lead
Accuracy and precision of the urinary lead method have been estimated from the
determination of a commercially available urinary material (Fisher Scientific
Urichem Level II) with lead values established by referee laboratories.
Precision at the "medical decision" level of approximately 100 ng/mL was 9% CV
(total CV, including within- and among-day components); with agreement of
+ 5 ng/mL with target values by reference laboratories.
-------
Urinary Cadmium
The reference material, SRM 2670, which would be the most appropriate material
with which to establish accuracy for urine cadmium determinations, is not
currently available to this laboratory. In order to evaluate method
precision, a series of urine pools were prepared from pooled "normal" urine,
which were spiked with: a) high purity cadmium (as acetate) to increase
cadmium concentration by approximately 5 ng/mL, or b) concentrates used to
prepare EPA quality control water samples (EPA samples 476-1,2,3).
Preliminary characterization runs for these materials were performed.
Precision at the "medical decision" level of approximately 5 ng/mL was
10% CV (total CV, including within- and among-day components). Performance on
both EPA and NBS reference water samples has consistently given agreement of +
10% of the certified target value in the concentration range 0-10 ng/mL.
-------
45
References
1. A Statistical Quality Control System for the Clinical Laboratory. In
Selection and Implementation of Methods in Clinical Chemistry, Commission
on Continuing Education, Council on Clinical Chemistry, American Society
of Clinical Pathology, Chicago, Illinois (March 1975).
2. Winefordner J and Long G. Limit of detection—a closer look at the IUPAC
definition," Anal Chem, 55 (7): 713A-719A (1983).
-------
46
PARAMETERS FOR DETERMINATION OF LEAD AND CADMIUM IN URINE
BY GRAPHITE FURNACE ATOMIC ABSORPTION
Wavelength
Lamp Current
EDL Power
Slit
Signal Mode
Recorder
Read Time
Inert Gas
Inert Gas Flow
Deuterium Background
Corrector
Furnace Type
TEMPERATURE PROGRAM:
DRY
CHAR
ATOMIZE
COOL
Pb
283 nm
8 ma
9.5 - 10 W
0.70 (ALT)'
Peak Height
ABS
5.0 sec
Argon
300 mL/min;
20 mL/min @
ATOMIZE
ON
PYROLYTIC-L'vov
150°C 35 sec,
5 sec RAMP
600°C 25 sec,
5 sec RAMP
2000°C 5 sec,
1 sec RAMP
20°C 10 sec,
1 sec RAMP
Cd
228 nm
6 ma
5 W
0.70 (ALT)
Peak Height
ABS
5.0 sec
Argon
300 mL/min;
20 mL/min @
ATOMIZE
ON
PYROLYTIC-L'vov
Same as Pb
450°C 25 sec,
5 sec RAMP
2000°C 5 sec
1 sec RAMP
20° C 10 sec,
1 sec RAMP
-------
URINE B2-MICR0GL0BULIM
1.0 Title Page:
B2~Microglobulin - approved method name.
B2 micro test - "popular" name.
B2m - abbreviation
Nay 12, 1983
2.0 Method Principles/Description
B2-micro test is a competitive radioimmunoassay. B2M in the sample
competes with a fixed amount of labelled B2m for the binding
sites of anti-B2M antibodies covalently bound to Sephadex particles.
The competitive capacity is then compared with that of B2M standards of
known concentrations.
3.0 Definitions
Human B2M is a low molecular weight protein (11,800 daltons) consisting
of a single polypeptide chain of 100 amino acids. The protein is present
on the surface of nucleated cells as the constant subunit of the classical
transplantation antigens. It is released into the body fluids as a result
of cell turnover.
4.0 Scope and Application
4.1 Clinical Significance
Normally, only trace amounts of B2M are excreted in final
urine. However, the excretion of B2M is markedly increased
after minor derangements of the functional integrity of the
proximal tubuli, e.g., after exposure to heavy metals, anticancer
drugs, aminoglycosides, and anti-inflammatory compounds. B2M is
also excreted in increased amounts in the urine of patients with
chronic pyelonephritis but not those with bacterial cystitis. The
serum level of B2M reflects the glomerular filtration rate more
accurately than that of serum creatinine. The production rate of
B2M is increased in patients with some nonrenal diseases,
particularly those involving the immune systems. In malignant
lymphoma and multiple myeloma, the serum B2M level can be
related to turnover cell load, prognosis, and disease activity.
The serum B2M level is correlated to disease activity also in
some nonmalignant diseases, including rheumatoid arthritis and
hepatitis.
-------
48
4.2 Limitations/Interferences
A.3 Normal Ranges
Serum Geometric x = 81 ug/L
Upper normal limit = 250 ug/L
Urine Age x(u&/L) Upper normal limit(ug/L)
20-39 1,565 2,000
40-59 1,894 2,600
60-79 1,999 3,100
4.4 Manpower Requirements
One person is required to process samples and complete a run.
4.5 Analytical Throughput
44 samples/day
5.0 Safety
5.1 Reagent Toxicity or Carcinogenicity
None
5.2 Radioactive Hazards
125J
5.3 Microbiological Hazards
None
5.4 Mechanical Hazards
None
5.5 Protective Equipment
Latex gloves, plastic-backed absorbent paper for desk, lab coat,
safety glasses.
5.6 Training
5.7 Personal Hygiene
Avoid direct contact with the radioactive material by wearing
protective clothing. Hands should be washed thoroughly with soap
after any contact with radioactivity.
5.8 Disposal of Wastes
Compliance with the CDC Office of Biosafety guidelines is
required. The procedure is as follows:
5.8.1 Place all dry, compactible waste (glassware, plastics,
etc.) in a 13- x 12- x 24-inch plastic bag. Secure top
with tape or rubber band.
5.8.2 Place all dry, noncompactible waste (paper, rubber gloves,
etc.) in a 13- x 12- x 24-inch plastic bag. Secure top
with tape or rubber band.
5.8.3 Isotope stock vials must be placed in small plastic bags
and secured.
5.8.4 All radioactive waste containers (bags, boxes, etc.) must
contain a label stating nuclide present, approximate
activity, and date.
5.8.5 All uncontaminated outer packaging from radiation materials
must have the warning labels removed or crossed out before
disposal in the regular trash.
-------
49
6.0 Sample Preservation and Handling
6.1 Sample Collection
6.1.1 Serum Samples
Blood should be collected by venipuncture, allowed to clot,
and the serum separated by centrifugation.
6.1.2 Urine Samples
The following procedure for urine collection is
recommended: the patient should first void the urinary
bladder, then drink a large glass of water and collect a
urine sample within 1 hour thereafter.
6.2 Sample Storage and Shipping Requirements
Serum samples can be stored for 1 week at 2°-8°C or for at
least 1 year at -20°C. Urine samples with a pH between 6 and 8
can be stored for 2 days at 2 -8°C or for at least 2 months at
-20°C.
6.3 Sample Handling
Urine should always be collected at high diuresis and the pH
adjusted with 1.0 m NaOH to between 6 and 8 before storage.
7.0 Apparatus and Equipment
7.1 Description
1 500-ml or 1,000-ml graduated cylinder
1 400-ml or 800-ml beaker
1 10-ml graduated pipette
1 2.0-ml repeating pipette
1 each 50-ul, 20-ul, and 1,000-ul micropipettes, with disposable
plastic tips
146 polystyrene centrifuge tubes with round bottoms, 12 x 75 mm.
Redistilled water, absorbent paper, plastic film, vortex mixer,
magnetic stirrer, magnetic stirring bar, centrifuge (swingout
bucket), horizontal shaker, and gamma counter.
7.2 Routine Calibration
7.2.1 Pipettes should be calibrated according to instructions
supplied by the manufacturer.
7.2.2 Gamma counter should be calibrated by counting a
certified 129j instrument standard.
7.3 Maintenance
-------
50
7.4 Trouble Shooting
Problem
Low activity
Low binding (B0)
Poor duplication
High nonspecific
binding
Flat curve
Possible Cause
Radioactive decay
Counter problem
Aliquot error
Improper incubation
conditions
Inadequate pelleting
Problem with super-
natant removal
Insufficient antibody
Aged tracer
Inadequate pelleting
Problem with super-
natant removal
Variable pipetting
Improper reagent use
Addition of primary
antibody to blank
.(reads same as "0"
binding)
Inadequate removal of
supernatant
Tracer deterioration
Insufficient standard
Excess antibody
Solution
Check expiration date of tracer.
Check counter efficiency.
Check volume of tracer used in assay.
Refer to manufacturer's recommendations
Equilibrate reagents to room temperature
Check centrifugation (time, recommended
g force, etc.)
Follow recommended decanting procedures
Check volume used in assay.
Check storage, expiration date.
Check expiration date of tracer.
Check centrifugation (time, G force,
etc.)
Follow recommended procedures for
supernatant removal.
Calibrate pipets and/or dispensers.
Equilibrate reagents to room
temperature and mix before use.
Do not add primary antibody to blank
Check for retained liquid in blank
tubes.
Check "0" binding, expiration date
of tracer and storage conditions.
Check standard addition.
Check antibody addition.
-------
51
Curve shift to right Insufficient standard
Excess tracer
Improper incubation
conditions
Curve shift to left
Low values
High values
Excess antibody
Excess standard
Insufficient tracer
Insufficient antibody
Insufficient aliquot
Improper sample storage
or use
Erratic curve
Excess sample aliquot
Inadequate pelleting
Erratic curve
Check standard addition.
Check tracer addition.
Refer to manufacturer's
recommendations.
Equilibrate reagents to room
temperature.
Check antibody addition.
Check standard addition.
Check tracer addition.
Check antibody addition.
Check volume used in assay.
Check proper storage conditions.
Do not acidify samples.
Check typical standard curve.
Check volume used in assay.
Check centrifugation and
decanting procedures.
Check typical standard curve.
-------
52
8.0 Reagents and Standard
8.1 Chemicals and Standards
Phadebas ® B2~micro Test Kit, Catalog number (Pharmacia
Diagnostics, Uppsala, Sweden)
8.2 Procedures for Evaluating New Lots of Chemicals and Standards
Reagents from Phadebas® B2M test packages with different lot
numbers should neither be pooled nor interchanged. Also, they are
not interchangeable with reagents in other tests from Pharmacia
Diagnostics. Overlapping analytical runs should be made to
confirm identical response with new reagent lots.
8.3 Reagent Preparation
8.3.1 Phadebas® B2M test buffer solution.
Add 5 riL (1 vial) Tween solution to 300 mL deionized water;
thereafter dissolve 8.3 g (1 vial) buffer substance powder
in this Tween-water solution.
8.3.2 Prepare Sephadex® - Anti-B2M Complex by adding 7.0 mL
deionized water. Allow the suspension to stand for 2 min.
Mix and put a magnetic stirring bar into the vial.
8.3.3 125I-B2M Solution
Reconstitute the by adding 5.5 mL deionized
water. Allow the solution to stand for 1 min. before
mixing.
8.4 Calibrator Preparation
Reconstitute the B2M calibrators by adding 1,000 uL deionized
water to each vial. Allow the solutions to stand for 1 min.
before mixing. To prepare a 1-ug/L calibrator, make a 1:10
dilution of the 10-ug/L calibrator with buffer solution.
9.0 Daily Operating Procedures
Label and arrange the test tubes. Each determination should be performed
in duplicate for both calibrators and unknowns. A calibration curve
should be prepared on each test occasion. Avoid dispensing solution onto
the walls of the tubes.
9.1 Pipette 50 uL of Phadebas® B2~micro buffer solution (= zero
calibrator) into tubes 1-2.
9.2 Pipette 50 uL of the B2~microglobulin calibrator solutions 1,10,
25, 75, 200, and 500 ug/L into tubes 3-14.
9.3 Pipette 50 uL of diluted Unknowns into tubes 15-16, etc.
9.4 Pipette 50 uL of the 125I-B2~n»icroglobulin solution into all
tubes, including tubes Ti and T2. Tubes Ti and T2 contain
only *-25I-B2-microglobulin and are used to determine the total
activity added. They are stoppered immediately and set aside.
9.5 Pipette 50 uL of the Sephadex® -Anti-B2~microglobulin complex
suspension into all tubes except Ti and T2.
The Sephadex® -Anti-B2~microglobulin complex suspension must
be stirred continuously on a magnetic stirrer while it is being
dispensed.
9.6 Mix (by vortexing) until the reaction solution turns a homogeneous
green. Cover the tubes with plastic film or aluminum foil and
incubate them on a shaker for 90 min. at controlled room
temperature.
-------
9.7
Separation
9.7.1 Add 2.0 mL Phadebas® B2~micro buffer solution into the
tubes.
9.7.2 Centrifuge the tubes at 2,000 X g for 10 min. Use a
swingout bucket.
9.7.3 Decant the supernatant by gently turning the tubes upside
down without interrupting the turning movement. Do not
shake the tubes! When the tubes are upside down, place
them on an absorbent paper for 5 sec.
9.8 Determine the bound radioactivity in the tubes and the total
activity (T^ and XŁ), using a gamma counter. When a gamma
counter with 50% efficiency is used, 1 min. counting time will be
inefficient. The background is determined by using an empty test
tube.
10.0 Quality Control
10.1 Quality Control Protocol
Normal and high B2M quality control samples of urine or serum
will be included in each run.
10.2 Performance Evaluation Samples
Blind report samples will be included on a random basis.
10.3 Blanks
An empty test tube will serve as a counting blank.
10.A External Proficiency Testing Program
None is available.
10.5 Special Requirements
None
11.0 Data Handling
11.1 Calculations
11.1.1 Calculations are performed by SPLINE function program on
LKB RACKGAMMA II .
11.1.2 SPLINE function fitting can only be done by using a
computer. An unknown concentration value is found by
exactly the same general principle as in linear
interpolation and polygonal interpolation. The curve
segment on which the unknown lies is found, and the
concentration is then calculated by using this equation.
11.2 Data Reporting Format
To be determined.
-------
54
12.0 Literature References
12.1 Berggard, I, and Beam, AG: Isolation and properties of a low
molecular weight B2~globulin occuring in human biological
fluids. J. Biol. Chem. (1968) 243:4095-4103.
12.2 Nakamuro, K, Tanigaki, N, and Pressman, D: Multiple common
properties of human B2-microglobulin and the common portion
fragment derived from HL-A antigen molecules. Proc Natl. Acad.
Sci. (1973) 70:2863-2865.
12.3 Grey, HM, Kubo, RT, Colon, S, et al: The small subunit of HL-A is
B^-microglobglin. J. Exp. Med. (1973) 138:1608-1612.
12.4 Peterson, PA, Rask, L and Linblom, JB: Highly purified
papain-solubilized HL-A antigens contain B2-microglobulin. Proc.
Natl. Acad. Sci. (1974) 71:35-39.
12.5 Wibell, L, and Karlsson, FA: The serum level of
-microglobulin—a low molecular weight protein. Prot. Biol.
Fluids (1975) 23:343-347.
12.6 Evrin, PE and Wibell, L: The serum levels and urinary excretion
of B2-microglobulin in apparently healthy subjects. Scand. J.
Clin. Lab. Invest. (1972) 29:69-74.
12.7 Kjellstrom, T, and Piscator, M: Quantitative analysis
of B2-microglobulin in urine as an indicator of renal tubular
damage induced by cadmium. Phadedoc Diagnostic Communications No.
1 (1979) Pharmacia Diagnostics, Uppsala, Sweden.
12.8 Fleming, JJ, Parapia, L, Morgan, DB, et.al: Increased
urinary B2-microglobulin after cancer chemotherapy. Cancer
Treat. Rep. (1980) 64:581-588.
12.9 Schentag, JJ, Sutfin, TA, Plaut, M.E., et al: Early detection of
aminoglycoside nephrotoxicity with urinary B2-microglobulin. J.
Med. (1978) 9:201-210.
12.10 Merle, LJ, Reidenberg, MM, Camacho, MT, et al: Renal injury in
patients with rheumatoid arthritis treated with gold. Clin.
Pharmacol. Ther. (1980) 28:216-222
12.11 Schardijn, G, Statius van Eps, LW, Swaak, AJG, et al: Urinary
B^-microglob^lin in upper and lower urinary tract infections.
Lancet (1979) 1:805-807.
12.12 Wibell, L, Evrin, PE and Berggard, I: Serum B2-microglobulin in
renal disease. Nephron. (1973) 10:320-331.
12.13 Kult, J, Lammlein, Ch, Rockel, A, et al: B2~Mikroglobulin im
Serum - ein Parameter des Glomerulumfiltrates. Dtsch. Med. Wschr.
(1974) 99:1686-1688.
12.14 Child, JA, Spati, B, Illingworth, S, et al:
Serum B2-microglobulin and C-reactive protein in the monitoring
of lymphomas: findings in a multicenter study and experience in
selected patients. Cancer (1980) 45:318-326.
12.15 Norfolk, D, Child, JA, Cooper, EH et al:
Serum B2-microglobulin in myelomatosis: potential value in
stratification and monitoring. Br. J. Cancer (1980) 42:510-515.
12.16 Simonsson, B, Wibell, L, and Nilsson, K: B2~microglobulin in
chronic lymphocytic leukemia. Scand. J. Haematol (1980) 24:174-180.
12.17 Spati, B, Child, JA, Kerruish, SM, et al: Behaviour of serum
B -microglobulin and acute phase reactant proteins in chronic
lymphocytic leukemia. Acta Haematol. (1980) 64:79-86.
12.18 Manicourt, 0., Brauman, H. and Orloff, S: Plasma and urinary
levels of B2-microglobulin in rheumatoid arthritis. Ann. Rheum.
Dis. (1978) 37:328-332.
12.19 Beorchia, S., Vincent, C., Revillard, J. P. et al: Elevation of
serum B2-microglobulin in liver diseases. Clin. Chim. Actra.
(1981) 109:245-255.
-------
13.1 Quality Control System
13.1.1 Definitions
13.1.2 Quality Control Statistics
QC statistics are computed by nested analysis of variance (ANOVA)
from replicate analyses of each QC pool. For urine assays two QC
samples will be included in each run. These samples were prepared
from a pool of urine from healthy volunteers. Aliquots of the pool
were dispensed into Wheaton vials and stored frozen for later use
as low QC samples. Aliquots of the pool after enrichment with
B2M calibrator were dispensed and frozen for later use as high QC
samples.
13.1.3 Quality Control Techniques
13.1.3.1 Run Format. Duplicate aliquots (within vial) of low and
high QC samples are included in each analytical run when
study samples are analyzed. When establishing QC limits
for these samples, quadruplicate aliquots from each QC
sample were analyzed in the same run. Twenty runs were
performed to establish QC limits before study samples
were analyzed.
13.1.3.2 Precision estimates for low and high urine QC pools
Coefficient of Variation
Sample MEAN STANDARD DEVIATION AMONG DAY(df) WITHIN VIAL(df)
Low Urine 52.4 ug/L 6.82 ug/L 27.1% (14) 13.0% (46)
High Urine 181.1 ug/L 61.53 ug/L 4.0% (11) 34.0% (24)
13.1.4 Control Charts and Display of Control Data. Levy-Jennings
charts of B2M concentration versus date are made for each
QC sample.
13.1.5 Laboratory Action Codes
13.1.6 Action Limits
13.1.7 Outlier Rules
13.1.8 Corrective Action Rules
13.1.9 Blind Quality Control
-------
URINARY CREATINE/PROTEIN
0000000
0000000
OOOBIXIO
aca~
DU PONT DISCRETE
CLINICAL ANALYZER
56
aca™ n/nc/1
TEST METHODOLOGY
criia
creatinine
INTENDED USE:
The CREA pack is used in the Ou Pont 3CB™ discrete
clinical analyzer to quantitatively measure creatinine in
serum or urine.
SUMMARY:
The creatinine (CREA) method employs a modification of
the kinetic Jaffe reaction reported by Larsen.' This method
has been reported to be less susceptible than conventional
methods to interference from non-creatinine, Jaffe-
positive compounds.1
Split sample comparison between the CREA method and
a conventional Jaffe procedure on the Autoanalyzer® gave
a correlation coefficient of 0.999 with no statistically signif-
icant bias.J Compared to a previous CREA method (Jaffe
end point with protein removal), the present method gave a
correlation coefficient of 0.994.2
®Registered trademark. Technicon Corp.. Tarrytown. N.Y.
PRECAUTIONS:
COMPARTMENT #6 CONTAINS 75 mL OF 10 N
NaOH; AVOID CONTACT; SKIN IRRITANT; RINSE
CONTACTED AREA WITH WATER.
USED PACKS CONTAIN HUMAN BODY FLUIDS-
HANDLE WITH APPROPRIATE CARE.
FOR IN VITRO DIAGNOSTIC USE
MIXING & DILUTION:
The aca™ analyzer automatically aspirates a 200 /iL
sample of body fluid from the sample cup and injects it into
the pack, along with 4.800 mL of Purified Water. The sam-
ple cup must contain a sufficient quantity of body fluid to
accommodate the 200 mL sample size plus the 120 jiL
"dead volume" of the cup. Precise filling of the cup by the
operator is not required. The micro sample cup insert, with
a total volume of 500 ^L and a "dead volume" of 10ML, may
also be used.
PRINCIPLES OF PROCEDURE:
In the presence of a strong base such as NaOH, picrate
reacts with creatinine to form a red chromophore. The rate
of increasing absorbance at 510 nm due to the formation
of this chromophore during a 17.07-second measurement
period is directly proportional to the creatinine concen-
tration in the sample.
Creatinine + Picrate . Red chromophore
(absorbs at 510 nm)
STORAGE INSTRUCTIONS:
Store under refrigeration (2—8°C). Do not freeze. Do not!
expose packs to temperatures above 35°C. Do not e*pose
packs to direct sunlight.
EXPIRATION:
Refer to EXPIRATION DATE on the tray label.
REAGENTS:
Compartment' Form Ingredient Quantity*
02,3, & 4 Liquid Picrate 0.11 mmol
#6 Liquid NaOH (for pH)
adjustment)0
a. Compartments are numbered 1 -7. with compartment H7 located closest
to pack (ill positron #2.
b. Nominal value at manufacture.
c. See PRECAUTIONS.
SPECIMEN COLLECTION:
Normal procedures for collecting and storing serum ndl
urine may be used for samples to be analyzed by the cocai
method.5 tREA
KNOWN INTERFERING SUBSTANCES4
• Serum Protein Influence
Serum protein levels exert a direct influence
CREA assay. The following should be taken*Into
-------
account when .this method is standardized as recom-
mended with protein-containing materials:
— Aqueous creatinine standards or urine specimens
will give CREA results depressed by approximately
0.7 mg/dL5 and will be less precise than samples
containing more than 3 g/dL protein.
— All urine specimens should be diluted with an
albumin solution to give a final protein concen-
tration of at least 3 g/dL. Du Pont Enzyme Diluent
(PN 790035-901) may be used for this purpose.
• High concentration of endogenous bilirubin (>20 mg/
dl) will give depressed CREA results (average de-
pression 0.8 mg/dL).6
• Grossly hemolyzed (Hb > 100 mg/dL) or visibly lipemic
specimens may cause falsely elevated CREA
results.2'7
• The following cephalosporin antibiotics at the
indicated serum concentrations have been shown to
have no measurable effect on CREA results:
Cephalosporin Drug Added (mg/dL)
Cefoxitin
Cefamandole
Cefazolin
Cephalexin
Cephaloridine
Cephalothin
Cephapirin
Cephradine
2.5
25
10
100
5
50
100
10
The single wavelength measurement used in this
method eliminates interferences from chromophores
whose 510-nm absorbance is constant throughout the
measurement period.
Each laboratory should determine the acceptability of
its own blood collection tubes and serum separation
products. Variations in these products may exist
between manufacturers and, at times, from lot to lot.
TEST STEPS
When running analytical test packs the operator need be
concerned only with loading the sample and appropriate
test packs into a properly prepared instrument. The 3C3
automatically advances the packs through the test steps
and prints the result. For details of sample preparation
and pack processing, refer to Operating Instructions Sec-
tion of the Instrument Manual.
Preset Creatinine Test Conditions
• Sample Size:
• Diluent:
• Test Temperature:
• Reaction Period
(Initiation to
measurement!:
• Measurement Period:
• Wavelength:
• Type of Measurement:
• Decimal Point
Location:
aca™ 1/II analyzer:
• Assigned Starting
Point:
• Scale Factor:
aca™ III analyzer:
• Assigned Offset C0:
• Linear Term Ci:
200 til
Purified Water
37.0 ± 0.1 °C
29 seconds
17.07 seconds
510 nm
Rate
000.0 mg/dL
990.0
0.2000 (mg/dL)/count"
-1.000 E1 (-1.0 x 10')
2.004 E-1 mg/dL"
The preset scale factor (linear term) was calculated from an absorbance
to concentration relationship (sensitivity) of 17.54 mA/min/(mg/dl)
Due to small differences in filters and electronic components between
instruments, the actual scale factor (linear term) may diffar from that
given above.
PROCEDURE:
TEST MATERIALS
Quantity Item Du Pont Cat. #
1 aca™ CREA Analytical Test Pack 701989901
702694901
702785000
1 Sample System Packet
or
1 Micro Sample System Packet
and
Micro Sample System Holders
Dylur® Photosensitive
Printer Paper
Purified Water
Cell Wash Solution
700036000
704209901
701864901
^Registered trademark, E. I. du Pont de Nemours & Co., Inc.
Wilmington, DE.
CALIBRATION
The general calibration procedure is described in the
Instrument Manual.
The following information should be considered when
calibrating the CREA channel:
e Range of Linearity:
• Reference Materials:
Suggested Calibration
Levels:
0—20 mg/dL
Protein containing primary
standards* or secondary
calibrators containing
protein.
20, 5. 1 mg/dL
-------
58
Starting Point (Offset
Co) Adjustment:
• Scale Factor (Linear
Term Ci) Adjustment:
• Count By
OCa™ l/il analyzer):
• Readout Units:
For 303™ I analyzer, use
the channel #8 adjustable
zero offset (ZO) for the last
two digits of the starting
point. If adjustment of the
first two digits of the start-
ing point is required, relace
the photometer method
switching board.
For aca™ II analyzer, use
adjustable starting point for
all four digits.
For aca™ III analyzer, enter
offset Co into Method
Memory.
May be required for dif-
ferent pack lots.
One (1)
The 303 prints out in 0.1
mg/dL increments.
Protein exerts a direct influence on the CREA method. Standard solu-
tions ol creatinine containing protein may be prepared as follows:
• Reagents
1. Creatinine. C,H,N,0. F. W. = 113 2. crystalline, dehydrated,
2. Purified Water.
3. Bovine albumin, crystalline.
• Bovine Albumin Solution (7.0 g/dL)
Dissolve 70.0 g of crystalline bovine albumin in about 900 mL of
Purified Water in a 1 -liter volumetric flask. Adjust the volume to 1 liter
with Purified Water.
• Creatinine Stock Solution (100 mg/dL)
Dissolve 500.0 mg of creatinine in 450 mL of the bovine albumin
solution in a 500-mL volumetric flask. Adjust the final volume to 500
mL with the bovine albumin solution.
• Creatinine Standards
Creatinine standard solutions may be prepared using aliquots from the
stock solution and bovine albumin solution as the diluent. These
solutions should be refrigerated at 2—8°C. Sodium azide (CAUTION:
Dangerous material. Handle with care.) may be added at a level of
10 mg/dL to inhibit the growth of micro-organisms.
NOTE
If the aca,M analyzer is calibrated with aqueous creatinine
standards, serum based controls and human, protain con-
taining specimens will analyze 0.6—0.8 mg/dL too high at all
levels.
• Creatinine Method Check. At least once daily run a
CREA test pack on a solution of known creatinine con-
centration such as an assayed control or calibration,
standard other than that used to calibrate the CREA
channel. For further details review the Quality Assur-
ance Section of the Chemistry Manual. The result ob-
tained should fall within acceptable limits defined by
the day-to-day variability of the system as measured
in the user's laboratory. (See SPECIFIC PERFOR-
MANCE CHARACTERISTICS for guidance.) If the
result falls outside the laboratory's acceptable limits,
follow the procedure outlined in the Chemistry Trouble-
shooting Section of the Chemistry Manual.
A standard deviation for five consecutive packs greater
than 0.15 mg/dL for a level of 1.0 mg/dL or greater than
0.68 mg/dL for a level of 20.0 mg/dL indicates a possible
system malfunction.
RESULTS:
The aca™ analyzer automatically calculates and prints
the CREA concentration in mg/dL using the general
scheme #2 illustrated in the Calculation of Results Section
of the Chemistry Manual.
Information specific to the CREA calculation is listed
below:
aca™ l/l! analyzer:
• Count By:
• Scale Factor:
303™ III analyzer:
• Linear Term:
One (1)
0.2000 (mg/dL)/counts
2.004 E-1 mg/dLa
LIMITATION OF PROCEDURE:
CREA readouts in excess of 20mg/dLshould be repeated
after diluting the sample with suitable protein base diluent
to produce a sample concentration within the range of
linearity. The resulting readout must then be multiplied by
the dilution factor to give the CREA concentration of the
undiluted sample.
The instrument reporting system contains error mes-
sages to warn the operator of specific malfunctions. Any
report slip containing a letter code or word immediately
following the numerical value should be held for follow-up.
Refer to the Instrument Manual.
QUALITY CONTROL
Two types of quality control procedures are recom-
mended:
• General Instrument Check. Refer to the Filter Bal-
ance Procedure and the Absorbance Test Method de-
scribed in the Instrument Manual. Refer also to the
ABS Test Methodology literature.
REFERENCE INTERVAL (Normal Range):*''
Males: 0.8 — 1.3 mg/dL
Females: 0.6 — 1.0 mg/dL
Each laboratory should establish its own referen
interval for CREA as performed on the analyzer. c®
f. Reference interval data from 200 apparently healthy individual
male, 129 temala) between the ages of 19 and 72. *
-------
59
SPECIFIC PERFORMANCE CHARACTERISTICS:'
REPRODUCIBILITY (Precision)
Within-Runh
MEAN
1.35 mg/dL
20.60 mg/dL
Day-to-Day'
MEAN
1.36 mg/dL
20.65 mg/dL
S. D.
0.05
0.12
S. D.
0.05
0.37
C. V. (%)
3.7
0.6
C. V. (%)
3.7
1.8
JL
20
20
N.
20
20
LINEARITY:
0—20 mg/dL
g. All SPECIFIC PERFORMANCE CHARACTERISTICS tests were run after
normal recommended equipment quality control checks were performed
(see Instrument Manual).
h. N test packs, in series with a sample cup containing lyophilind serum
base material, were used.
t. One test pack per day for N days. A fresh sample of lyophilized serum
base material was used each day.
BIBLIOGRAPHY:
'Larsen, K., C/in. Chem. Acta., 41, p. 209 (1972).
2Kawas, E. E.. Richards, A. H., and Bigger, R., "An Evaluation of a Kinetic
Creatinine Test for the Ou Pont 3C3/' Trover Clinic and Hopkins County
Hospital, Madisonville, KY, February, 1973.'
JTigtz, N. W , "Fundamentals of Clinical Chemistry," W. 8. Saunders Co.,
Philadelphia, PA, 1970. p. 723.
* Supplementary information pertaining to the effects of various drugs and
patient conditions on in vivo or in vitro diagnostic levels can be found in
"Drug Interferences with Clinical Laboratory Tests." Cfin. Chem.. 21,
April, 1975. (This is an update of an earlier compendium — Clin. Chem.,
18, October. 1972.)
5The Du Pont Company, Instrument Products. Automatic Clinical Analysis
Division. Technical Services Laboratory Report. May, 1974
*Watkins, fl„ Feldkamp, C. S.. Thibet*, R. J., and Zak, B., CUn. Chem,. 21,
p. 1002 (1975). Presented at the 9th International Congress on Clinical
Chemistry, July, 1975.
7Westgard, J. 0., "Effect of Hemolysis and Lipemia on 303 Creatinine
Method, 0.200 mL Sample Size," University of Wisconsin. Madison.
Wl, October, 1972.1
"Gadsden, R. H., and Phelps, C. A., M. T.. "A Normal Range Study of Amylase
in Urine and Serum on the Du Pont 303," Medical University of South
Carolina, March, 1978. *
j. Reprints available from the Ou Pont Co., Clinical Systems Division.
Du Pont Company • Clinical Systems Division • Wilmington, DE19898
Clinical Systems
PN 703708 ft/11/82 700C48
(SMI)
-------
60
05ut»u0^
aca™
DU PONT DISCRETE
CLINICAL ANALYZER
aca™ 2/3S
TEST METHODOLOGY
UP
URINARY PROTEIN
INTENDED USE:
PRECAUTIONS:
UP packs are used in the Du Pont 303™ discrete clini-
cal analyzer to quantitatively measure protein in urine.
SUMMARY:
COMPARTMENTS #2 AND #3 IN UP-1 AND UP-2
PACKS EACH CONTAIN 65 OF 14 MOL/L NaOH:
AVOID CONTACT; SKIN IRRITANT; RINSE CON-
TACTED AREA WITH WATER.
The UP method allows direct quantitation of protein in
most urine samples within the normal and abnormal range.
The UP method is an adaptation of the turbidimetric method
of Iwata and Nishikaze.1 Split-sample comparison between
the UP method and biuret methods showed good correla-
tion (see SPECIFIC PERFORMANCE CHARACTERISTICS).
PRINCIPLES OF PROCEDURE:
The UP method uses a two-pack, end-point technique to
measure urinary protein. The UP-1 pack provides a sample
blank at 540 nm. In the UP-2 pack, benzethonium chloride
(BEC) precipitates urinary protein in an alkaline medium.
Light scattering by the precipitate causes a decrease in light
transmission. The decrease in light transmission is mea-
sured as absorbance at 540 nm. The absorbance difference
between the UP-1 and UP-2 pack is related to the total
protein concentration in the sample by means of a standard
curve or mathematical function.
Protein BEC
NaOH
Protein Precipitate
(Scatters light at 540 nm)
REAGENTS:
UP-1 (Blank)
Compartment'
Form
Ingredient
Quantity''
Liquid
EDTA and Microbial
Inhibitor
#2 & «3
Liquid
NaOHc
#4
Liquid
Surfactant and
Microbial Inhibitor
UP-2 (Reaction)
Compartment*
Form
Ingredient
Quantity11
01
Liquid
EOTA and Microbial
Inhibitor
#2 & *3
Liquid
NaOHc
tt4
Liquid
Surfactant and
Microbial Inhibitor
#5 & *6
Liquid
Beniethonium
Chloride'
45 »mol
a Compartments are numbered 1-7. with compartment »7 located closest
to pack (ill position #2.
b. Nominal value at manufacture
c. Sea PRECAUTIONS
COMPARTMENTS #5 AND #6 IN UP-2 PACKS
EACH CONTAIN 50 mL OF 0.45 MOL/L BENZETHO-
NIUM CHLORIDE; AVOID CONTACT; SKIN IRRITANT-
CORROSIVE TO MUCOUS MEMBRANES; RINSE
CONTACTED AREA WITH WATER.
USED PACKS CONTAIN HUMAN BODY FLUIDS-
HANDLE WITH APPROPRIATE CARE.
FOR IN VITRO DIAGNOSTIC USE
USE LIMITATIONS:
UP-1 AND UP-2 TEST PACK LOTS WHICH ARE SHIPPpq
TOGETHER MUST BE USED TOGETHER.
MIXING AND DILUTION:
The UP test is performed with two test packs which must
be placed behind a sample cup in the order UP-1, UP-2. The
aca™ discrete clinical analyzer automatically aspirates a
400 juL sample of urine and injects it into each test pack
along with 4.600 mL of Purified Water. The sample cup
must contain a sufficient quantity of sample to accommo-
date the total 800 mL sample size plus the 120 mL "dead
volume" of the cup. Precise filling of the cup by the operator
is not required.
STORAGE INSTRUCTIONS:
Store under refrigeration (2-8°C). Do not freeze. Do not
expose packs to temperatures above 35°C or to direct
sunlight.
EXPIRATION:
Refer to EXPIRATION DATE on the tray label.
SPECIMEN COLLECTION;
Normal procedures for collecting urine may be used for
samples to be analyzed by the UP method. Specimens
stored at 4°C with no additives are stable for at least three
days1. Specimens stored under toluene or those containin
sodium hydroxide (5%) or boric acid (100 mg/m|_) are
-------
61
acceptable. Specimens stored at room temperature with no
additives showed an increase (*»10%) in the protein level
over a three-day period.
KNOWN INTERFERING SUBSTANCES:
• Magnesium at levels greater than 20 mg/dL will
depress urinary protein results. Patients on intra-
venous MgSO.i therapy may attain urinary magnesium
levels high enough to depress results by as much
as 50%.:
• Hydrochloric acid (0.1 N) or boric acid (^200 mg/mL)
cause destruction of protein and should be avoided as
additives at these concentrations1.
• Bilirubin at a level of 1.9 mg/dL[32.5 >imol/L]did not
interfere1. Bilirubin added to a level of 20 mg/dL
[342 Mmol/L] increased the apparent protein level by
76 mg/L at a level of 200 mg/L and by 128 mg/L at a
level of 1300 mg/L.
• The following substances at the levels shown had no
effect on the UP method:
Ammonia,
180 Mg/dL [100 mmol/L]
Ascorbic acid, 100 mg/dL [5.7 mmol/L]
Creatinine,
Glucose,
Phosphorus,
Urea,
Uric acid,
20 mg/dL [1.8 mmol/L]
2.5 g/dL [138 mmol/L]
1 g/L [32 mmol/L]
1.7 g/dL [600 mmol/L]
50 mg/dL [3 mmol/L]
Hemoglobin interference (see ANALYTICAL SPECIFICITY).
PROCEDURE:
TEST MATERIALS:
Quantity Item
1 pair UP-1, UP-2 test packs
and
Graph Paper: GR DAd
1 Sample System Packet
Dyluic® Photosensitive
Printer Paper
Purified Water
Cell Wash Solution
Du Pont Cat. It
70S213901
701989901
700038000
704209901
701864901
® Registered trademark. E. I. du Pont de Nemours & Co.. Inc., Wilming-
ton, DE.
d. Graph paper is packaged in each carton of UP-2 test packs. Each sheet
of graph paper and each carton label has the letter code "GR DA" fol-
lowed by a single digit number. For proper calibration, the letter/number
code on the graph paper must match that on the carton label.
TEST STEPS:
tration units. The actual mechanical travel of the test pack
through the instrument is described in detail in Section III of
the Instrument Manual for the aca™ analyzer.
Preset Urinary Protein Test Conditions
• Sample Size:
• Diluent:
• Test Temperature:
• Reaction Period
(initiation to
measurement):
• Wavelength:
• Type of
Measurement:
• Decimal Point
Location:
303™ II analyzer
• Assigned Starting
Point®:
• Scale Factor:
• Count By:
aca™ III analyzer
• Assigned Offset
Co:
• Assigned Linear
Term Ct:
• Assigned Logit-Log
Function Terms, C:
and Cj:
800 mL (400 ML/pack)
Purified Water
37.0 ± 0.1°C
46 seconds
540 nm
Two pack, end point
0000.
9999. mA
0.1000 m A/count
One (1)
Co, Ci, C:, Cj are match-
ed to each lot of UP test
packs.
See heading of graph paper
packaged in cartons.
e. For 2-pack methods on aca™ II analyzers, the starting point must be set
below zero. Only a below zero starting point triggers the error circuitry
which differentiates starting point printouts from result printouts on the
petient report slip.
CALIBRATION:
The general calibration procedure is described in the
Calibration/Verification chapter of the Instrument Manu-
als for the aca™ II and aca™ III discrete clinical analyzers.
Calibration procedures should be followed for each new lot
of test packs and must be repeated every three months for
any one lot of test packs.
When running test packs, the operator need be con-
cerned only with loading the sample and appropriate test
pack(s) into a properly prepared instrument. The aca™
discrete clinical analyzer automatically advances the
pack(s) through the test steps. The aca™ II analyzer prints
the result in milliabsorbance (mA) units which must be
converted by the operator to concentration units using a
previously prepared calibration curve or a mathematical
function. The 303™ III analyzer prints the result in concen-
The UP channel on the 303™ analyzer should be cali-
brated over the assay range using five calibrators analyzed
in duplicate.
• Assay Range: 60-2400 mg/L
• Suggested Calibration 90, 500, 1100, 1650. 2250
Levels: mg/L
-------
• Reference Material: Primary standards or
secondary calibrators such
as Du Pont 3Ca™ Urinary
Protein Calibrator (P/N
790559901)
To calibrate the 3.CS.™ II analyzer for the UP method,
construct a calibration curve on the graph paper provided
according to the instructions in the Calibration/Verification
chapter, Immunoassay paragraph of the Instrument Man-
ual for the aca™ II analyzer. Adjustment of the starting
point and scale factor is not required for calibration of
aca™ II analyzers.
To calibrate the 303™ III analyzer for the UP method,
follow the instructions in the Calibration/Verification chap-
ter, Immunoassay paragraph of the Instrument Manual for
the aca™ III analyzer. The theoretical constants for the
logit-log function are matched to each UP pack lot and are
given on the top of the graph paper.d Adjustment of the
OFFSET, (G>) and LINEAR TERM, (Ci) may be required to
calibrate the instrument.
If Du Pont aca™ urinary protein calibrators are being
used, prepare them according to the instructions in the
calibrator insert sheet.
QUALITY CONTROL:
Two types of quality control
recommended:
procedures are
• General Instrument Check. Refer to the Filter Bal-
ance Procedure and the Absorbance Test Method
described in the Instrument Manual. Refer also to the
ABS Test Methodology literature.
• Urinary Protein Method Check. At least once daily
run a UP test on a solution of known protein concen-
tration such as an assayed control or calibration
standard other than that used to calibrate the UP
channel. For further details review the Quality Assur-
ance Section of the Chemistry Manual. The result
obtained should fall within acceptable limitsdefined by
theday-to-dayvariability.of the system as measured in
the user's laboratory. (See SPECIFIC PERFORMANCE
CHARACTERISTICS for guidance.) If the result falls
outside the laboratory's acceptable limits, follow the
procedure outlined in the Chemical Troubleshooting
Section of the Chemistry manual.
A possible system malfunction is indicated when analy-
sis of a sample with five consecutive tests gives a coeffi-
cient of variation greater than 6.8% when calculated from
mg/L results (or 8.8% when calculated from mA results) at
a level of 340 mg/L, or gives a coefficient of variation
greater than 8.7% when calculated from mg/L results (or
5.7% when calculated from mA results) at a level of 1400
mg/L. Refer to the procedure outlined in the Troubleshoot-
ing Section of the Chemistry Manual.
RESULTS:
For each UP test on the 3Ca™ II analyzer, two results will
be listed on the printout in the order UP-1 result, UP-2
result and both will be preceded by the letters GR DA. The
first result is the preset starting point. The second result is
the sample measurement in milliabsorbance (mA) units
The urinary protein concentration in mg/L must be deter-
mined from the calibration curve constructed according to
instructions in the Calibration/Verification chapter, Immu-
noassay paragraph of the Instrument Manual.'
The 3CarM III analyzer automatically calculates and
prints the concentration of urinary protein in mg/ L using the
logit-log function described in the Field Evaluation Report1
Only one result is printed for each pair of UP packs.
The calibration curve must be constructed on the correct graph paper
The OPERATOR MUST VERIFY THAT THE CORRECT GRAPH PAPER |§
BEING USED Each sheet of graph paper and each carton label has the
letter code 'GR OA" followed bv a single diqit numoer The letter/
number code on the graph paper must match that on the carton label and
the entry for "DATE PLOTTED" must be within the previous 3 months
LIMITATION OF PROCEDURE:
Readouts in excess of 2400 mg/L should be repeated
after diluting the sample with Du Pont Purified Water to
produce a sample concentration within the assay range
The resulting readout must then be multiplied by the
appropriate dilution factor to give the UP concentration of
the undiluted sample.
Results less than 60 mg/L should be reported as "less
than 60 mg/L" instead of the numerical value. On aCarM lit
analyzers the Linear Limit error code "LL~ will follow
results less than 60 mg/L Foraca™ II analyzers, 60 mg/L
is the lowest value shown on the graph paper.
aca™ II Analyzers:
The reporting system contains error messages to warm
the operator of specific malfunctions. It is characteristic!
that the first printout of a 2-pack method will be the startinr
point followed by the code A. The second printout is the
measured value for the sample in milliabsorbance (mAI
units.
When one pack of a 2-pack method is decoded in iht
photometer, the computer is automatically programmed tc
receive pack 2 of that method as the next pack. Howeven
circumstances where the next pack may not be the secont
pack of that test pair are possible, e.g.,
1. A single pack of a 2-pack method was used to chec'
the starting point during calibration.
2. One pack of a 2-pack method fell off the transport pin
3. Only one pack of a 2-pack method was loaded inti
the input tray.
In these cases, if the next test processed is also a 2-pac
method, ERRONEOUS RESULTS WILL BE PROOUCED 01
BOTH TESTS' This will be quite evident because the start
ing point and A error code will be printed for pack 2 0f tn
second 2-pack method.
Examples
Casei 1 & 3
Pack S«gu»rc»
SAL-1
UP-1
UP-2
Printout
SAL ¦ 999.8]
GR OA 5is]
Iwill vary with
sampia)
GR OA
9999
-------
63
Case 2:
Pack Sequence
SAL-1
SAL-29
UP-1
UP-2
Printout
SAL 999 8A
CDGDC OOP
GR OA 9999A
GR DA 9999A
g. SAL-2 was removed from the transport chain before it reached the photo-
tometer.
To return the computer to proper sequence:
1. Toggle the MOTOR HOLD RESET switch on the read-
out board, or
2. Decode a header in the photometer from any 2-pack
method, or
3. If the next test processed is a single pack method, the
computer automatically resets and prints out the
proper answer.
NOTE
These example printouts apply to 303™ II ana-
lyzers only. If cases 1 or 3 occur on aca™ III ana-
lyzers, the error message "SECOND PACK MISS-
ED" is displayed and the filling station shuts down.
To reset the filling station, remove the UP-1 pack
from under the decode head in the filling station
and push the ERROR OVERRIDE button. The SAL-
1 pack is processed to the photometer where the
error message "MISSING PACK" is displayed and
printed on the report slip. If Case 2 occurs on
303™ III analyzers, the error message "MISSING
PACK" is displayed and printed on the report slip.
Any report slip containing another code or word imme-
diately following the numerical value should be held for
follow-up. Refer to the Instrument Manual for more details.
The UP method should not be used on instruments with
round log amplifiers. The location of the log amplifier is
shown in the Instrument Manual (303™ III analyzers have
only square log amplifiers). Round log amplifiers can be
identified by their round shape and gold color.
REFERENCE INTERVAL1:
Less than or equal to 165 mg/day
Less than or equal to 135 mg/L
Twenty-four-hour urine collections were obtained from
195 apparently healthy adult individuals. This population
consisted of laboratory personnel and their families and
was nearly equally distributed between male (48%) and
female (52%). One hundred and fifty-five (155) individuals
were from southwestern Pennsylvania and forty (40) were
from northcentral Texas.
The reference interval was derived non-parametricallyby
determining the 95th percentile.
Each laboratory should establish its own reference inter-
val for UP as performed on the aca™ analyzer.
SPECIFIC PERFORMANCE CHARACTERISTICS:11'"
ASSAY RANGE:
60-2400 mg/L
Control Material
Urine Pool
Level I
Level II
URI-CHEM*
Level I
Level II
REPRODUCIBILITY':
Standard Deviation (% CV)
Mean (mg/L) Within-Day Between-Day
126
2164
475
988
2.1 (1.7)
53 (25)
1.4(0.3)
3.2(0.3)
40(3.2)
69 .13 2)
3.6(0 8)
5 5 (0.6)
Uri-Chem Controls: Fisher Diagnostics. Orangeburg, NJ.
CORRELATION:
Regression Statistics'
Comparative Method
Slope
Intercept
Gel filtration-biuret'
0.98
-76
0 989
Phosohotungstic acid-biuret'
0 97
-86
0.976
Trichloroacetic acid-biuret'
0 94
48
0.990
Coomassie Blue
0.96
50
0.986
Sulfosalicylic acid-turbidimetric
1.04
110
0 968
Trichloroacetic acid-turbidimetric
1.54
-58
0.926
h. All SPECIFIC PERFORMANCE CHARACTERISTICS tests were run after
normal recommended equipment quality control checks were performed
(see Instrument Manual).
i. Specimens at each level were analyzed in duplicate twice a day for
twenty days. The within-day and between-day standard deviations were
calculated by the analysis of variance method
j. Model equation for regression statistics is: OC3rM analyzer result! =
Slope ^[comparative method result) + Intercept.
ANALYTICAL SPECIFIC!TY::
Recovery of various proteins by the UP method is shown
below.
Protein
% Recovery
Albumin
96
Gamma globulin
77
Transferrin
103
Orosomucoid
64
B-lipoprotem
92
Tamm-Horsfall
232
Lysozyme
11
Hemoglobin
10
BIBLIOGRAPHY:
'Iwata. J. and Nishikaze. 0 . New micro-turbidimetric method for determina-
tion of protein in cerebrospinal fluid and urine, din. Chem. 25:1317-1319
(1979).
:Gadsden. R.H.. Ritzmann, S.E., Aguanno, J.J. Finney. MM. Savory. J.,
and Savory, M G.. An Evaluation of the Urinary Protein Method for the
Du Pont aC3rM Discrete Clinical Analyzer, Du Pont Company. Wilmington.
DE (October. 1982)k
'Doetsch. K. and Gadsden, R.H . Determination of urinary total protein bv
use of gel filtration and a modified biuret method, in Selected Methods of
ClinicalChemistry, Vol 8, G.R Cooper. Ed., p 179-188. American Associa-
tion for Clinical Chemistry, Washington, DC. 1977
4Savory. J.. Pu. PH. andSunderman. Jr.. F W , A biuret method for determi-
nation of protein in normal urine. Ctm. Chem 14 1160-1.171 (1968)
'Tieti. N W .Fundamentals of Clinical Chemistry, p 363. W 8 SaundersCo.
Philadelphia. PA. 1976
k. Reprints available from the Du Pont Company. Clinical Systems Division.
Du Pont Company • Clinical Systems Division • Wilmington, DE 1 9898
Clinical Systems
17093424 II 2 82 700C 216
E 544n3
-------
64
quality
assurance
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gi«»«»iW3w^M assurance
4-1. General.
To assure accurate and precise results from the
aca , each method must be properly set up in the
user's lab and thereafter must -be maintained in
control. The set-up procedure, either calibration
for non-enzyme methods or verification for enzyme
methods, is accomplished when the aca is origi-
nally installed or when a method is first put into
routine use. Thereafter, a daily quality control
program should be used to indicate changes in the
aca system that may significantly affect the re-
sults of patient samples. This section of the manual
presents Du Pont's recommendations for these two
aspects of Quality Assurance.
4-2. Calibration of Mon-Enzyme
Methods.
A non-enzyme method can be set up following the
calibration procedure in Section 5 of the aca Instru-
ment Instruction Manual using standards prepared
as described in the Test Methodology literature in
the second half of this manual. Each new pack lot
received by the laboratory should be calibrated.
Since calibration requires two or three packs at each
level, the precision of a new pack lot should be
examined during this procedure. If the precision
is unacceptable, refer to Chart 5-10 in Section 5.
When new calibration values are entered in the
aca, the calibration should be verified by running
several packs on the standards used. The calibration
may also require verification during troubleshooting
or after the replacement of some instrument
components, notably the photometer filters.
4-3.Verificat:on of Enzyme Methods.
Du Pont does not recommend lot-to-lot slope
Calibration of aca enzyme channels because there
vare no enzyme standards and lot-to-lot slope
variations are generally less than ± 5% as measured
in the user's laboratory. However, in some cases
offset adjustments may be necessary.
Specific data will be found in the Calibration sec-
tion of each Test Methodology literature. When
pack lots of an enzyme test are changed, the chan-
nel should be verified using the procedure given
in Section 5 of the aca Instrument Instruction
Manual. The recommended materials and the ex-
pected precision and linearity will also be found in
the Test Methodology literature.
Enzyme verification materials can be either com-
mercial control products or serum pools. They
should be stable and have bottle values assigned by
the user. If a lyophilized control product is used,
at least three vials should be pooled to minimize the
effect of vial-to-vial variation.
Bottle values can be correctly assigned only after
the verification materials have been assayed using
at least five pack lots. Preliminary bottle values
can be estimated by assaying the materials with the
first pack lot; these values are used for the veri-
fication of pack lots received before the bottle
values are correctly established. The slope of aca
readout versus bottle value for the first pack lot is,
of course, 1.00. When a second pack lot is received,
it should be verified using the bottle values esti-
mated with the first pack lot. As mentioned above,
the slope variations from lot-to-lot should be less
than ±5%, i.e., less than a 10% total range; thus,
the slope of the second pack lot must be within
10% of the slope of the first pack lot. Since the
slope of the first pack lot is 1.00, the second could
be as low as 0.90 or as high as 1.10. The third pack
lot received should also be verified using the pre-
liminary bottle values estimated with the first pack
lot. The slope of the third pack lot must be both
within 10% of that of the first pack lot and within
10% of that of the second.
For example, if the first slope is 1.00 and the sec-
ond is 1.06, then the third must be at least 0.96
(within 10% of the highest slope, 1.06) and at most
1.10 (within 10% of the lowest slope, 1.00). Each
of the subsequent pack lots must be within 10%
of all the previous lots.
4-1
-------
The preliminary bottle values can be corrected
once the slopes of at least five pack lots have been
determined. The preliminary value should be
multiplied by the center of the range of observed
slopes, i.e., the average of the highest and lowest
slopes, to obtain the corrected bottle value. Future
verifications using the new values should result in
slopes between 0.95 and 1.05.
Table 4-1 provides a summary of the procedure for
determining bottle values.
When a new lot of control product or a new serum
pool is used, a new "corrected" bottle value may
be assigned by a crossover study using 20 packs
from one pack lot and the following equation:
new bottle value - 10P3ck for "ew material x oldbotllevalue
10 pack mean for old material
Instrument bias between aca's must be considered
if a laboratory chooses to use a commercial con-
trol material having a bottle value that was not
assigned using their aca. Slope differences as
great as 10% can be obtained when two instruments
are directly compared. For example, if the same
control product was assayed with one pack lot on
two different, properly functioning aca 's, it is
possible that the slope on one instrument could be
0.95 and on the other, 1.05. If that control product
was assayed on the first instrument with several
pack lots, the range of slopes might be 0.90 to 1.00;
with the same pack lots the range of slopes on the
second instrument would be 1.00 to 1.10.
4-4. Quality Control Program.
Du Pont recommends that a sample of known
activity or concentration (control) be analyzed at
least once daily on the aca after acceptable
performance has been demonstrated by the general
instrument checkout procedures, i.e., filter balance
and an ABS check. A control analysis should be
run for each method to be used during the day.
The results of the control analysis should be im-
mediately recorded and if the results are outside
acceptable limits, the cause of the out-of-control
situation should be found and corrected before any
more patient data is reported.
The control sample should be a material other than
~hat used to calibrate (verify if an enzyme method)
the aca. If the control and calibration samples
were identical, this could result in undetected
error. If lyophilized quality control samples are
used, the reconstitution technique recommended
by the manufacturer's insert sheet or accompany-
ing literature should be carefully followed. A fresh
vial of lyophilized control should be used each day.
Hydration times should be kept as constant as
possible, since the activities of some constituents
change as the hydrated sample ages.
If only one control is used, a concentration or
activity at the clinical decision making level, usually
the upper limit of the normal range, is recom-
mended. If a second concentration of activity is
desired, a level near the upper limit of the range of
linearity stated in the aca Test Methodology
literature is recommended.
4-5. PREPARING A QUALITY CONTROL
CHART.
In the following procedure it is assumed that the
method has been properly set up as described
previously in this section. The suggested technique
of data handling and presentation is only one of
several that may be used.
The quality control «mple should be treated as a
patient's serum oi. the aca. The tests should be
repeated for 20 to 30 days with the same lot of test
packs and control product. The individual deter-
minations are used to-calculate the mean (x) value
for the control, and the day-to-day standard devi-
ation (SD) is then calculated using the following
equation:
/ 2 (x'- x)J
SD = i
"V N- 1
With some electronic calculators the following
equivalent formula for SD is easier to use:
VZ (xJ) -NxJ
:
N- 1
Where:
x^= Individual determinations
x = the mean of the individual determinations
N ¦ Number of determinations
4-2
-------
66
quality assurance
aca
Table 4-1. Bottle Value Determination.
Procedure Example
1.
Estimate bottle values for verification
materials with the initial pack lot. The
slope of this pack is, by definition, 1.00.
1.
Estimated bottle values are V! = 138 and
V2 = 26
m = 1.00 (m = slope)
2.
When a new pack lot arrives, assay the
verification materials with the new packs.
Plot aca printout (y axis) vs estimated
values from step 1 (x axis).
2.
The slope obtained is 1.06.
Slope Specifications
minimum acceptable slope =
highest previous m - 0.10
maximum acceptable slope =
lowest previous m +0.10
previous m = 1.00
min. slope = 1.00 -0.10 = 0.90
max. slope = 1.00 + 0.10 = 1.10
1.06 is acceptable because it is within
the range of 0.90 to 1.10
3.
When another pack lot arrives, repeat step 2.
3.
The slope obtained is 0.98
Slope Specifications
Same as in step 2.
highest previous m = 1.06
lowest previous m = 1.00
min. slope = 1.06 - 0.1 = 0.96
max. slope = 1.00 + 0.1 = 1.10
0.98 is acceptable because it is within
the range of 0.96 to 1.10
4.
Repeat step 2 with each new pack lot,
always plotting aca printout vs bottle
values estimated in step 1. After at
least five different pack lots have been
used, proceed to step 5.
4.
The slopes obtained are between the previous
high slope 1.06 and the previous low slope
0.98 and are, therefore, acceptable.
5.
Calculate the correct bottle values by
multiplying the estimated values by the
average of the highest acceptable slope
and the lowest acceptable slope.
5.
highest m = 1.06 -61
lowest m = 0.98
1.06 + 0.98 _ -j ^
2
138 x 1.02 = 140.8
26 x 1.02 = 26.5
6.
When new pack lots arrive, plot aca printout
vs the correct bottle values. The slopes are
acceptable.if they fall within the range of 0.95
to 1.05
•¦I/.
4-3
-------
67
aca
SU'ONr turowariC
quality assurance
LLi
D
—J
<
>
_i
O
cc
H
Z
O
u
116
113
110
107
104
x = 110 mg/dŁ
SD = 3 mg/dŁ
PRIMED
BACKFLUSH
DILUENT
#1 CHANGED
l I
NEW
PACK LOT
(C4196A)
I I i
+2SD
+1SD
¦1SD
-2SD
i I
i
i
i r i I i i i i f i I I I I i r i i i i
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
DAYS
J/79A
703C/70
Figure 4-1. Example of Glucose Quality Control Chart.
A quality control chart of the type described by
Barnett1 and shown in figure 4-1 can then be pre-
pared. Horizontal lines representing x and action
limits (frequently ±2 SD) are drawn. The horizontal
axis is graduated to represent days of the month.
When a quality control test result falls farther away
from the mean than the action limits, determination
should be made whether the outlying point was
caused by a systematic problem or was the result
of normal statistical fluctuation. The trouble-
shooting charts in Section 5 will help. Each user
should decide on action limits appropriate for a
particular test. These could differ based on the
level of confidence in control desired by the user.
Typical action limits are ±2 SD. Limits of ±1 SD
1 Barnett, R. M., M. D., "Clinical Laboratory Statistics."
Little, Brown and Co., Boston, (1971), p. 77.
might be appropriate where small systematic
changes have a significant impact.
4-6. INITIATING THE PROGRAM.
When the laboratory initiates a quality control
program with the aca, control must be assured
during the initial 30-day period over which aver-
age values and typical SD are being determined.
Tentative x may be obtained by a one-time
analysis of a five-vial pool of the control product.
The average of a five-pack determination on this
pool should give a reasonable estimate of the
average (bottle) value to be expected. At this time
the within-run precision should be assured as
acceptable (consult the Test Methodology literature
for typical performance characteristics). Tentative
4-4
-------
action limits may be obtained from any of the
following criteria:
1. Past performances of an alternate
methodology.
2. Tonk's Allowable Limits of Error2 —
Generally given as
+ % normal range spread ^qq
Mean of normal range
3. State of the art CV3
4. Specific performance characteristics given
in the aca test methodology literature.
Typical performance characteristics are obtained
from one laboratory with a given control product
and test pack lot. Day-to-day precision in the user's
laboratory should approximate that stated in
the Test Methodology literature. If day-to-day pre-
cision established during the 30-day trial period
period is unacceptable or if one test result is out-
side ±3 standard deviation, consult troubleshooting
charts 5-1 or 5-2 in Section 5.
4-7. THE PROGRAM IN OPERATION,
The quality control chart shown in figure 4-1 is
used to assess the performance of the method
during any current month. Thus present perfor-
mance is judged relative to past performance.
Changes in the system that may affect daily per-
formance are noted on the chart (the value of
accurate record keeping is evident). At the end of
the month, the x and SD are recalculated to
accommodate changes in the control lot or lot-to-
1 Tonks, D., Clin. Chem., 9, 217 (1963).
3 Barnett, R. M., Amer. J. Clin. Path., 50, 671 (1968).
lot variability in the test packs. A cumulative SD
(over several months) can be used as a more refined
measure of day-to-day precision to be expected
from the system.
If quality control results are normally distributed,
95% of the data points (19 out of 20) should fall
within ±2 SD of the mean. If more than one point
within 10 determinations falls outside ±2 SD limits,
consult troubleshooting Charts 5-1 or 5-2 in
Section 5. Note that 99.7% of the data points
(299 out of 300) should fall within ±3 SD of the
mean. Thus, only one determination outside these
limits indicates the need to immediately consult
the troubleshooting charts.
Du Pont strongly recommends that the laboratory
never completely run out of pack lots, buffers, or
control products that have performed satisfactorily
in the past, e.g., try not to run completely out of
the pack lot presently in use before a new pack
lot arrives and is found to be acceptable. New
pack lots should be calibrated or verified when
they arrive in a laboratory and when they can be
directly compared to a satisfactory lot. For non-
enzyme methods, the new Ci and Co may be cal-
culated and noted in the chemistry log sheets or
printed out on Dylux paper. When the old lot is ex-
hausted, the new Ci and Co can be entered in the
aca, and they can then be verified.
When a new quality control product or a new lot
of control product is introduced, its performance
should be evaluated in a 30-day trial while the
system is known to be in control. The new x and
SD can be established and compared directly to
the values of the old material during this trial.
4-5/4-6
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69
ACA QUALITY CONTROL FORMAT
Quality control materials: Fisher Diagnostics Urichem Human Urine
(Lyophilized) Chemistry Control, Levels I and II, 25 mL/bottle.
Name Pack Code Sample Size. uL
1. Urinary Protein (UP) 800 uL#
2. Urinary Creatinine (CREA) 200 uL##
Total Urine Required = 1,000 uL, or 1 mL
# a 2-pack method, reflects 2- to 400-uL samples
## urine is diluted 1:20 for analysis
A. PRESTUDY PROTOCOL: To characterize instrument after calibration verified
by manufacturer.
The protocol for urinary protein and creatinine is essentially the same as
that for the serum analytes. Urinary protein, however, is a 2-pack
method; pack #1 is a sample blank and pack //2 is the test pack. Only
Level II of the Urichem controls has urinary protein; a 1:4 dilution with
deionized water will provide a second level assessment in the normal
range. Urinary creatinines must be diluted 1:20 with 3 g/dL albumin to be
assayed on the ACA. (Calibration is based on serum creatinine and verified
with urinary creatinine pools. J'
Goal: Ten running days, 2 runs per day (AM and PM)
2 pools per run (Levels I and II for
creatinine, Level II undiluted, and
diluted 1:4 for urinary protein)
1 sample per pool, 2 packs per analyte
The Urichem controls are lyophilized in 25-mL quantities. One bottle of
each level may be rehydrated with deionized water and dispensed in smaller
aliquots, which are then frozen and used as required.
Level I: Rehydrate with 25 ml deionized water and mix well. Prepare 1:20
dilutions for CREA assay by diluting 50-uL aliquots of Level I with
950 uL of 3 g/dL human albumin. Mix well, freeze aliquots at -20°C.
Level II: Rehydrate with 25 ml deionized water. Prepare 10 1:20
dilutions for CREA assay as for Level I. Prepare 10 1,900-uL
aliquots for UP (undiluted) assay. Dispense 10 500-uL aliquots and
to each aliquot add 1,500 uL deionized water. Mix well. Freeze all
aliquots at -20°C.
For each run, thaw one aliquot of each level or dilution. Mix contents of
vials, fill cups, and arrange packs as follows:
Level 1 - 1:20 dilution
Cup 1-2 packs CREA = 400-uL sample
-------
Level 2 - 1:20 dilution
Cup 1-2 packs CREA = 400-uL sample
Level 2 - 1:4 dilution
Cup 1-2 pack-sets UP = 1,600-uL sample
Level 2 - undiluted
Cup 1-2 pack-sets UP = 1,600-uL sample
Total packs consumed = 80 packs for creatinine and 80 pack-sets for urinary
protein
Quality control materials required = 20 aliquots or dilutions per pool per
analyte
B. WITHIN—STUDY PROTOCOL:
Pack lot variation will be minimized by having only one lot of packs per
analyte, if possible, and one lot of each quality control pool.
1. Based on a maximum of 27 21-analyte profiles in 1 day, 4 sets of
single packs for the normal level pool will be used:
a. at beginning of day
b. after 7-8 profiles C21 packs/profile)
c. after 7-8 more profiles
d. at end of day
1 vial of Level I will be prepared for each set.
2. CAP survey materials will be analyzed at least once during any study,
possibly more often, if the surveys have been received during the
course of a study.
Urine Chemistry - Survey U, Series 1
CREA, UP - 2 packs or pack-sets per
analyte per specimen
1. Specimen 1
2. Specimen 2
3. Blind duplicates
Blind duplicate samples will be collected in the field and presented
in runs by the Special Activities group - two per day will be run.
These samples may also consist of external control materials or pools
for each analyte.
-------
4. Replicate samples
Two replicates of samples scheduled for a day will be randomly
repeated within that same day, if sample size permits.
BETWEEN-STUDIES PROTOCOL
Single packs will be analyzed for all analytes, using Levels I and II
pools of Urichem.
Urichem, Level 1 - Cup 1 - 1:20 dilution, CREA
Level 2 - Cup 1 - 1:20 dilution, CREA
Cup 2-1:4 dilution, UP
Cup 3 - Undiluted, UP
If any analyte is out of control, analysis will be repeated to verify and
check calibration.
-------
HAIR ARSENIC/CADMIUM/LEAD
METHOD FOR SCREENING FOR SELECTED INORGANIC ELEMENTS IN ACID DIGESTS OF HUMAN
HAIR BY INDUCTIVELY COUPLED ARGON PLASMA EMISSION SPECTROSCOPY
1.0.00 PRINCIPLE
Inorganic elements that may be found in human hair are derived from one or
more of the following sources: (a) deposition during the synthesis of hair in
the follicles; (b) external contamination of the hair from sweat; (c) external
contamination from water and/or hair treatments (e.g., shampoos, hair condi-
tioners, "permanent wave" solutions, tints and dyes); (d) external contamina-
tion from particulates or aerosols suspended in the atmosphere, it is hypothe-
sized that a mild washing procedure will remove inorganic elements that are
due to external deposition but will not damage the hair matrix to such an
extent that the inorganic elements incorporated in the hair as it is growing
are removed by the washing. The hypothesis is that such inorganic elements as
are found in such a washed hair sample are in some way related to the chronic
exposure or "body burden" of those elements.
Before analysis, the hair matrix is destroyed by digesting the sample in
nitric acid and hydrogen peroxide.This frees the elements from the complex
organic hair matrix. The elements in the acid digest are then identified by
using inductively coupled argon plasma emission spectroscopy. The sample to
be analyzed is aspirated into the plasma, where the extreme temperatures of
the plasma excite the atoms. As these excited atoms return to a lower energy
level, they emit energy at characteristic spectral wavelengths that may be
detected by the sophisticated optical bench within the instrument. By
comparing the intensity of emission in a sample with the intensity of known
standards, the concentration of a particular element in a sample can be
estimated.
2.0.00 SAMPLE COLLECTION AND PREPARATION
2.1.00 EQUIPMENT. REAGENTS. AND SUPPLIES
The following items are needed for hair collection:
a) plastic zip-lock bags with labels affixed for storing
the collected samples
b) several pairs of high-quality stainless steel surgical
scissors or plastic scissors for cutting the hair
c) several plastic combs
d) several aluminum or plastic hair clips
e) 70* isopropyl alcohol (and appropriate containers) for
cleaning scissors, clips, and combs
-------
73
2.1.00 continued
f) surgical gloves to fit the person(s) collecting samples
g) information questionnaires to be filled out by those donating
samples
The following are needed for hair washing, drying, weighing, and digestion:
a) facilities with Class 100 air supply
b) analytical balance capable of weighing accurately to a tenth of a
milligram (0.0001 gram)
c) aluminum digestion block shaped and drilled to hold Teflon diges-
tion tubes
d) hot plate to maintain the aluminum digestion block at 80-100°C
e) 15-mL round-bottom Teflon tubes with screw-caps, calibrated for
10-mL volume
f) Gppendorf pipets and pipet tips
g) high-quality stainless steel surgical scissors or plastic scissors
h) surgical gloves
i) 15-mL conical-bottom plastic disposable capped centrifuge tubes
(Falcon #2095)
j) disposable 15- x 100 mm plastic petri dishes (Falcon #1010)
k) miscellaneous Teflon bottles and beakers
1) nitric acid, G. Frederick Smith, double-distilled from Vycor
m) hydrogen peroxide, Mallinckrodt, 30%, analytical reagent grade
n) sodium lauryl sulfate (1.0 g/100 mL) or ammonium lauryl sulfate
(1.0 mL Agree shampoo/100 mL) in deionized water
o) deionized water and Milli-Q deionized water
-------
74
2.2.00 CLEANING OF LABORATORY SUPPLIES
The disposable plastic "labware" obtained from Falcon Plastics can usually
be used as obtained. The Division of Environmental Hazards and Health
Effects screens each new lot of such labware for inorganic contamination
prior to use. All other plastics, glassware, and Teflon labware must be
washed before being used.
The washing procedure involves two stages. The first stage is a detergent
wash to remove much of the inorganic contamination as well as any organic
residues. The second stage is an acid wash to remove the last traces of
inorganic contamination.
The wash procedure is as follows. Labware to be cleaned is soaked for
3-24 hours in a 2% solution of Isoclean Decontamination Solution (Isolab,
Inc.). After this detergent soak, the labware is rinsed thoroughly with
deionized water and then soaked overnight (12-20 hours) in 30%
hydrochloric or nitric acid (J.T. Baker analyzed reagent grade acid).
After this acid soak, the labware is thoroughly rinsed (minimum of six
rinses) with deionized water (Milli-Q reagent grade water) and placed in a
clean work station providing Class 100 air for drying. The clean
glassware is stored in the Class 100 work station or is packaged in
plastic bags to prevent contamination before being used.
2.3.00 SAMPLE COLLECTION AND STORAGE
The individual collecting hair specimens should wear surgical gloves for
his/her own personal hygienic protection and to avoid contaminating the
hair samples with sweat from his/her hands. The gloved hands should be
dipped in 70% isopropyl alcohol and dried between collections from
different donors to prevent the transfer of scalp infection from one donor
to the next. Likewise, combs, scissors, and hair clips should be soaked
in 70% isopropyl alcohol between donors. The availability of several sets
of combs, scissors, and hair clips facilitates the soaking, drying, and
using cycles for these items.
Hair is cut from the occipital region of the scalp, between the top of the
ears and the nape of the neck. Only the two inches of hair closest to the
scalp, representing 2-6 months' recent growth, is used. The hair is cut
so that the cuts show as little as possible.
Fasten hair that is not being cut out of the way with the aluminum (or
plastic) hair clips. Cut 8-20 strands of hair from 10-20 different sites
in the occipital region of the scalp with stainless steel surgical
scissors (or plastic scissors, if available). Save only the two inches of
hair growing next to the scalp. About 0.5 gram of hair is required for
duplicate analyses of a hair sample, but a 1-gram sample is preferred to
permit reanalysis of samples that have unusually high or low values for an
element of interest.
-------
2.3.00 continued
The collected hair is placed in a clean Zip-Lock plastic bag, and the
donor's name and identification number are written on the label of the
bag. The questionnaire completed by the donor (also showing the donor's
name and identification number) is stapled to the sample bag.
When the samples and questionnaires are received in the laboratory, they
are stored in a clean, dust-free environment at room temperature until
they are prepared for analysis.
2.4.00 SAMPLE WASHING
The hair sample is transferred from the collection/storage bag to a 15- x
100-mm disposable plastic petri dish and covered with a 1.0% solution of
either sodium lauryl sulfate or ammonium lauryl sulfate in deionized water
(approximately 25 mL of detergent solution is required). The petri dishes
are occasionally agitated (gently) to insure thorough contact of all the
sample with the detergent solution. After 30 min, the detergent is poured
off and the sample is rinsed (6 times) with deionized water (Milli-Q
reagent grade). The samples are then left to dry in the uncovered petri
dishes in a clean work station (Class 100 air).
2.5.00 SAMPLE WEIGHING
A 200-mg (approximate) portion of each washed, dried hair sample is
accurately weighed (to the nearest tenth of a milligram) into a screw-cap
Teflon digestion tube. Duplicate portions are taken from each sample.
The tubes are capped after receiving the sample.
2.6.00 SAMPLE DIGESTION
The capped Teflon tubes containing the weighed hair sample are transferred
to a digestion hood designed to provide a down-sweep of Class 100 air. A
1.0-mL aliquot of concentrated nitric acid (G.F. Smith double-distilled)
is added to each tube with an Eppendorf pipet. The tubes are capped and
the digestion is continued at room temperature until the hair is in
solution (about 2 hours). The tubes are then transferred to an aluminum
digestion block, placed in a digestion hood, and heated overnight (16-20
hours) at 80-110 C.
After the acid digestion, the tubes are chilled in an ice bath and
carefully uncapped (with gloved hands to avoid bums from acid vapors
escaping under pressure). A 0.5-mL aliquot of 30% hydrogen peroxide is
added to each tube with an Eppendorf pipet, and the tubes are capped and
returned to the digestion block for 1.5-2.0 hours at 110°C. The tubes
are then cooled to room temperature, and the volume of the samples is
brought to the 10-mL calibration mark with deionized water (Milli-Q
reagent grade). The samples are then poured into 15-mL disposable
polystyrene centrifuge tubes (Falcon #2095) and capped tightly until
analyzed.
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76
2.6.01 NOTES ON SAMPLE DIGESTION
About 15 hair samples can be accurately weighed in duplicate and
prepared for digestion in the early afternoon. The hair must have
time to digest at room temperature before being heated to prevent a
violent reaction. If this step is omitted, some samples react so
vigorously that they boil out of the closed tubes at 80°C. By
starting the digestion at room temperature and waiting until all the
hair is in solution, we have never lost a sample because of boil-over.
The acid digest is yellow-green and clear (no particulate matter).
The nitric acid oxidizes the protein, and the peroxide oxidizes the
lipid material. This is not a complete digestion to carbon dioxide
and water.
All manipulations of samples (except weighing) should be done in a
hood or clean work station (Class 100 air) to minimize contamination.
At least four blank digestion tubes are prepared (containing no hair)
for each digestion operation. A minimum of two samples of a solid
hair quality control sample (described in a later section) are
included in each digestion operation.
3.0.00 ANALYSIS BY ICAP EMISSION SPECTROSCOPY
Digested hair samples are analyzed by using a Jarrel-Ash Plasma AtomComp 1160
spectrometer connected to a Digital Equipment Corporation PDP 11/34 computer.
The Jarrell-Ash fixed cross-flow nebulizer is connected to a Gilson Minipuls 2
peristaltic pump to assist in sample aspiration and nebulization. The
principle of the method is presented in Section 1.0.00.
3.1.00 EQUIPMENT. REAGENTS.AND SUPPLIES
The following are needed for the analytical procedure:
a) Jarrell-Ash Plasma AtomComp 1160 with PDP 11/34 computer
b) Gilson Minipuls 2 peristaltic pump
c) Argon, high purity
d) J.T Baker Instra-Analyzed Atomic Spectral Standards (guaranteed
1000 + 10 ppm from 99.99% pure metals or salts) for the following
elements: Al, As, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb,
Sb, Sn, Se, Sr, Ti, V, Zn
-------
77
3.1.00 continued
e) Aldrich atomic absorption standard solutions (1,000 ppm) for the
following elements: Au, Tl, P, Y
f) National Bureau of Standards' Standard Reference Materials for the
following: calcium carbonate (1.2488 g/500 mL); lithium carobonate
(2.6624 g/500 mL); sodium chloride (1.2713 g/500 mL); potassium
chloride (0.9535 g/500 mL)
g) J.T. Baker Ultrex hydrochloric acid
h) G. Frederick Smith nitric acid, double-distilled from Vycor
i) J.T. Baker Ultrex acetic acid
3.2.00 CLEANING OF LABORATORY EQUIPMENT AND SUPPLIES
The procedure for the cleaning of laboratory supplies is the same as that
described in Section 2.2.00.
The nebulizer/torch assembly from the ICAP is cleaned weekly under normal
operating conditions used for hair analysis. If the instrument has been
used for the analysis of trace elements in other matrices, then the
cleaning protocol described for those matrices is used. The standard
cleaning procedure for the nebulizer/torch assembly is as follows. The
assembly is removed from the instrument, disassembled, and soaked in a
warm solution (2%) of Isoclean Decontamination solution for 3-24 hours.
This is followed by 6 rinses in Milli-Q reagent grade water and gently
shaken to remove excess water. After the instrument has been assembled
and installed, the argon used for the instrument is allowed to flow
through the torch for several minutes to accomplish final drying. Too
much moisture in the torch will prevent plasma from forming.
3.3.00 INSTRUMENT CALIBRATION AND SAMPLE ANALYSIS
The instrument is calibrated by using five mixtures of standards. These
calibrator solutions are designated UAT1, WAT2, UAT3, WAT4, and V1AT5.
They are prepared from the stock standards (1,000 ppm) of each individual
element. These solutions are prepared as follows:
WAT1; 15 mL G.F. Smith nitric acid diluted to 500 mL with Milli-Q reagent
grade water. This is the blank calibrator solution.
WAT2: 15 mL G.F. Smith nitric acid and 5.0 mL of the individual stock
standards (1000 ppm) for the following elements: Ca, Cd, Co, Cu, Mg, Mn,
Pb, Zn. The volume is brought to 500 mL with Milli-Q reagent grade water.
-------
78
3.3.00 continued
WAT3; 15 mL G.F. Smith nitric acid and 5.0 mL of the individual stock
standards (1,000 ppm) for the following elements: Al, Ba, Be, Fe, Li, Mo,
Na, Ni, Sb, Sn, Sr, Ti, Tl. The volume is brought to 500 mL with Milli-Q
reagent grade water.
WAT4: 15 mL G.F. Smith nitric acid and 5.0 mL of the individual stock
standards (1,000 ppm) for the following elements: As, B, Cr, P, Se. The
volume is brought to 500 mL with Milli-Q reagent grade water.
WAT5: 15 mL G.F. Smith nitric acid and 5.0 mL of the individual stock
standards (1,000 ppm) for the following elements: Au, V, Y. To this is
added 50 mL of the potassium stock standard (1,000 ppm). The volume is
brought to 500 mL with Milli-Q reagent grade water.
The calibrator solutions contain 10 ppm of each element except for
potassium, which is at 100 ppm.
The calibrator solutions prepared as described above are used with ACT =
CDCS (analytical control table CDCS), which uses the spectrum shifter for
background correction. When ACT = CDC1 is used, 7.5 mL of Ultrex acetic
acid is added to each calibrator solution before the final volume is
brought to 500 mL. The acetic acid in these is added to correct for the
nonspecific carbon interference in the digested hair samples. The
spectrum shifter is not used in ACT = CDC1.
ACT = CDCS provides the following parameters:
two burns per sample
five seconds per burn
spectrum shifter at 11
ACT = CDC1 provides the following parameters:
three burns per sample
ten seconds per burn
spectrum shifter at zero
acetic acid in calibrator solutions
The ICAP is calibrated in the intensity mode by using the appropriate ACT
and the appropriate calibrator solutions. After being calibrated, the
instrument is set to the concentration mode. The calibrator solutions are
then read in the concentration mode as a check of the calibration. The
pooled digested hair quality control sample (described in a later section)
is analyzed as a check for between-day precision for each element.
-------
79
3.3.00 continued
A standard solution containing 2 ppm of each element is analyzed
periodically to monitor calibration. The system is washed between samples
and standards to prevent cross-contamination. Analytical results for
standards, blanks, digestion blanks, unknown samples, pooled hair digest
quality control samples, and other quality control samples are printed out
in the concentration mode as "ppm." Values recorded for duplicates are
averaged to calculate a final value for a given sample.
3.A.00 QUALITY CONTROL
Since this method is considered an experimental method, still under
development and evaluation, no formal quality control protocol has been
established. At present, two quality control samples are used: a sample
of hair obtained from one individual and a pool of excess hair digests.
The pooled hair digest sample is used to monitor within-day and
between-day precision. The solid hair sample has been rendered as
homogeneous as we are able to make it, and it is used to monitor the
overall reproducibility of the method, from weighing through analysis (the
washing step is not included). As our experience and resources permit,
additional quality control materials and procedures will be developed.
3.5.00 REPORTING OF ANALYTICAL RESULTS
This method is considered an experimental method, still under development
and evaluation.
-------
Table 1
ANALYTICAL GOALS SUMMARY
ANALYTE
QUALITY CONTROL
MATERIALS
MATRIX
LINEARITY
ESTIMATED
PRECISION
DETECTION
LIMIT
SAMPLE
THROUGHPUT
Whole Blood
Lead
Erythrocyte
Protoporphyrin
Whole Blood
Cadmium
Urinary
Arsenic
Urinary
Cadmium
Urinary
Lead
.Urine
B2-Microglobulin
Urinary
Creatinine
Urinary
Protein
1. Spiked Whole Blood
2. Internal Controls
1. Standards (ProtoIX)
2. Internal Controls
1. Spiked Whole Blood
2. Internal Controls
1. Spiked Urine
2. Internal Controls
1. Calibrators
2. Internal Controls
1. Calibrators
2. Internal Controls
1. Calibrators
2. Internal Controls
1. Calibrators
2. Internal Controls
1. Calibrators
2. Internal Controls
Bovine Blood 0 -
Bovine Blood
Aqueous 0 -
Human/Bovine
Human Blood 0.5
Bovine Blood
Human Urine 0 -
Commercial
Human Urine 0 -
Commercial
Human Urine 0 -
Commercial
Human Urine 1 -
Human Urine
Bovine Alb Soln 0 •
Commercial
Bovine Alb Soln 60
Commercial
100 ug/dL
100 ug/dL
-10 ng/mL
250 ng/mL
10 ng/mL
120 ng/mL
500 ug/mL
20 mg/mL
-2400 mg/L
10% CV
@ 15 ug/dL
7% CV
@28.5 ug/dL
10% CV
@ 6.9 ng/mL
5% CV
@ 245 ng/mL
10% CV
@ 4.6 ng/mL
9% Cv
@ 109 ng/mL
14% CV
0 40.0 ug/L
4% CV
@1.40 mg/dL
3% CV
@ 130 mg/L
2.0 ug/dL 40 samples/day
1 ug/dL 100 samples/day
0.25 ng/mL 40 samples/day
4.0 ng/mL 20 samples/day
0.1 ng/mL 20 samples/day
3.0 ng/mL 20 samples/day
1 ug/mL 40 samples/day
0.15 mg/dL 80 samples/hour
60 mg/L 80 samples/hour
Hair Not Available
Arsenic/Cadmium/Lead
Under Under
Development Development
-------
81
METHOD MODIFICATIONS
All analytical methods were as outlined in the preceding sections of this
appendix, with the following modifications.
URINARY LEAD - A 1.6% weight by volume solution of ammonium phosphate in
4% by volume nitric acid was used in place of a 1% solution of ammonium
phosphate in nitric acid as a matrix modifier.
URINARY BETA-2-MICR0GL0BULIN -.10 mg of sodium carbonate was added to each
10 ml of urine specimen. This replaced the use of 1.0 m NaOH to adjust the pH
to between 6 and 8 before storage.
HAIR CADMIUM AND LEAD - Because of the limited sample size, the amount ot
sample weighed and digested was reduced by 50%, with a corresponding reduction
in the volumes of the digestion reagents (nitric acid, hydrogen peroxide, and
deionized water). The resulting decrease in the volume of digest necessitated
a change in the analytical sampling scheme (a 2-bum sequence using ACT CDC 1
replaced the normal 3-burn sequence). Samples were washed in either a 1% SDS
solution or in 95% ethanol (statistical analysis has shown that there is no
significant difference between these two methods of washing in our laboratory).
The concentrations of several elements (e.g., Fe, Cr, Cu, Ni) in the SDS-wash
of sample 312-1052 suggest a possible "steel" contamination. Although the Pb
and Cd results do not seem to be affected, contamination may have occurred
during the collection, preparation, and/or analysis of this specimen.
Lead values below 5.0 ug/g of hair are less than five times the detection
limit of the method and are reported for comparative purposes only. All
cadmium values are above five times the detection limit for cadmium.
HAIR ARSENIC - The modifications of the washing and digest method were the
same as above for lead and cadmium. No ICAP values for arsenic are reported
because all were below the detection limit of the method. The acid digests of
the SDS-washed hair specimens were analyzed by using graphite furnace atomic
absorption with L'vov platform and matrix modifier. The conditions chosen
were similar to those described by W. Slavin et al., Atomic Spectroscopy, 4:
69-86, 1983, and F.J. Fernandez and R. Giddings, Atomic Spectroscopy, 3:
61-65, 1982, for arsenic in bioglogical materials. Calibration was done by
adding inorganic arsenic standards to a pooled hair digest. Instrumental
conditions were:
DRY 200 C 5 sec
CHAR 800 C 5 sec
ATOMIZE 2400 C 1 sec
COOL 20 C 1 sec
RAMP 30 sec
RAMP 20 sec
RAMP 5 sec
RAMP 10 sec
HOLD
HOLD
HOLD; 0 flow
HOLD
-------
82
An arsenic EDL was used as a resonance Line source; conventional (deuterium)
background correction was used. The applicability of conventional background
correction was tested by absorbance measurements of the unspiked pool digest,
with and without deuterium correction. The measured absorbance difference for
this pool was about 0.03 AU, well within the correction capability of this
system (maximum about 0.20 AU). The specimen digest were diluted 1:1 with a
matrix modifier (1,000 ppm in nickel—as nitrate—and 4% v/v in nitric axid).
The method of average slopes was used to calculate results, with specimens
bracketed between two hair digest standard addition curves to compensate for
any sensitivity change or instrument drift with time.
-------
83
QUALITY CONTROL DATA FOR
ANALYTICAL RUNS
Twenty-five blood specimens collected from children in East Helena, Montana
were anaylzed for blood lead. The specimens were processed in two analytical
runs. Values reported were means of duplicate determinations. One aliquot of
each of three bench control pools was included in each run. Additional
aliquots of one of these pools were included as blind controls, four in the
first run and two in the second run. All control means were within the 95%
limits shown below except the mean for the 83-33 pool in the second run, which
was within the 99% limits. Units below for blood lead are ug/dl.
QC Mean Limits
Pool UCL99 UCL95
83-33 7.03 6.51
DE8A03 17.09 16.15
DE8C01 37.74 36.53
blind 7.03 6.51
Mean LCL95 LCL99
4.87 3.23 2.71
13.19 10.22 9.28
32.70 28.88 27.67
4.87 3.23 2.71
Fifty-two blood specimens were analyzed for cadmium in six analytical runs.
All values reported were means of duplicate determinations. Included in each
run were two aliquots of one bench control pool and one aliquot of another
bench control pool. All means were within the 95% limits given below (blood
cadium units are ng/ral):
QC Mean Limits
Pool UCL99 UCL95 Mean LCL95 LCL99
High 8.95 8.58 7.43 6.28 5.92
PB83 0.797 0.674 0.286 -0.102 -0.225
Twenty-five blood specimens from children were analyzed for erythrocyte
protoporphyrin (EP) in one analytical run. Four EP determinations were also
made on each of four bench control pools. All means were within the 95%
limits given below. Three of the ranges were within the 95% limits; the
fourth was outside the 99% limits. Uith eight QC parameters, the probability
of at least one being outside the 99% limits by chance is about 8%, which is
larger than the 2% probability level used within the Division of Environmental
Health Laboratory Sciences to warrant rejection of an analytical run. Six
samples from an uncharacterized blind control pool were also included in the
anaylytical run. These had a CV of 6.86%. Unints for EP in the table below
are ug/dl.
QC Mean Limits Range of
Quadruplicate
Values Within Run
Pool
UCL99
UCL95
mean
LCL95
LCL99
UCL99
UCL95
2681
55.5
54.6
52.7
49.0
48.1
6.80
5.61
2781
129.6
126.7
117.7
108.7
105.8
17.46
14.41
2881
182.3
178.9
168.4
157.9
154.6
20.30
16.75
1882
38.7
37.9
35.5
33.1
32.4
5.27
4.34
-------
84
Ninety urine specimens were anaylzed for arsenic in 11 analytical nans. All
values were means of duplicate determinations. One aliquot from each of two
bench contol pools included in each run. .In addition, one to three aliquots
of one of these pools were included in the first four runs as blind controls.
All means were within the 95% control limits shown below, except the 476-1
pool mean in the fourth run, and one of the three blind control means (476-1
pool) for the third run both of which were within the 99% control limits. All
units for urinary arsenic below are ng/ml.
QC Mean Limits
Pool UCL99 UCL95 Mean LCL95 LCL99
base 12.98 10.86 4.16 0 0
476-1* 38.79 35.26 24.13 12.99 9.47
*Bench and blind control
Seven pairs of blind split duplicates were also included in the analytical
runs. The table below shows the results of measurements on the duplicate
aliquots. Units for urinary arsenic are ng/ml.
Pair Run Number(s) Concentration 1 Concentration 2
1
7.
10
22.5
28.3
2
5,
3
4.0
16.1
3
3,
4
4.0
. 4.0
4
3,
3
4.0
4.0
5
1,
1
7.1
32.7
6
1,
2
4.0
4.0
7
2,
2
10.2
19.3
In four of these split duplicate pairs, the two values varied considerably.
In each case, the value for the duplicate specimen (concentration 2) was
higher than for the original specimen from which it was aliquoted
(concentration 1). Thus, some contamination in the aliquoting process may
account for the discrepancies. Similarly, values approaching the detection
limit of the method (i.e., 4 ug/mL) are subject to a great deal of analytical
variability, as reflected in the base pool control.
Forty urine specimens were analyzed for lead in two anayltical runs. All
values were means of duplicate determinations. One aliquot of each two bench
control pools was placed at the beginning and at the end of each run. In the
first run, one aliquot and, in the second run two aliquots of one of these
pools were also included as blind controls. All means were within the 99%
control limits given below, except the means for the base urine pool at the
beginning of the first run and for the base pool at the beginning and end of
the second run, which were within the 99% control limits. Units below for
urinary lead are ng/ml.
QC Mean Limits
Pool UCL99 UCL95 Mean LCL95 LCL99
base 7.71 7.11 5.20 3.29 2.69
476-1* 42.42 39.33 29.58 19.83 16.74
*Bench and blind control
-------
85
Three pairs of "blind" split duplicates were also included in the analytical
runs. The table below shows the results of the determinations on the
duplicate aliquots. Units for urinary lead are ng/ml.
Pair Run Number(s) Concentration 1 Concentration 2
1 1,1 2.8 3.3
2 1, 2 2.8 2.8
3 2, 1 3.1 2.8
Fifty urine specimens were analyzed for cadmium in two analytical runs. All
values were means of duplicate determinations. One aliquot from each of two
bench control pools was included at the beginning and the end of each run. In
addition, two aliquots from one of these pools were also included in each run
as blind controls. All means were within the 95% control limits given below.
Unit for urinary cadium are ng/ml.
QC Mean Limits
Pool UCL99 UCL95 Mean LCL95 LCL99
base 0.553 0.501 0.338 0.176 0.125
476-1* 8.59 7.95 5.91 3.88 3.23
*Bench and blind control
Four pairs of blind split duplicates were included in the analyses for urinary
cadmium, one member of each pair in each of the two runs. The table below
shows the results on duplicate aliquots. All units for urinary cadium are in
ng/mL.
Pair Run Number Concentration 1 Concentration 2
1 1, 2 1.6 1.4
2 2, 1 0.3 0.3
3 2, 2 0.5 0.5
4 1, 1 0.6 0.4
Forty-five urine specimens were analyzed for beta-2-microglobulin in three
analytical runs. Three samples from each of two bench control pools were
included in each run. All means were within 95% control limits. The ranges
for the 1436 pool in the first and second runs were outside the 99% limits;
the range for the third run was inside the 95% limits. With four QC
parameters being monitored, the probability of one or more being outside the
99% control limits for any given run is about 4%, which is larger than the 2%
probability level used within the Division of Environmental Health Laboratory
Sciences to warrant rejection of an analytical run. Consequently, the runs
shquld be considered in control. Units below for beta-2-microglobulin are
ug/1.
QC Mean Limits Range of Triplicate Values
Witin Run
Pool UCL95 Mean LCL95 UCL99 UCL95
1435 85.62 54.57 23.52 39.38 31.64
1436 295.15 222.23 149.51 80.87 64.97
-------
86
Eighty-five urine specimens were analyzed for creatine in two analytical
runs. Two samples from each of two bench control pools were included in each
run. Two samples of one of these pools were also included in each run as
blind controls. For creatine, all means and ranges were within the 957.
control limits shown below. Units for urinary creatine are mg/dl.
QC Mean Limits
Pool UCL99 UCL95 Mean
Urichem I 135.77 134.53 130.60
Urichem II* 96.42 95.34 91.90
*Bench and Blind Control
LCL95 LCL99
126.67 125.43
88.46 87.38
Range of Duplicate
Values. Within Run
UCL99 UCL95
2.30 1.75
4.31 3.28
Forty-five urine specimens were analyzed for total protein in two analytical
runs. Two samples from each of two bench control pools were included in each
run. Two samples of one of these pools were also included in each run as
blind controls.
For urinary total protein, all means and ranges were within the 951 limits
below except the first run mean and the second run range for one of the bench
controls, which were outside the 99% limits. With six QC parameters, the
probability of one or more being outside the 99% limits for a given run is
about 6%, so the runs should be considered in control. Units for urinary
total protein are mg/1.
QC Mean Limits
Range Limits
Pool
Urichem II*
Urichem II
(Diluted 1:4)
*Bench and blind controls
UCL99 UCL95 Mean
587.60 583.21 569.33
144.92 144.26 142.20
LCL95 LCL99
555.45 551.05
140.14 139.48
UCL99 UCL95
41.19 31.35
3.64 2.77
Forty-five hair specimens were analyzed for lead and cadmium by the ICAP in
four analytical runs. Five specimens of one hair-digest bench-control pool
were included in each run. Both cadmium and lead are measured simultaneously
by the ICAP, so 4 QC parameters are monitored, two means and two ranges. All
means were within the 99% control limits given below except the mean for Pb in
the third run, which was within the 99% control limits. All ranges were
within the 95% limits shown below, except the range for Pb in the first run,
Which was outside the 99% limits. The probability of at least one of four QC
parameters being outside the 99% limits in a given run is about 4%. By the
multiple pool run accept/reject rules used within the Division of
Environmental Health Laboratory Sciences, all four runs are considered in
control. Units in the table below for are ug/g.
QC Mean Limits Range of Quintuplet
Values. Within Run
Analyte UCL99 UCL95 Mean LCL95 LCL99 UCL99 UCL95
Cd 0.8087 0.7135 0.4126 0.1117 0.0165 0.3723 0.3124
Pb 4.494 4.165 3.126 2.087 1.758 1.858 1.559
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87
The hair specimens were not large enough to run split duplicates to estimate
run precision. Consequently, six pairs of duplicate hair digests from
children of CDC employees were also included in the runs. The CV*s for these
duplicates averaged 13.5% for lead and 10.6% for cadmium.
Twenty-one hair digest samples were analyzed for arsenic by atomic absorption
spectrophotometry. No certified reference materials were available for
arsenic in hair to serve as bench or blind controls. Two EPA water quality QC
samples were measured for arsenic. These are aqueous samples with dilute
nitric acid, with target values of 20 and 40 ng/ml. Analytical results (mean
of triplicate determinations) were 20.6 ng/ml and 35.9 ng/ml, respectively.
Thus, although no quantitative quality control limits were available, the
analysis gave values within 3.0% and 10.2% of the target values, respectively.
Three pairs of duplicate hair digests from digests form children of CDC
personnel were also included to check run precision. All determinations of
these were below detection limits (250 ng/g) for arsenic.
In summary, all of the analytical systems used for generating the data for
this study appeared to be stable and in control.
-------
RANGES OF VALUES FROM NORMAL INDIVIDUALS
The CDC laboratories analyzing
information on ranges of values
Analyte
Blood lead
Erythrocyte protoporphyrin
Blood cadmium
Urinary arsenic
Urinary lead
Urinary cadmium
Urinary beta-2-microglobulin
Urinary creatine
Urinary total protein
Hair lead
Hair cadmium
Hair arsenic
he specimens provided the following
from normal individuals:
Range
0-40 ug/dl (adults)
<25 ug/dl (children)
<35 ug/dl (<16 yr)
<60 ug/dl (>16 yr)
0-5 ng/ml
0 - 100 ng/ml
0 - 100 ng/ml
0-5 ug/ml
0 - 250 ug/1
1-2 g/1/24 hr (males)
0.8 - 1.6 g/1/24 hr (females)
<135 mg/1
0.511 - 27.6 ug/g (males)
0.454 - 5.93 ug/g (females)
0 - 2.00 ug/g (males)
0 - 0.478 ug/g (females)
Values pending
The creatine normal ranges reported were based on a 24-hour urine sample.
Since the specimens collected in this study were first morning voids, the
normal ranges are not strictly applicable. In addition, values of urinary
creatine less than 40 mg/dL are considered too low for purposes of normaliz
other urinary analyte concentrations.
-------
Appendix 21
Quality Assurance and Quality Control for
Laboratory Analyses of Environmental Samples
1.0 Soils. Vacuum Dusts
Additional soil quality assurance measures were undertaken by
HDHES because recovery information obtained by sending standard
reference materials as blind field samples to EPA's contract
laboratory showed poor recoveries by the Superfund laboratory.
Table A shows precision and accuracy information for nine
reference materials. Lead values, which were most essential in
this study, gave overall recoveries (neglecting low standard
concentrations1) of 65.1%, 84.0% and 103.6% for VERSAR,
MDHES-ICP, and MDHES X-ray, respectively. X-ray analyses of
arsenic, copper, and zinc were adversely affected by a tungsten
interference. After the samples were analyzed, it was discovered
that a new tungsten-carbide grinding chamber had lost sufficient
amounts of tungsten during the grinding of high silicate soils to
interfere with the analyses of the above three elements. A
correction for the above effect, though possible, had not been
made during calibrations because the manufacturer had conjectured
the grinder would sustain no loss of material.
Table B shows correlations for the eight elements of interest
reported in this study for 29 field samples. A high correlation
exists between MDHES ICP and X-ray results, as for most elemental
comparisons. However, T-tests show that the arsenic, cadmium,
silicon, and titanium results differ statistically between the
methods. These differences were expected for arsenic because of
the tungsten interference described above.
East Helena soils were found to be high in lead concentrations—
considerably higher than the background lead levels of most
reference materials; comparisons against standards with elevated
lead values were, therefore, appropriate.
-------
2.0 Vegetables
Vegetation sample analyses were performed by MDHES using ICP/AAS
instrument techniques. About 223 samples were dried, ground with a
Wiley mill, digested by sealed bomb methods to prevent loss of
volatile elements, and analyzed chemically.
Twenty-five samples were sent to an EPA Superfund contract laboratory
by chain-of-custody procedures to provide sample security. Table C
shows analytical comparisons between the MDHES and VERSAR, Inc.,
laboratories. Low t-values show the data to correlate favorably;
i.e., each laboratory reports the same data for each element.
However, large standard deviations here allow for this statistical
data comparability. MDHES sent VERSAR several reference materials
with the vegetable samples. Analytical recoveries based on these
samples are listed in Table D, which compares precision and accuracy
information for both laboratories. Reference samples were selected
to present low, intermediate, and high elemental concentrations
suitable to concentrations of vegetables analyzed in this study. The
EPA sludge reference material lists what appear to be consensus
values for the elements listed with large acceptable ranges; arsenic,
for example, lists a mean of 17.0 ppm, with a range of 0 to 88.9, for
the 95% confidence interval.
MDHES performance was superior overall to that of VERSAR. Any
notable deviations in recoveries from 100% were found for those cases
in which differences in small numbers tended to influence the average
(overall) recovery in an artificial manner. For lead in vegetation,
for this study, MDHES recovery was essentially 96% to 98%, VERSAR's
recovery was 73% to 88%. Here again, recovery is a measure of
laboratory accuracy.
3.0 Handwash Analyses
Laboratory recoveries and measures of instrumental accuracy were
acceptable for handwash analyses. Lead recoveries averaged 100.8 ±
6.6%, (11=57). Table E shows data for a limited number of blind
repeat handwash analyses. Many ICP data points in the table reflect
the detection limit problems noted above. Table F lists quality
control comparisons for four EPA reference solutions sent to the
MDHES laboratory as blind handwash samples. The MDHES analyzed each
reference solution three times, with the results shown. EPA
acceptance and warning limits are listed.
4.0 MDHES Chain-of-Custody Forms
See Forms A and B attached.
-------
Table A
Precision and Accuracy Data for Soils
Between Laboratories
and Methods
Ref.
No.
Reference
Library-
Recovery
Al%
As (ppm)
Cd (ppra)
Cu (ppra)
Pb (ppm)
Si (%)
Ti (ppm)
Zn (ppm)
NBS River Sed.
2.264- 0.04
66
10.2+ 1.5
109+ 19
714+ 28
1720+ 170
1645
-VERSAR Inc.
0.389
37
5.0
97.8
520
26.9
105
1570
% recovery
17.2
56.1
49.0
89.7
72.3
91.3
1645
-MDHES Lab (n=4)
2.34+ 0.02
64.9+ 1.7
20.4+ 5
112+ 6
680+ 55
20.6+ 7.3
426+ 9
1664+ 91
X recovery
103.5
98.3
200
102.8
95.2
96.7
1645
-MDHES XRF (n=31)
1.98+ 0.088
67.3+ 4.7
4.3+ 3.4
266+ 26
709+ 6
22.34+ 0.18
1057.43
1706+ 45
X recovery
87.6
102.0
42.2
244
99.3
99.2
NBS Fly Ash
61+ 6
1.45+ 0.06
128+5
70+ 4
8600+ 1100
210+ 20
1633
-MDHES Lab (n=5)
12.8+ 0.5
64+ 3
N.D.
128+ 3
35+ 13
23+ 1
6181+ 68
232+ 20
X recovery
104.9
100
50.0
71.9
110.5
1633
-MDHES XRF (n=*3)
14.6+ 0.5
44+ 4
7.3+ 1.4
219+ 90
59+ 5
22.8 0.18
7264+ 128
384+ 161
X recovery
72.1
503
171
84.3
84.4
183
NBS Urban Part.
3.42+ 0.11
115+ 10
75+ 7
609+ 27
6550+ 80
12.52
4760+ 140
1648
-MDHES XRF (n=13)
3.48+ 0.13
125+ 5
59+ 7
661+ 18
7270+ 61
12.56+ 0.02
4546+ 60
4495+ 77
X recovery
101.8
108.7
78.7
108.5
111
100.3
94.4
US6S Andesite soil
9.07+ 0.18
0.84+ 0.27
0.061+ 0.008
60+ 6
36+ 5
27.67+ 0.27
6340+ 300
88+ 2
AG VI
-MDHES XRF (n=3)
9.35+ 0.30
N.D
7.2+ 3.7
124+ 52
47+ 3
27.51+ 0.24
6072+ 152
267+ 101
X recovery
103.1
11803
207
130.6
99.4
95.8
303
USGS Basalt
7.30+ 0.12
1.5
0.12
137+ 6
4+ 2
23.3+ 0.4
16000+ 500
102+ 7
BHVO
-VERSAR Inc.
0.443
0.5
0.05
67.8
0.6
34.1
1510
14.6
X recovery
6.1
49.5
15
146.4
9.4
14.3
BHVO
-MDHES Lab (n=l)
5.1
1.1
0.11
130
13.8
15.9
12521
135
X recovery
69.9
73.3
91.7
94.9
345
68.2
78.3
132
BHVO
-MDHES XRF (n=l)
7.61
6.8
2.25
164
7.5
23.8
15980
144
X recovery
104.2 618
2045
119.7
188
102.1
99.9
141
-------
Table A (Continued)
Precision and Accuracy Data for Soils Between Laboratories and Methods
Reference
Ref. Library
Ho. Recovery Al% As (ppm) Cd (ppm) Cu (ppm) Pb (ppm) Si ¦ (%) Ti (ppm) Zn (ppm)
USGS Diabase
8.14+
0.06
1.2
0.1
84
24.2
6200+
200
87+ 21
W2
MDHES Lab (n=3)
7.64- 0.42
5.34- 4.6
N.D
1114- 5
11.8+
0.7
21.74- 0.8
5658+ 53
93+ 9.9
% recovery
93.4
441.7
132
89.7
91.3
106.9
W2
MDHES XRF (n=l)
8.33
N.D.
1.4
116
26
23.0
6099
90
% recovery
102.3
1400
138
95.0
98.4
103.4
CANMET Blend
1296
467
11720
4604
21.3
59940
PDS02
MDHES Lab (n=2)
6.58+
0.21
14074- 66
4934- 103
12529+
1127
4403+
13
20.4+ 1.13
6590+
110
60836+ 228
% recovery
108.6
105.6
106.9
95.6
95.8
101.5
PDS02
MDHES XRF (n=8)
6.86+
0.06
11494- 27
4434- 6
10977+
118
4425+
66
21.96+ 0.03
6840+
54
61887+ 1037
88.7
94.9
93.7
96.1
103.1
103.2
CANMET Soil
8.07+
0.18
1.24- 0.2
0.184- 0.14
74- 1
214- 4
24.99+ 0.23
8600+
200
124+ 5
S02
VERSAR Inc.
2.12
0.7
0.1
2.75
3.4
29.1
259
40.1
% recovery
26.3
58.3
55.6
39.3
16.2
116.4
3.0
32.3
EPA Soil
6.37
2070
52
2140
1310
27.0
2130
1730
4778
VERSAR Inc.
0.745
1800
35
2220
1400
36.9
290
1100
% recovery
11.7
87.0
67.3
103.7
106.9
136.7
13.6
63.6
MDHES Lab (n=3)
6.34+
0.14
20494- 149
9.464- 149
2303+
33
1247+
106
30.76+ 1.25
1612+
30
1481+ 137
% recovery
99.5
99.0
18.2
107.6
95.2
113.9
75.7
85.6
MDHES XRF (n=2)
6.73+
0.02
20124- 32
22+- 2
2185 +
10
1310+
11
27.17+ 0.00
2521+
40
1332+ 8
% recovery
105.7
97.2
42.3
102.1
100.0
100.6
118.4
77.0
Ave. VERSAR Rec. 15.3% 67.1% 57.3% 70.6% 52.6% 133.2% 8.7% 50.4%
71.6%*
58.1%*
65.1%*
Ave. MDHES-LAB
Rec.
91.6%
154.3%
103.9%
107.4%
136.2%
91.9%
79.3%
105.5%
102.7%*
107.9%*
84.0%*
Ave. MDHES-XRF
Rec.
100.8%
181.1%
1785%
148.0%
115.6%
100.1%
99.4%
138.0%
93.7%*
64.5%*
103.6%*
* See reference b. in Table 11 Summary
-------
TABLE A SUMMARY
Concentration Range Avg. Percent Soil Recoveries
E. Helena Study Soils8 MDHES MDHES
Min.
Mean
Max.
ICP
XRF
VERSAR
A1
(%)
1.7
6.6
12.8
91.6
100.8
15.3
As
(ppm)
A.9
118
1,909
102.7b
93. 7b
71.6b
Cd
Cppm)
0
20
160
107.9b
64.5b
58. lb
Cu
(ppm)
0.1
220
11,700
107.4
148.0
70.6
Pb
(ppm)
3.1
527
7,965
84.0b
103.6b
65. lb
Si
(%)
12.8
30.0
37.1
91.9
100.1
133.2
Ti
(ppm)
763
2939
35,600
79.3
99.4
8.7
Zn
(ppm)
3
584
14,600
105.5
138.0
50.4
Soils considered were front/rear, side, and play.
Percent recoveries here are averages calculated without those
recoveries associated with detection limit problems or problems in
which percent recoveries were large because of differences between
two small reported values.
-------
TABLE B
MDHES LABORATORY QUALITY ASSURANCE COMPARISON BETWEEN AAS, ICP, AND XRF ANALYSES
OF 29 DUPLICATE SOIL MATERIALS FROM AREAS 1, 2, and 3
Elements
Mean
Std. Dev.
Mean Diff.
2-Tail
Corr. Prob.
Deg. 2-Tail
T ValveFreedom Prob.
A1 - Lab
A1 - XRF
65962
65170
6781
8184
792
0.773 0.000
0.82
28
0.420
As - Lab
As - XRF
51
110
46.0
53.4
-59.7
0.478 0.009
-6.29
28
0.000
Cd - Lab
Cd - XRF
6.92
20.9
14.0
14.1
-14.0
0.743 0.000
-7.49
28
0.000
Cu - Lab
Cu - XRF
141.9
127.0
216.3
160.3
14.9
0.912 0.000
0.84
28
0.410
Pb - Lab
Pb - XRF
527.8
485.6
750.9
624.2
42.2
0.970 0.000
1.08
28
0.288
Si - Lab
Si - XRF
295310
310403
26137
22869
15093
0.800 0.000
-5.14
28
0.000
Ti - Lab
Ti - XRF
2375
2800
658
684
-425
0.884 0.000
-7.05
28
0.000
Zn - Lab
Zn - XRF
409.0
441.0
477.1
455.6
-32.0
0.921 0.000
-0.92
28
0.365
-------
TABLE C
Laboratory Quality Assurance Comparison Between Vegetation Analyses Done by MDHES and VERSAR
Mean 2-Tail Deg. 2-Tail
Elements Mean Std. Dev. Diff. Std. Dev. Corr. Prob t-valve Freedom Prob.
As
As
VERSAR
MDHES
2.839
3.704
9.372
11.867
-0.864
2.550
0.999
0.000
-1.40
16
0.182
Cd
Cd
VERSAR
MDHES
3.888
8.858
9.492
16.934
-4.970
13.250
0.626
0.003
-1.68
19
0.110
Cu
Cu
VERSAR
MDHES
21.6725
30.735
51.769
63.252
-9.062
34.645
0.837
0.000
-1.17
19
0.257
Pb
Pb
VERSAR
MDHES
43.650
59.215
118.373
137.257
-15.565
33.009
0.977
0.000
-2.11
19
0.048
Zn VERSAR 90.990 151.247
-4581.2 20476.6 0.521 0.018 -1.00 19 0.330
Zn MDHES 4672.15 20555.0
-------
TABLE D
QUALITY ASSURANCE BLIND SAMPLE COMPARISON BETWEEN REFERENCE VEGETATION MATERIALS
AND EPA'S CONTRACT (SUPERFUHD) LABORATORY AND MDHES RESULTS
A1
As
Cd
Cu
Pb
Si
Ti
Zn
NBS 1571®
VERSAR
% Recovery
MDHES (n = 5)
% Recovery
NBS 1573b
VERSAR
% Recovery
MDHES
% Recovery
EPA S Ludge
VERSAR
% Recovery
MDHES
% Recovery
Avg. VERSAR Rec.
Ave. MDHES Rec.
60.3
220
1200
3070
12275
10+2
6.5
65
11+1.5
110
0.27+0.05
0.5
0.32
118.5
17.0C
2.5
14.
3.
22,
AO
83.8
0.11+0.01
0.54
491
0.37+0.15
336
3
11.0
367
2.0
67
19.1
68
356
18.2
95.3
405
166
12+1
11.3
94.1
13.2+1.9
110
11.1
10.0
90.1
9.0
81.1
1080
1000
92.6
1050
97.2
92.3
96.1
45+3
33
73.3
43.4+7.2
96.4
6.3=0.3
4.1
65.1
4.0
63.5
526
463
88.0
514
97.7
75.5
85.9
N.D.
2.65
1.18%
0.85%
16.1%
11.3%
8.8
77
70
1875
25+3
18.7
74.8
28.6+9.5
114.4
62+6
53
85.4
55
88.7
1320
1070
81.1
1415
107.2
80.4
103
a Orchard leaves
^ Tomato leaves
c EPA's: x + t .95 (df)s for As is 0 - 88.9 implying great uncertainty.
-------
TABLE E
Results of Blind Repeat Analysis of Handwash Samples
East Helena Lead Study
Sample #
Alc
Asc
Cdc
Cuc
Pbc
Sic
Tic
Znc
312-117-2-21-98
0.010d
0.001
0.005d
0.020
0.60
0.010d
0.010
0.070
312-486-l-21-ab
0.052
-
0.005d
0.005d
0.025d
-
0.005d
0.072
312-001-1-21-13
0.020
0.003
0.005d
0.080
0.130
0.010d
0.005d
0.240
312-484-5-21-7b
0.037
0.002
0.005d
0.067
0.068
-
0.005d
0.234
312-051-2-21-03
0.020
0.0005d
0.005d
0.010
0.060
0.010d
0.005d
0.010
312-484-l-21-8b
0.042
0.0005d
0.005d
0.005d
0.058
-
0.005d
0.012
312-151-2-21-9®
0.020
0.001
0.005d
0.010
0.025d
-
0.005d
0.060
312-482-l-21-6b
0.010d
0.001
0.005d
0.010
0.025d
-
0.010
0.060
312-221-1-21-03
0.020
0.0005d
0.005d
0.005d
0.025d
-
0.005d
0.005d
312-500-l-21-lb
0.020
0.0005d
0.005d
0.005d
0.025d
-
0.005d
0.005d
312-145-1-21-93
0.020
0.0005d
0.005d
0.010
0.025d
0.010d
0.005d
0.080
312-481-2-21-2b
0.07
0.0005d,
0.0005d,0.0005d
0.005d
0.005d
0.025d
-
0.005d
0.076
a Original sample number.
b Sample number assigned for repeat analysis.
c All values in ug/ml (ppm).
d These values are half (1/2) of the instrumental detection limit and were assigned to these samples by AQB when analytical
results were below the instrumental detection limit.
-------
TABLE F
Quality Control Comparison of Four EPA-Certified Reference Solutions Injected into MDHES Laboratory Sample Flow
Alc
Asc
Cdc
Cuc
Pbc
Tic
Znc
EPA Certified WP004(l)a
MDHES 481-1-21-5/WP004(1)
MDHES 484-3-21-2/WP004(1)
mdhes 487-2-2i-8/,wpoo4(i)
EPA Acceptance Limits
EPA Warning Limits
0.05
0.09
0.08
0.14
0-.156
.0195-.126
EPA Certified WP004(2)a 0.450
MDHES 489-1-21-2 0.48
MDHES 489-2-21-a 0.43
MDHES 490-2-21-8 0.60
EPA Acceptance Limits .263-.652
EPA Warning Limits .345-.569
EPA Certified WP006(l)b 0.504
MDHES 491-l-21-l/WP006(l)b 0.546
MDHES 493-3-21-8/WP006(l)t> 0.520
MDHES 495-2-21-2/WP006(l)b 0.574
EPA Acceptance Limits .412-.582
EPA Warning Limits .433-.560
0.018
0.019
0.018
o.oir
.0013-.0342
.0055-.030
0.060
0.060
0.059
0.063
.0185-.101
.0294-.0904
0.044
0.043
0.044
0.041
.0258-.0615
.0303-.057
0.0015
0.005
0.005
0.005
0-.0074
0-.00463
0.008
0.005
0.010
0.005
0-.0359
0-.0272
0.013
0.005
0.010
0.010
.00594-.0199
.0091-.016
0.0325
0.030
0.030
0.028
.0243-.0369
.0259-.0354
0.050
0.043
0.050
0.070
.0191-.085
.0304-.0742
0.0521
0.047
0.050
0.051
.0438-.0593
.0457-.0573
0.018
0.025
0.025
0.025
0-.0502
0-.0425
0.120
0.094
0.110
0.110
.0679-.172
.0821-.158
0.157
0.162
0.150
0.114
.130-.181
.136-.175
EPA Certified WP006(2) 0.730 0.235 0.039 0.339 0.43
MDHES 497—1-21-7/WP006(2) 0.779 0.120 0.036 0.334 0.442
MDHES 499-2-21-6Art>006(2) 0.718 0.120 0.040 0.340 0.420
MDHES 500-2-21-9/WP006(2) 0.736 0.225 0.035 0.345 0.408
EPA Acceptance Limits .570-.913 .137-.329 .029-.045 .284-.387 .354-.511
EPA Warning Limits .613-.870 .161-.305 .031-.043 .297-.374 .373-.492
O.OOS
0.005
0.005
0.005
0.010
O.OOS
0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.012
0.005
0.010
0.005
0-.0457
0-.033
0.080
0.074
0.070
0.090
.041—126
.0556-.Ill
0.075
0.179
0.170
0.182
.156-.192
.161-.187
0.418
0.416
0.408
0.409
.364-.467
.377-.455
a Values in parentheses denote concentration of Standard: (1) = Concentration #1
(2) = Concentration #2
k WP006(1) was diluted by a factor of 10 to produce a standard within the working range of the State
Laboratory's ICP system. The certified values have been adjusted to reflect this.
c All analytical results are in ug/ml (pptn).
-------
FORM A
CHAIN-OF-CUSTODY
-------
FORM B
CIIAIN-OF-CUSTODY
Proj. Cod* Project Nome /\rea
East Helena Lead Study
/ Analysis ^
/*&/ REMARKS
/Cv
/<$/
/•> lu Ho.
Dust
1
Soil
Vegetation
Other
—
—
—
—
¦¦
Kcl(i:i)ulshc4 by: (Sl'jntture)
lUle/Tlcu
1
j Received tys (Signature)
RelInqulsliei) l>y: (Slgnituro)
Date/Time
Received bys (Signature)
fc;llni|:ijsiiL"J L-y; {Sl'j'Ulurc)
J. .
kccclvcJ ty: (Sl<|njlurt:)
ftc 1 (fi«4U 1 »licd by; (Signature)
Ditc/(Imc
Received by: (Sljnjture)
I I i
r\i
o
Ufslrliiutloii.' tcccuptnlii Shlpre.'ili Ctfiy >.u CoorJImlMr Field files
-------
RT1/2474/79—OIF
APPENDIX 22
SYSTEMS AND PERFORMANCE AUDIT OF THE
EAST HELENA LEAD EXPOSURE STUDY
by
R. C. Shores
EPA Task Manager: W. F. Barnard
EPA Contract No. 68-02-3767
Work Assignment 79
Prepared for
Quality Assurance Division
Environmental Monitoring Systems Laboratory
U. S. Environmental Protection Agency
Environmental Research Center
Research Triangle Park, North Carolina 27711
February 1984
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27709
-------
one.
TABLE OF CONTENTS
Secti on Page
1 INTRODUCTION 1
2 SUMMARY 2
2.1 Summary of Systems Audit 2
2.2 Summary of Performance Audit 3
3 AUDIT SYSTEMS VERIFICATION ... 5
3.1 High Volume Sampler Flow Measurement 5
3.2 Dichotomous Sampler Flow Measurement 6
4 AUDIT PROCEDURES 7
4.1 System Audit Approach 7
4.2 Performance Audit Approach 7
5 AUDIT RESULTS 9
5.1 System Audit Results 9
5.2 performance Audit Results 15
-------
LIST OF ILLUSTRATIONS
Figure Page
1 Chain of Custody Particulate Filters/Samples,
State of Montana 11
2 Hi-Vol Data Record Form, State of Montana 12
Tab! e
1 Summary of Performance Audit Results from Particulate
Samplers 4
2 High Volume Sampler Performance Audit Results 16
3 Dichotomous Sampler Performance Audit Results 18
i i
-------
PREFACE
Since 1972 the Environmental Protection Agency (EPA) has been en-
gaged in an on-site performance audit program of various monitoring
groups throughout the United States and in several foreign countries.
An on-site audit program is one part of an overall quality assurance
program. The purposes of this audit program are twofold. First,
agencies are furnished a means of rapid self-evaluation of the specific
operation under study. The second objective of the program is to pro-
vide EPA with a continuing index of the validity of data reported to
air quality data banks. The first, from a participant standpoint, is
the more important. Used along with information obtained from an in-
ternal quality control program, the conclusions from the performance
audits can be quite meaningful. However, results from any single set
of audit data should not be construed as absolute indicators of data
quality.
This program is being coordinated through the Quality Assurance
Division (QAD) .of the Environmental Monitoring Systems Laboratory
(EMSL), Environmental Research Center, Research Triangle Park, North
Carolina 27711. Comments or questions about the program should be sent
to the above address.
iii
-------
SECTION 1
INTRODUCTION
The purpose of this report is to document the findings of the sys-
tems and performance audit evaluations of the office, laboratory, and
field monitoring sites constituting the East Helena Lead Exposure Study
located in Helena and East Helena, Montana. The audit was organized
and coordinated through the Quality Assurance Division of the Environ-
mental Protection Agency (EPA) Environmental Monitoring Systems Labora-
tory.
During the period spanning September 12 through September 15,
1983, personnel of the Research Triangle Institute (RTI) visited the
East Helena Lead Smelter Exposure Study. Although the complete expo-
sure study involves a wide variety of professional disciplines, RTI's
audit was concerned only with the particulate sampling network. The
sampling network consisted of nine high volume and two dichotomous sam-
plers. The office, laboratory, and monitoring network constituting the
East Helena Lead Exposure Study audit was conducted in accordance with
recommendations given in the EPA publication Quality Assurance Handbook
for Air Pollution Measurement Systems, Volume II, Ambient Air Specific
Methods.
-------
SECTION 2
SUMMARY
The results of the systems and performance audits are contained in
this section. Section 2.1 summarizes the results of the systems audit,
and Section 2.2 summarizes the results of the performance audit.
2.1 SUMMARY OF SYSTEMS AUDIT
The systems audit conducted at the East Helena Lead Smelter Expo-
sure Study was divided into four major areas:
o Review of the project documentation including the quality
assurance and procedures manual.
o Review of management organization, data archiving, and
recordkeeping.
o Chain of custody for samples, and
o Monitoring site criteria evaluation.
Project documentation (Quality Assurance and Procedures Manual),
specific to this study was not completely finished and was not availa-
ble for inspection. The Environmental Program Manager reported that
the monitoring program was still being modified, and as new information
became available, the Quality Assurance and Procedures Manual would
become final. During the interim, the State of Montana Air Monitoring
Quality Assurance and Procedures Manual was being used as a reference
for sampler operation. This manual was available and adequately
covered all necessary aspects specified in the Quality Assurance Hand-
book for Air Pollution Measurement Systems, Volume I & II, including
both high volume and dichotomous samplers. The State of Montana Air
Monitoring Quality Assurance and Procedures Manual has been reviewed
and approved by the EPA Region VIII Quality Assurance Coordinator.
2
-------
The Air Quality Group of the Montana Department of Health and
Environmental Sciences is wel 1-organized and appears to work together
satisfactorily. Data archiving and recordkeeping is organized and up-
to-date. Data forms being used by the Air Quality Group satisfactorily
document all information necessary to record valid samples. All data
and recordkeeping are placed in cardboard file boxes and stored at the
Air Quality Group's office for at least three years. The following
individuals are responsible for the Montana Department of Health and
Environmental Science particulate monitoring network data: ¦
o David Maughan - Environmental Program Manager
o Jerry Schneider - Quality Assurance Coordinator
o James Olsen - Air Monitoring Supervisor
o Chuck Homer - Environmental Specialists
o Diane Ertman - Laboratory Technician
o Ben Myren - Contracted; Site Operator
A complete chain-of-custody has not been maintained for the par-
ticulate samples. Since the sample is handled by several people from
the time of exposure, who was responsible for each sample at any one
point in time is not recorded.
Monitoring site criteria have not been maintained at the Hastie
Site. This site has been operating for several years and during this
time a crab apple and several pine trees have grown enough to cause air
flow obstruction. Several other sites are located in close proximity
to East Helena streets, but because of the low volume of traffic, this
is probably not a problem.
2.2 SUMMARY OF PERFORMANCE AUDIT
The East Helena Lead Exposure Study contained nine sites consist-
ing of nine high volume and two dichotomous samplers. Table 1 sum-
marizes the results of the flow rate audit conducted on these samplers.
The last column to the right indicates each sampler's performance based
on the criteria outlined in Section 5.
3
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TABLE 1. SUMMARY OF PERFORMANCE AUDIT RESULTS FROM PARTICULATE SAMPLERS
SITE NAME AND
SAROAD NUMBER
DATE OF
AUDIT
SAMPLER
TYPE
S/N
FLOW
TYPE
*AUDIT
SATISFACTORY/
UNSATISFACTORY
TOWNSEND SITE
27 0720 017 F05
9-13-83
High Volume
12733
N/A
S
SCHNEIDER SITE
9-13-83
Hiqh Volume
338
N/A
S
27 0860 007 F02
Dichotomous
123
Total
b
Coarse
b
Fi ne
b
DUDLEY SITE
9-13-83
Hiah Volume
12294
N/A
s
27 0860 725 F05
Dichotomous
190
Total
6
Coarse
5
Fine
b
SOUTH SITE
27 0860 716 F02
9-13-83
High Volume
9378
N/A
s
DARTMAN FIELD SITE
27 0860 724 F02
9-13-83
High Volume
15708
N/A
s
HADFIELD EAST SITE
27 0860 719 F07
9-14-83
High Volume
139216
N/A
s
HADFIELD WEST SITE
27 0860 719 F02
9-14-83
High Volume
139218
N/A
s
FIRE HALL SITE
27 0860 714 F02
9-14-83
High Volume
16189
N/A
s
HASTIE SITE
27 0860 002 F02
9-14-83
High Volume
9091
N/A
s
* S = satisfactory; U = unsatisfactory
4
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SECTION 3
AUDIT SYSTEMS VERIFICATION
Verification of the accuracy of RTI's particulate audit system
was conducted prior to the audit trip. The particulate auditing system
consisted of a Reference Flow (ReF) device and a dry gas meter. The
verification of this system was conducted in RTI's Standards Laboratory
on September 8, 1983. The accuracy of the ReF device used in auditing
the high volume samplers (serial no. 272), was established through
calibration with the RTI Roots meter, serial no. 7807141, a primary
standard.
The Singer ten liter per revolution dry gas meter used in auditing
the dichotomous samplers was calibrated in the RTI Standards Laboratory
against a one cubic foot per revolution wet test meter (s/n 11AH2).
Air flows were drawn simultaneously through the dry gas meter and the
wet test meter with a vacuum pump. Flow rates through the meters were
determined at least three times using an electronic timer for each air
flow level generated. Measurements were based on five revolutions of
the wet test meter (5.0 ft^). Temperature, pressure, and water vapor
corrections were appropiately applied across both meters and incor-
porated into the calibration calculations.
The wet test meter is periodically calibrated at the EPA QAD/EMSL.
The gasometer used to calibrate the wet test meter is a primary stan-
dard.
3.1 HIGH VOLUME SAMPLER FLOW MEASUREMENT
Instrument verified: Reference Flow (ReF) device
Serial No.: 272
Location: RTI Standards Laboratory
Date of verification: September 8, 1983
Calibrated by: R. Shores
5
-------
Calibration Equation:
Qstd = 0.2645 UP Pa/Ta)1/2 + 0.1157
where:
Qstd = ^0w rate at standard conditions (temperature =
298.16 K; pressure = 760 mm Hg) in cubic meters/minute
aP = pressure drop across the Ref device in inches of water
Pa = atmospheric pressure (mm Hg)
Ta = atmospheric temperature in degrees Kelvin (273.16 + °C)
3.2 DICH0T0M0DS SAMPLER FLOW MEASUREMENT
Instrument verified: 10 liter per revolution dry gas meter
Serial No.: 80SH583660
Location: RTI Standards Laboratory
Date of verification: September 8, 1983
Calibrated by: R. Shores
Calibration Equation:
Qstd = 0.9343 (Qind) [(298.16 x Pa)/(760 x Ta)]
where:
Qstd = fl°w rate at standard conditions (temperature =
298.16 K; pressure = 760 mm Hg) in liters/minute.
Qind = ^ow rate that 1S indicated by the dry gas meter at
meter conditions.
Pa = atmospheric pressure (mm Hg)
Ta = atmospheric temperature (K)
0.9343 = correction factor for dry gas meter versus standard wet
test meter (s/n 11AH2)
6
-------
SECTION 4
AUDIT PROCEDURES
Systems audit procedures have been determined in accordance with
recommendations given in Environmental Protection Agency's (EPA's)
Quality Assurance Handbook for Air Pollution Measurement Systems,
Volume 11. Performance audit procedures for particulate samplers con-
sist of an audit of the samplers inlet flow rate. Sections 4.1 and 4.2
discusses the audit procedures used during the audit for the systems
and performance audit, respectively.
4.1 SYSTEM AUDIT APPROACH
The systems audit of the office, laboratory, and field sites of
the East Helena Lead Smelter Exposure Study was conducted in accordance
with the recommendations given in the EPA publication Qua! i ty Assurance
Handbook for Air Pollution Measurement Systems, Volume II, emphasizing
the guidelines for systems audits given in Section 2.0.11, "Systems
Audit Criteria and Procedures for Ambient Air Monitoring Programs."
The approach taken in conducting the systems audit followed these
steps:
o Review of the project's documentation including quality
assurance and procedures manual,
o Review of managment organization, data archiving, and record-
keeping,
o Chain of custody for samples, and-
o Monitoring site criteria evaluation.
4.2 PERFORMANCE AUDIT APPROACH
The performance audits of the field sites constituting the East
Helena Lead Smelter Exposure Study were conducted in accordance with
the recommendations given in the EPA publication Qua! ity Assurance
Handbook for Air Pollution Measurement Systems, Volume II. emphasizing
the guidelines for performance audits given in Section 2.0.12, "Audit
7
-------
Procedures for Use by State and Local Air Monitoring Agencies" and a
draft version of the EPA document entitled, Inhalable Particulate
Network Operations and Quality Assurance Manual, March 1983. The
specific approach taken for the two types of samplers audited are
described in the following subsections.
4.2.1 High Volume Samp!ers
A Reference Flow (ReF) device was used to audit the flow rate
calibration of the high volume particulate samplers. The ReF device is
a limiting orifice device which yields audit checks of sampler flow
rate by measuring the pressure drop across the orifice using a water
manometer. This pressure drop is translated into a flow rate at either
standard or actual conditions using the applicable calibration equa-
tion. Traceability is established by calibrating the ReF device using
a Roots meter, a primary standard.
4.2.2 Dichotomous Samplers
A dry gas meter was used to measure the total and fine flow rates
and the coarse flow rate was determined as the difference between the
total and fine flow rates. After clean filters were installed and the
rotameters were properly set the dry gas meter was connected to the
sampler's inlet. An actual flow rate was obtained by measuring a
volume of air over an interval of time and applying the appropriate
calibration factor for that meter. A total flow rate was obtained
first, and by capping off the coarse flow vacuum line under the virtual
impactor (and filter holder), a fine flow rate was obtained. The
coarse flow rate was determined by subtracting the fine flow rate from
the total flow rate.
8
-------
SECTION 5
AUDIT RESULTS
During the period of time spanning September 13 through September
15, 1983, RTI personnel conducted systems and performance audits at
eight sites surrounding the American Smelting and Refining Company
(ASRCO) located in East Helena, Montana.
Data for each portion of the audit are presented in this section.
Each component of the systems audit, as described in Section 4, is dis-
cussed in Section 5.1. The particulate samplers' audit data are com-
pared in terms of percent difference of flow rate. The particulate
sampler audit results are given in Section 5.2. The following criteria
based on average percent difference in flow rate may be used as a
measure to evaluate the performance of a particulate sampler:
(1) Satisfactory = | Percent Difference | <_ 10 percent
(2) Unsatisfactory = | Percent Difference | > 10 percent
5.1 SYSTEMS AUDIT RESULTS
Project documentation (quality assurance and procedure manual)
specific to this study was not available for inspection. The State of
Montana Environmental Program Manager reported that the monitoring pro-
gram was still being modified, and as new information became available,
the quality assurance and procedures manual would become final. During
the interim, the State of Montana Air Monitoring Quality Assurance and
Procedures Manual was used as a reference for sampler operation. This
manual was available and adequately covered all necessary aspects
specified in the Quality Assurance Handbook for Air Pollution Measure-
ment Systems, Volume I & II, including both high volume and dichotomous
samplers. The State of Montana Air Monitoring Quality Assurance and
Procedures Manual has been reviewed and approved by the EPA, Region
VIII Quality Assurance Coordinator.
9
-------
Figure 1 represents the chain-of-custody being maintained for
filters/samples collected as part of the East Helena Lead Smelter Expo-
sure Study. However, this chain-of-custody is not being documented.
From the time of exposure, the sample is handled by several people and
who was responsible for the sample at any one point in time is not
recorded. The hi-vol data record form is placed with the filter prior
to exposure and remains with the filter thereafter. Because the chain-
of-custody becomes important after the filter has been exposed, the hi-
vol data record form could also provide the necessary chain-of-custody.
To provide this chain-of-custody, the hi-vol data record form should
include a sign-in and out of possession section. This section would
provide for initials, date and time, both in and out of possession for
each set of initials. This section would provide each sample a record
of who was responsible for that sample from exposure to data archiving.
Strips are cut from samples for further chemical analysis. The same
hi-vol data record form can be copied and the copy could then become
the strip's chain-o'f-custody form. Figure 2 represents a hi-vol data
record form which remains with a sample from exposure to data archiv-
ing. This form establishes the link between serial numbers stamped
onto each filter and the SAROAD identification number, flow rate, and
date of the sample. Each filter serial number is also recorded in the
filter weight log which contains the sample collection information and
the filter weight information.
The chain-of-custody procedure presently being maintained for sam-
ple collected as part of the East Helena Lead Smelter Study will not
affect the quality of the samples. However, this chain-of-custody does
allow for the potential of tampered samples with no specific individual
being responsible.
5.1.1 Towsend Site (Visited 9-13-83)
One high volume sampler, which is bolted to a four feet by eight
feet piece of plywood mounted on scaffolding six feet above the ground
is located at the Towsend Site, is located fifty-five feet southwest of
Townsend road and twenty-one feet east of the Artisan Furniture
10
-------
Filter Preparation
(Laboratory)
C. Homer
o Receives Filters
o Q/C Conducted
o Deliver to D. Ertman
D. Ertman
o Receives Filters
o Filter Conditioning
o Determine Tare Weight
o Documentation
o Deliver to C. Homer
C. Homer
o Receives FiIters
o Ship to Field Site
Filter Exposure Post Exposure Weighing
(Field Si to) (Laboratory)
Site Operator
o Receives Filters
o Initial Entry of Data
on data form
(See Figure 2)
o Filter Exposure
o Complete Entry of Data
on Data Form
o Deliver Sample to
C. Homer
C. Homer
o Receives Samples
o Q/C Conducted
o Calculation of Total Air
Volume Sampled
o Deliver to D. Ertman
D. Ertman
o Receives Samples
o Filter Conditioning
o Determine Gross and Net
Weights
o Calculate Concentration
(Mg/m3)
o Cuts Sample Strips for
Further Chemical Analysis
o Deliver to C. Homer
C. Homer
o Receives Samples
o Archives Samples in Storage
Boxes
o Tabulates Data
FIGURE 1. CHAIN OF CUSTODY,
PARTICULATE FILTERS, STATE OF MONTANA
-------
DATE
ADDITIONAL DETERMINATIONS
PAIUJETEH | VALUE (ug/M3)
HI - VOL DATA RECORD
Montana Pcpartncnt of Health
and Environncnta1 Sciences
A\n QUALITY BUREAU PHONE: 4J9-34S4
REV. 12/76
Site
WITAHA CI TV
07 E 9 905 F05
(City or Town)
SCHOOL
(Simpler Location]
Operator
I i
i Iter No. _ | Sampler No.
Tine or Flokneter
Date Meter Reading Reading
Start :
End:
CC?-?SNT5 (continued)
lE'lARkS (use Codes*, or actual values if known)
KIND VIS. SKY HUM. TE'!P. FRHSS'JRg
Start: ~~
End:
Codes*
li'INO: 1-calm 2-light 3-gusty 4-strong
VISIBILITY: S-clear 6-ha:v
SKY: 7-clear 8-scattered 9-overcast
HUMIDITY: A-dr)- B-moderate C-hurid D-rain or snow
TEMP. (°F): E: < 20 F:20-40 C:41-e0 H:61-80 I:>o0
Unusual activities or conditions near the sair.plinp
s i t e :
Initial CFM
Total Sampling Time_
Air Volume
Filter Cross Weight
Filter Tare height
N
;t Particulate Weight
em.
b
i
Particulate Concentration
uc'M* 1
FIGURE 2. HI-VOL DATA RECORD FORM, STATE OF MONTANA
Final CFM_
hrs.
_min.
M3
12
-------
Upholster building. Southeast of the site, twenty-one feet away is a
barn-like building that is four-to-five feet above the sampler; how-
ever, this stucture should not cause any air flow obstruction, because
it is relatively small (approximately 10 ft x 20 ft). The only point
source in close proximity is a wood stove chimney in the upholstery
building. Because the chimney is so close to the sampler, a biased
sample could result in periods with certain meteorological conditions.
The surrounding area may be described as a subdivision neighborhood
with paved streets.
5.1.2 Schneider Site (Visited 9-13-83)
One high volume sampler is located at the Schneider site. The
dichotomous sampler located at this site is inside the Schneider resi-
dence and is part of an indoor pollution study. The high volume sam-
pler is bolted to a four feet by eight feet piece of plywood and is
mounted on scaffolding six feet above the ground. The site is sixty
feet southeast of Tejon Lane and one hundred and twenty feet southwest
of the Schneider residence. The only air flow obstruction may be
caused by two two-story colonial style homes which are at least one
hundred feet away from the site. The only point sources in close
proximity to the site are wood stove chimneys. The surrounding area
may be described as a subdivision neighborhood with paved streets.
5.1.3 Dudley Site (Visited 9-13-83)
One dichotomous and one high volume sampler are located at the
Dudley site. The high volume sampler is bolted to a four feet by eight
feet piece of plywood and is mounted on scaffolding six feet above the
ground. The dichotomous sampler was mounted on two inch by four inch
planks extended off one edge of the platform approximately two feet,
with a distance between the two samplers of six feet. This site is
located on the back property line of a one-half acre lot. A one story
ranch house is located forty-five feet north of the site, and the site
is one hundred and twenty-five feet south of Dudley Road. South and
southwest of the site are vacant lots, overgrown with weeds. The
13
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surrounding area may be described as a subdivision neighborhood with
paved streets.
5.1.4 South Site (Visited 9-13-83)
One high volume sampler, which is bolted to a four feet by eight
feet piece of plywood mounted on scaffolding, six feet above the ground
is located at the South site . This site is located only two thousand
feet southeast of the Lead Smelter. The surrounding area is open
fields and scrub brush with no air flow obstructions.
5.1.5 Dartman Site (Visited 9-13-83)
One high volume sampler bolted to a four feet by eight feet piece
of plywood mounted on scaffolding six feet above the ground is located
at the Dartman site. This site is located in a field one hundred and
fifty feet west of a lightly traveled dirt road. East of the site a
row of trees borders the field one hundred and twenty-five feet away.
The area is a combination of pastures, overgrown pastures, and houses
with no air flow obstructions.
5.1.6 Hadfield East and West Site (Visited 9-14-83)
Two high volume samplers, bolted on opposite ends of a six feet by
twelve feet platform which is mounted on scaffolding ten feet above the
ground, are located at this site. The sampler mounted on the east side
of the platform is designated "East," and the sampler mounted on the
west side is designated "West". This site is located thirty-six feet
southwest and seventy-two feet southeast of Cleveland Avenue and Main
Street, respectively. This site is surrounded by buildings, but
because of the height of the sampler there is no air flow obstruction.
This site is approximately five hundred and fifty-feet northwest of the
1ead smel ter.
5.1.7 Hastie Site (Visited (9-14-83)
One high volume sampler which is bolted on a four feet by six feet
piece of plywood mounted on scaffolding fifteen feet above the ground
-------
is located at this site. This site is located approximatley four hun-
dred and twenty feet northeast of the lead smelter. A crab apple and
several pine trees have overgrown this site, causing severe sitting
criteria violations. The trees cover one ninety degree gradient eleven
to twenty-three feet from the sampler and three to ten feet above the
sampler. Because the trees are directly between the sampler and the
lead smelter, collected ambient air concentrations may be lower than
surrounding concentrations. This site has only recently been overgrown
by the crab apple and pine trees. The impact of the trees upon the
collected ambient air data may best be evaluated through comparison to
the past years of data collected at this site.
5.2 PERFORMANCE AUDIT RESULTS
Eight high volume and two dichotomous samplers were audited.
Table 1 contains the audit results for the high volume samplers, and
Table 2 contains the audit results for the dichotomous samplers.
15
-------
TABLE 2. HIGH VOLUME SAMPLER PERFORMANCE AUDIT RESULTS
Site Name Serial Plate
(Date of Audit) Number Number
Audit Flow
(std. m^/inin)
Sampler Flow
(std. m^/niin)
Difference
std. m^/rnin
Percent Avg.
TOWNSEND
(9-13-83)
12733
NP
18
13
10
7
1.707
1.482
1.371
1.263
1.026
1.650
1.415
1.312
1.194
0.945
-0.057
-0.067
-0.059
-0.069
-0.081
3.3
4.5
4.3
5.5
7.9
-5.1
SCHNEIDER
(9-13-83)
338
NP
18
13
10
7
1.682
1.465
1.352
1.227
1.017
1.659
1.435
1.323
1.195
0.972
-0.023
-0.030
-0.029
-0.032
-0.045
1.4
2.0
2.1
2.6
4.4
-2.5
DUDLEY
(9-13-83)
12294
NP
18
13
10
7
1.470
1.294
1.195
1.102
0.915
1.408
1.233
1.131
1.044
0.870
-0.062
-0.061
-0.064
-0.058
-0.045
- 4.2
-4.7
- 5.4
- 5.3
- 4.9
-4.9
SOUTH
(9-13-83)
9378
NP
18
13
10
7
1.473
1.298
1 .185
1 .092
0.923
1.486
1.288
1.152
1.015
0.794
+0.013
-0.010
-0.033
-0.077
-0.129
+ 0.9
-0.8
- 2.8
- 7.1
-14.0
-4.8
-------
TABLE 2. HIGH VOLUME SAMPLER PERFORMANCE AUDIT RESULTS
(cont'd)
Site Name Serial Plate Audit Flow Sampler Flow Difference
(Date of Audit) Number Number (std. m^/min) (std. m^/inin) std. m^/min Percent Avg.
DARTMAN FIELD
(9-13-83)
15708
NP
18
13
10
7
1.470
1.302
1.204
1.089
0.911
1.441
1.253
1.159
1.034
0.800
-0.029
-0.049
-0.045
-0.055
-0.111
- 2.0
-3.8
- 3.7
- 5.1
-12.2
-5.4
HADFIELD EAST
(9-14-83) 139216
NP
18
13
10
1.482
1.305
1.219
1.103
1.434
1.233
1.116
0.982
-0.048
-0.072
-0.103
-0.121
- 3.2
- 5.5
- 8.4
-11.0
-7.0
HADFIELD WEST
(9-14-83) 139218
NP
18
13
10
7
1.396
1.332
1.234
1.127
0.942
1.468
1.257
1.143
1.030
0.836
+0.072
-0.075
-0.091
-0.097
-0.106
+ 5.2
- 5.6
- 7.4
- 8.6
-11.3
-5.5
FIRE HALL
(9-14-83)
16189
NP
18
13
10
1.374
1.239
1.164
1.067
1.316
1.143
1.044
0.995
-0.058
-0.096
-0.120
-0.072
- 4.2
- 7.7
-10.3
- 6.7
-7.2
HASTIE
(9-14-83)
9091
NP
18
13
10
1.403
1.236
1.154
1.065
1.444
1.293
1.205
1.116
+0.041
+0.057
+0.051
+0.051
+ 2.9
+ 4.6
+ 4.4
+ 4.8
+4.2
-------
TABLE 3. DICHOTOMOUS SAMPLER PERFORMANCE AUDIT RESULTS
Site Name
(Date of Audit)
Sampler Model Flow
and S/N Type
Audit Flow
(act. L/min)
Sample Flow
(act. L/inin)
Act.
Difference
L/min
Percent
SCHNEIDER
(9-13-83)
Sierra
S/N 123
TOTAL
FINE
COARSE
16.87
15.26
1.61
16.67
15.00
1.67
-0.20
-0.26
+0.06
- 1.2
- 1.7
+ 3.7
DUDLEY
(9-13-83)
Sierra
S/N 190
TOTAL
FINE
COARSE
15.65
14.09
1.56
16.67
15.00
1.67
+1.02
+0.91
+0.11
+ 6.5
+ 6.5
+ 7.1
-------
APPENDIX 23
ESTIMATING THE AMOUNT OF SOIL INGESTED BY YOUNG CHILDREN
THROUGH TRACER ELEMENTS
Reported by
Sue Binder, M.D.*
David Sokal, M.D.*
David Maughan, M.A.^
1Division of Environmental Hazards
and Health Effects
Center for Environmental Health
Centers for Disease Control
Public Health Service
U.S. Department of Health and
Human Services
Atlanta, Georgia 30333
^Air Quality Board
Montana Department of Health
and Environmental Sciences
Helena, Montana
-------
CONTENTS
Page
Abstract 3
Background 3
Methods 5
Results y
Discussion 12
Conclusions and Recommendations 16
Tables 17
Plots 29
References 3b
-------
ABSTRACT
In this pilot study, we modified methods used in estimating the amount of soil
ingested by ruminants to measure soil ingested by children. Using aluminum,
silicon, and titanium as tracers, we estimated soil ingestion for 5y children
aged 1-3 years from East Helena, Montana. Estimated daily soil ingestion
based on aluminum and silicon concentrations were 121 and 184 mg/day,
respectively; the estimate based on the titanium concentration was about 10
times higher, 1,834 mg/day. We do not consider these estimates accurate
measures of soil ingestion, although the method we used is a reasonable
approach that, to our knowledge, has not been used before for humans.
Refinement of this method and a better understanding of the metabolism of
aluminum, silicon, and titanium will lead to more accurate estimates of soil
ingestion in toddlers.
BACKGROUND
An assessment of the human health risk associated with toxic materials in the
environment is only as good as the estimate of exposure. Relatively "good"
estimates are available for the amount of air breathed or water ingested.
However, estimates of the amount of dirt and dust ingested during normal
activities are based on little objective evidence, and they vary widely.
This study estimates soil ingestion in children by measuring aluminum,
silicon, and titanium in soil, dust, and feces.
- 2 -
-------
Previous estimates of soil ingestion in children
Previous estimates of the soil ingestion of children have generally been
guesses or a combination of guesswork, and measurements. Lepow et al. measured
a mean of 10 milligrams (mg) dirt on the hands of 22 children. They guessed
that a child puts its hands into its mouth approximately 10 times a day for a
daily soil intake of 100 mg/day.* The National Research Council estimated
that young children ingest an average ot 40 mg/day of street dust.^ Day et
al. measured 5-50 mg of dirt transferred from a child's hand to a sticky sweet
and estimated that a daily intake ot 2-20 sweets would lead to a dirt intake
3
of 10-1,000 mg. Kiinbrough et al. estimated that the average amount of soil
ingested per day is 1 gram (g) for children aged V to 18 months, 10 g for 18
to 42 months, 1 g for 42 months to 5 years, and 100 mg for 5 years through
adulthood.
Estimates or soil ingestion in ruminants
Veterinary scientists have developed methods for measuring soil ingestion by
grazing animals. Using nonabsorbed elements as tracers, they have measured
fecal excretion of elements that have high concentrations in soil. Silicon
and titanium have usually been used for this purpose, and estimates of soil
ingestion in ruminants, such as cows, have ranged from .42 to 4219
g/day.^,b We adapted this method for estimating the amount of soil ingested
by children.
- 3 -
-------
METHODS
Collection and processing of stool samples
As part of a health study of residents living near a lead smelter in East
Helena, Montana, during the summer of 1984, we collected 3-day fecal specimens
from 70 children ages 1 to 3 who were not toilet trained. Parents
participating in the stool collection part of the study were given a free,
3-day supply of one brand ot disposable diapers. Parents placed all used
diapers in large plastic bags for daily collection by study personnel. They
were also asked to wipe the child's bottom with the diaper itself and to
refrain from using creams or lotions on the child's bottom for the 3-day
collection period.
Stool was scraped from the diaper liner and put in a clean plastic bottle.
The diaper liner was separated from the diaper and cleaned in distilled water
to remove adherent stool. The rinse water was added to the stool material in
the plastic bottle, and the bottles were stored in a walk-in refrigerator at
4°C for several months before being freeze-dried. Some of the samples
required additional drying in an oven. The stool samples were then
inspected, hair, small pieces of white plastic material (thought to originate
from the diaper cleaning process), and raisins were the only identifiable
objects. These were removed, and the samples were weighed and then
- 4 -
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homogenized to a fine powder in a food blender with stainless steel blades.
(Neither an SPEX shatterbox nor an herb grinder worked adequately.) The powder
was pressed to b-g pellets and analyzed for aluminum, silicon, and titanium by
x-ray fluorescence (XRF). (Since no stool reference standards exist and since
no National Bureau of Standards biological reference materials contain silicon
or titanium and few contain aluminum, the XRF results could not be verified.
Reagent-grade stock solutions can be used to calibrate inductively coupled
plasma atomic emission spectrometry (ICP). For this reason, ICP analysis was
performed on fecal material remaining after the pellets were made. Quality
assurance was assessed by adding precise quantities of aluminum, silicon, and
titanium to stool and assessing recovery (Table 1)).
Collection and processing of soil and dust samples
In addition to stool samples, yard soil and indoor dust samples were collected
frou most of the participating children's houses. After sod and grass were
removed, four soil samples from the front yard and one from the back were
obtained from the top 1 inch of ground with a stainless steel corer and were
composited in the field. Grab dust samples were collected from vacuum cleaner
bags in participants' homes. Aluminum, silicon, and titanium concentrations
in soil and housedust were analyzed by XRF; reference materials are available
for XRF analysis of these elements in soil and housedust.
- 5 -
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Algorithm for determining soil Ingestion
Estimates of daily soil ingestion for each child i (I-L) based on aluminum,
on silicon, and on titanium (M. ) were calculated according to the
i, e
following formula:
Mi,e = fl'e * Fj
i.e
where M, = estimated soil ingestion for child i based on element e in
i,e
mg/day,
f. = concentration of element e in fecal sample for child i in mg/g
1, e
= daily fecal dry weight for child i in g/day, and
s. « concentration of element e in child i's yard soil in mg/g
i.e
Because stool weights in this study were so much less than expected (as low as
1.8 g/day), F^ was replaced with 15 g/day, an estimate of the daily fecal
dry weight for all children in this age group. This estimate was derived from
reanalysis of data presented in a 1S>79 paper.^ (Seventeen infants aged
13-24 months produced a mean of 1.7 stools per day. Average stool weight was
35 g, of which 73.8% was water. The calculated mean dry weight of the stools
was approximately 15 g/day.)
- 6 -
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In addition to aluminum, silicon, and titanium that children ingest as part of
their normal diets, certain materials provide concentrated sources of these
elements. For example, baked goods can contain large amounts of aluminum,
beer is a saturated solution of silicon, and paint contains large quantities
of titanium. If a child has consumed a concentrated source of one element,
the estimated amount of soil ingested based on that element will be higher
than the estimates based on the other two elements. An alternative method of
estimating soil ingestion i% to use the minimum of the three soil ingestion
estimates for each child. Using the same notation as before, according to
this method, we find:
M, = Minimum (M. )
i i,e
where I-L = estimated soil ingestion for child i in mg/d, and
t'l. = estimated soil ingestion for child i based on element e in mg/d.
*•»®
Questionnaire
A questionnaire was administered as part of the Childhood Lead Study. It
Included questions about demographic information and childhood habits (see
Montana Cooperative Agreement Report). An additional questionnaire was
administered to parents of participants in this soil ingestion study. It
included questions about foods eaten during the study by participating
children and activities during the days of the study.
- 7 -
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RESULTS
Study population
Three-day stool samples and questionnaires were obtained from 70 children.
Labels on 2 stool samples were lost; the 68 remaining samples were analyzed by
XRF. Sixty-five of these samples were also analyzed by ICP. (Three samples
were lost after XRF was completed.) Unless otherwise noted, all statistics in
this report describe these 65 children. The mean age of children studied was
1.6 years. Sixty-five percent were male (Table 2). There was no significant
difference in age between males and females.
Stool samples
Mean and median daily fecal dry weight were 7.3 grams/day and 6.7 grams/day,
respectively (Table 3). Sixty-five stool samples were analyzed by both XRF
and ICP (in the absence of suitable reference materials). The correlation
between the two methods is good for silicon and titanium but poor for aluminum
(Table 4, Figures 1-3). Analysis of the residuals revealed no systematic
errors. Unless otherwise stated, the ICP values are used in the analyses that
follow.
The mean, median, and geometric mean fecal concentrations of aluminum,
silicon, and titanium are shown in Table 5. The distributions of elements in
the b5 stools are highly skewed; only silicon appeared to oe normally
distributed with logarithmic transformation.
- 8 -
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Soil and dust samples
Front and back yard composite soil samples were obtained for 59 of the o5
children with ICP stool data; dust samples were available for 45 children.
Botn soil and indoor vacuum grab sample data were available for 42 of these
children (Table 6).
Mean, median, and geometric mean soil and dust concentrations and standard
deviations are shown in Table 7. Soil silicon and titanium and dust titanium
concentrations were log normally distributed. Correlations between yard soil
and indoor dust aluminum, silicon, and titanium concentrations were .14, .la,
and .04, respectively.
Estimated soil ingestion by individual elements
Estimates of soil ingestion based on aluminum, silicon, and titanium were
calculated for the 59 children with both composite yard soil and ICF data
(Table a, Plots 4, 5, and b). The arithmetic mean of soil ingestion estimates
based on aluminum (181 mg/day) is similar to the one based on silicon (184
mg/day), but tne estimate based on titanium (1,834 mg/day) is tenfold higher.
As would be expected, the arithmetic mean estimate based on the minimum of the
three estimates for each child (108 mg/day) is lower than that calculated on
any individual element (Table 9, Plot 7).
- 9 -
-------
Soil ingestion estimates calculated with mean soil element concentrations
Soil ingestion estimated with arithmetic mean soil concentrations of aluminum,
silicon, and titanium in all the children's yards (Table 10) is not
appreciably different from soil ingestion estimated with the concentrations in
each child's yard (Table 10). Estimates of soil ingestion based on the
geometric means of soil element concentrations will be lower than those based
on the arithmetic means.
Analysis of dust ingestion estimates
Table 11 shows the estimates of dust ingestion obtained when one assumes that
all of each element present in stool derives from housedust only. Because
concentrations of aluminum, silicon, and titanium in dust tended to be lower
than concentrations in soil, estimates of dust ingestion are higher than
estimates of soil ingestion. In this analysis, the relative amounts of dust
and soil ingestion cannot be estimated.
Questionaire data
There was no apparent relationship between responses to questions about
childhood behaviors that might be associated with increased soil ingestion,
such as furniture mouthing, paint chip ingestion, etc., and estimated soil
ingestion by any of the above methods. Dietary histories obtained by
questionnaire were not complete enough for meaningful analysis.
- 10 -
-------
DISCUSSION
Soil ingestion estimates
On the basis of our data and calculations, we estimated that mean daily soil
ingestion in the East Helena children is between lOtf mg/day (based on the
minimum estimate of all elements for each child) to 1,&34 mg/day (based on
titanium). The estimates of soil ingestion for the children based0on aluminum
and silicon are an order of magnitude less than the estimate based on
titanium. To determine which estimate best represents the amount of soil a
child ingests, we would have to know which element is the best tracer for soil
ingestion and that information is not available.
It is difficult to explain why the soil ingestion estimate based on titanium
is so much larger than that for either of the other elements. Although
titanium is present in paint, houses in East Helena did not have chipping or
peeling paint. One would expect the estimate from aluminum to be highest,
since aluminum occurs so commonly in foods. Titanium does not occur in large
y
quantities in drinking water or foods, including milk, and it is present in
children's vitamins in only minute quantities (personal communication, Squibb
Pharmaceutical Company). It is present in some toothpastes, especially gels,
as the ingredient of lowest concentration (<2.5%); however, the exact amounts
of titanium in toothpaste are proprietary information (personal communication,
Colgate-Palmolive Company). Titanium does not leach from diapers (unpublished
data, Center for Environmental Health, Centers for Disease Control) and is not
present in baby powder (personal communication, Johnson and Johnson Company;.
- 11 -
-------
It is unlikely that stool samples were contaminated with titanium during
processing. (We were unable to elute titanium from plastic gloves, sample
jars, and similar items.) We think it likely that a combination of
differences in absorption or metabolism of these elements in children compared
with adults and unrecognized sources of titanium (in diet or in the laboratory
processing of stools) are responsible for the relatively high soil ingestion
estimate based on titanium.
In addition to the assumption that aluminum, silicon, and titanium are neither
introduced into nor lost from samples during processing and that ail urine and
feces were completely removed from the diapers, we needed to make several
other assumptions in applying our algorithm (Table 12). These are summarized
below.
Sources of Ingested soil and dust
The algorithm assumes that children ingest predominantly soil from their own
yards and that concentrations of elements in composite front and back yard
soil samples are representative of overall concentrations in the yard. In
East Helena, the calculation of soil ingestion with the study population's
mean yard soil concentrations of elements (Table 11) instead of each child's
individual values (Table y) had little effect on estimated soil ingestion; in
Other areas this assumption may be more important.
- 12 -
-------
Because garages, cars, and places in the house that are inaccessible Co
children may be vacuumed with a household vacuum cleaner, grab samples may not
be representative of dust that children may ingest. The magnitude of
housedust ingestion, like that of soil ingestion, is unknown. In East Helena,
dust concentrations of elements (from vacuum cleaner grab samples) tended to
be lower than soil concentrations. Thus, if all of a child's intake of a
given element came from dust, he or she would need to eat much more dust than
soil to achieve a given fecal excretion. Children probably eat a combination
of soil and dust. In East Helena, the algorithm used, whicn does not
distinguish between soil and dust ingestion, will, if dust ingestion is
ignored, overestimate the amount of soil ingestion.
Fecal weight
Dry fecal sample weights in this study were much lower than we had expected.
Some of the reported fecal weights were so low that they were hard to accept
as valid (Table 4). Although we reviewed the diaper processing procedure and
could not identify any step at which stool samples were lost, it is possible
that not all of the stool was recovered from the diapers or that some
specimens were misplaced before the weighing. It is also possible that
parents did not submit all soiled diapers.
- 13 -
-------
Soil ingestion calculated by the algorithm is directly proportional to fecal
weight. Because evidence that the fecal sample weight data were inaccurate,
we chose to use an estimate of 15 grams per day as the average fecal weight in
toddlers. (See page 7 for how this number was derived.) In reality, daily
fecal weight varies among toddlers; use of a single fecal weight estimate for
all children is a potential major source of bias in the calculated ingestion
estimates.
Metabolism and dietary sources of aluminum, silicon, and titanium
Studies have shown that aluminum, silicon, and titanium can be absorbed from
the gastrointestinal tracts of adults, although usually in small
8-14
amounts. Studies of lead metabolism show that absorption of elements
2
nay be significantly different in children and adults. Unless young
children were in negative metabolic balance for these elements, however,
absorption of these elements in urine did not lead to an underestimate of soil
ingestion in our study. In children who are still wearing diapers, mixing of
urine and feces will decrease the importance of gastrointestinal absorption on
soil ingestion estimates obtained by using the algorithm. (Studies in older
children and adults will need to take the possibility of absorption and
urinary excretion into account.)
Contrary to our assumptions, dietary intake of aluminum, silicon, and titanium
is not negligible when compared with potential intake of these elements from
soil. The average adult dietary intakes of aluminum, silicon, and titanium
are 15 rag/day, 7 mg/day, and .3 mg/day, respectively. The presence of
these elements in food will lead to an overestimation of the amount of soil
children eat.
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CONCLUSIONS AND RECOMMENDATIONS
The estimates of soil ingestion generated in this report range over an order
of magnitude. It is difficult to select the best measure of soil ingestion in
toddlers on basis of the results, given the assumptions we had to make for
this estimate. We need a better understanding of the metabolism of aluminum,
silicon, and titanium in children. Studies of aluminum, silicon, and titanium
levels in the feces of volupteers; studies of the excretion of these elements
by hospitalized children; repetition of our study in an area with homogenous
soil, and controlled studies of the methodology used are possible next steps.
Careful attention to study methods and monitoring of dietary intake will
undoubtedly lead to better estimates of soil ingestion. We hope that this
study will stimulate further studies of soil ingestion by young children.
Use of trade names is for identification only and does not constitute
endorsement by the Public Health Service or Dy the U.S. Department of
Health and Human Services.
- 15 -
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Table 1. Recovery of aluminum, silicon, and titanium from spiked pooled stool
samples, analyzed by ICP.*
Element
Amount added
(ug/g)
Number of
samples
Mean amount S.D.
recovered (ug)
iH
Percent
recovered
Method A+ Method BIf.
Aluminum
0
250
500
3
3
5
575
7bZ
1012
52.1
68.5
26.1
y3
94
77
87
Silicon
Titanium
0
750
1500
0
229
456
3
3
7
3
3
5
2053
2753
3075
325
509
766
175.0
151.0
125.0
20.7
31.0
33.3
98
87
92
98
93
63
80
97
* Alternative methods of calculating recovery and their advantages and disadvantages
have been discussed in the literature 15,16 -j^q methods have been used in
this table.
+ Method A:
% recovery
observed spiked conc.
x 100.
observed unspiked conc. + spike conc.
If Method B:
% recovery ¦ observed spiked conc. - observed unspiked conc. x 100.
spike conc.
ug/g » micrograms of element per gram of stool
- 16 -
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Table 2. Age and sex distribution of study participants.
Age (years) Sex
Male Female Total
1.0-1.49 24 11 35
1.5-1.99 7 7 14
2.0-2.4y 9 5 14
2.5-3.0 2 0 2
Total 42 23 65
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Table 3. Mean daily fecal mass for male and female toddlers.
Sex
JL
Mean
(§/d)
Median
(g/d)
Geo.- mean S.D.
(g/d; (g)
Range
(g/d)
Male
42
7.5
7.1
6.9 3.G
1.9-15.7
Female
23
6.9
b.O
6.1 3.4
l.b-17.2
Total
65
7.3
6.7
6.6 3.2
1.8-17.2
g/d = grams per day
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Table 4. Comparison of fecal aluminum, silicon, and titanium concentrations
measured by XRF and ICP.
Mean concentrations (mg/g) Correlation Coefficient
Element XRF ICP (R)
Aluminum 0.47 0.83 0. 19
Silicon 3.24 3.65 0.92
Titanium 0. 29 0.33 0.97
mg/g = milligrams of element per gram of stool
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Table 5. Fecal aluminum, silicon, and titanium concentrations (mg/g) by ICP
for the 65 children with stool ICP data.
Element
Mean
(mg/g)
Median
(rag/g)
Geometric
mean (mg/g)
Standard
deviation
(mg/g)
Range
(mg/g)
Aluminum
0.83
0.53
0.60
0.82
0.11- 5.10
Silicon
3.e>5
2.3b
2.50
3.45
0.66-14.88
Titanium
0.34
0.12
0.08
0.54
<0.01- 2.83
mg/g = milligrams of element per gram stool
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Table t>. Number of soil and dust samples available for the 65 children with
stool ICP data.
Soil Sample
Present Absent Total
Present 42 3 45
Dust sample
Absent 17 3 2U
Total 59 6 65
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Table 7. Aluminum, silicon, and titanium concentrations determined (by XRF)
in composite front-back yard soil samples and dust vacuum cleaner-bag samples.
Geometric Standard
Sample
Mean
Median
mean
deviation
Range
Sample
size
(mg/g)
(mg/g)
(mg/g)
(mg/g)
(mg/g;
Soil
Aluminum
59
6b. 61
66.95
66.03
9.22
45.14-111.41
Soil
Silicon
59
302.52
302.19
301.94
18.34
243.06-332.23
Soil
Titanium
59
2.98
2.93
.42
2.95
2.38- 4.01
Dust
Aluminum
45
33. o5
34. 19
29.91
13.77
5.37- 64.23
Dust
Silicon
45
202.43
202.54
198.77
38.94
115.81-319.41
Dust
Titanium
45
2. 72
2.77
2. b7
0.52
1.23- 3.84
mg/g
= milligrams of
element
per gram of
stool
- 22 -
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Table a. Estimated daily soil ingestion based on aluminum, silicon, and
titanium for the 59 children with both stool ICP and yard soil data.
Estimation
method
Mean Median
(mg/d) (mg/d)
Geometric
mean (mg/d)
Standard
deviation
(mg/d)
Range
(mg/d)
95th per-
centile
Aluminum
181 121
12«
2U3
25-1,324
584
Silicon
184 136
130
175
31- 79y
578
Titanium
1,834 618
401
3,091
4-17,076
9,590
mg/d = milligrams of soil
per day
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Table 9. Estimated daily soil ingestion from the minimum of calculated
ingestion based on aluminum, on silicon, and on titanium.
Element
producing
the lowest 95c^
ingestion
estimate
Number of
children
% of
children
Mean
(mg/d)
Median
(mg/d)
Geometric
mean (mg/d)
S.D.
(mg/d)
Range
(mg/d)
percentile
(mg/d)
Al
21*
30**
100
y3
90
43
2 5-209
204
Si
24*
41**
159
112
11U
168
37-708
675
Ti
14*
24**
34
14
lb
3o
4- 97
97
Total
59
100
108
88
65
121
4-708
38b
*Number of children with the soil ingestion estimate for that element as the iowest of the
estimates for aluminum, silicon, and titanium.
**Percentage of children with the soil ingestion estimate for that element as the lowest o
the estimates for aluminum, silicon, and titanium.
mS/d ¦ Milligrams of soil per day
- 24 -
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Table 10. Estimated daily soil ingestion based on mean soil concentrations*
of aluminum, silicon, and titanium for the 65 children with stool ICP data.
Estimation
Mean
Median
Geometric
S.D.
Range
method
(mg/d)
(mg/d)
(mg/d)
(mg)
(mg/d)
Aluminum
186
120
136
185
26-1,149
Silicon
181
127
127
171
33-738
Titanium
1,705
549
399
2,711
5-14,221
*See Table 7
for mean
soil concentrations.
mg/g = millig
rams of
soil per day
- 25 -
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Table 11. Estimated of daily dust ingestion (assuming no soil ingestion) for
the 45 children with stool 1CP and dust data.
Estimation
method
Mean
(mg/d)
Median
(rag/d)
Geometric
mean (mg/d)
S.D.
(mg/d)
Range
(mg/d)
Aluminum
515
289
326
634
33-3,474
Silicon
286
197
205
287
48-1,265
Titanium
2,1)68
871
510
3,006
5-13,923
mg/d = milligrams of dust per day
- 26 -
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Table 12. Effect of
study.
Assumption
various assumptions
on estimated soil
ingestion in this
Effect on estimated
soil ingestion if
assumption not true
1. Removal of feces and urine from diapers is complete.
Decreased
2. Individual child's yard's composite soil concentra-
tions reflect his or her ingested soil better than
mean soil concentrations for area.
3. Dust ingestion is negligible (effect on sum of dust and
soil ingestion).
4. Daily stool output averages 15 g per child.
5. Al, Si, and Ti are unabosrbed from the g.i. tract.
6. Dietary Al, Si, and Ti are negligible.
7. Aluminum, silicon, and titanium were neither added to
nor lost from the specimens during processing.
Increased
or decreased
Increased
Increased or
decreased
Increased
Decreased
Increased or
decr'eased
- 27 -
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References
1. Lepow ML, Bruckman L, Rubino RA, Markowitz S, Gillette M, Kapish J. Role
of airborne lead in increased body burden of lead in Hartford children.
Environmental Health Perspectives 1974;7:99—102.
2. National Research Council. Lead in the human environment. Washington,
D.C.: National Research Council, 1980.
3. Day JP, Hart M, Robinson MS. Lead in urban street dust. Nature
1975;253:343-345.
4. Kimbrough RD, Falk H, Stehr P, Fries G. Health implications of
2,3,7,8-tetrachlorodibenzodioxin (TCDD) contamination of residential
soil. Journal of Toxicology and Environmental Health 1984;14:47-93.
5. Field AC, Purves D. The intake .
- 28 -
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15. Plaut D, Blanchard R, Mac^ueen J. Letter to the editjr. American
Journal of Medical Technology 1972;38:274-5.
16. Provost LP, Elder RS. Interpretation of percent recovery data. American
Laboratory December 1983:57-63.
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