-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development. U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide range of energy-related environ-
mental issues.
EPA REVIEW NOTICE
This report has been reviewed by the participating Federal Agencies, and approved
for publication. Approval does not signify that the contents necessarily reflect
the views and policies of the Government nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
This document is available to the public through the National Technical Informa-
tion Service, Springfield. Virginia 22161.
-------
EP.A-600/7-84-095a
.^ept^moer 1984
ENVIRONMENTAL ASSESSMENT OF A
FIRETUBE BOILER FIRING
COAL/OIL/WATER MIXTURES
Volume II
Data Supplement
by
R. DeRosier
Acurex Corporation
Energy & Environmental Division
555 Clyde Avenue
P.O. Box 7555
Mountain View, California 94039
Contract No. 68-02-3188
Project Officer
R.E. Hall
Combustion Research Branch
Energy Assessment and Control Division
Industrial Environmental Research Laboratory
Research Triangle Park, North Carolina 27711
OFFICE OF RESEARCH AND DEVELOPMENT
US. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20460
-------
ABSTRACT
This report is a compendium of detailed emission and test data obtained
from field tests of a firetube industrial boiler burning a coal/oil/water
(COW) mixture. The boiler was tested while burning COW fuel, and the COW with
soda ash (sodium carbonate) added to serve as an S02 sorbent. The test data
includes preliminary equipment calibration data, boiler operating data for
both tests, fuel analysis results, and complete flue gas emission measurement
and laboratory analysis results. Flue gas emission measurements included
continuous monitoring for criteria gas pollutants; gas chromatography (GC) of
gas grab samples for volatile organics (Cj-Cg); EPA Method 5 for particulate;
controlled condensation system for sulfur oxide emissions; and source
assessment sampling system (SASS) for total organics in two boiling point
ranges (100 to 300°C and >300°C), organic compound category information using
infrared spectrometry (IR) and low resolution mass spectrometry (LRMS),
specific quantitation of the semivolatile organic priority pollutants using
gas chromatography/mass spectrometry (GC/MS), liquid chromatography (LC)
separation of organic extracts into seven polarity fraction with total organic
and IR analyses of eluted fractions, flue gas concentrations of trace elements
by spark source mass spectrometry (SSMS) and atomic absorption spectroscopy
(AAS), and biological assays of organic sample extracts. All field and
laboratory data for these measurements are included in the volume.
11
-------
CONTENTS
1. INTRODUCTION 1-1
2. PRELIMINARY EQUIPMENT CALIBRATION DATA 2-1
3. BOILER OPERATING DATA 3-1
4. SAMPLING DATA SHEETS 4-1
4.1 CONTINUOUS EMISSION MONITORING DATA (ACUREX) 4-2
4.2 CONTINUOUS EMISSION MONITORING DATA (ADELPHI) 4-8
4.3 FIELD DATA SHEETS FOR SASS AND CONTROLLED
CONDENSATION (ACUREX) 4-9
4.4 SUMMARY OF FIELD DATA FOR METHOD 5 (ADELPHI) 4-28
5. ANALYTICAL LABORATORY RESULTS ' 5-1
5.1 ULTIMATE FUEL ANALYSES 5-2
5.2 PARTICULATE EMISSIONS FROM SASS SAMPLES 5-3
5.3 SULFUR OXIDE EMISSIONS FROM CONTROLLED CONDENSATION
SAMPLES 5-6
5.4 TRACE ELEMENT ANALYSES 5~8
5.5 TOTAL CHROMATOGRAPHABLE ORGANICS (TCO), GRAVIMETRIC
ORGANICS (GRAV), INFRARED (IR) SPECTRA, AND GAS
CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) OF TOTAL
SAMPLE EXTRACTS 5-24
5.6 LIQUID CHROMATOGRAPHY (LC) SEPARATION, INFRARED (IR)
SPECTRA OF LC FRACTIONS, AND LOW RESOLUTION MASS
SPECTROMETRY (LRMS) OF SELECTED LC FRACTIONS AND TOTAL
SAMPLE EXTRACTS 5-35
5.7 G! - Ce CHROMATOGRAPHY 5-60
5.8 RADIOMETRIC ANALYSES 5-72
5.9 BIOLOGICAL ASSAY REPORT 5-74
ill
-------
SECTION 1
INTRODUCTION
The purpose of this data supplement is to document data in greater
detail than was practical in Volume I (Technical Results) of this report. It
is intended to provide sufficient detail for researchers to perform their own
analysis of the data obtained. Readers are referred to the technical volume
for objectives, description of the source emission results, interpretation,
and conclusions.
The remaining sections of this data supplement contain the following
information:
2 Preliminary Equipment Calibration Data
3 Boiler Operating Data. Water, stack, air, and fuel temperatures,
and water and fuel flowrates.
4 Sampling Data Sheets. Continuous flue gas monitor data; operating
data for EPA Method 5 (for particulate mass emissions), controlled
condensation (for SOg and $03 sampling), and SASS (for particulate,
trace element and organic sampling).
5 Analytical Laboratory Results. Ultimate analysis of the
coal-oil-water mixtures used in the test program; SASS particulate
emissions, sulfur oxides emissions hy turbidometric analysis; trace
element emissions by Spark Source Mass Spectrometry (SSMS) analysis and
1-1
-------
Atonic Absorption Spectroscopy (AAS) analysis; Total Chronatographable
Organic (TCO) and gravimetry results; infrared (IR) spectra of total
sample extracts; determination of organic compounds by Gas
Chroriatography/Mass Spectrometry (GC/MS); liquid chromatography (LC)
separation with IR spectra of LC fractions; low resolution mass
spectrometry (LRMS) of selected fractions and total sample extracts;
radiometric analysis results; and biological assay reports on the SASS
train sample extracts.
1-2
-------
I-* 1000
nelfM 1i«w«tu'-«
SECTION 2
PRELIMINARY EQUIPMENT CALIBRATION DATA
K»t»r >
Ortf1c» Nl9**h«11c
'MBIT CjllbritIM N*t*r
CSKtro! KMulr
, JJ
,, -,« ^ j> «
lulb
CALIBRATION DATA
Dry Test
fteUr
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(In. w?.)
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2.6
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2-1
-------
0 , $ ' JO
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PFTtfi CALIBRATION DATA
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2-2
-------
SECTION 3
BOILER OPERATING DATA
Temperatures
Water return
Water supply
-- Stack
Air
Fuel
Flowrates
-- Water
-- Fuel
3-1
-------
BOILER OPERATING CONDITIONS: TEST 1 (COW)a
Temperatures (°F)
Minutes
into
test
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
Water
return
166.6
167.7
170.1
172.2
175.2
177.7
179.1
180.0
180.0
181.0
182.0
183.9
185.8
-_C
195.1
194.4
Water
supply
244
244
248
249
249
252
253
255
255
256
257
260
262
275
270
.9
.0
.8
.2
.7
.6
.3
.8
.0
.6
.7
.2
.8
.2
.4
Stack
276
279
286
287
288
291
292
295
295
297
299
302
306
319
315
Air
77
77
77
74
73
74
74
73
73
73
73
74
73
72
72
Fuel
77
77
78
79
78
78
78
78
78
76
76
76
76
74
75
Coal
40
51
57
56
56
54
59
54
41
42
48
43
38
40
40
Flows
H20 flow
(gal /mi n)
285
290
288
296
297
297
296
297
298
294
296
290
285
--
283
298
.2
.3
.8
.3
.0
.5
.9
.1
.0
.9
.5
.3
.2
.3
.2
Fuel
(kg/I
420
431
438
433
434
431
431
431
430
432
433
435
433
440
438
436
435
f 1 ow
.5
.8
.4
.1
.1
.2
.3
.9
.4
.1
.3
.3
.6
.8
.6
aSummary of computer printout averaged every 20 minutes.
bAveraged using offscale readings = 441 kg/hr.
cUnable to average due to poor quality copy.
3-2
-------
ROILER OPERATING CONDITIONS: TEST 2 (COM+SA)"
Temperatures (°F)
Minutes
into
test
20
40
60
80
100
120
140
160
180
200
Water
return
173
173
172
173
173
176
176
178
181
184
Water
supply
248
249
246
245
244
245
245
245
246
249
Stack
325
339
343
344
348
356
365
373
384
399
Air
76
75
75
74
74
75
75
75
75
76
Fuel
88
89
91
91
90
92
90
91
91
89
Coal
45
48
48
49
'50
51
52
47
49
52
Flows
H20 flow
(gal /"ri n)
277.2
289.8
293.8
294.9
293.9
303.1
306.6
305.8
305.8
308.8
^;h;!;w
430.0
420.1
435.5
438.8
433.0
435.7
440.0
435.3
434.4
389.9
aSummary of computer printout averaged every 20 ninutes,
bAveraged using offscale = 441 kg/hr.
3-3
-------
SECTION 4
SAMPLING DATA SHEETS
Page
4.1 CONTINUOUS EMISSION MONITORING DATA (ACUREX) 4~2
02, C02, CO, NOX
4.2 CONTINUOUS EMISSION MONITORING DATA (AOELPHI) 4-8
N2, 02, Excess Air, C02, H20, S02
4.3 FIELD DATA SHEETS FOR SASS AND CONTROLLED CONDENSATION (ACUREX) 4-9
4.4 SUMMARY OF FIELD DATA FOR METHOD 5 (AOELPHI) 4-28
4-1
-------
i.l CONTINUOUS EMISSION MONITORING DATA (ACUREX)
4-2
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CONTINUOUS EMISSION MONITORING DATA (ADELPHI)
CONTINUOUS MONITORS APELPHIa
Test 1 (COW)
Tine
ninutes
40
6U
80
100
120
140
160
180
20U
220
240
260
280
300
320
340
360
N2
percent
76.8
76.5
76.5
76.6
76.6
76.5
76.5
76.5
C
76.5
76.5
76.4
76.5
76.4
c
76.2
76.3
02
percent
3.3
2.4
2.7
3.1
3.0
3.1
3.1
3.1
c
3.1
3.1
2.8
2.8
2.3
c
2.0
1.8
C02
H20
percent percent
12.7
13.2
13.1
12.9
12.9
12.9
12.9
12.9
C
12.9
12.9
13.1
13.1
13.6
c
13.8
13.9
6.4
6.7
6.6
6.5
6.5
6.5
6.5
6.5
c
6.5
6.5
C
6.6
6.7
6.8
6.9
7.0
S02
ppn
1,324
1,484
1,389
1,299
1,286
1,285
1,296
1,296
__C
1,288
1,309
c
1,338
1,425
1,620
1,640
1,548
E.A.h
percent
19.7
13.9
15.6
18.0
17.8
18.1
18.0
17.9
__c
18.2
18.0
16.5
16.3
12.9
__c
10.9
10.2
Test 2 (COW+SA)
20
40
60
80
100
120
140
160
180
200
76.5
76.6
76.5
76.5
76.5
76.5
76.4
76.4
76.5
76.9
2.9
3.0
3.0
2.8
3.0
2.9
2.7
3.1
3.8
2.7
12.9
13.0
13.1
13.2
13.1
13.2
13.3
13.1
13.1
13.0
6.8
6.5
6.5
6.5
6.5
6.5
6.6
6.5
6.4
6.4
44
138
68
40
39
34
51
30
2
35
16.9
17.7
17.3
16.3
17.9
16.6
15.5
18.5
43.1
15.7
aAverage of previous 20 minutes of data.
Percent excess air 100 (79 x 02) / (%N - 79 x 02),
71 7T
cUnab1e to read from printout.
4-8
-------
4.3 FIELD DATA SHEETS FOR SASS AND CONTROLLED CONDENSATION (ACUREX)
4-9
-------
P 1 ant
Date
f i PI+,
jg-l? -
Sample Location
Sample Type
Run Number /-
Operator
f<
Ambient Temperature_
Barometric Pressure
Static Pressure. (H20)_
Filter Number(s)
Leak Check; Initial at
Final at
Hg,
Hg,
CFM
\»r H
FIELD DATA
Implnger Volumes
Initial Final
$?P
yiTD
3UO
SIHca Gel
.0/
Page ./__ of '-
Probe Length and
Nozzel l.D. (No.)2 (,
Assumed Moisture k
Molecular Height, Dry, (M.)
Meter Box Number^
Meter Coefficient
« Factor Q
K «
K(NdJ*~^
( )
Traverse
Point
Number
Clock Time
(24-hr)
Clock
Sampling
Time, mln
Gas Meter
Reading
Velocity
Head
foPc).
(n. H20
Orifice Pressure
Differential
. In, HzO
Desired
Actual
Stack
Temperature °F
Probe
Imp1 nger
Organic
Module
Oven
Gas Meter
In
Out
Pump
Vacuum
In. Hg
Av
15
'0
a.
66
37
7ft
ol
in
73
SL
77
.51
f/7
3006
6+
/OS
V/7
39.
r/
Comments:
-------
Pane 2of
Traverse
Point
Number
Clock Time
(24-hr)
Clock
Sampling
Time, mln
Gas Meter
Reading
(Vffl). ft 3
Inlt-
Velocity
Head
(6PS).
In. H20
Orifice Pressure
Differential
(AH), In. HgO
Desired
Actual
Temperature °F
Stack
Probe
Impinger
Organic
Module
Oven
Gas Meter
In
Out
Pump
Vacuum
In. Hg
Avg.
VfiP
/SO
5^3-0
-37
50
76
03
M-
Vo?
'7//F
f/O
So
3/5"
//?
,35"
7
11
if-
it!
Run Ho. _/.jr
Date
Sawpl ing Lorat ion
-------
plant
Date
ISOKINECTIC NOZZLE CALCULATION
AND
SAMPLING RATE CALCULATION
Performed by P.
Sample
Test No./Type /-
Nd
/AHTsy
\KTmAP/
where: Nj = Nozzel diameter (Inches)
Average pressure differential across the
orifice meter (in. ^0)
Temperature stack gas, average (°F)
Temperature of gas meter, average (°F)
Stack gas velocity pressure (in ^0)
/ (2*1 V (*^° + 460) V25
\fa*+± <^* 460)(.S ))
AH
Ts
Tm
AP
Nd
;?/
^to
54
Nd
T«
TS
AP
AH
K(Nn)4
4-12
-------
ISOKINECTIC SAMPLING WORKSHEET
P1 ant Ay>PL PA', i1p,v.t c e <. 'i TV, Reformed by
Date 2- l?-<*l
Sample Location
Test No./Type /
K = 782.687 (Cp)2 (1-BWO)2 Ps Md
K 2 M P
o s m
where: K = Contant of fixed and assumed parameters (dimensionless)
Pitot coefficient (dimensionless)
Water vapor in the gas stream
(proportion by volume)
Absolute stack gas pressure (in. Hg)
Molecular vcight, stack gas dry
(Ib/lb-mole)
Orifice coefficient (dimensionless)
Molecular weight, stack gas wet
(Ib/lb-mole) Md(l-BwQ) + 18(BWQ)
Abolute meter pressure (in. Hg)
782.687 (.ViSO2 (l-^oXJ2 &.<,(.) (*trt
(?.-?v^ b».it (&si&
CP
Bwo
PS
"d
KO
Ms
Pm
K
n*>$-
.cV
3o.(.L
3*1.0
S.n^s-
^8r.»^
^0-U(p
30.JS-V
4-13
-------
ISOKINETIC PERFORMANCE WORKSHEET & PARTICIPATE CALCULATIONS
Plant /3?x.j.f±u Z^v, ' A-.VC- /s^Aft A»> Performed by_
Date J-/*-*/
Sample
Test No./Type /-g.
Barometric Pressure (in. Hg)
Meter volume (std),
itec£iy\ /(yOoiu)
17 64 I }{
i)/*.-*,)
"i\
-yaast>/ \i2£Z.) + 46° /
Volume of liquid collected (grams)
Volume of liquid at standard
Vlc x 0.04707
condition (scf)
Stack gas proportion of water vapor
vw std , facr)
W 3 L U WI d tU ^^^^^^^» «^^^^M«M
Molecular weight, stack gas
(Ib/lb-mole)
(% C02x 0.44) + (X 02x 0.32)
(/y.p x 0.44) + fe.:i x 0.32)
Molecular weight, stack gas
(Ib/lb-mole)
Mdfl-B..,-) + 18(8,.-), (a?j^)(
WO WO .^-^^
Absolute stack pressure (in.
. , "stack <1"- H20'
'b I3.6
dry
+ (X N2+ 2 CO x 0.28)
+ (a;^ + - x 0.28)
wet
Hg)
(30-tiM + tit
I J.O
'b
Vm std
Vlc
Vwstd
Bwo
Md
Ms
%
««-.
,..<9
*
o-c#
3«v 5*/»-
«..,»
J3t-> 4-6
7602/5/81/Rev 1
4-14
-------
Temperature stack gas, average (°F)
Stflrlr w»l&6
4$.^*
/. ^«^
=2'?^
7602/5/81/Rev 1
4-15
-------
FIELD DATA
Caye / of
Plant
Date
Sample Location
Sample Type
Run Number 2-
Operator.
Ambient Temperature,
Barometric Pressure
Static Pressure. (H20)
Filter Nunber(s)
Leak Check; Initial at ' Hg,
Final at " Hg.
CFM
CFH
Impinqer Volume's
Initial Final
.5Ofr
.S1TC>
Silica Gel
Probe Length and Type
Nouel l.D. (Ho.).
Assumed Moisture
Molecular Weight. Ory,
Meter Box Number
Meter Coefficient yC = S. 7W
« Factor
K(Nd)
AH = K
,041 cA
(
Clock Time
(34-hr)
Clock
Traverse
Point
Number
Gas Meter
Reading
(Vm). ft
Velocity
Head
(&PS),
In. R20
Orifice Pressure
Differential
(&H). In. H20
Desired
Actual
Temperature °F
Stack
Probe
Implnger
Organic
Module
Oven
Gas Meter
In
Out
Pump
Vacuum
In. Hg
Avg.
>top
/.s
Z5
,3
33(
7/
0.6
335
73
7
"75"
c^'/-^
o-r
f/3
73
5-7
73
33>
70
SG
0-5"
71
/.I
76
67
70
71
Connents:
-------
Page "Z- of 2-
Traverse
Point
Number
Clock Time
(24-hr)
Clock
Sampling
Time, rain
Gas Meter
Reading
JfJW~
.33
70
6S
550
IK)
73
75"
I
H-*
-J
£8
Hun Nu.
Oate
Sdinpling Location
Loniiii'iil v:
-------
Plant_
Date
ISOK1NETIC PERFORMANCE WORKSHEET & PARTICULATE CALCULATIONS
. Performed by -^C,JN
Sample Location
Test No./Type .0 -£?*£
Barometric Pressure (in. Hg)
Meter volume (std),
(±)h*&\
\a/\Tm + 460/
/Q*M)\ /(3/.«0 + <*J# \
17 61 if 13'6
\(_^2fr) / y **±J + 46o /
Volume of liquid collected (grams)
Volume of liquid at standard condition (scf)
Vlc x "0.04707
Stack gas proportion of water vapor
Vw std . ( )
Vw std + Vm std < } + ( >
Molecular weight, stack gas dry
(Ib/lb-mole)
(% C02x 0.44) + (% 02x 0.32) 4 (% N2+ % CO x 0.28)
( HT.O x 0.44) + (}J x 0.32) + (ig-3 + ' x 0.28)
Molecular weight, stack gas wet
(Ib/lb-mole)
Md(l-Bwo) + 18(BWO), (:£*) d-^±) + 18('Q.
3.it>-o4-
MX)> 12.
C,i*i:+.t+iT rCt. 1
;
S)*i';±isn£
.&4
30- -13
39.J.-T
nD- /
7602/5/81/Rev 1
4-18
-------
Temperature stack gas, average (°F)
Stack velocity (fps) .....
_ /rcav9 * 46°
85 19 fC I f/AP 1 / s^ .
w.iy iupj tvars j i p
V s S
/(**«£) + 460
OC 40 l,-fi3j\l \ I . . . . .. , .
' ( H '3^J J(.VM)fe**')
Total sample time (minutes)
Nozzle diameter, actual (inches)
Percent isokinetic (X)
17.33 (Ts + 460)(Vw std + Vm std)
6 Vs Ps Nd2
17.33 (Sr6^+ 460)((/^.^6) + (^-^)
( -»T )(37-J^ )( <5b / )( 2.^.<)
Area of stack (ft2) »= 3.1416
*r2 + 144 , ir (Js~l 144
Stack gas volume at standard conditions (dscfm)
60 (1 - 8 )VS A. / 528 \ / Ps \
wo avg J /T^ ayg + 4go i i 29 92 i
60 (1 - * -_,4j. ^»
* t ^^ ^^
? 7:14-
-7*
£,*£"
^-ci
/%*?
ptri
4-19
7602/5/81/Rev
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD DATA SHEET
Plant
Date
Sample Location
Run No. ,
Operator _T^_..^
Ambient Temperature ~ f*> ^,
Barometric Pressure ~.-/>. 'O~L
Meter Box Number oo
Meter Orifice Coefficient_
Meter a Factor >,oi?
Clock Time
(24-hr)
clock
pling
Time,
min O
Gas
Meter
Reading
(vm). ft3
Init.
Temperature (°F)
Stack
Probe
Filter
Skin
Out
Recirc
Water
Exit
Coil
Dry Gas
Meter
In
Out
6.0
10
10
It
4*0
'T
10
70
Jo
t,*/
1,0
"Z.
I/
1*0
1 1
V*
1*7 V
) to
Average
(eO
1)2.1
1
.0
4-20'
-------
COHTROLLEO CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET
Checkpoint
Supervisor
Initials
QA !
Inspector ;
Remarks
LABORATORY PREPARATION
* Inspect and clean CCC. Both filter holder and CCC
are cleaned with hot chromic acid solution and
D.I. H20.
Rinse with acetone and air dry CCC.
Place Tissuequartz filter in filter housing.
Check seal between end of joint and filter.
Do not use grease on joints.
Inspect and clean all glass joints.
L-/
SITE SETUP-
Rinse the inside of probe prior to run.
Rinse probe with acetone until rinse solution is
clear.
Perform leak test.
Leak rate must be less than 80 ml/min (0.003 cfm).
Thermocouple leads attched to probe and filter.
t CCC water bath held at 60°C (140°F) +1°C.
Leak test train.
Probe temperature maintained at 316°C (600°F)
* Gas temperature out of filter holder held at
228°C (550°F).
Fresh solutions placed in impingers.
Fresh absorbent replaced in final impinger.
Adjust flowrate in system to 8 1pm.
4-21
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET -- Continued
Checkpoint
SAMPLING RUN
Turn vacuum pump on just before inserting probe
in stack.
Check seal between probe and port to prevent any
outside air from entering stack.
Run test for 1 hour or until coils are frosted to
1/2 or 2/3 their length.
* After run, cap both ends of probe and lay in
horizontal position.
Rinse the CCC coils into the modified Erlenmeyer
flask with a maximum of 40 ml D.I. HjO.
Was any of the solution lost ( n ml estimated)?
After probe has cooled, it is rinsed with a maximum of
40 ml D.I. HgO into a 25-ml Erlenmeyer flask.
- Was any solution lost ( & ml estimated)?
Clean support equipment priot to next run.
- Save filter for titration.
Initials
Supervisor
QA
Inspector
C-^~
-
-^
u^
< ""~^
.-^-
<-
Remarks
W
_
Conroents:
4-22
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
LABORATORY CHECKPOINTS
Checkpoint
LABORATORY ANALYSIS
dean glassware prior to titration.
Use Sromphenol Blue indicator.
Is the NaOH buret protected with a CO 2
absorbent tube?
When was NaOH standardized last (Date )?
Filter any solution that has suspended particulate.
t Use same number of indicator drops for each
titration.
Perform indicator blank on a volume of D.I. H20
equal to sample aliquot used.
Indicator blank added to HgSOd mi Hi -equivalents
found.
Perform all analyses in triplicate.
DATA ANALYSIS VERIFICATION
Obtain and titrate test samples from main laboratory.
Initials
Supervisor
QA
Inspector
^
v^-
^
JJ Iff
^^
L^-
-"-
\^~
L^
^
j
Remancs
i
'"""' !
*~
~*~
Comments:
4-23
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD DATA SHEET
Plant
Date
/'t,,,,f
Sample Location
Run No.
Operator
<>Tf>«_)'
Ambient Temperature
Barometric Pressure
Meter Box Number
o o 1
r*~)Q
Meter Orifice Coefficient_
Meter a Factor (.ofL
Clock Time
(24-hr)
clock
pnng
Time,
min
Gas
Meter
Reading
(Vm).
Init.
Temperature (°F)
Stack
Probe
Filter
Skin
Out
Recirc
Water
Dry Gas
Meter
Exit
Coil
In
Out
no
39'if
Average
: 2:
(,0
Tf
71
4-24
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET
Checkpoint
Supervisor
Initials
QA
Inspector
Remarks j
LABORATORY PREPARATION
Inspect and clean CCC. Both filter holder and CCC
are cleaned with hot chromic acid solution and
D.I. H20.
Rinse with acetone and air dry CCC.
Place Tissuequartz filter in filter housing.
Check seal between end of joint and filter.
t Do not use grease on joints.
Inspect and clean all glass joints.
SITE SETUP
Rinse the inside of probe prior to run.
t Rinse probe with acetone until rinse solution is
clear.
Perform leak test.
Leak rate must be less than SO ml/min (0.003 cfm).
Thermocouple leads attched to probe and filter.
t CCC water bath held at 60°C (140°F) +1°C.
Leak test train.
Probe temperature maintained at 316°C (600°F)
+17oc.
* Gas temperature out of filter holder held at
228°C (550°F).
Fresh solutions placed in Impingers.
Fresh absorbent replaced in final impinger.
Adjust flowrate in system to 8 1pm.
A
4-25
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
FIELD CHECKPOINT SHEET -- Continued
Checkpoint
SAMPLING RUN
t Turn vacuum pump on just before inserting probe
in stack.
Check seal between probe and port to prevent any
outside air from entering stack.
Run test for 1 hour or until coils are frosted to
1/2 or 2/3 their length.
After run, cap both ends of probe and lay in
horizontal position.
Rinse the CCC coils into the modified Erlenmeyer
flask with a maximum of 40 ml D.I. HjO.
Was any of the solution lost ( $ ml estimated)?
After probe has cooled, it is rinsed with a maximum of
40 ml O.I. HjO into a 25-ml Erlenmeyer flask.
Was any solution lost ( S? ml estimated)?
Clean support equipment priot to next run.
- Save filter for titration.
Initials
Supervisor
QA
Inspector
-
-
&~- £*-
&~^
~-
c^
L^
^
Remarks
"7 *4- -*_J*__*
U3-\o % Srv*
Comnents:
4-26
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
LABORATORY CHECKPOINTS
Checkpoint
LABORATORY ANALYSIS
Clean glassware prior to titration.
Use 3romphenol Blue indicator.
Is the NaOH buret protected with a C02
absorbent tube?
When was NaOH standardized last (Date )?
Filter any solution that has suspended participate.
t Use same number of indicator drops for each
titration.
Perform indicator blank on a volume of O.I. H20
equal to sample aliquot used.
Indicator blank added to H2$04 milli-equivalents
found.
Perform all analyses in triplicate.
DATA ANALYSIS VERIFICATION
t Obtain and titrate test samples from main laboratory.
Initials
Supervisor
QA
Inspector
-
^-^
//A
^
~
^-
~^
-^"
^
Remar
-------
-.1 SUMMARY OF FIELD DATA FOR METHOD 5 (AOELPHI)
ADELPHI CENTER FOR ENERGY STUDIES
A division of
ADELPHI RESEARCH CENTER, INC.
OR JOHN P OOOHER
DIRECTOR
AOELPHI UNIVERSITY
GARDEN CITY, L.I., NEW YORK 11530
516-294-8700 ext 7618-7619
October 22, 1981
Mr. Bob De Rosier
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
Dear Mr. DeRosier:
Please find enclosed the particulate information you requested:
wt of sample collected 0.7240g
wt of water collected 12g
Dry Gas Volume 6.872 ft3
Stack Temperature 375 °F
Temperature at meter 70 ° F
If you have any questions, please call me.
Sincerely ,
Terry Kanabrocki
/mh
Enclosure
4-28
-------
SECTION 5
ANALYTICAL LABORATORY RESULTS
Page
b.l ULTIMATE FUEL ANALYSES 5-2
5.2 PARTICULATE EMISSIONS FROM SASS SAMPLES 5-3
5.3 SULFUR OXIDE EMISSIONS FROM CONTROLLED CONDENSATION SAMPLES 5~6
5.4 TRACE ELEMENT ANALYSES 5-8
5.5 TOTAL CHROMATOGRAPHABLE ORGANICS (TCO), GRAVIMETRIC ORGANICS (GRAV),
INFRARED (IR) SPECTRA, AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)
OF TOTAL SAMPLE EXTRACTS 5-24
b.6 LiqUID CHROMATOGRAPHY {LC) SEPARATION, INFRARED (IR) SPECTRA OF LC
FRACTIONS, AND LOW RESOLUTION MASS SPECTROMETRY (LRMS) OF SELECTED LC
FRACTIONS AND TOTAL SAMPLE EXTRACTS 5-35
5.7 Cj - C6 CHROMATOGRAPHY 5~60
5.8 RADIOMETRIC ANALYSES 5-72
5.9 BIOLOGICAL ASSAY REPORT 5-74
5-1
-------
s.i "LT:MA:E FUEL ANALYSES
LABORATORY CERTIFICATE
CURTIS & TOMPKIIS'S.LTD.
n-v
»OCiC"f*
- C3H E>S X STTS -CONSULTING
AMPLXRS INSPECTOR*
t»0 DIVISION STREET
AN FRANCISCO. CALIF 94IO3 *'
U.S.A. "
TttvteM M1B! M1-18K1
Jo. SlhlOO
Prelimmirj No. 6b&7
For ACURZX CORPORATION
Report on 5 samples of Fuel Product
Reported 8/13/81
Sampled
Received 7/07/8!
Project No. 773^.12, 7/06/81, Blanket Subcontract RB59186A,
Releese No- 2.
DRY BASIS EXCEPT AS NOTED
COW
813895
1st 2nd 3rd
Test Test Test
COW+SA
613593
1st 2nd 3rc
Test Test Test
Carbon (C), % 78.02
Ei-drogen (H), £ 8.60
Oxygen (0), (by
difference),$ 6.70
Nitrogen (B), % 0.75 0.77 0.75
Sulfur (Z), % 1.93 1.90 1.95
Heating Value:
BTU/Pound 15,985
Specific Gravity,
75°F (es rec'd) ----- l.Ool
Ash,
l+.OO
Moisture ,%
(as rec'd) ....... 15. 60
71.71*
7.83
U.65
0.72 0.72 Q.Ik
1.89 1.90 1.90
13,705
1.131
13.17
15.5U
5-2
SAMPLES DISCARDED 30DAYS AFTER RECEIPT UNLESS OTHERWISE REQUESTED
matt wpliu only to 0i« «m»)« « «»»». to»«rtp«td ««J to «> Meworily hitficifM.or tht quilt) 01 condition of tppvtntly id«ntk*l or
M«MtorATamrtial MMKttoiI fcTS«tt, tfcf p3Jl« «* »h Ubomloty. Utii report » a-bmlned ind .ccepied for tht Mdu«ve u« of th.
uid
condition ffc*l it kw>< to be vwd. In whole or in p*rt, in »ny ad*vtifing or publicity mitta without prior
-------
5.2 PARTICULATE EMISSIONS FROM SASS SAMPLES
5-3
-------
ACUREX
Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CUSTOMER
CMEA
DATE
CUSTOMER CONTRACT NO. 307605..C2
RESULTS REPORT TO C. Castaldlni
ADDRESS .
Adelphi I 23.776 dscm
ACUREX CONTRACT NO A81-04-J1Q4.
TELEPHONE __
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PARAMETER
Ash Weight
398
lOy
18.9052
399
3u
13.8240
420
In
11. 7542
_
Fi 1 ters
2.4102
407
Probe
1.3175
T-. :
!
- :
UNITS
g
01
Form EED-057 4 80
ANALYST _
REVIEWER
-------
ACUREX
Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CUSTOMER QMEA
CUSTOMER CONTRACT NO. 307605.62
RESULTS REPORT TO C. Castaldini
ADDRESS
DATE
ACUREX CONTRACT NO.
TELEPHONE
A81-04-004
Adelphi 2
6.142 dscm
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
Ash weignt
439
lOy
3.2482
438
3p
0.2809
437
lu
0.7805
Filters
15.6518
441
Probe
2.7962
V
g
01
I
01
Ir/irn H ll (f,l 4 BO
ANALYST
Ml VII WbH
-------
5.3 SULFUR OXIDE EMISSIONS FROM CONTROLLED CONDENSATION SAMPLES
CONTROLLED CONDENSATION SYSTEM (CCS)
LABORATORY DATA SHEET
P1 ant
Date
Sample Location _J_j~.
Run No.
Method
Analyst r..:.. ^ * 1~ -
Date Lab Analysis Completed ^ - .. -
Titration Data
-
Titrant
i _^. _
Normality . ,..' Indicator \
Sample
Description
Sample No.
Vol. of Sample r.'x
Vol. of Aliquot
Vol. of Titrant
Used
Average Vol. of
Titrant Used
Probe,
Nozzle
and
Filter
Rinse
;-, 5:7.
? --i
/ 1 . j
.< » "
.cr~
G/R
Coil
Rinse
?i;;vr
", r.~
i C , ^
''" ,<-x
t 5
Impinger
Contents
and
Rinse
* .
, t'-
- ^
-' i - . -
*
, C .V
?-
r.-
L
.-
H20
Blank
*, ;i-,^
_,'*" ;"* -
r
i .
t ^
c ,'
c ;
**'
,f
356
H?02
Blank
x. i t j"~
-'-">
f V J
*N
r - *
Calculations
Vol. of Gas Sampled (VM) -,...: ft3, Avg. Meter Temp (TM) -,. - °Ft
Meter Pressure (P^) .-;'-_ ..- r "Hg, Meter a Factor ,.c , ; . dimensTonless
-
li"' '«. = .:<*'' ' "i --TI*
, ** <*f . , .^ ,
-------
CONTROLLED CONDENSATION SYSTEM (CCS)
LABORATORY DATA SHEET
Date , - , -x
Sample Location _j._-
Run No. . e
.,. Analyst -. . . . " .. ,
Date Lab Analysis Completed _-__
Method
Titrant
Titration Data
- - Normality .< _' Indicator
Sample
Description
Sample No.
Vol. of Sample
Vol. of Aliquot
Vol. of Titrant
Used .
Average Vol. of
Titrant Used
Probe,
Nozzle
and
Filter
Rinse
*;: ^-s-
C ^* i '"^
^
6/R
Coil
Rinse
-"i '- r~-~-
I,..7
-«*
s * »
I s-
* 1 V "**
-x
Impinger
Contents
and
Rinse
'"Cl"
c . r
t *'-
t
n-
/
. t-
H20
Blank
'. "; 1-- "
j.:,-
,i ^
»
:.:
r "
^ «--
-
3%
H202
Blank
-i _ t'- -
-I - -,
r
1
r
' -
Calculations
Vol. of Gas Sampled (VM)
Meter Pressure (P^) . , - -
~~ * ~~ J*l . C V-
ppm 504 =
f ,
^ / » _ '" C '
"**-^ /
-*- -; %.»-'
ppm S02 = _._
,-.'- ft3. Avq. Meter Temp (TM) '--OF,
"Hg, Meter a Factor ,. , j dimensionless
i
48.15 ( , MgSO.)( , TM+460)
PPM_ * ( )
SO. 96 ( , VM)( , PH) * >0!;
^
.o
;-^'-,- 48.15 (,.--, MgS02)(_^, TM+460)
PPM _ * la]
50^ 64 ( ,. ' , VM)( _ .-- , PMJ v- "
£ n n
5-7
-------
~^4CE ELEMENT ANALYSES
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES. 228 NORTH LA SALLE STREET, CHICAGO. ILLINOIS 60601
AREA CODE 312 726-6434
Reply to
Instrument Analysis Division
490 Orchard Street
Goiden, CO 80401
Phone: 303-278-9521
October 29, 1981
Mr. Larry Waterman
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
Re: IAD #97-H479-116-05
Re-logged in from 97-G452-116-30
Analytical Report
In an October 2, 1981, phone conversation (L. Waterman/J. Oldham) we were
asked to analyze five (5) samples for Sulfur that had1>een reported
originally as part of IAD Batch #97-6452-116-30. These samples were either
impinger solutions or ash in which Sulfur had been determined as a major
component by spark source mass spectrometry (SSMS).
Five millilitres of the impinger solutions and sO.5 g of the ash samples were
fused with sodium carbonate in platinum. The fusion was dissolved in hot water,
acidified, and the Sulfur precipitated as barium sulfate which was filtered
out and weighed. Data from these gravimetric determinations were calculated
to give results in weight per cent (wt %) Sulfur and are presented in
Table No. I.
Table No. I
Sample
Adelphi
Adelphi
Adelphi
Adelphi
Adelphi
2
1
- 1
- 2
- 2
lu &
3 &
Imp.
3 &
Imp.
filter
10w
1
10W
1
Sulfur
16
1.1
1.3
11
0.40
If there are any questions concerning these results, please call.
Bruce A. Hale
Section Supervisor
5-8
M. L. Jacobs, Ph.D., Mngr.
Instrumental Analyses Div.'-a"- " ' >\
OVER 40 BRANCH LABORATORIES STRATEGICALLY LOCATED IN PRINCIPAL COAL MINING AREAS,
TIDEWATER AND GREAT LAKES PORTS, AND RIVER LOADING FACILITIES
Charter Member
-------
Reply ro
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES- 729 NORTH LA SALL£ STREET, CHICAGO .LLtNOiS «080' A»EA CODE 313 729-8434
INSTRUMENTAL ANAirSIS DIVISION '4335 WEST 44IH Av£NU£ GOICEN COlOSAOO VjtO' =«CSE ;C3 J78-95J!
To= Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
Date June 23, 1931
P. O. No.: Subcontract SW 59159A
Sample No.: 813896 SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
SPAR
Adelphi (OW(42/
42/16)
Analyst J. Oldham
IAD No 97-G452-116-30
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium 0.7
Thorium 0.4
Bismuth
Lead 4
Thallium 0.4
Mercury NR
Gold
Platinum
Indium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
Dysprosium
ELEMENT
Terbium
Gadol inium
Europium
Samarium
Neodymi urn
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodi urn
Note: Sar
CONC.
0.05
0.4
0.6
0.3
1
2
22
0.3
0.4
0.05
STD
0.04
0.08
nple thei
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
anally ashed at
CONC
1
0.5
0.6
3
8
2
0.7
3
4
0.6
1
6
3
8
2
MC
10
6
ELEMENT
Vanadium
Ti tanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Al uminum
Magnesium
Sodium
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryll ium
Lithium
Hydrogen
CONC.
15
MC
0.3
MC
MC
1
MC
35
MC
>20
92
>51
=7
NR
NR
NR
2
0.3
1
NR
450"" for 1 hour prior to
STD Internal Standard
NR Not Reported anal vsi s
All elements not detected<0.02ppm y Approved:
MC - Maior Component >100ppm
INT interference 3 y
-------
Repiv to
COMMERCIAL TESTING & ENGINEERING CO.
3EHERAL OFFICES 238 NORTH LA SALLE STREET, CHICAGO. ILLINOIS 80801 AREA CODE 312 72«-8«34
INSTRUMENTAL ANALYSIS DIVISION. '4335 WEST MTH AVENUE GOLDEN, COLORADO 80401 'HONE 303-278-9521
TO: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
P. O. No.: Subcnontract SW 59159A
Sample No.;
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Iridium
Osmi urn
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
zTt
M«fCI '^0*
Adelphl-1 3+ SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
lOy 2.0456/
32 7292q CONCENTRATION IN PPAA WEIGHT
CONC.
80
17
0.7
420
10
NR
2
0.3
1
0.2
2
0.3
2
3
Dysprosium 4
STD Internal Standard
NR Not Reported
All elements not detected <
MC Maior Component
(NT Interference
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymi urn
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodi urn
O.lppm
CONC.
2
5
2
19
25
12
53
42
430
3
2
0.3
0.7
0.6
STD
0.7
<0.2
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromi urn
5-10
Approved:
CONC.
10
12
150
30
200
35
20
110
43
9
30
150
31
38
7
MC
310
100
Date- June 18, 1981
Analyst- J- Oldham
IAD No. 97-G452-116-30
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryl 1 i urn
Lithium
Hydrogen
CONC.
460
MC
53
MC
MC
51
>570
MC
MC
>91
MC
>230
= 14
NR
NR
NR
21
2
13
NR
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES 336 NORTH LA SALLE STREET. CHICAGO ILLINOIS 60601 AREA CODE 312 *2»8*34
INSTRUMENTAL ANALYSIS DIVISION. '4335 WEST 44TH AVENUE &CHDEN. COLORADO 80*01 PHQNE 5032789521
To: Mr. R. Belletto
Acurex Corporation
4BS uyae Avenue
Mountain View, CA 94042
P. 0. No,: Subcontract SW 59159A
Sample No.:
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Iridium
Osmi urn
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
Dysprosium
Adelphi-1
Filter +lu
14/1644 g
CONC.
68
18
0.7
230
10
NR
5
0.8
2
0.5
6
0.8
4
5
8
STD Internal Standard
NR Not Reported
All elements not detected <
MC - Major
Component
SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
1 .74947
CONCENTRATION IN PPM WEIGHT
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymi urn
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
O.lppm
CONC.
1
5
2
20
27
22
120
100
MC
6
2
6
4
STD
0.3
1
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromi urn
5-11
Approved: /.
/
CONC.
t
27
22
62
140
390
83
17
12
160
48
64
160
58
400
33
MC
MC
280
J/'Trf (
/I '
Date June 18, 1981
Analyst J. Oldham
IAD No 97-G452-116-30
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryl 1 ium
Lithium
Hydrogen
./ /^^ s.
CONC
480
MC
56
MC
MC
24
>600
MC
MC
>96
MC
>250
=150
NR
NR
NR
91
16
140
NR
^^A
INT Interference " ( / > '
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES- 128 NORTH LA SALLE STREET, CHICAGO ILLINOIS 80601 AREA COOE 312 728-B434
INSTRUMENTAL ANALYSIS DIVISION. '4335 W£ST 44TW AVENUE. GOLDEN. COLORADO 80401 PHONE 303-278-9521
To: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
P. O. No.: Subcontract SW 59159A
Sample No.: Adelphi-1 XAD SPARK SOURCE AAASS SPECTROGRAPHIC ANALYSIS
3/130 g
3 CONCENTRATION IN PPM WEIGHT
Date. June 18, 1981
Analyst J. Oldham
IAD No 97-G452-116-30
ELEMENT CONC.
Uranium
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES- 228 NORTH LA SALLE STREET. CHICAGO ILLINOIS 80801 AȣA CODE 3'2 738-8434
INSTRUMENTAL ANALYSIS DIVISION '4335 WIST 44TH AVENUE GOlOEN COLOBAOO 40401 PHONE 303-378-95J'
To: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
Date June 18, 1931
Analyst J. Oldham
P. O. No.: Subcontract SW 59159A
Sample No.: Adelphi-1 ImplSPARK SOURCE AAASS SPECTROGRAPHIC ANALYSIS IAD No 97-Q452-116-30
100/1780 ml CONCENTRATE IN ug/ml
ELEMENT CONC.
Uranium <0.003
Thorium <0.004
Bismuth
Lead 0 . 3
Thallium
Mercury NR
Gold
Platinum
Iridium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
Dysprosium
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymi urn
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
CONC.
<0.001
£0.001
0.02
<0.001
<0.001
<0.001
0.004
0.004
STD
<0.001
0.02
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC. ELEMENT
Vanadium
0 2 Titanium
Q 002 Scandium
0.007 Calcium
<0.001 Potassium
0.003 Chlorine
<0.001 Sulfur
<0.008 Phosphorus
<0.01 Silicon
0 003 Aluminum
0.001 Magnesium
0.004 Sodium
MC Fluorine
0.6 Oxygen
MC Nitrogen
0.002 Carbon
MC Boron
0.5 Beryllium
MC Lithium
Hydrogen
CONC
0.009
0.05
<0.001
0.7
0.7
0.03
>3
0.2
0.4
0.2
0.08
>1
=0.2
NR
NR
NR
0.001
0.06
NR
STO Internal Standard
NR Not Reported
All elements nor detected< O.OOlug/ml
MC - Major Component >10ug/ml
INT Interference
5-13
Approved:
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES: 238 NORTH LA SALLE STREET. CHICAGO ILLINOIS 80601 AREA CODE 311 7S«-8«34
INSTRUMENTAL ANALYSIS DIVISION. '4335 WEST 44TH AVENUE. GOLDEN. COLORADO 80401. PHON6 303-J78-952I
To: Mr. R. Belletto
Acurex Corporation -
485 Clyde Avenue Date June 19, 1981
Mountain View, CA 94042
Analyst- J, Old ham
P, O. No.: Subcontract SW 59159A
Sample No.: Adelphi-1 Imp SPARK SOURCE AAASS SPECTROGRAPHIC ANALYSIS 'AD No. 97-G452-116-30
1 Blank-290ml CONCENTRATION IK pg/wl
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thai 1 i um
Mercury
Gold
Platinum
Iridium
Osmi um
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Holmium
Dysprosium
CONC. ELEMENT CONC.
< 0.007 Terbium
<0.009 Gadolinium
Europium
<0.004 Samarium
Neodymi um
NR Praseodymium
Cerium
Lanthanum
Barium 0.005
Cesium
Iodine
Tellurium
Antimony
Tin
Indium STD
Cadmium
Silver 0.2
Palladium
Rhodi um
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttri um
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
CONC. ELEMENT
Vanadium
Titanium
Scandium
0.002 Calcium
Potassium
<0.001 Chlorine
Sulfur
0.003 Phosphorus
<0.003 Silicon
*0.003 Aluminum
Magnesium
<0.001 Sodium
0.07 Fluorine
0.006 Oxygen
0.004 Nitrogen
<0.001 Carbon
0.03 Boron
0.001 Beryllium
0.01 Lithium
Hydrogen
CONC.
<0.001
0.003
<0.001
0.3
0.2
0.02
2
0.2
0.3
0.05
0.1
>3
10.04
NR
NR
NR
<0.001
NR
STO Internal Standard
NR Not Reported
All elements not detected< Q.OOlyg/ml
MC Maior Component >10iig/fnT
!NT Interference
5-14
Approved:
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
QENERAU OFFICES- 228 NORTH LA SALLE STREET CHICAGO ILLINOIS 90601 AREA COOE l!3 '28.843*
INSTBUMENIAl ANALYSIS DIVISION, U33S WEST «TH AvENUf OOltEN COLORADO 80401 SHONE 303 278-9521
T°: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
P. O. No.: Subcontract SW 59159A
Sample No.: 813892 Adel phiSRARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
(OW+SA(38.6/39
.2/14.5/7.7)
Date June 18, 1981
Analyst J. Old ham
IAD No.- 97-G452-116-30
CONCENTRATION IN PPM WEIGHT
ELEMENT CONC.
Uranium 14
Thorium 4
Bismuth
Lead 2
Thallium 100ppm
INT Interference
Heterogeneous
5" 15
Approved:
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
(36NERAL OFFICES aS8 NORTH LA SAILE STREET, CHICAGO. ILLINOIS 80901 AREA CODE 312 728-8*3*
iNSTRUMENTAl ANALYSIS DIVISION. '*33S WEST MTH AVENUE. GOLDEN. COLORADO 80*01 PHONE 303-278-9521
Mr. Jim Steiner
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
P.O. NO.; Subcontract SW 591 59A
Sample No.:Adelphi-2 $pARK SOURCE ,^55 SPECTROGRAPHIC ANALYSIS
3+IOy """ r.H.
CONCENTRATION IN PPM WEIGHT
Date:july 24, 1981
Analyst- j. oidham
IAD No- 97-G452-1 16-30
ELEMENT
Uranium
Thorium
Bismuth
Lead
Thallium
Mercury
Gold
Platinum
Iridium
Osmi urn
Rhenium
Tungsten
Tantalum
Hafnium
lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
CONC.
34
4
£0.2
440
3
NR
0.9
0.2
2
0.3
0.9
1
Dysprosium 2
STD Internal Standard
NR - Not Reported
All elements not detected <
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymi urn
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmi urn
Silver
Palladium
Rhodium
0.1 ppm
CONC.
0.4
3
0.6
5
5
2
45
44
450
1
2
0.3
0.6
0.6
STD
0.3
2
5-16
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromi ne
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobalt
Iron
Manganese
Chromium
4i
Approved: ///>
CONC.
24
5
62
31
68
21
21
12
20
10
14
63
29
99
2
MC
320
49
ys/
f , X j
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodium
Fluorine
Oxygen
Ni trogen
Carbon
Boron
Beryllium
Lithium
Hydrogen
//
//^^JSZ^nf*--^-
CONC.
480
900
6
MC
MC
540
>590
410
MC
>95
MC
>240
= 8
NR
NR
NR
72
2
13
NR
&
MC AAaior Component
!NT Interference
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
GENERAL OFFICES- J28 NORTH LA SALLE STREET. CHICAGO ILLINOIS 80601 AREA CODE 312 '56.8*31
INSTRUMENTAL ANALYSIS DIVISION. '4335 WEST 44TM AVENUE GOLDEN COLORADO 80401 'HONE 303-278-9521
To: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
P. O. No.: Subcontract SW 59159A
ztt.
Date June 19, 1981
Analyst J. Oldham
Sample No.: Adelphl-2 1
+ Filter 1
16.4323g
ELEMENT CONC.
Uranium 8
Thorium
Bismuth
Lead 170
Thallium 2
Mercury NR
Gold
Platinum
Iridium
Osmium
Rhenium
Tungsten 2
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
Dysprosium
STD - Internal Standard
NR Not Reoorted
All elements not detected <
MC Major Component
INT Interference
y SPARK SOURCE AAASS SPECTROGRAPHIC ANALYSIS
CONCENTRATION IN PPM WEIGHT
ELEMENT
Terbium
Gadolinium
Europium
Samarium
Neodymium
Praseodymium
Cerium
Lanthanum
Barium
Cesium
Iodine
Tellurium
Antimony
Tin
Indium
Cadmium
Silver
Palladium
Rhodium
0.1 ppm
CONC. ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
0.5 Strontium
5 Rubidium
4 Bromine
220 Selenium
0.6 Arsenic
0.4 Germanium
<0.1 Gallium
0.4 Zinc
0.3 Copper
STD Nickel
Cobalt
0.4 Iron
Manganese
Chromium
5-17
,X
Approved: ^ /
/
CONC.
f
25
1
3
3
15
20
20
5
25
5
7
26
12
17
2
MC
20
83
f
7J/J*
/ i
IAD No. 97-G452-116-3C
ELEMENT
Vanadium
Titanium
Scandium
Calcium
Potassium
Chlorine
Sulfur
Phosphorus
Silicon
Aluminum
Magnesium
Sodi urn
Fluorine
Oxygen
Nitrogen
Carbon
Boron
Beryl 1 ium
Lithium
Hydrogen
f
/? '
s
y^-^-^^c/~
CONC
68
270
<0.1
MC
MC
MC
>560
160
MC
>90
420
>230
-140
NR
NR
NR
21
0.3
6
NR
/?
'Is i
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
OENERAL OFFICES 538 NORTH LASALLE STREET. CHICAGO. ILLINOIS 806O1 AREA CODE 311 758-843*
INSTRUMENTAL ANALYSIS DIVISION, '4335 WEST 44TH AVENUE. GOLDEN. COLORADO 80401. PHONE- 303-278-9521
Mr. R. Belletto
485 Clyde Avenue
Mountain View, CA 94042
Date: June 19, 1981
P. O. No.: subcontract SW 59159A
Sample No.: Adelphi-2 SPARK SOURCE AAASS SPECTROGRAPHIC ANALYSIS
XAD 3/130g
y CONCENTRATION IN PPM WEIGHT
Analyst- J. Qldham
)AD No. 97-G452-116-30
ELEMENT CONC.
Uranium
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
SENERAL OFFICES: 326 NORTH LA SALLE STREET. CHICAGO ILLINOIS 80601 AREA CODE 312 '26-9*34
INS1RUMNTAI ANALYSIS DIVISION. '4335 WEST "IH AVENUE GOLCEN. COLORADO 80^01 ?MONE 303278.9521
To: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
June 13, 19?:
P. O. No,: Subcontract SW 59159A
Sample No.: Adel phi -2 lstSPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
Imp 100/940ml
CONCENTRATION IN pg/ml
Analyst J. Oldham
IAD No 97-G452-116-30
ELEMENT CONC.
Uranium 4
= 0.1
NR
NR
NR
<0.001
o.o:
NR
STD Internal Standard
NR - Not Reported
All elements not detected< O.QOlyg/ml
AAC - Major Component >10ug/ml
(NT Interference
Heterogeneous
5~ 19 Approved:
-------
COMMERCIAL TESTING & ENGINEERING CO.
AENEOAl OFFICES 356 NORTH LA SALLE STBEET. CHICAGO ILLINOIS 8O80I AREA CCOE 315 119-943*
INSTtUMENTAl ANALYSIS DIVISION. '4J3S WEST 44TH AVENUE. GOLDEN. COLORADO B0401 PHOHl 303.27S.9S2I
Reply to
0 Mr. R. Belletto
Acurex Corporation
435 Clyde Avenue
Mountain View, CA 94042
P. O. No.: Subcontract SW 59159A
Sample No.: CMEA Filter SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS IAD No 97-452-116-30
Datl> June 23, 1981
Analyst J. Oldham
Blank
ELEMENT CONC.
Uranium < 0.001
Thorium < 0.002
Bismuth
Lead 0.003
Thallium
Mercury NR
Gold
Platinum
Iridium
Osmium
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Holmium
Dysprosium
STO Internal Standard
NR - Not Reported
Atl elements not detected <
MC Maior Component
INT Interference
2
CONCENTRATION IN yg/CITl
ELEMENT CONC.
Terbium
Gadolinium
Europium
Samarium
Neodymi um
Praseodymium
Cerium <0.001
Lanthanum <0.001
Barium 0.02
Cesium
Iodine <0.001
Tellurium
Antimony
Tin
Indium STD
Cadmium
Silver
Palladium
Rhodi um
ELEMENT
Ruthenium
Molybdenum
Niobium
Zirconium
Yttrium
Strontium
Rubidium
Bromine
Selenium
Arsenic
Germanium
Gallium
Zinc
Copper
Nickel
Cobal t
Iron
Manganese
Chromi um
* Heterogeneous
2 5-20
0.001 yg/cm Approved: ^/
t
CONC. ELEMENT
Vanadium
Titanium
Scandium
*0.005 Calcium
< 0.001 Potassium
0-002 Chlorine
<0.001 sulfur
0-04 Phosphorus
<0.001 silicon
<0-001 Aluminum
Magnesium
< 0.001 sodium
°-01 Fluorine
°-002 Oxygen
0.002 Nitrogen
°-001 Carbon
°-05 Boron
< 0.001 Beryllium
<0-001 Lithium
Hydrogen
W^&~
CONC
cO.OOl
0.02
<0.001
MC
>2
0.1
0.02
0.3
MC
>0.2
0.8
0.5
=0.06
NR
NR
NR
0.2
<0.001
0.002
NR
^^
-------
Reply to
COMMERCIAL TESTING & ENGINEERING CO.
OENE9AL OFFICES 331 NO'TM L» »*LLE ST»EET. CHICAGO IH.INOIS »0«0i ABE* CODE Jl3 ?3(-«*3<
IMSTtUMENIAl ANALYSIS DIVISION. 'AUS WEST M'M AV(Mu( GOlCtN COIOSAOO KMOI VMOME M3 J7I-9S2!
TO: Mr. R. Belletto
Acurex Corporation
485 Clyde Avenue
Mountain View, CA 94042
p O. No.: Subcontract
S»mple No.: XAD Blank
SW 59159A
SPARK SOURCE MASS SPECTROGRAPHIC ANALYSIS
CONCENTRATION IN PPM WEIGHT
Date June 23, 1981
Analyst J. Oldham
(AD No 97-G452-116-30
ELEMENT CONC
Uranium <0.3
Thorium <0.4
Bismuth
Lead 0.2
Tha 11 i urn
Mercury NR
Gold
Platinum 0.5
Iridium
Os mi urn
Rhenium
Tungsten
Tantalum
Hafnium
Lutetium
Ytterbium
Thulium
Erbium
Hoi mi urn
Dysprosium
ELEMENT CONC.
Terbium
Gadol inium
Europium
Samarium
Neodymi um
Praseodymium
Cerium
Lanthanum 0.1
Barium n
Cesium
-------
ACUREX
Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CMEA
CUSTOMER
CUSTOMER CONTRACT NO. 307605.62
RESULTS REPORT TO C. Castalo'inl
ADDRESS
DATE -
ACUREX CONTRACT NO. A81-04-004
TELEPHONE
Adelphi 1 23.776
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PARAMETER
Hq Aliquot
Hq Blank
Hq
As Aliquot
As
Sb Aliquot
Sb
398
10u+3u
16
< 10
0.011
_
.
_
_
420
Filter +lu
830
< 10
0.28
_
_
_
424
XAD
7
8
-------
ACUREX
Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CUSTOMER CMEA
CUSTOMER CONTRACT NO. 307605.62
RESULTS REPORT TO C. Castaldini
ADDRESS
DATE
ACUREX CONTRACT NO. A81-04-004
TELEPHONE
Adelphi 2 6.142 dscm
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PAPAMPTFR
Hg Aliquot
Hg blank
Hg
As Aliquot
As
Sb Aliquot
Sb
439
10y+3uFHR
< 10
< 10
< 10
_
_
-
-
437
Filter+ly
< 10
< 10
< 10
-
_
_
_
436
XAO
38
8
63
_
_
_
_
440
Imp 1
440
170
0.33
_
_
_
_
448
Imp 2+3
14
50
< 0.2
< 10
< 2
< 10
< 2
-
ng
ng
ug/dscni
ug/L
ug/dscm
ug/L
ug/dscm
en
i
CO
I I,IHI I I ll if,' -1 Wi
ANAI YSI
Ml VII WI.H
-------
TOTAL CHROMATOGRAPHABLE ORGANICS (TCO), GRAVIMETRIC ORGANICS (GRAV),
INFRARED (IR) SPECTRA, AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)
OF TOTAL SAMPLE EXTRACTS
ACUREX
Corporation
Energy & Environmental Division
May 22, 1981
Lab ID Number: A81-04-004
Customter P.O. Number: 307605
CMEA
MS #2-2260
Attention: C. Castaldini
Sample: 5 SASS Trains, received April 7, 1981
The above referenced samples were received and analyzed per Level 1 protocol
Arsenic and antimony were determined by furnace AAS as per directive of
B. Higginbotham.
Polynuclears were determined by a modified method 625. 1 ul of a sample
was injected-on a SE-54 J+W 30 meter capillary column using Grob injection.
The column was held at 35° C for 5 minutes then ramped at 10° C/minute to
300° C.
Prepared by: ^-rug /t*6g>faf Athorized by:_
Greg\N/col1 William L. Fitch, Ph.
Manager, Organics Technical Director
5-24
485 CLYDE AVENUE. MOUNTAIN VIEW. CA 94042 PHONE (415) 964-3200 TELEX: 34-6391 TWX 910-3796593
-------
£\ACUREX
"ITA Corporation
ANALYSIS LABORATORIES
CUSTOMER
DATA REPORTING FORM
CMEA
DATE
CUSTOMER CONTRACT NO.
307605.62
RESULTS REPORT TO r. fa«tt-al/Hn1
ADDRESS
Adelphl 1 23.776 dscm
ACUREX CONTRACT NO.
TELEPHONE
A81-04-004
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PAP* UPTCti
GRAV Aliquot
GRAV Blank
GRAV
TCO Aliquot
TCO Blank
TCO
Fluoranthene Aliquot
Phenanthene Aliquot
Fluoranthene
Phenanthene
Other Poylnuclears
398
10p+3p
< 4
< 4
< 200
_
_
_
< 1
< 1
< 0.05
< 0.05
< 0.05
420
Filter +Tu
< 4
< 4
< 300
M
.
< 1
< 1
< 0.07
< 0.07
< 0.07
424
XAO
87
19
2900
1.4
1
< 40
1
3
0.05
0.1
< 0.05
427
OMC
< 4
< 4
< eoo
< 0.1
< 0.1
< 4.
< 1
< 1
< 0.04
< 0.04
< 0.04
mq
mg
ig/dscm
mg
mg
jg/dscm
ng
ng
g/dscm
g/dscm
g/dscm
UI
I
to
en
4 SO
ANALYST
HEVItWER
-------
IR REPORT
SAMPLE:
A81-04-004-424 Adelphi I XAD
INM Rmtaf
- U.'1)
3460-3040
2910
2850
1770
1468
1450
1280
1130
1080
970
Intimity
w
s
s
s
m
m
s
s
s
m
AMignnwnt Comrrwnti
0-H (COOH)
C-H Alkane
C-H Alkane
C=0 (COOH)
C-H Alkane
C-H Alkane
C-0 (COOH)
C-0
C-0
C-C Alkane
__
5-26
-------
IR REPORT
SAMPLE:
A81-04-004-398 Adelphi 1 10u+ 3-
ta-'1)
limrniry
CommwiB
3440-2960
w
0-H (COOH)
2910
C-H Alkane
2850
C-H ATkane
5-27
-------
R REPORT
lAMPLf: A81 -04-004-420 Adelphi 1 Filter + ly
*-£*-
3480-2960
2920-2360
imtmity
m
s
AMignmtnt Cemmtntt
0-H (COOH) Weak Spectrum
C-H Alkane
1
*.
5-28
-------
IR REPORT
SAMPLE: AST-04-004-427 Adelphi 1 OMC
T
Inttmity
Anigpnwnt
Ce«nni«nO
3540-3000
m
0-H (COOH)
Weak Spectrum
2920-2320
C-H Alkane
5-29
-------
ACUREX
Corporation
ANALYSIS LABORATORIES
DATA REPORTING FORM
CMEA
DATE
CUSTOMER
CUSTOMER CONTRACT NO. 307605^62 ACUREX CONTRACT NO.
RESULTS REPORT TO C. Castaldini TELEPHONE
ADDRESS
Adelphi 2 6.142 dscm [
A81-04-004
SAMPLE ID (CUSTOMER)
SAMPLE ID (LAB)
PARAMETER
GRAV Aliquot
GRAV Blank
GRAV
TCO Aliquot
TCO Blank
TCO
Phenanthene Aliquot
Phenanthene
Other Polynuclears
439
10u+3u+FH[
8
< 4
2000
-
-
-
< 1
< 0.3
< 0.3
437
lu+Ftltei
7
< 4
2000
-
-
-
< 1
< 0.3
< 0.3
436
XAD
48
19
5000
0.3
1
< 200
4
0.7
< 0.2
435
OMC
< 4
< 4
< 700
< 0.1
< 0.1
< 20
< 1
< 0.2
< 0.2
_ _ .
_^^^«^^__
UNITS
mg
mg
ug/dscm
mg
mg
ug/dscm
ng
ug/dscm
ug/dscm
Ul
I
Form EED-057 4 80
ANALYST _
REVIEWER
-------
IR REPORT
SAMPLE:. AST-04-004-439 Adelphi 2 10u+3y
Www Nvntar
- <-1>
3620-2980
2910
2840
730
Intimity
w
s
s
w
Anignmwtt Comments
0-H (COOH)
C-H Alkane
C-H Alkane
C=0 (COOH)
..
5-31
-------
IR REPORT
SAMPLE:
A81-04-004-437 Adelphi 2 Filter + In
WmNMftfc*
. fa-'1)
3460-2960
2920
2840
noo
970
inanity
w
5
S
m
w
Assignment Cemtntna
0-H (COOH)
C-H Alkane
C-H Alkane
C-0
C-C Alkane
*
5-32
-------
IR REPORT
SAMPLE:
HIM Nwntar
<-1)
3440-3000
2920
2840
1730
1270
1070
970
Intimity
w
s
s
m
w
w
w
Acsignmwtt Comments
0-H (COOH)
C-H Alkane
C-H Alkane
C=0 (COOH)
C-0
C-0
C-C Alkane
t
v
5-33
-------
R REPORT
SAMPLE: A81 -04-004-435 Adelphi 2 OMC
"^X"
3440-2980
2920-2390
'
Inttmity
S
m
Anignmwit Cemmmti
0-H (COOH) Weak Spectrum
C-H Alkane
.
5-34
-------
5.6 LIQUID CHROMATOGRAPHY (1C) SEPARATION, INFRARED (IR^ SPECTDfl OF LC
FRACTIONS, AND LOW RESOLUTION MASS SPECTROMETRY (LR*S) ^ r.ELECTE-> LC
FRACTIONS AND TOTAL SAMPLE EXTRACTS
5-35
-------
SAMPLE: Adelphi 1 XAD #424
Total Sampla1
TakaniarLC*
Racovmd^
TCO
«t
< 1
-
-
CRAV
*
68
34
23
TCO + GRAV
Total m|
68
34
23
COMMtlBtlCN
mg/dscm
2.9
1.4
0.97
en
i
CO
Oi
FnctlM
1
2
3
4
6
B
1
Sum
TGO In mi
Found In
Friction
Blink
Cor-
ractid
.
Total4
CRAV in m|
Found In
Fraction
7
6.0
5.2
1.8
<0.8
3.2
1.4
25
Blank
<1
<0.8
=0.8
<1.2
<0.8
<0.8
<0.8
<6
Cor-
rtrtad
7
6.0
5.2
<0.8
<0.8
3.2
1.4
23
Tola!4
14
12
10
< 2
< 2
6
3
45
TCO *
CRAV
Total mi
14*
12
10
<2
<2
6
3
45
Caneantratlan
mg/dscm
0.59
0.50
0.42
< 0.08
< 0.08
0".25
0.13
1.9
1. Quantity in antln tampto, ditfrmlnid bafora LC
2. Portion of whola nmpla utid for LC. actual n»|
X Quantity rtcovartd from LC column, actual m|
4. Total m| computed bach to total nmplt
-------
IR REPORT
AMPLE:
Adelphi 1 XAD 424 FT
Ilfan Bomb*
< I-'1)
2920
2840
1740
1270
.
t-
_
_
_
fmraity
s
s
Auigprntnt CommmB
CH- Aliphatic hydrocarbons
CH Aliphatic Hydrocarbons
m | C=0 Aliphatic hydrocarbons
m
-
--
CO Aliphatic hydrocarbons
"
..
-- ._
.
.-
5-37
-------
IR RE?CHT
SAMPLE: Adelphi 1 XAD 424 F2
Wm 1um6«f
(-')
2920
2840
1730
1270
intimity
S
S
S
S
A*signm*nt Commmti
CH Aliphatic oxygen compounds
CH Aliphatic oxygen compounds
C=0 Aliphatic oxygen compounds
CO Aliphatic oxygen compounds
»*
5-38
-------
IR REPORT
SAMPLE: Adelphl 1 XAD 424 F3
WIM Numbw
I-1)
2920
2860
1730
1280
Imtmity
S
S
S
S
Assignment Comments
CH Aliphatic oxygen compounds
CH Aliphatic oxygen compounds
C=0 Aliphatic oxygen compounds
CO Aliphatic oxygen compounds
*
_.
-
5-39
-------
IR REPORT
SAMPLE: Adelphl 1 XAD 424 F4
Wen Horab«
Imsmity
Auignnwnt
Comm«nt»
No Peaks
5-40
-------
IR REPORT
SAMPLE:
Adelphi 1 XAD 424 F5
Wm Nnmbi
- to'1)
tnttraity
Assignment
Commfenti
No Peaks
5-41
-------
IR REPORT
KAMPLE:
WfM iBtnNr
- fa-*1)
2860
1110-1070
.
_
.:
-'
Adelphi 1 XAD
Imtraity
s
m
"
424 F6
_
Auignmtnt Commanti
CH Alcohols
CO Alcohols
.
.,
m..
-"
*" __
-
"
_ , .__
5-42
-------
1R REPORT
: Adelphl 1 XAD 424 F7
tomb*
C-'1)
Inttraity
Commtna
No Peaks
5-43
-------
LRMS REPORT
SAMPLE: 424 FT Adelphi 1 XAD
Major Cattgoriis
lnt*niity
1
Category
Aliphatic hydrocarbons
,
MW Ring.
Sub-Cat»3ori«, Specific Compounds
Intimity
Compoiition
Oth«r
5-44
-------
REPORT
SAMPLE:
424 F2 AdelPhi
Major CattgoriM
Intaniiry
Crtt^ory
None found
MW Rang*
Su^Cittgori«, Seteifte Campourwfa
Intimity
Category
m/t
Composition
Other
5-45
-------
REPORT
SAMPLE: 424 F3 Adelphi 1 XAD
Cittgorits
Inunsity
Gregory
None found
MWRang*
Sub-Cattgoriw. Sp*efie Compound*
Inunsty
m/t
Cornpoiirion
Other
5-46
-------
SAMPLE: Adelphi 2 XAD 0436
Total Sunplt1
TikinforLC*
Rwovtrtd'
TCO
mi
< 1
-
-
GRAV
m|
29
20
12
TCO + GRAV
Totting
29
20
12
Conctntratkon
mg/dscm
4.7
3.3
2.0
en
4^.
-4
Frictltn
1
2
3
4
B
B
1
Sum
TCO In mi
Fovntf in
Friction
Blink
Cor-
roilon
«
Tow*
GRAV to m|
Foond In
Fraction
<1
4.0
6.2
1.6
<0.8
1.4
<0.8
13
Blink
<1
<0.8
<0.8
<1.2
<0.8
<0.8
<0.8
<6
Cor-
M..J-J
r*ciwi
<1
4.0
6.2
<0.8
<0.8
1.4
<0.8
12
Tntil4
<1
6
' 9
<1
, 1
2
<1
18
TCO +
GRAV
Totil mi
*
< 1
6
'9
< 1
< 1
2
- 1
18
Conctntntion
mg/dscm
<0.2
1
1
-0.2
<0.2
0.3
-------
IR REPOflT
IAMPLE: Adelphl 2 XAD 436 F1
Romb*
ttrcxnity
Axignnwnt
Commtnts
2940-2380
:H Aliphatic hydrocarbons
5-48
-------
IR REPORT
SAMPLE: Adelphi 2 436 F2
WIM flumb«
<«1)
2920
2840
1730
1270
Intensity
S
S
S
S
Assignment Commmti
CH Aliphatic oxygen compounds
CH Aliphatic oxygen compounds
C=0 Aliphatic oxygen compounds
CO Aliphatic oxygen compounds
*
.».
*
5-49
-------
IR REPORT
SAMPLE: Adelphi 2 XAD 436 F3
Wm Narebv
(-1)
2920-2380
1730
1270
tiraraity
S
m
m
Assignment Comments
CH Aliphatic oxygen compounds
C=0 Aliphatic oxygen compounds
CO Aliphatic oxygen compounds
»
-*
5-50
-------
IR REPORT
SAMPLE: Adelphi 2 XAD 436 F4
WIM Numbw
to.1)
3460-2380
, '
Intimity
S
-
Aijignm«nt Commcna
OH, CH Aliphatic oxygen compounds
' '
-
5-51
-------
fl REPORT
SAMPLE: Adelphi 2 XAD 436 F5
_-
Wm fUmfcv
- to.'1)
-
. f
_,
InttrnJty
.
Aiiignmmt Commtnts
No Peaks
_
.
.
-"
._ * ._
1
5-52
-------
IR REPORT
tAMPLE; Ade1phi 2 XAD 436 F6
WIM Numbw
Auignm«nt
Commmts
No Peaks
5-53
-------
IR REPORT
tAMFLE. Adelphl 2 XAD 436 F7
Wnt Hamb«
tnttnsity
Auigpnwnt
Commtnti
No Peaks
5-54
-------
LRMS REPORT
SAMPLE: 436 FT Adelphi 2 XAD
Major CitvgortM
Intensity
1
Cmoory
Aliphatic hydrocarbons
MW Rin^a
Sub-Cmgoriw, Specific Compounds
Inttnsty
m/t
Composition
Othwr
5-55
-------
LRMS REPORT
SAMPLE:
436 F2 Adelphi 2 XAD
Major Cittgoriw
inunsity
Category
None found
MW Rang.
Sub-Cjt»9orit«. Sptefie Campoundi
Inttnsicy
Cttegorf
m/t
Composition
Other
5-56
-------
LRMS REPORT
SAMPLE: 436 F3 Adelphl 2 XAD
VUjof Cirtgori**
Inunsity
Category MW Rang*
None found
1
Sub-CatBgoriw, Soacific Compounds
Intimity
Category
m/«
Composition
..
i
Other
5-57
-------
LRMS REPORT
SAMPLE: 439 Adelphi 2 lOu and 3u
Major CaTtgoriw
taunrity
1
Cntgory MW Rang*
Aliohatic hydrocarbons
Sub-CitB5or itj. Sp*«fic Compound*
Intensity
Category
1
-
m/t
Composition
--
Oth*r
5-58
-------
LRMS REPORT
SAMPLE:
Major C*ttgori*s
437 Adelphi 2 1u and filter
Inuniity
1
1
Category
Phenols
Aliphatic hydrocarbons
MW Rang*
Sub-CangortM, Specific Compound*
Intensity
Canqory
m/t
Composition
Oth«r
5-59
-------
C, - C6 CHROMATOGPAPHY ^
Cal
Cal
Sample
Sample
Sample
Sample
2/12/81
Time
0810
1429
1103
1127
1239
1405
Chrona-
COW Test 1, ppm
togram # C^ C2
1 I
7 fa\
3
4
5
6
15.1
'9 80.5
3.1
1.8
2.4
14.6
131
1.4
1.1
»
C3 C4
15.6 15.2
T74~ 1337!
1.5
1.1
1.3
1.4
C5
15.6
817!
w «
C6
15.9
~4T"
--
"* ""*
Soda Ash Injection
Cal
Cal
Blank
Sample
Sample
2/13/81
Time
0951
1328
0827
1002
1023
Chroma -
togran # Cj C2
10
13
9
11
12
\
1 avc
15
J 80
3
1
1
.1
.5
.3
.8
.3
14
139
0
--
.6
.5
.5
Test 2, ppm
C3
15.6
173
0.2
--
C4
15.2
132
2.0
1.3
C5
15.6
82.5
C6
15.9
43
5-60
-------
-
t
1
1
1
-i
£
t
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ANALYSES
SAFETY SPECIALISTS, Inc.
3284 F Edward Avanua. Stnta Ctira. California 96060
ASSAY REPORT
Taiephon* (408) 988-1111
Acurex Corporation
485 Clyde Avenue
Attn: Ms. Linda Bohannon, M/S 0-1212
Mountain View, California 94042
Date: June 3, 1981
Date Samples Received: 4/30/81
Customer Order No.: RB59185A, Rel. 10
SSI No.
Client Description
Activity
Gross Alpha
pCi/gram
Gross Beta
pCi/gram
C
D
E
Adelphi - 1, ly + filter
Adelphi - 1, 3y + 10y
Adelphi - 2, filter rinse +
10y + 3u
Adelphi - 2, lu + filter
224.1 ± 47.7 128.8 ± 38.6
196.7 ± 20.0 79.3 ± 11.3
93.7 ± 8.5
24.8 ± 4.8
40.5 ± 14.7
16.3 ± 2.9
Analyst:Pamela S. Shreve
Approved: T. C. Noble, Director
Safety and Health Services Division
*The ± values are the two sigma Poisson standard deviation of the counting
error
w * i w i *
The £ values are equal to or less than three sigma of the counting error.
5-72
-------
SAFETY SPECIALISTS, Inc.
3284F Edward Avenue, Santa Clara, California 95050 Teieohone (403) 988-11'
ASSAY REPORT
Acurex Corporation
Attn: Mr. Larry Waterland
485 Clyde Avenue
Mountain View, California 94042
Date: August 7, 1981
Date Samples Received: 4/30/81
Customer Order No.: RB59185A. Rel. 1C
SSI No.
Client Description
Activity
Gross Gamma
p Ci/o
C
D
E
Adelphi - 1, IP and 1, filter
Adelphi - 1, 3u and 1, 10y
Adelphi 2, filter rinse; 2, 10y
and 2, 3u
Adelphi 2, ly and 2, filter
553
183
219
300
292
293
100 ± 290
Analyst: Pamela S. Snreve
Approved: T. C. Noble, Director
Safety and Health Services
*The ± values are the two sioma Poisson standard deviation of the counting
error.
The < values are equal to or less than three sigma of the counting error.
5-73
-------
GENETICS ASSAY NO. 5806
SAFETY NO. 7032
MUTAGENICITY EVALUATION OF
424 (ADELPHI 1 XAD EXTRACT)
IN THE
EPA LEVEL 1
AMES SALMONETCA7MICROSOME
PLATE TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
Litton
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: SEPTEMBER 1981
BIONETICS 5-74
-------
ffl
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salmone11 a/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. Tne
data was evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of cnenr-
cals representing various chemical classes3. This assay is one of severa:
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test, material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Act of 1979. Deviations from standard procedure shall be
fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
BIONETICS
Utton 5-75
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TABLE OF CONTENTS
Page No.
PREFACE i
I. ASSAY SUMMARY 1
II. OBJECTIVE : 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Microorganisms 4
B. Media 4
C. Activation System 5
1. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
8. Mutagenicity Test . 6
1. Nonactivation Assay '. . . 6
2. Activation Assay 6
C. Control Compounds 8
D. Recording and Presenting Data 8
VI. RESULTS 9
A. Interpretation 9
B. Tables . . . . 9
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA 12
A. Surviving Populations 12
B. Dose-Response Phenomena 12
C. Control Tests 12
D. Evaluation Criteria for Ames Assay 13
1. Strains TA-1535 and TA-1537 13
2. Strains TA-98 and TA-100 13
3. Pattern 13
4. Reproducibility 13
E. Relation Between Mutagenicity and
Carcinogenicity 14
F. Criteria for Ranking Samples in the Ames Assay . . 14
VIII. REFERENCES 15
m
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BIONETICS 5_76
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5806
1. Identification: 424 (Adelphi 1 XAD extract)
2. Date Received: June 28, 1981
3. Physical Description: Clear, yellow liquid.
C. Type of Assay: EPA Level 1 Ames Salmonella/Microsome Plate Test
D. Assay Design Number: 401 (EPA Level 1)
E. Study Dates:
1. Initiation: August 22, 1981
2. Completion: September 7, 1981
F. Supervi sory Personnel:
A. Study Director: D.R. Jagannath, Ph.D.
G. Evaluation:
The test material, 424 (Adelphi 1 XAD extract), was evaluated
for mutagenic activity over a concentration range of 10 ul/plate
to 200 ul/plate in the Ames Salmonella/microsome assay. With-
out metabolic activation, the test results were negative for
all test strains. With metabolic activation, strains TA-1537,
TA-98 and TA-100 were all positive. TA-1535 was negative but
was evaluated at only one test concentration because of limited
sample. The minimum effective concentration (MEC) was found
to be 10 MVplate in strain TA-98 with activation. This was
equivalent to 49 ug organics per plate. This was evaluated
as essentially on the borderline between high and moderate
(H/M) mutagenicity categories defined for the Level 1 Ames
assay1.
Submitted by:
Study Director
Reviewed by:
m
Litton
jfar : D.R. Jagannat
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
BIONETICS
'Date
DavT9 J. Brusick, Pt^D.
Director,
Department of Molecular
Toxicology
ate'
5-77
-------
II. OBJECTIVE
The objective of this study was to determine the genetic activity of 424
(Adelphi 1 XAD extract) in the Salmonella/ microsome assay with and without
the addition of mammalian metabolic activation preparations. The genetic
activity of a sample is measured in these assays by its ability to revert
the Salmonella indicator strains from histidine dependence to histidine
independence. The degree of genetic activity of a sample is reflected
in the number of revertants that are observed on the histidine-free medium.
CH
BIONETICS 5-78
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m
Utton
III. TEST MATERIAL
A. Description
The test material was received as a clear, yellow solution in methylene
chloride. The sample contained 17.0 milligrams of organic material in ar
undetermined volume of methylene chloride. No information on the sampling
parameters (such as the equivalent volume of stack gas represented by
the sample) was provided.
B. Handling and Preparation
The test material was received at LBI on June 28, 1981. The sample was
assigned LBI safety number 7032 and LBI assay number 5806. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene chloride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 3.5 ml of DMSO,
giving a sample concentration of 4.9 mg organics per ml DMSO. The sample
was transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 2.56 ml of test material was used between three trials.
In all trials, varying sized aliquots of the test material were added
directly to the mixtures to give the desired concentration. The amount
of sample used was 0.36 ml for Trial 1, 1.08 ml for Trial 2 and 1.12 ml
for Trial 3.
BIONETICS 5"79
-------
IV.
A.
MATERIALS
Indicator Microoraanisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
TA-1535
TA-1537
TA-98
TA-100
Gene
Affected
his G
his C
his D
his G
Additional Mutations
Repair
A uvr B
A uvr B
A uvr B
A uvr B
IPS
rfa
rfa
rfa
rfa
R Factor
-
pKMlOl
pKMlOl
Mutation Type
Detected
Base-pair
substitution
Frameshift
Frames hi ft
Base-pair
substitution
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced DNA damage (uvrB-).
The rfa- mutation makes the strains more permeable to many large molecules.
The uvrB- mutation decreases repair of some types of chemically or physi-
cally damaged DNA and thereby enhances the strain's sensitivity to some
mutagenic agents. The resistant transfer factor plasmid (R factor) pKMlOl
in TA-98 and TA-100 is believed to cause an increase in error-prone DNA
repair that leads to many more mutations for a given dose of most mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, which is a convenient marker to detect the presence of plasmid
in the cells.
All indicator strains are kept at 4°C on minimal medium plates supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 ug/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (his, rfa uvrB. bio) and for the presence of plasmid. For each
experiment, an inoculum from the stock culture plates is grown overnight
at 37°C in nutrient broth (Oxoid CM67) and used.
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
Litton
BIONETICS
5-80
-------
overlay agar consisted of 0.6% purified agar with 0.05 niM rvstidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et z\_/-
C.
1.
Activation System
59 Homogenate
A 9,000 x cj supernatant prepared from Sprague-Dawley adult male rat live-
induced by Aroclor 1254 (Ames et al_.9) was purchased from Bionetics Labora-
tory Products, Litton Bionetics, Inc. and used in these assays.
2. S9 Mix
S9 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 uliters
m
Litton
5-81
BIONETICS
-------
V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are tested
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 pi per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor.
Because the amount of test material was potentially limiting, a preliminary
range-finding trial was run using a single plate per dose with strain TA-98
without metabolic activation. This trial, as did the two subsequent trials,
used the recommended dose levels of 200, 100, 50, and 10 ul per plate.
B. Mutagenicity Testing
The procedure used was based on the paper published by Ames et. aj[.9 and
was performed as follows:
(1) Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the follow-
ing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and 0.05 mM
biotin.
0.01 ml to 0.2 ml of a solution of the test chemical to give
the appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B, Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his* revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
(2) Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of £9 mix (see IV C,
Activation System) to the tubes in place of 0.5 ml of phosphate buffer
which was added in nonactivation assays. All other details were similar
to the procedure for nonactivation assays.
A detailed flow diagram for the plate incorporation assay is
^^.^ provided in Figure 1.
Ltd BIONETICS 5-82 6
Litton
-------
Aliquot of
saline
AMES ASSAY [PLATE INCORPORATION METHOD]
Molten [45°C] overlay agar
appropriately supplemented
0.5 ml
0.01 - 0.2 ml
Test, positive or solvent
control chemical
0.1 mi
Aliquot of an overnight culture
of bacteria! 109 cells I ml]
-S-9
0.5 ml S-9 mix {hepatic
+ S-9- homogenate from PCS
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37*C for 48 hours
The numbers of revertants/plate counted
Data analyzed
Interpretation/Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
5-83
-------
C. Control Compounds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b' of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were performed in the absence and presence of S9 mix. The solvent
used to prepare the stock solution of the test material is given in the
Results section of this report. All dilutions of the test material were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were also
used and assayed concurrently with the test material. The concentrations
and specificities of these compounds to specific strains are given in
the following table:
Concentration
per plate Salmonel"
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent (pg)
Water
D i methyl -
sulf oxide
Ethanol
10.0
10.0
50. D
Strains
TA-1535,
TA-98
TA-1537
la
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
ffl
Litton
5-84
BIONETICS
-------
E
Utton
VI. RESULTS
A. Interpretation
The test material, sample 424, (Adelphi 1 XAD extract), was initially
tested in strain TA-98 without activation. This trial served as a range-
finding study to help select doses for subsequent testing with all four
strains. The dose range for all trials was 200, 100, 50 and 10 ul per
plate using a single plate per dose. Trial 2 was with strain TA-98 with
activation and strain TA-100 both with and without activation. Trial 3
employed strains TA-1535 and TA-1537 both with and without activation.
The nonactivation results for all strains tested were negative. Slight
increases were observed at 100 ul/plate with TA-1537, TA-98 and TA-100
but they were not close to the criteria set for designating a positive
response.
The activation test results for strains TA-1537, TA-98, and TA-100 were
all positive by established criteria. The mutagenic response was generally
dose-related over the range of concentration tested. TA-1535 was not
active at the single dose of 200 ul/plate that was tested.
The minimum effective concentration (MEC) was found to be 10 ul per plate
with strain TA-98 with activation. This concentration was equivalent to
49 ug or organics per plate. This value was essentially on the borderline
between high and moderate (H/M) mutagenicity using IERL-EPA Level 1
criteria for the Ames bioassay1.
Solvent control and positive control values were within acceptable ranges.
These results achieved the assay acceptance criteria and provided confi-
dence in the assumption that the recorded data represented typical respon-
ses to the test material.
B. Tables
This report is based on the data provided in Tables 1 and 2.
5-85
BIONETICS
-------
RrSULTS
TAMLE t
A. NAME OR CODE DESIGNATION OF THE IEST COMPOUND: 424IADELPHI 1 XAD EXTRACT)
R. SOLVENT: OHSO
c. TEST INITIATION DATES: 08/22/01 on/25/ni
0. TtST COMPLETION DAU: 09/01/81
E. S-9 L01»: S9-11
NOTE: CONCENIRATIONS ARE GIVEN IN MICROLITEHS PER PLATE
en
oo
TEST
NONACTIVATION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL**
POSITIVE CONTROL**
TEST CO WOUND
10.000 UL
50.000 UL
100*000 UL
200.000 UL
ACTIVATION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL**
SPECIES
...
___
-_-
-.
RAT
RAT
* RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
10.000 UL
50.000 UL
100.000 UL
200.000 UL
» i
RAT
RAT
RAT
RAT
TISSUE
___
...
___
---
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
R C V C R
IA-98
1 2
43
44
053
732
64
63
70
2B
40
39
1772
1R31
H3
260
316
437
TA-98 2-NITROFLUORENE
TA-100 SODIUM
A/ IDE
TA-100
3123
139
123
IB55
1748
162
1R8
221
121)
122
126
2271
2161
142
214
255
216
* * *
10 UC/PLATE TA-98 2-ANTHRAHINE 2.5 UG/PLATt
10 UG/PLATE TA-100 2-ANTHRAHINE 2.5 UG/PLATE
-------
RCSUITS
TABLE 2
Ol
00
-a
A. NAME OR CODE DESIGNATION OF THE IEST COMPOUND: 424 CADELPMI 1 KAD EXTRACT)
it. SOLVENT: OMSO
C. TEST INITIATION DATES! 09/03/81
0. TEST COMPLETION DATE: 09/01/81
r. s-9 LOT*: s9-n
NOTE: CONCENTRATIONS ARC GIVEN IN NICROLITCRS FER PLATE
REVERTANTS PER PLATF
TEST SPF.CICS
NONACTI VAIION
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL'*
POSITIVE CONTROL**
TEST COMPOUND
10.000 UL
50.000 UL
100.000 UL
200.000 UL
ACTIVATION
SOLVENT CONTROL RAT
SOLVENT CONTROL RAT
POSITIVE CONTROL*" RAT
POSITIVE CONTROL*** RAT
TEST COMPOUND
10.000 UL RAI
50.000 UL RAT
100.000 UL RAT
200.000 UL RAT
* i
TA-133S SODIUM AZIOE
TISSUE
-
LIVER
LIVER
LIVCR
LIVER
LIVER
LIVER
LIVER
LIVER
TA-1535
1 2
18
2fl
1041
954
-
-
-
41
15
14
5B6
369
-
-
-
15
TA-13J? 9-AMINOACR10INE
TA-153T
3 123
7
12
R23
349
11
IT
20
»
n
10
29T
1 13
41
37
46
26
10 DC/PLATE IA-1535 2-ANINR AMINE 2.5 UG/PLATT
50 UC/PLATF IA-1537 2-ANTIIRAMINF 2.5 UG/PLAU
SOLVENT 50 UL/PLAIE
- INDICATES ICSI WAS NOT RONE
-------
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in this protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semisolid overlay. Because the test material and the
cells are incubated in the overlay for approximately 2 days and a few
cell divisions occur during the incubation period, the test is semi quanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplieating DNA.
The combined incubation of the test article and the cells
in the overlay permits constant exposure of the indicator
cells for approximately 2 days.
A. Surv iving Popu1at1ons
Plate test procedures do not permit exact quantitation of the number of
cells surviving chemical treatment. At low concentrations of the test
material, the surviving population on the treatment plates is essentially
the same as that on the negative control plate. At high concentrations,
the surviving population is usually reduced by some fraction. Our protocol
will normally employ several doses ranging over two or three log concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria.
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, no increases may
be observed over the dose range selected. Conversely, if the lowest
dose employed is highly cytotoxic, the test material may kill any mutants
that are induced, and the test material will not appear to be mutagenic.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
LD BIONET1CS 5-88 12
Litton
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Negative controls consisted of the test material solvent in the over",ay
agar together with the other essential components. The negative contro"
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
D. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semi quantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will'be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), to some extent there is a built-in redundancy in the microbial
assay. In general, the two strains of a set respond to the same mutagen
and such a pattern is sought. Generally, if a strain responds to a mutagen
in nonactivation tests, it will do so in activation tests.
4. Reproducibility
If a test material produces a response in a single test that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
E5-89 13
BIONETICS
Litton
-------
E.
Relation Between Mutagenlclty and Carcinogem'clty
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et aj.4 show an extremely good correlation between results of
microbial mutagenesis tests and j_n vivo rodent carcinogenesis assays.
All evaluations and interpretation of the data to be presented in the
final report will be based only on the demonstration, or lack, of muta-
genic activity.
F.
Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section D above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (ND)
Solids
(MEC in ug/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in ul/plate)
20-200
>200
Concentration of organic extracts is based upon organic content (ug
organics per plate) and not volume (ul extract per plate) of sample
tested.
ffl
Litton
BIONETICS
5-90
14
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VIII. REFERENCES
1. Brusick, D.J., et ah: IERL-RTP Procedures Manual: Level 1 Envl^or-
mentalI Assessment Biological Tests. EPA Contract No. 66-02-2581,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington. MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Environmental Assessment:
Limits of Bioassay Resolution and Detectability. EPA Contract
No. 68-02-2681, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
4. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmonel1 a/microsome test: Assay of
300 chemicals. Proc. Nat. Acad. Sci., USA 72:5135-5139, 1975.
5. Ames, B.N. , Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. Ames, B.N., Lee, F.D., and Durston, W.E.: An improved bacterial
test system for the detection and classification of mutagens and
-carcinogens. Proc. Nat. Acad. Sci., USA 70:782-786, 1973.
7. Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D.: Carcinogens
are mutagens: A simple test system combining liver homogenates for
activation and bacteria for detection. Proc. Nat. Acad. Sci., USA
70:2281-2285, 1973.
8. McCann, J., Springarn, N.E., Kobori, J. and Ames, B.N.: Detection
of carcinogens as mutagens: Bacterial tester strains with R factor
plasnrids. Proc. Nat. Acad. Sci. USA 72:979-983, 1975.
9. Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting
carcinogens and mutagens with the Salmonel1 a/mammalian-microsome
mutagenicity test. Mutation Res., 31:347-364, 1975.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase off. coli partial
purification and some properties. J. Biol. Chem., 218:97-106, 1966.
BIONETICS 5-91 15
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Ltd BIONETICS
Litton
GENETICS ASSAY NO. 5806
LBI SAFETY NO. 7032
CYTOTOXIC EVALUATION OF
424 (ADELPHI 1 XAD EXTRACT)
IN THE
RODENT CELL (CHO)
CLONAL TOXTclTY ASSAY
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94092
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: SEPTEMBER 1981
5-92
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Chinese
hamster ovary cell (CHO) clonal toxicity assay as described in "lER^-RT^
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1).
The data was evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The CHO clonal toxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of
influent and effluent streams from industrial and energy-producing
processes. This assay has been well validated with a wide range of posi-
tive and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test, material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good
Laboratory Practices Act of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
Ltj BIONETICS 5-93
Litton
-------
TABLE OF CONTENTS
PREFACE .
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
ASSAY SUMMARY
OBJECTIVE
TEST MATERIAL
A. Description
EL Handling and Preparation
MATERIALS
A. Indicator Cells
B. Media
C. Controls
EXPERIMENTAL DESIGN
A. Dose Selection
B. Clonal Toxicity Assay
ASSAY ACCEPTANCE CRITERIA
RESULTS
A. Interpretation
B. Tables and Figures
ASSAY EVALUATION CRITERIA
REFERENCES
Page No.
.... i
.... 1
.... 2
.... 3
.... 3
.... 3
.... 4
.... 4
.... 4
.... 4
.... 5
.... 5
.... 5
.... 7
.... 8
.... 8
.... 8
11
.... 12
m
Litton
BIONETICS
5-94
ii
-------
I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5806
1. Identification: 424 (Adelphi 1 XAD extract)
2. Date Received: June 28, 1981
3. Physical Description: Transparent liquid with a yellow
tint.
C. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
D. ASSAY DESIGN NUMBER: 442
E. STUDY DATES:
A. Initiation: August 20, 1981
B. Completion: August 27, 1981
F. SUPERVISORY PERSONNEL:
A. Study Director: Brian C. Myhr, Ph.D.
B. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material caused a slight increase in toxicity with
increasing concentrations up to 10 ul/ml. The material was
completely lethal to the cells at the maximum applicable dose
of 20 Ml/ml. The EC50 was estimated graphically to be 13 jjl/ml
which was equivalent to 63.2 ug of organics per ml. This sample
was therefore evaluated to be in the moderate (M) toxicity
category defined for the IERL-EPA Level 1 CHO clonal toxicity
bioassay.1
CB
Litton
Submitted by:
Study Director
Brian Myhr, Ph.!
Associate Director,
Department of Molecular
Toxicology
BIONETICS
Date
5-95
Reviewed by:
Javid J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of sample 424 (Adelphi 1 XAD extract) to cultured Chinese hamster cells
(CHO-K1 cell line). The measure of cytotoxicity was the reduction in
colony-forming ability after a 24-hour exposure to the test material.
After a period of recovery and growth, the number of colonies that
developed in the treated cultures was compared to the colony number in
unexposed vehicle control cultures. The concentration of test material
that reduced the colony number by 50% was estimated graphically and
referred to as the EC50 value. Standard EPA Level 1 toxicity evaluation
criteria for the CHO clonal toxicity assay were used to rank the toxicity
potential of the test material.
m
Litton
BIONETICS 5"96
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III. TEST MATERIAL
A. Description
The test material was received as a clear yellow tinted solution in
methylene chloride. The sample contained 17.0 milligrams of organic
material in an undetermined volume of methylene chloride. No infor-
mation on the sampling parameters (such as the equivalent volume of
stack gas represented by the sample) was provided.
B. Handling and Preparation
The test material was received at LBI on June 28, 1981. The sample was
assigned LBI safety number 7032 and LBI assay number 5806. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent-exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene chloride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 3.5 ml of DMSO,
giving a sample concentration of 4.86 nog organics per ml DMSO. The sample
was then transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 0.45 ml of test sample was used in the CHO assay. The
maximum concentration of 20 Ml/ml was obtained by adding 0.12 ml of sample
to 5".88 ml of F12 medium; this resulted in 2% (v/v) DMSO in the medium
and effectively limited the concentration of test material that could be
assayed. Only two plates were dosed at the top dose in order to conserve
sample. Another 0.12 ml aliquot of sample was used to prepare the 10 ul/ml
test concentration. An additional 0.21 ml of test sample was used to
prepare a series of dilutions in DMSO from which 1:100 dilutions into
growth medium were performed to obtain the lower assayed concentrations.
Thus, except for the 20 pi/ml test concentration, the final DMSO concen-
tration was constant at 1% (v/v).
E5-97
B1ONETICS
Litton
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IV. MATERIALS
A. Indicator..Cells
The indicator cells for this study were Chinese hamster CHO-K1 cells (ATCC
No. CCL 61) obtained from Flow Laboratories, Inc., Rockville, MD. This
cell type was derived from ovarian tissue and has spontaneously transformed
to a stable, hypodiploid line of rounded, fibroblastic cells with unlimited
growth potential. Monolayer cultures have a fast doubling time of 11 to
14 hours, and untreated cells can normally be cloned with an efficiency
of 80% or greater. Laboratory stock are maintained by routine serial
subpassage. Cells are cultivated in Ham's F-12 nutrient medium at 37°C
in 5 percent C02 with saturated humidity. Stocks are continually observed
macroscopically and microscopically for possible microbial contamination.
Laboratory cultures are periodically checked by culturing and staining
methods for the absence of mycoplasma contamination. Laboratory cultures
are discarded every three months and new cultures started from mycoplasma-
free, long-term frozen cultures.
B. Media
The CHO-K1 cell line has an absolute requirement for proline and therefore
must be maintained in culture medium containing sufficient amounts of this
ami no acid. Ham's F12 medium, which contains 3 x 10-4 M L-proline was used,
supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 units/ml of
penicillin, 100 ug/ml of streptomycin, and 0.9 ul/ml of amphotericin B.
A 10X formulation of Ham's F10 is available commercially and-was used for
the 'testing of aqueous test samples in order to avoid the dilution of
medium components. This medium contains 1 x 10-4 L-proline and was sup-
plemented in the same manner as F12, except that kanamycin at 40 ug/ml
is included for additional protection against bacterial contamination.
Both media formulations support the growth and cloning of CHO cells
equally well.
C. Controls
The negative control consists of three untreated cultures carried through
the same experimental time period as the treated cells. The test material
was supplied as a solution in an organic vehicle (usually DMSO) and was
diluted into the medium to provide each test concentration, three vehicle
control cultures containing the organic solvent at 1% or 2% by volume are
prepared. Particulate test samples were suspended in small amounts of
DMSO, but DMSO was not included in the negative control because the maxi-
mum content in the treated cultures is only 0.42%. The average number of
colonies in the negative control establishes the cloning efficiency of
the CHO cells used in the assay, and the appropriate negative or vehicle
controls provide the reference points for determining the effects of dif-
ferent concentrations of the test material on cell survival.
LU BIONETICS 5-98
Litton
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V. EXPERIMENTAL DESIGN
A. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following dose ranges are usually tested for different
sample forms. Aqueous samples, suspensions, or slurries are tested from
600 pi/ml to 3 ul/ml, usually in six dose steps. Eight doses are often
used when the amount of test sample is limited to provide a more precise
description of toxicity in the event of sharp dose-response curves. Dry,
particulate material is dissolved or suspended in DMSO, diluted into growth
medium, and tested at six dose levels from 1000 ug/ml to 3 ug/ml. Samples
that are solvent-exchanged into DMSO are tested from 20 ul/ml (2% DMSO
in growth medium) to 0.2 (jl/ml, also in six dose steps. A second dose
study is performed with an adjusted dose range if the EC50 was not located
properly in the initial test. However, EC50 values greater than 1000 ug/ml
for particulate material, 600 ul/ml for aqueous samples, or 20 ul/ml for
organic solutions will not be determined. .
This sample, number 424 (Adelphi 1 XAD extract) was tested at eight dose
levels. The concentrations started with the maximum applicable dose (MAD)
of 20 ul/ml and included 10, 6, 3, 1, 0.6, 0.3, and 0.1 ul of test material
per ml of culture medium. The corresponding concentration of organics
at the MAD level was 97.2 ug/ml; the lower doses were equivalent to 48.6,
29.2, 14.6, 4.9, 2.9, 1.5, and 0.5 ug/ml.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase were tryp-
sinized with 0.1% trypsin plus 0.01% versene for 4 minutes and the density
of the resulting cell suspension determined by hemocytometer. A number
of 60-mm culture dishes were then seeded with 200 cells and 4 ml of culture
medium per dish. The cultures were incubated for approximately 6 hours
at 37°C in a humidified atmosphere containing 5% C02 to allow attachment
of the cells. The 6-hour attachment period was used in order to avoid
cell division and the subsequent formation of two-cell colonies prior to
treatment.
If the medium was aspirated from the cultures and 4 ml medium containing
the test material applied. Three cultures were exposed to each test con-
centration. After an exposure time of 24 hours at 37°C, the medium was
removed by aspiration and each culture washed three times with approxi-
mately 4 ml aliquots of Dulbecco's phosphate buffered saline (pre-warmed
to 37°C). Fresh culture medium (5 ml) was placed in each dish and incuba-
tion at 37°C is continued for an additional 6 days to allow colony develop-
ment.
If the test material caused a color change in the culture medium, the pH
of the medium containing the high dose would be determined at the time of
treatment. The pH at the lowest dose that results in a slight color change
Ltd BIONETICS 5~"
Lrtton
-------
would also be recorded. At the end of the treatment period, the pH values
of the discarded media from the two described treatments would be recorded
again. No sample related pH effects were noted.
After the incubation period, the medium was drained from the cultures
and the surviving colonies fixed with 100% ethanol and stained with
Giemsa. Colonies were counted by eye; tiny colonies of approximately
50 cells or less were arbitrarily excluded from the counts.
CB
Litton
BIONET1CS 5"100
-------
VI. ASSAY ACCEPTANCE CRITERIA
The assay is considered acceptable for evaluation of the test results if
the following criteria are met:
The average cloning efficiency of the CHO-K1 cells in the
negative controls is 70% or greater, but not exceeding
115%.
The distribution of colonies in the treated cultures is
generally uniform over the surface of the culture dish.
The data points for each test concentration critical to
the location of the EC50 are the averages of at least two
treated cultures.
A sufficient number of test concentrations are available
to clearly locate the EC50 within a toxicity region as
defined under Assay Evaluation Criteria.
If the EC50 value is greater than 1000 ug/ml, 600 uliters
of aqueous sample/ml, or 20 uliters Of nonaqueous sample/ml,
the plotted curve does not exceed 110% of the negative
control.
HB B.ONET.CS
Litton
-------
VII. RESULTS
A. Interpretation
The application of sample 424 (Adelphi 1 XAD extract) to the CHO cell
cultures caused a rapid lowering of the number of cells able to form
colonies as the concentration increased above 10 pi/ml. Relative sur-
vival values were calculated as the ratio of colonies formed in treated
cultures to the colonies formed in the appropriate vehicle control, and
these relative survival values were plotted against the concentration of
test material. As shown in Figure 1, the relative survival decreased
gradually in the 0.1 to 10 ul/ml range and dropped to zero at the 20 ul/ml
dose level.
The concentration expected to kill 50 percent of the cells (ECs0) was
found to be 13 pi of test material per ml of culture medium. This con-
centration was equivalent to 63.2 M9 of organic material per ml of
culture medium. This value placed the test material in the moderate (M)
toxicity range defined for the IERL-EPA CHO clonal toxicity bioassay.
The cells used for the assay were in logarithmic growth phase and were
100% viable. About 77% of the seeded cells formed colonies and in both
the 1* and 2% DMSO vehicle control, slightly less than 72% of the cells
formed colonies. Colony growth was normal and well distributed on the
culture dishes. These results achieved the assay acceptance criteria
and provided confidence in the assumption that the recorded data repre-
sented typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Table 1 and Figure 1.
LJj BIONETJCS 5-102
Litton 8
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TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: 424 (Adelphi 1 XAD
Extract
Description of Sample: Transparent
liquid with a yellow tint
LBI Assay No.: 5806
EC50 Value: 63.2 ug organics/ml
(13.0 Ml/ml)
Toxicity
Classification: Moderate (M)
pH Alterations: None
Date Received: June 28. 1981
Test Date: August 20, 1981
Vehicle: DMSO
Comments on
Treatment: Sample prepared in DMSO
at a concentration of 4.86 ug
organics/pl
Cell Type: CHO-K1
Cells Seeded per Dish: 200
COLONY COUNTS
Sample
NCb
VC,1*C
VC,2%
Test
Test
Test
Test
Test
Test
Testx
Testd
Applied
'Concentration
Ml /ml
10
20
0.1
0.3
0.6
1.0
3.0
6.0
10.0
20.0
Dish
#1
157
154
145
145
119
122
132
123
129
117
0
Dish
#2
160
131
140
130
131
137
130
119
121
106
0
Dish
#3
144
145
144
133
134
130
110
120
115
121
Average
Count
153.7
143.3
143.0
136.0
128.0
129.7
124.0
120.7
121.7
114.7
0
Relative
Survival
_»
100.0
100.0
94.9
89.3
90.5
86.5
84.2
84.9
80.0
0
Cloning
Efficiency
76.9
71.7
71.5
Relative to 2% VC for 20 ul/ml treatment and to 2% VC for other treatments.
bNC = Negative Control, F12 medium.
SfC = Vehicle Control, percent DMSO given.
dOnly two plates dosed to conserve limited test material.
m
Utton
BIONETICS
5-103
-------
HO r
130
r 70
FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
424 (ADELPHI 1 XAD EXTRACT)
100
CONCENTRATION, yl/ml
5-104
10
-------
VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentrations of test material that -ecuces
the colony-forming ability of CHO cells to 50% of the vehicle or negative
control value. EC50 values are determined graphically by fitting a curve
by eye through relative survival data plotted as a function of the loga-
rithm of the applied concentration. Each data point normally represents
the average of three culture dishes. In order to indicate the variability
of the data, the high and low colony counts for each concentration are
used to calculate the relative survivals, and the range is shown by a
bar at the position of the plotted average. If no bar is shown, the
variability was within the size of the symbol. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
Solids
Toxicity (ECSO in ug/ml)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids
(EC50 in ul/ml)
<6
6 to 60
60 to 600
>600
Nonaqueous Liquias
(EC50 in pi/ml)
<.2
.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level Environmental Assessment Biological Tests"!
Criteria for nonaqueous liquids are tentative and under evaluation.
If the organic or solids content is known, the sample is evaluated
under the solids criteria.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as L/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
Toxicity
High
Moderate
Low
Nondetectable
EC50 In
Liters/ml (L/ml)
<10
10-100
100-1000
>1000
EC50 In
Dry Standard Cubic Feet/ml (DSCF/ml)
<0.35 DSCF
0.35-3.5
3.5-35
>35
5-105
BIONET1CS ..
Utton n
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IX. REFERENCES
1. Brusick, O.J., et a_L: IERL-RTP Procedures Manual: Environmental
Assessment Biological Tests. EPA Contract No. 68-02-2681, Technical
Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Environmental Assessment:
Limits of Bioassay Resolution and Detectability.EPA Contract
No. 68-02-268, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
CH 5-106
BIONET.CS
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GENETICS ASSAY NO.
LBI SAFETY NC.
MUTAGENICITY EVALUATION OF
436 (ADELPHI 2 XAD EXTRACT)
IN THE
EPA LEVEL 1
AMES SALMONELTA7MTCROSOME
PLATE TEST
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94042
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: SEPTEMBER 1981
LU BIONETICS 5-107
Utton
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Ames
Salflionella/microsome mutagenesis assay as described in "IERL-RTP Proce-
dures Manual: Level 1 Environmental Assessment Biological Tests"1. The
data was evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting"2.
The Ames Salmonella/microsome mutagenesis assay has been shown to be a
sensitive method for detecting mutagenic activity for a variety of chemi-
cals representing various chemical classes3. This assay is one of several
recommended by EPA to identify, categorize and rank the pollutant potential
of influent and effluent streams from industrial and energy-producing pro-
cesses. This assay has been well validated with a wide range of positive
and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good Labora-
tory Practices Act of 1979. Deviations from standard procedure shall be
fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
LU BIONETICS 5-108
Litton
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TABLE OF CONTENTS
Page No.
PREFACE -;
I. ASSAY SUMMARY 1
II. OBJECTIVE , .... 2
III. TEST MATERIAL ... 3
A. Description 3
B. Handling and Preparation ... . 3
IV. MATERIALS 4
A. Indicator Microorganisms .'.... 4
B. Media 4
C. Activation System 5
I. S9 Homogenate 5
2. S9 Mix 5
V. EXPERIMENTAL DESIGN 6
A. Dose Selection 6
B. Mutagenicity Test 6
1. Nonactivation Assay 6
2. Activation Assay 6
C. Control Compounds 7
D. Recording and Presenting Data 7
VI. RESULTS 9
A. Interpretation 9
B. Tables 9
VII. ASSAY ACCEPTANCE AND EVALUATION CRITERIA 12
A. Surviving Populations ....... 12
B. Dose-Response Phenomena 12
C. Control Tests 12
D. Evaluation Criteria for Ames Assay 13
1. Strains TA-1535 and TA-1537 13
2. Strains TA-98 and TA-100 13
3. Pattern 13
4. Reproducibility 13
E. Relation Between Mutagenicity and
Carcinogenicity 14
F. Criteria for Ranking Samples in the Ames Assay . . 14
VIII. REFERENCES 15
_ BIONETICS 5-109
Litton
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I. ASSAY SUMMARY
A. Sponsor: Acurex Corporation
B. Material (Test Compound): Genetics Assay Number: 5809
1. Identification: 436 (Adelphi 2 XAD Extract)
2. Date Received: June 28, 1981
3. Physical Description: Clear, colorless liquid
C. Type of Assay: EPA Level 1 Ames Sal monell a/Mi crosome Plate Test
Assay Design Number: 401 (EPA Level 1)
Study Dates:
1. Initiation: August 22, 1981
2. Completion: September 7. 1981
Supervi spry Personne 1 :
A. Study Director: D.R. Jagannath, Ph.D.
Evaluation:
D.
E-
G.
The test sample, 436, was mutagenic in the Ames Salmonella/
mi crosome assay. The sample was mutagenic for frameshift tester
strains TA-1537 and TA-98 under activation (+S9) conditions.
Strain TA-100 was reverted by the sample but the response did
not meet the established criteria for a positive effort. Strain
TA-1535 was not affected by sample 436 at the single concentra-
tion evaluated. The minimum effective concentration (MEC)
was 10 pi/plate with strain TA-98. This was equivalent to
13 ug organics/plate. This response was evaluated to be in
the high (H) mutagenicity range defined for the IERL-EPA Level 1
Ames bioassay1.
Submitted by:
Study Director
Reviewed by:
. . _
D.R. Jagannattf, Ph.D.
Section Chief,
Submammalian Genetics,
Department of Molecular
Toxicology
ate
David J. Brusick, Ph.D.
Director,
Department of Molecular
Toxicology
m
Litton
BIONETICS
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II. OBJECTIVE
The objective of this study was to determine the genetic activity of
sample 436 (Adelphi 2 XAD extract) in the Salmonel1 a/microsome assay
with and without the addition of mammalian metabolic activation prepara-
tions. The genetic activity of a sample is measured in these assays oy
its ability to revert the Salmonella indicator strains from histidine
dependence to histidine independence. The degree of genetic activity of
a sample is reflected in the number of revertants that are observed on
the histidine-free medium.
5-111
BIONETICS
Litton
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III. TEST MATERIAL
A. Description
The test material was received as a clear, colorless solution in methylene
chloride. The sample contained 4.7 milligrams of organic material in an
undetermined volume of methylene chloride. No information on the sampling
parameters (such as the equivalent volume of stack gas represented by
the sample) was provided.
B. Hand!ing and Preparation
The test material was received at LBI on June 28, 1981. The sample was
assigned LBI safety number 7033 and LBI assay number 5809. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent exchanging the sample
into dimethylsulfoxide (DMSO). The sample was transferred with methylene
chloride rinses into a graduated conical tube. The methylene chloride
was gradually evaporated (50°C under a stream of nitrogen) and DMSO was
sequentially added. The sample was brought to volume in 3.5 ml of DMSO,
giving a sample concentration of 1.3 mg organics per ml DMSO. The sample
was transferred to a glass vial and sealed with a teflon-coated rubber
septum.
A total volume of 2.56 ml of test material was used between three trials.
In the trials, varying aliquots of the test material were added directly
to the test mixtures to give the desired concentration. The amount of
sample used was 0.36 ml for Trial 1, 1.08 ml for Trial 2 and 1.12 ml for
Trial 3.
BIONETICS 5"U2
Litton
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IV.
A.
MATERIALS
Indicator Microorganisms
The Salmonella typhimurium strains used in this assay were obtained from
Dr. Bruce Ames, University of California at Berkeley.4-8 The following
four strains were used.
Strain
Designation
TA-1535
TA-1537
TA-98
TA-100
Gene
Affected
his G
Ms C
Mi D
Mi G
Additional Mutations
Repair
A uvr B
A uvr B
A uvr B
A uvr B
LPS R Factor
rfa
rfa
rfa pKMlOl
rfa pKMlOl
Mutation Type
Detected
Base-pair
substitution
Frameshift
Frameshift
Base-pair
substitution
All the above strains have, in addition to the mutation in the histidine
operon, mutation (rfa-) that leads to defective lipopolysaccharide coat,
a deletion that covers genes involved in the synthesis of vitamin biotin
(bio-) and in the repair of ultraviolet (uv) - induced DNA damage (uvrB-).
The rfa- mutation makes the strains more permeable to many large molecules.
The uvrB- mutation decreases repair of some types of chemically or physi-
cally damaged DNA and thereby enhances the strain's sensitivity to some
mutagenic agents. The resistant transfer factor plasmid (R factor) pKMlOl
in TA-98 and TA-100 is believed to cause an increase in error-prone DNA
repair that leads to many more mutations for a given dose of most mutagens.8
In addition, plasmid pKMlOl confers resistance to the antibiotic ampi-
cillin, which is a convenient marker to detect the presence of plasmid
in the cells.
All indicator strains are kept at 4°C on minimal medium plates supplemented
with a trace of biotin and an excess of histidine. In addition, the
plates with plasmid-carrying strains contain ampicillin (25 \sg/ml) to
ensure stable maintenance of plasmid pKMlOl. New stock culture plates
are made as often as necessary from the frozen master cultures or from
single colony reisolates that were checked for their genotypic character-
istics (his, rfa uvrB. bio) and for the presence of plasmid. For each
experiment, an inoculum from the stock culture plates is grown overnight
at 37°C in nutrient broth (Oxoid CM67) and used.
B.
Media
The bacterial strains were cultured in Oxoid Media #2 (Nutrient Broth).
The selective medium was Vogen Bonner Medium E with 2% glucose.10 The
ffl
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BIONETICS
5-113
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overlay agar consisted of 0.6% purified agar with 0.05 mM histidine,
0.05 mM biotin and 0.1M NaCl according to the methods of Ames et al_.9
C.
1.
Activation System
S9 Homogenate
A 9,000 x 3 supernatant prepared from Sprague-Dawley adult male rat liver
induced by Aroclor 1254 (Ames et a_K9) was purchased from Bionetics Labora-
tory Products, Litton Bionetics, Inc. and used in these assays.
2.
S9 Mix
59 mix used in these assays consisted of the following components:
Components
Concentration per Milliliter
S9 Mix
NADP (sodium salt)
D-glucose-6-phosphate
MgCl2
KC1
Sodium phosphate buffer
pH 7.4
Organ homogenate from rat
liver (S9 fraction)
4 umoles
5 umoles
8 umoles
33 umoles
100 umoles
100 M
ffl
Litton
BIONETICS
5-U4
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V. EXPERIMENTAL DESIGN
A. Dosage Selection
Test strategy and dose selection depend upon sample type and sample avail-
ability. The Level 1 manual1 recommends solids to be initially tested
at the maximum applicable dose (MAD) of 5 mg per plate and at lower con-
centrations of 2.5, 1, 0.5, 0.1 and 0.05 mg per plate. Liquids are testec
initially at the MAD of 200 ul per plate, and at lower concentrations of
100, 50 and 10 ul per plate. Samples are retested over a narrower range
of concentrations with strains showing positive results initially. Alter-
nate dose are employed if sample size is limiting or at the direction of
the sponsor
A preliminary range-finding test was run with strain TA-98 using the
recommended doses of 200, 100, 50, and 10 ul per plate. Subsequent testing
with the other strains utilized these dos6 levels. These doses were
equivalent to 260, 130, 65, and 13 ug of organics per plate.
B. Mutagem'city Testing
The procedure used was based on the paper published by Ames et. a_L9 and
was performed as follows:
1. Nonactivation Assay
To a sterile 13 x 100 mm test tube placed in a 43°C water bath the fol-
lowing was added in order:
2.00 ml of 0.6% agar containing 0.05 mM histidine and
0.05 mM biotin.
0.01 ml to 0.2 ml of a solution of the test chemical to
give the appropriate dose.
0.1 ml to 0.2 ml of indicator organism(s).
0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates
(see IV B Media). After the top agar had set, the plates were incubated
at 37°C for approximately 2 days. The number of his+ revertant colonies
growing on the plates were counted with an automatic colony counter and
recorded.
2. Activation Assay
The activation assay was run concurrently with the nonactivation assay.
The only difference was the addition of 0.5 ml of S9 mix (see IV C, Acti-
vation System) to the tubes in place of 0.5 ml of phosphate buffer which
was added in nonactivation assays. All other details were similar to
the procedure for nonactivation assays.
CH BIONETICS 5~U5
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A detailed flow diagram for the plate incorporation assay is provided in
Figure 1.
C.
ContrQL_Cgmp_p_unds
A negative control consisting of the solvent used for the test material
was also assayed concurrently with the test material. For negative con-
trols, step 'b1 of Nonactivation Assays was replaced by 0.05 ml of the
solvent. The negative controls were employed for each indicator strain
and were performed in the absence and presence of S9 mix. The solvent
used to prepare the stock solution of the test material is given in the
Results section of this report. All dilutions of the test material were
made using this solvent. The amount of solvent used was equal to the
maximum volume used to give the appropriate test dose.
Specific positive control compounds known to revert each strain were
also used and assayed concurrently with the test material. The concentra-
tions and specificities of these compounds to specific strains are given
in the following table:
Concentration
per plate Salmonella
Assay
Nonactivation
Chemical
Sodium azide
2-Nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent (yg)
Water
Dimethyl-
sulfoxide
Ethanol
10.0
10.0
50.0
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Dimethyl-
sulfoxide
2.5
For all strains
D.
Recording and Presenting Data
The number of colonies on each plate were counted and recorded on printed
forms. These raw data were analyzed in a computer program and reported
on a printout. The results are presented as revertants per plate for
each indicator strain employed in the assay. The positive and solvent
controls are provided as reference points.
m
Litton
BIONETICS
5-116
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AMES ASSAY (PLATE INCORPORATION METHOD]
Molten [45*C] overlay agar
appropriately supplemented
.01 - .2 ml
Test, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacteria [ 109 cells /ml]
Aliquot of
saline
0.5 ml
-S-9
0.5 ml S-9 mix [hepatic
S-9 homogenate from PCS
pretreated rat plus
necessary cofactors
Overlay poured on selective
bottom agar medium
Plated incubated at 37°C for 48 hours
The numbers of revertants/piate counted
Data analyzed
Interpretation/ Conclusion
Figure 1 AMES SALMONELLA/MICROSOME MUTAQENESIS ASSAY
5-117
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VI. RESULTS
A. Interpretations
The test material, sample 436, was tested in strains TA-98, TA-100, TA-1535
and TA-1537 both with and without the addition of metabolic activation.
The tests were conducted at concentrations of 10, 50, 100 and 200 yl/plate
except for strain TA-1535 which was tested only at 200 ul/plate. The
assay was conducted in three steps. Trial 1 was a range-finding assay
using strain TA-98 without activation. Trial 2 utilized strain TA-98
with activation and strain TA-100 both with and without activation.
Trial 3 was with strains TA-1535 and TA-1537 both with and without acti-
vation.
The response in all strains without metabolic activation was negative.
With activation, strains TA-98 and TA-1537 were mutagenic under the assay
conditions and both showed a dose-related increase in the number of revert-
ants. Strain TA-100 was reverted by the test material but the response
did not meet the criteria for a positive effect. Strain TA-1535 was
negative at the single dose tested. There was indication of sample related
toxicity at the maximum applicable dose of 200 pi/plate.
The minimum effective concentration (MEC) was observed to be 10 ul/plate
in TA-98 with activation. This was equivalent to 13 ug of organic material
per plate. This response was categorized as high (H) mutagenic activity
using the IERL-EPA Level 1 criteria for the Ames assay1.
Solvent and positive control values were within acceptable ranges. These
results achieve the assay acceptance criteria and provide confidence in
the assumptions that the recorded data represented typical responses to
the test material.
B. Tables
This report is based on the data provided in Tables 1 and 2.
LD BIONETICS 5"118
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RESULTS
T*HLC I
A. NAME OR CODE DESIGNATION OF THE TEST COMPOUND: 436
-------
RESOLIS
TABLE ?
NAME OR CODE DESIGNATION OF IMC ICST COMPOUND: 436IADELPHI 2 IAD EXTRACT}
SOLVENT: DHSO
TEST INITIATION DATES: 09/03S81
TEST COMPLETION DATE: 09/07/B1
S-9 LOIi: S9-11
CONCENTRATIONS ARC GIVEN IN MICROLIICRS PER PLATE
A.
B.
C.
D.
C.
NOTE:
TEST
NONACTIVATION
REVERTANTS FER PLATE
SPECIES TISSUE TA-1535 TA-1531
123 123
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONIROL**
POSITIVE CONTROL**
TEST COMPOUND
10.000
50.000
100.000
200.000
ACTIVATION
UL
UL
UL
UL
1R
28
1041
954
31
7
12
B23
349
13
19
23
25
SOLVENT CONTROL
SOLVENT CONTROL
POSITIVE CONTROL***
POSITIVE CONTROL***
TEST COMPOUND
10.000 UL
SO. 000 UL
100.000 UL
200.000 UL
RAT
RAT
RAT
RAI
RAT
RAT
RAT
RAT
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
LIVER
15
14
5fl6
369
-
-
-
15
a
ID
29T
113
211
35
38
4A
TA-1335 SODIUM AZIOE
TA-15J7 9-AMINOACRIDINE
SOLVENT 50 UL/PLATE
- INDICATES TEST HAS NOT DONE
10 UC/PLATE
50 U6/PLATE
TA-1535
TA-153T
2-ANTHRAMINE
2-ANTHRAHINE
2.5 US/PLATE
2.5 US/PLATE
-------
vn- ASSAY ACCEPTANCE AND EVALUATION CRITERIA
Statistical methods are not currently used, and evaluation is based on
the criteria included in tnis protocol.
Plate test data consists of direct revertant colony counts obtained from
a set of selective agar plates seeded with populations of mutant cells
suspended in a semi sol id overlay. Because the test material and the
cells are incubated in the overlay for approximately 2 days and a few
cell divisions occur during the incubation period, the test is semiquanti-
tative in nature. Although these features of the assay reduce the quanti-
tation of results, they provide certain advantages not contained in a
quantitative suspension test:
The small number of cell divisions permits potential
mutagens to act on replication DNA, which is often more
sensitive than nonreplieating DNA.
The combined incubation of the test article and the cells
in the overlay permits constant exposure of the indicator
cells for approximately 2 days.
A. Surviving Populations
Plate test procedures do not permit exact quantisation of the number of
cells surviving chemical treatment. At low concentrations of the test
material, the surviving population on the treatment plates is essentially
the same as that on the negative control plate. At high concentrations,
the surviving population is usually reduced by some fraction. Our protocol
will normally employ several doses ranging over two or three log concen-
trations, the highest of these doses being selected to show slight toxicity
as determined by subjective criteria,
B. Dose-Response Phenomena
The demonstration of dose-related increased in mutant counts is an impor-
tant criterion in establishing metagenicity. A factor that might modify
dose-response results for a mutagen would be the selection of doses that
are too low (usually mutagenicity and toxicity are related). If the
highest dose is far lower than a toxic concentration, no increases may
be observed over the dose range selected. Conversely, if the lowest
dose employed is highly cytotoxic, the test material may kill any mutants
that are induced, and the test material will not appear to be mutagenic.
C. Control Tests
Positive and negative control assays were conducted with each experiment
and consisted of direct-acting mutagens for nonactivation assays and
mutagens that require metabolic biotransformation in activation assays.
m 5-121
BIONETICS 12
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Negative controls consisted of the test material solvent in the overlay
agar together with the other essential components. The negative control
plate for each strain gave a reference point to which the test data was
compared. The positive control assay was conducted to demonstrate that
the test systems were functional with known mutagens.
The following normal range of revertants for solvent controls are generally
considered acceptable.
TA-1535: 8-30
TA-1537: 4-30
TA-98: 20-75
TA-100: 80-250
0. Evaluation Criteria for Ames Assay
Because the procedures to be used to evaluate the mutagenicity of the
test material are semi quantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base. Most data sets will be evaluated using the following
criteria.
1. Strains TA-1535 and TA-1537
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to three times the solvent control value will
be considered to be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a test material
that produces a positive dose response over three concentrations with
the highest increase equal to twice the solvent control value for TA-98
and TA-100 will be considered to be mutagenic.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain
(G-46), to some extent there is a built-in redundancy in the microbial
assay. In general, the two strains of a set respond to the same mutagen
and such a pattern is sought. Generally, if a strain responds to a mutagen
in nonactivation tests, it will do so in activation tests.
4. Reproducibility
If a test material produces a response in a single test that cannot be
reproduced in additional runs, the initial positive test data lose signi-
ficance.
The preceding criteria are not absolute, and other extenuating factors
may enter into a final evaluation decision. However, these criteria
will be applied to the majority of situations and are presented to aid
those individuals not familar with this procedure. As the data base is
increased, the criteria for evaluation can be more firmly established.
LLJ BIONETICS 5"122 13
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E- Relation Between Mutagenlcity and Carcinogeniclty
It must be emphasized that the Ames Salmonella/Microsome Plate Assay is
not a definitive test for chemical carcinogens. It is recognized, however,
that correlative and functional relations have been demonstrated between
these two endpoints. The results of comparative tests on 300 chemicals
by McCann et al_.4 show an extremely good correlation between results of
microbial mutagenesis tests and ni vivo rodent carcinogenesis assays.
All evaluations and interpretation of the data to be presented in the
final report will be based only on the demonstration, or lack, of muta-
genic activity.
F- Criteria for Ranking Samples in the Ames Assay
The goal of EPA Level 1 Ames testing is tq rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples are first identified
as mutagenic or nonmutagenic by the criteria in Section 0 above and
then ranked using the mutagenicity categories presented in the table
below. The lowest concentration giving a positive response in any strain,
with or without metabolic activation, is identified as the minimum effec-
tive concentration (MEC) for that sample. The mutagenicity of the sample
is evaluated as high (H), moderate (M), low (L), or nondetectable (ND)
according to the evaluation criteria developed in the Level 1 manual1
and summarized below. Samples with no detectable activity at the maximum
applicable dose (MAD) are ranked nondetectable (ND).
Ames Assay Mutagenicity Ranking Criteria1
Mutagenic
Activity
High (H)
Moderate (M)
Low (L)
Not Detectable (NO)
Solids
(MEC in pg/plate)
<50
50-500
500-5000
>5000
(MEC
<2
2-20
Liquids3
in pi/plate)
20-200
>200
Concentration of organic extracts is based upon organic content ((jg
organics per plate) and not volume (ul extract per plate) of sample
tested.
E5-123
BIONETICS
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VIII. REFERENCES
1. Brusick, D.J., et aj_.: IERL-RTP Procedures Manual: Level 1 Environ-
ment^ Assessment Biological Tests. EPA Contract No. 68-02-2681,
Technical Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bioassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Environmental Assessment:
Limits of Bioassay Resolution and Detectability.EPA Contract
No. 68-02-2681, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
4. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N.: Detection of
carcinogens as mutagens in the Salmone11 a/microsome test: Assay of
300 chemicals. Proc. Nat. Acad. Sci., USA 72:5135-5139, 1975.
5. Ames, B.N., Gurney, E.G., Miller, J.A. and Bartsch, H.: Carcinogens
as frameshift mutagens: Metabolites and derivatives of 2-acetylamino-
fluorene and other aromatic amine carcinogens. Proc. Nat. Acad.
Sci., USA 69:3128-3132, 1972.
6. Ames, B.N., Lee, F.D., and Durston, W.E.: An improved bacterial
test system for the detection and classification of mutagens and
carcinogens. Proc. Nat. Acad. Sci., USA 70:782-786, 1973.
7. Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D.: Carcinogens
are mutagens: A simple test system combining liver homogenates for
activation and bacteria for detection. Proc. Nat. Acad. Sci., USA
70:2281-2285, 1973.
8. McCann, J., Springarn, N.E., Kobori, J. and Ames, B.N.: Detection
of carcinogens as mutagens: Bacterial tester strains with R factor
plasmids. Proc. Nat. Acad. Sci. USA 72:979-983, 1975.
9. Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting
carcinogens and mutagens with the Salmone11 a/mammalian-microsome
mutagenicity test. Mutation Res., 31:347-364, 1975.
10. Vogel, H.J. and Bonner, D.M.: Acetylornithinase of E. coli partial
purification and some properties. J. Biol. Chem., 218:97-106, 1966.
Ltd BIONETICS 5'124 15
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GENETICS ASSAY NO. 5809
LBI SAFETY NO. 7022
CYTOTOXIC EVALUATION OF
436 (ADELPHI 2 XAD EXTRACT)
IN THE
RODENT CELL (CHO)
CLONAL TOXICITY~AS'SAY
_ ^^^^ i^B^^^^^^^^^IBBW ^
FINAL REPORT
SUBMITTED TO:
ACUREX CORPORATION
485 CLYDE AVENUE
MOUNTAIN VIEW, CALIFORNIA 94092
SUBMITTED BY:
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
LBI PROJECT NO. 22064
REPORT DATE: SEPTEMBER 1981
E5-125
BIONETICS
Litton
-------
PREFACE
This assay conforms to the standard EPA Level 1 procedure for the Chinese
hamster ovary cell (CHO) clonal toxicity assay as described in "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment Biological Tests" (1),
The data was evaluated and formatted as recommended in "Level 1 Biological
Testing Assessment and Data Formatting" (2).
The CHO clonal toxicity assay has been shown to be a sensitive method for
detecting cytotoxic activity for a variety of chemicals representing
various chemical classes (3). This assay is one of several recommended
by EPA to identify, categorize and rank the pollutant potential of
influent and effluent streams from industrial and energy-producing
processes. This assay has been well validated with a wide range of posi-
tive and negative control chemicals and complex environmental samples.
All procedures and documents pertaining to the receipt, storage, prepa-
ration, testing and evaluation of the test material shall conform to
Litton Bionetics, Inc. standard operating procedures and the Good
Laboratory Practices Act of 1979. Deviations from standard procedure
shall be fully documented and noted in the report.
All test and control results in this report are supported by fully docu-
mented raw data which are permanently maintained in the files of the
Department of Molecular Toxicology or in the archives of Litton Bionetics,
Inc., 5516 Nicholson Lane, Kensington, Maryland 20895. Copies of raw
data will be supplied to the sponsor upon request.
BIONETICS 5"126
Utton
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TABLE OF CONTENTS
Page No.
PREFACE i
I. ASSAY SUMMARY 1
II. OBJECTIVE 2
III. TEST MATERIAL 3
A. Description 3
B. Handling and Preparation 3
IV. MATERIALS 4
A. Indicator Cells 4
B. Media 4
C. Controls . 4
V. EXPERIMENTAL DESIGN 5
A. Dose Selection 5
B. Clonal Toxicity Assay 5
VI. . ASSAY ACCEPTANCE CRITERIA 7
VII. RESULTS 8
A. Interpretation . .
B. Tables and Figures
VIII. ASSAY EVALUATION CRITERIA 11
IX. REFERENCES 12
[B BIONETICS -
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I. ASSAY SUMMARY
A. SPONSOR: Acurex Corporation
B. MATERIAL (TEST COMPOUND): GENETICS ASSAY NUMBER: 5809
1. Identification: 436 (Adelphi 2 XAD extract)
2. Date Received: June 28, 1981
3. Physical Description: Clear, transparent liquid
C. TYPE OF ASSAY: Rodent Cell (CHO) Clonal Toxicity Assay
D. ASSAY DESIGN NUMBER: 442
E. STUDY DATES:
1. Initiation: August 19, 1981
2. Completion: August 26, 1981
F. SUPERVISORY PERSONNEL:
A. Study Director: Brian C. Myhr, Ph.D.
B. Laboratory Supervisor: Robert Young, M.S.
G. EVALUATION:
The test material caused only a slight increase in toxicity at
the maximum applicable dose (MAD) of 20 ul/ml (26.8 u§ of
organics per ml). Since no tested dose produced 50 percent
killing of the cells, the EC50 could not be calculated. How-
ever, the sample was ranked by IERL-EPA evaluation criteria
as being excluded from the high toxicity category and as
having moderate (M) toxicity or less. The inability to test
higher concentrations was attributable to the extremely small
quantity of test material supplied (4.7 mg).
ffl
Utton
Submitted by:
Study Director
D
Brian Myhr,
Associate Director,
Department of Molecular
Toxicology
ate
B1ONETICS
5-128
Reviewed by:
David J. Brusick, Ph.D.
Di rector,
Department of Molecular
Toxicology
Date
-------
II. OBJECTIVE
The objective of this study was to determine and rank the cytotoxicity
of sample 436 (Adelphi 2 XAD Extract) to cultured Chinese hamster cells
(CHO-K1 cell line). The measure of cytotoxicity was the reduction in
colony-forming ability after a 24-hour exposure to the test material.
After a period of recovery and growth, the number of colonies that devel-
oped in the treated cultures was compared to the colony number in unexposed
vehicle control cultures. The concentration of test material that reduced
the colony number by 50% was estimated graphically and referred to as
the EC50 value. Standard EPA Level 1 toxicity evaluation criteria for
the CHO clonal toxicity assay were used to rank the toxicity potential
of the test material.
E5-129
BIONETICS
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III. TEST MATERIAL
A. Description
The test material was received as a clear, transparent solution in
methylene chloride. The sample contained 4.7 milligrams of organic
material in an undetermined volume of methylene chloride. No infor-
mation on the sampling parameters (such as the equivalent volume of
stack gas represented by the sample) was provided.
B. Handling and Preparation
The test material was received at LBI on June 28, 1981. The sample was
assigned LBI safety number 7033 and LBI assay number 5809. The sample
was stored at +4°C in the dark.
Pretest sample preparation consisted of solvent-exchanging the sample into
dimethylsulfoxide (DM50). The sample was transferred with methylene chloride
rinses into a graduated conical tube. The methylene chloride was gradually
evaporated (50°C under stream of nitrogen) and DMSO was sequentially added.
The sample was brought to volume in 3.5 ml of DMSO, giving a sample concen-
tration of 1.34 mg organics per ml DMSO. The sample was then transferred
to a glass vial and sealed with a teflon-coated rubber septum.
A total volume of 0.45 ml of test sample was used in the CHO assay. The
maximum concentration of 20 Ml/ml was obtained by adding 0.12 ml of sample
to 5.88 ml of F12 medium; this resulted in 2% (v/v) DMSO in the medium
and effectively limited the concentration of test material that could be
assayed. Only two plates were dosed at the top dose in order to conserve
sample. Another 0.12 ml aliquot of sample was used to prepare the 10 ul/ml
test concentration. An additional 0.21 ml of test sample was used to
prepare a series of dilutions in DMSO from which 1:100 dilutions into
growth medium were performed to obtain the lower assayed concentrations.
Thus, except for the 20 ul/ml test concentration, the final DMSO concen-
tration was constant at 1% (v/v).
[H B.ONET.CS
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IV- MATERIALS
A- Indicator Cells
The indicator cells for this study were Chinese hamster CHO-K1 cells (ATCC
No. CCL 61) obtained from Flow Laboratories, Inc., Rockville, MO. This
cell type was derived from ovarian tissue and has spontaneously transformed
to a stable, hypodiploid line of rounded, fibroblastic cells with unlimited
growth potential. Monolayer cultures have a fast doubling time of 11 to
14 hours, and untreated cells can normally be cloned with an efficiency
of 80% or greater. Laboratory stock are maintained by routine serial
subpassage. Cells are cultivated in Ham's F-12 nutrient medium at 37°C
in 5 percent C02 with saturated humidity. Stocks are continually observed
macroscopically and microscopically for possible microbial contamination.
Laboratory cultures are periodically checked by culturing and staining
methods for the absence of mycoplasma contamination. Laboratory cultures
are discarded every three months and new cultures started from mycoplasma-
free, long-term frozen cultures.
B. Media
The CHO-K1 cell line has an absolute requirement for proline and therefore
must be maintained in culture medium containing sufficient amounts of this
ami no acid. Ham's F12 medium, which contains 3 x 10-4 M L-proline was used,
supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 units/ml of
penicillin, 100 ug/ml of streptomycin, and 0.9 ul/ml of amphotericin B.
A 10X formulation of Ham's F10 is available commercially and-was used for
the testing of aqueous test samples in order to avoid the dilution of
medium components. This medium contains 1 x 10-4 L-proline and was sup-
plemented in the same manner as F12, except that kanamycin at 40 ug/ml
is included for additional protection against bacterial contamination.
Both media formulations support the growth and cloning of CHO cells
equally well.
C. Controls
The negative control consists of three untreated cultures carried through
the same experimental time period as the treated cells. The test material
was supplied as a solution in an organic vehicle (usually DMSO) and was
diluted into the medium to provide each test concentration, three vehicle
control cultures containing the organic solvent at 1% or 2% by volume are
prepared. Particulate test samples were suspended in small amounts of
DMSO, but DMSO was not included in the negative control because the maxi-
mum content in the treated cultures is only 0.42%. The average number of
colonies in the negative control establishes the cloning efficiency of
the CHO cells used in the assay, and the appropriate negative or vehicle
controls provide the reference points for determining the effects of dif-
ferent concentrations of the test material on cell survival.
LJU BIONETICS 5"131
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V. EXPERIMENTAL DESIGN
A. Dose Selection
Unless the approximate toxicity is already known or the sample size is
limiting, the following dose ranges are usually tested for different
sample forms. Aqueous samples, suspensions, or slurries are tested from
600 pi/ml to 3 pi/ml, usually in six dose steps. Eight doses are often
used when the amount of test sample is limited to provide a more precise
description of toxicity in the event of sharp dose-response curves. Dry,
particulate material is dissolved or suspended in DMSO, diluted into growth
medium, and tested at six dose levels from 1000 ug/ml to 3 pg/ml. Samples
that are solvent-exchanged into DMSO are tested from 20 ul/ml (2% DMSO
in growth medium) to 0.2 pi/ml, also in six dose steps. A second dose
study is performed with an adjusted dose range if the EC50 was not located
properly in the initial test. However, EC50 values greater than 1000 pg/ml
for particulate material, 600 pi/ml for aqueous samples, or 20 ul/ml for
organic solutions will not be determined.
Sample 436 (Adelphi 2 XAD extract) was tested at eight concentrations.
The concentrations used started with the maximum applicable dose (MAD)
of 20 pi/ml and included 10, 6, 3, 1, 0.6, 0.3, and 0.1 pi of test
material per ml of culture medium. The MAD of 20 pi/ml was equivalent
to 26.8 pg of organics per ml of culture medium. The lower dose levels
were equivalent to 13.4, 8.04, 4.02, 1.34, 0.80, 0.40 and 0.13 pg of
organics per ml.
B. Clonal Toxicity Assay
Cells from monolayer stock cultures in logarithmic growth phase were tryp-
sinized with 0.1% trypsin plus 0.013 versene for 4 minutes and the density
of the resulting cell suspension determined by hemocytometer. A number
of 60-mm culture dishes were then seeded with 200 cells and 4 ml of culture
medium per dish. The cultures were incubated for approximately 6 hours
at 37°C in a humidified atmosphere containing 5% C02 to allow attachment
of the cells. The 6-hour attachment period was used in order to avoid
cell division and the subsequent formation of two-cell colonies prior to
treatment.
The medium was aspirated from the cultures and 4 ml medium containing
the test material applied. Three cultures were exposed to each test con-
centration. After an exposure time of 24 hours at 37°C, the medium was
removed by aspiration and each culture washed three times with approxi-
mately 4 ml aliquots of Dulbecco's phosphate buffered saline (pre-warmed
to 37°C). Fresh culture medium (5 ml) was placed in each dish and incuba-
tion at 37°C is continued for an additional 6 days to allow colony develop-
ment.
If the test material caused a color change in the culture medium, the pH
of the medium containing the high dose would be determined at the time
of treatment. The pH at the lowest dose that results in a slight color
EH BIONETICS 5"132
Litton
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change would also be recorded. At the end of the treatment period, tne
pH values of the discarded media from the two described treatments would
be recorded again. No sample related pH effects were noted.
After the incubation period, the medium was drained from the cultures
and the surviving colonies fixed with 100% ethanol and stained with
Giemsa. Colonies were counted by eye; tiny colonies of approximately
50 cells or less were arbitrarily excluded from the counts.
E5-133
BIONETICS
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VI. ASSAY ACCEPTANCE CRITERIA
The assay is considered acceptable for evaluation of the test results if
the following criteria are met:
The average cloning efficiency of the CHO-K1 cells in the
negative controls is 70% or greater, but not exceeding
115%.
The distribution of colonies in the treated cultures is
generally uniform over the surface of the culture dish.
The data points for each test concentration critical to
the location of the EC50 are the averages of at least two
treated cultures.
A sufficient number of test concentrations are available
to clearly locate the EC50 within a toxicity region as
defined under Assay Evaluation Criteria.
If the EC50 value is greater than 1000 ug/ml, 600 uliters
of aqueous sample/ml, or 20 uliters of nonaqueous sample/ml,
the plotted curve does not exceed 110% of the negative
control.
LLJ BIONET1CS 5'134
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VII. RESULTS
A- Interpretation
The application of sample 436 (Adelphi 2 XAD extract) to the CHO cell
cultures caused only a small decrease in the number of cells able to
form colonies as the concentration increased to the maximum applicable
dose (MAD) of 20 ul/ml. Relative survival values were calculated as the
ratio of colonies formed in treated cultures to the colonies formed in
the appropriate vehicle control, and these relative survival values were
plotted against the concentration of test material. As shown in Figure 1,
the relative survival decreased only to about 88% for the 20 ul/ml treatment.
Since none of the tested doses caused killing approaching 50% of the cells,
an EC50 could not be calculated. However, a value greater than 20 ul/ml
(26.8 ug organics/ml) excluded the sample from the high toxicity category
and placed it in the moderate range or even less toxicity, based on the
IERL-RTP evaluation criteria.1 The plotted results suggested the possi-
bility of an ECSO less than 100 ul/ml (134 ug organics/ml), so the sample
could have been evaluated as moderately toxic or being on the moderate/low
toxicity borderline had sufficient sample been available. The toxicity
of the sample was therefore evaluated as undetermined but moderate (M)
or less.
Testing and evaluating materials such as this sample indicate the need
to supply sufficient quantities of test material to ensure adequate testing.
While minimum testable sample size is often a function of the biological
activity of the sample, efforts should be made to supply at least 100 mg
of SASS train organics for combined Ames and CHO testing.
The cells used for the assay were in logarithmic growth phase and were
99.4% viable. About 99% of the cells seeded and cultured in F12 medium
formed colonies. The cloning efficiency of the 1% vehicle control was
82.9% and the 2% vehicle control, 75.4%. Colony growth was normal and
well distributed on the culture dishes. These results achieved the assay
acceptance criteria and provided confidence in the assumption that the
recorded data represented typical responses to the test material.
B. Tables and Figures
This report is based on the data provided in Table 1 and Figure 1.
OB B.ONETICS 5-135
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TABLE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
Sample Identity: 436 (Adelphi 2 XAD
Extract
Description of Sample: Clear^,
transpa re nt]i qu id
LBI Assay No.: 5809
Date Received: June 28, 1981
Test Date: August 19. 1981
Vehicle: DMSO
Cell Type: CHO-K1
Cells Seeded per Dish: 200
EC50 Value: >26.8 ug organics/ml
(>20 pi/ml)
Toxicity
Classification: Undetermined, but
Moderate (M) orTess
pH Alterations: None
Comments on
Treatment: Sample prepared in DJ1SO
at a concentration of 1.34 pg
organics/ul.
COLONY COUNTS
Applied
'Concentration Dish
Sample Ml/ml #1
NCb f
vc.ir1
VC,2%
Test
Test
Test
Test
Test
Test
Test.
Testd
«»
10
20
0.1
0.3
0.6
1.0
3.0
6.0
10.0
20.0
184
164
157
160
155
166
160
147
154
166
135
Dish
#2
187
175
148
166
179
169
166
162
164
160
130
Dish
#3
182
158
147
158
145
150
156
171
143
153
"""*""""""
Average
Count
184.3
165.7
150.7
161.3
159.7
161.7
160.7
160.0
153.7
159.7
132.5
Relative
Survival
...
100.0
100.0
97.3
96.4
97.6
97.0
96.6
92.8
96.4
87.9
Cloning
Efficiency
92.2
82.9
75.4
Relative to 2% VC for 20 Ml/ml treatment and to 1% VC for other treatments.
NC = Negative Control, F12 medium.
= Vehicle Control, percent DMSO given.
Only two plates dosed to conserve limited test material.
m
Litton
BIONETICS
5-136
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FIGURE 1
RODENT CELL (CHO) CLONAL TOXICITY ASSAY
EC50 DETERMINATION
436 (ADELPHI 2 XAD EXTRACT)
CONCENTRATION, pi/ml
5-137
10
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VIII. ASSAY EVALUATION CRITERIA
The EC50 value represents the concentrations of test material that reduces
the coTony-forming ability of CHO cells to 50% of the vehicle or negative
control value. EC50 values are determined graphically by fitting a curve
fay eye through relative survival data plotted as a function of the loga-
rithm of the applied concentration. Each data point normally represents
the average of three culture dishes. In order to indicate the variability
of the data, the high and low colony counts for each concentration are
used to calculate the relative survivals, and the range is shown by a
bar at the position of the plotted average. If no bar is shown, the
variability was within the size of the symbol. Statistical analysis is
unnecessary in most cases for evaluation.
The toxicity of the test material is evaluated as high, moderate, low,
or nondetectable according to the range of EC50 values defined in the
following table.
Solids
Toxicity (EC50 in ug/ml)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids
(EC50 in Ml /ml)
<6
6 to 60
60 to 600
>600
Nonaqueous Liquids"
(EC50 in ul/ml)
<.2
.2-2
2-20
>20
Evaluation criteria formulated by Litton Bionetics, Inc. for IERL-RTP
Procedures Manual: Level Environmental Assessment Biological Tests.1
Criteria for nonaqueous liquids are tentative and under evaluation.
If the organic or solids content is known, the sample is evaluated
under the solids criteria.
Another evaluation scheme is proposed for extracts obtained from SASS
train gas volumes. The proportion of the total gas volume corresponding
to the volume of extract used in the bioassay is calculated and expressed
as I/ml of culture medium (or DSCF/ml of culture medium). A criterion
of 1000 L/ml is set as the limit for nondetectable toxicity. This gas
volume corresponds to the average volume breathed by humans over a 2-hour
period. The subsequent toxicity ranges are defined by 10-fold dilution
steps to conform to standard procedure. The toxicity ranges are defined
in the following table for liter and dry standard cubic feet units:
EC50 In ECSO In
Toxicity Liters/ml (L/ml) Dry Standard Cubic Feet/ml (DSCF/ml)
High 1000 >35
m ,,
BIONETICS n
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IX. REFERENCES
1. Brusick, D.J. , et a_l_.: IERL-RTP Procedures Manual: Envi ronmenta'
Assessment Biological Tests. EPA Contract No. 68-02-2681, Technical
Directive No. 501, Litton Bionetics, Inc., Kensington, MD,
September 1980, 177 pp. In press.
2. Brusick, D.J.: Level 1 Bi'oassay Assessment and Data Formatting.
EPA-600/7-80-079, Litton Bionetics, Inc., Kensington, MD, April 1980,
100 pp.
3. Brusick, D.J. and Young, R.R.: Level 1 Environmental Assessment:
Limits of Bioassay Resolution andDetectability.EPA Contract
No. 68-02-268, Technical Directive No. 301, Litton Bionetics,
Inc., Kensington, MD, February 1981, 56 pp. In press.
E 5-139 12
BIONETICS
Litton
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TECHNICAL REPORT DATA
(Please read Imirucnum on the re: era' before completing!
£pA-600''7-84-095b
3. RECIPIENT'S ACCESSION NO.
-:T_£ ANO SJBT.TLE
Environmental Assessment of a Firetube Boiler Firing
Coal/Oil/Water mixtures; Volume II. Data Supple-
ment
S. REPORT DATE
September 1984
6. PERFORMING ORGANIZATION CODE
? AUT«ORlS>
R. DeRosier
8. PERFORMING ORGANIZATION REPORT NO.
TR-81-89/EE
9 OERFOSMING ORGANIZATION NAME AND ADDRESS
.A cur ex Corporation
Energy and Environmental Division
P. O. Box 7555
Mountain View, California 94039.
10. PRC'GRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-02-3188
12 SPONSORING AGENCY NAME ANO ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final; 2/81 - 11/83
14. SPONSORING AGENCY CODE
EPA/600/13
is. SUPPLEMENTARY NOTES IZRL-RTP project officer is Robert E. Hall. Mail Drop 65, 919/
541-2477. Volume I is the Technical Results.
16. ABSTRACT
volume is a compendium of detailed emission and test data from field
tests of a firetube industrial boiler burning a coal/ oil /water (COW) mixture. The
boiler was tested while burning COW fuel, and COW with soda ash added (COW+SA)
to serve as an SO2 sorbent. The test data include: preliminary equipment calibration
data, boiler operating data for both tests, fuel analysis results, and .complete flue
gas emission measurement and laboratory analysis results. Flue gas emission mea-
surements included: continuous monitoring for criteria gas pollutants; gas chroma-
tography (GC) of gas grab samples for volatile organics (C1-C6); EPA Method 5 for
particulate; controlled condensation system for SO2 emissions; and source assess-
ment sampling system (SASS) for total organics in two boiling point ranges (100 to
300 C and > 300 C), organic compound category information using infrared spectro-
metry (IR) and low resolution mass spectrometry (LRMS), specific quantitation of
the semivolatile organic priority pollutants using gas chromatography/mass spectro-
metry (GC/MS), liquid chromatography (LC) separation of organic extracts into
seven polarity fractions with total organic and IR analyses of eluted fractions, flue
gas concentrations of trace elements by spark source mass spectrometry (SSMS) and
atomic absorption spectroscopy (AAS), and biological assays of organic extracts.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSAT1 Field/Group
Pollution
Fire Tube Boilers
Coal
Fuel Oil
Water
Sodium Carbonates
Assessments
Flue Gases
Pollution Control
Stationary Sources
Coal/Oil/Water Mixture
Environmental Assess-
ment
13B
ISA
21D
07B
14 B
21B
3. OlSTfliBUTlON STATEMENT
Release to Public
19. SECURITY CLASS ITtta Report)
Unclassified
21. NO. OF PAGES
178
20. SECURITY CLASS fTnit page)
Unclassified
22. PRICE
EPA form 2220-t (9-73)
5-140
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